Acid Phosphatases Do Not Contribute to the Pathogenesis of Type A Francisella tularensis

The intracellular pathogen Francisella tularensis is the causative agent of tularemia, a zoonosis that can affect humans with potentially lethal consequences. Essential to Francisella virulence is its ability to survive and proliferate within phagocytes through phagosomal escape and cytosolic replication. Francisella spp. encode a variety of acid phosphatases, whose roles in phagosomal escape and virulence have been documented yet remain controversial. Here we have examined in the highly virulent (type A) F. tularensis strain Schu S4 the pathogenic roles of three distinct acid phosphatases, AcpA, AcpB, and AcpC, that are most conserved between Francisella subspecies. Neither the deletion of acpA nor the combination of acpA, acpB, and acpC deletions affected the phagosomal escape or cytosolic growth of Schu S4 in murine and human macrophages, despite decreases in acid phosphatase activities by as much as 95%. Furthermore, none of these mutants were affected in their ability to cause lethality in mice upon intranasal inoculation. Hence, the acid phosphatases AcpA, AcpB, and AcpC do not contribute to intracellular pathogenesis and do not play a major role in the virulence of type A Francisella strains.The Gram-negative bacterium Francisella tularensis is a highly infectious, facultative intracellular pathogen that causes tularemia, a widespread zoonosis affecting humans. Human tularemia is a fulminant disease that can be contracted by exposure to as few as 10 bacteria, the pneumonic form of which can lead to mortality rates as high as 25% if untreated (35). Three subspecies of F. tularensis, Francisella tularensis subsp. tularensis (type A), Francisella tularensis subsp. holarctica (type B), and Francisella tularensis subsp. mediasiatica, are recognized, among which strains of the first two subspecies can cause tularemia in humans (15). While type B strains are geographically distributed all over the northern hemisphere, the highly virulent type A strains are restricted to North America and account for the most-severe cases of the disease. Francisella novicida, a species of low virulence in humans but high virulence in rodents, has been used extensively as a surrogate model of F. tularensis pathogenesis, based on the assumption that it uses conserved virulence mechanisms (4, 7, 8, 19, 23, 25-29, 31, 41-45, 47). As a facultative intracellular pathogen, F. tularensis is capable of infecting and proliferating in a variety of host cell types, including hepatocytes, epithelial cells, and mononuclear phagocytes (15). Macrophages constitute an important target for infection in vivo (21), and the pathogenesis of F. tularensis depends on the bacterium''s ability to survive and replicate within these host cells (15). Upon phagocytosis, Francisella ensures its effective survival and proliferation via rapid phagosomal escape followed by extensive replication in the cytosol (11, 14, 20, 42), thereby segregating itself from the degradative endosomal system and its associated bactericidal activities. Phagosomal escape is a tightly regulated process whose efficiency depends on conditions encountered within the early phagosome (12, 41), such as vacuolar acidification, although some controversy remains as to whether Francisella-containing phagosomes are significantly acidified prior to membrane disruption (13). Regardless of such discrepancies, phagosomal escape is an essential step in Francisella intracellular pathogenesis, since it is a prerequisite for cytosolic replication. Indeed, Francisella mutants that are defective in phagosomal escape do not grow intracellularly and are attenuated in vivo (6, 24, 43-45), and a belated phagosomal escape delays intracellular proliferation of the highly virulent type A strain Schu S4 (12).Much effort has focused on identifying bacterial factors that contribute to phagosomal escape. Several genes located within a 30-kb chromosomal locus known as the Francisella pathogenicity island (FPI) (31) are required for proper phagosomal escape of F. novicida (43, 44) and the attenuated F. tularensis subsp. holarctica live vaccine strain (LVS) (6, 24), since transposon insertions or targeted deletions in iglC, iglD, and pdpA affect the translocation of the mutants to the cytosol. Based on the homology of some FPI proteins with components of type VI secretion systems in other pathogens (30, 36), the FPI likely encodes a secretion apparatus that is required for phagosomal disruption. Yet a true understanding of FPI functions and the characterization of actual Francisella effectors of phagosomal escape are lacking. In addition to the FPI, Mohapatra et al. have recently reported for F. novicida that the acid phosphatases AcpA, AcpB, AcpC, and Hap are required for phagosomal escape and virulence in mice (27, 29). Acid phosphatases, which are ubiquitous in nature and hydrolyze phosphomonoesters at acidic pHs, have been associated with the survival of intracellular parasites within phagocytes through inhibition of the respiratory burst (1, 3, 9, 22, 37-40), suggesting that they act as virulence factors. In Francisella, a prominent role was established for AcpA, an unusual, respiratory-burst-inhibiting enzyme exemplifying a novel family of acid phosphatases (18, 37). AcpA accounts for most of the acid phosphatase and phospholipase activities in the outer membrane fraction of F. novicida (29). These reports assigned acid phosphatases a role in phagosomal escape yet contradicted a previous study by Baron et al., who concluded that AcpA was not required for the intracellular growth or virulence of F. novicida (4). While the acpA mutants were constructed differently in these studies, the acid phosphatase activity associated with AcpA was abolished in both situations. A proposed explanation for these conflicting results was that the truncated AcpA generated by Baron et al. remained functional as a phospholipase C (37), an activity that would be required for phagosomal escape and virulence (27). Yet this hypothesis has not been tested, leaving the role of AcpA in Francisella virulence a controversial matter.All studies of Francisella acid phosphatases have been carried out with F. novicida (4, 27, 29, 37), raising the question of significance with regard to the virulent F. tularensis subspecies. In particular, recent whole-genome comparisons between F. novicida and the different Francisella tularensis subspecies have highlighted important intervening sequence (IS)-mediated genome rearrangements in F. tularensis subsp. holarctica and F. tularensis subsp. tularensis strains relative to F. novicida (10). Such rearrangements have disrupted large numbers of open reading frames (ORFs), thereby creating pseudogenes (10) and likely inactivating many functions in virulent F. tularensis strains. For example, Mohapatra et al. (29) have reported that the virulent type A strain Schu S4 is missing a homolog of one of the two hap genes (FTN_0022) present in F. novicida, raising the question of conservation of acid phosphatase-encoding genes in virulent strains. Because phagosomal escape is an essential stage of the Francisella intracellular cycle that is common to F. novicida and F. tularensis, we have postulated that factors required to promote this process must be conserved between these organisms. Here we have compared acid phosphatase-encoding genes in F. novicida and virulent F. tularensis subspecies, and we have generated deletion mutants of the most conserved genes in Schu S4 in order to test their role in the phagosomal escape and pathogenesis of the highly virulent F. tularensis subspecies. We demonstrate that most acid-phosphatase-encoding genes are disrupted in virulent strains and that the most conserved loci are not required for phagosomal escape and virulence.     

Vaccination with an Attenuated Strain of Francisella novicida Prevents T-Cell Depletion and Protects Mice Infected with the Wild-Type Strain from Severe Sepsis

Francisella tularensis is the causative agent of zoonotic tularemia, a severe pneumonia in humans, and Francisella novicida causes a similarly severe tularemia in mice upon inhalation. The correlates of protective immunity, as well as the virulence mechanisms of this deadly pathogen, are not well understood. In the present study, we compared the host immune responses of lethally infected and vaccinated mice to highlight the host determinants of protection from this disease. Intranasal infection with an attenuated mutant (Mut) of F. novicida lacking a 58-kDa hypothetical protein protected C57BL/6 mice from a subsequent challenge with the fully virulent wild-type strain U112 via the same route. The protection conferred by Mut vaccination was associated with reduced bacterial burdens in systemic organs, as well as the absence of bacteremia. Also, there was reduced lung pathology and associated cell death in the lungs of vaccinated mice. Both vaccinated and nonvaccinated mice displayed an initial 2-day delay in upregulation of signature inflammatory mediators after challenge. Whereas the nonvaccinated mice developed severe sepsis characterized by hypercytokinemia and T-cell depletion, the vaccinated mice displayed moderated cytokine induction and contained increased numbers of αβ T cells. The recall response in vaccinated mice consisted of a characteristic Th1-type response in terms of cytokines, as well as antibody isotypes. Our results show that a regulated Th1 type of cell-mediated and humoral immunity in the absence of severe sepsis is associated with protection from respiratory tularemia, whereas a deregulated host response leading to severe sepsis contributes to mortality.The causative agent of respiratory tularemia, Francisella tularensis, is a gram-negative intracellular bacterium. There are four closely related subspecies of F. tularensis, F. tularensis subsp. tularensis (type A), F. tularensis subsp. holarctica (type B), F. tularensis subsp. mediasiatica, and “F. tularensis subsp. novicida,” and type A is the most virulent subspecies in humans (20). This pathogen is capable of causing acute respiratory infection following inhalation of as few as 10 organisms (10, 48). This extremely low infectious dose, the ease of transmission via the aerosol route, and the wide host range have led the CDC to recognize this pathogen as a potential bioweapon (56). Since the fully virulent strains of F. tularensis are highly infectious, much of our knowledge about Francisella pathogenesis has been obtained by using the attenuated live vaccine strain (LVS) derived from a type B strain of F. tularensis or Francisella novicida. Although attenuated for humans, F. novicida is virulent in mice and results in a disease that closely resembles human tularemia. Despite continuous efforts, an effective vaccine for tularemia has not been developed yet. This highlights the need for understanding the virulence mechanisms of Francisella, as well as the correlates of protective immunity, in order to devise effective therapeutics for use against tularemia.Primary respiratory infections with Francisella cause a delay in the initial innate immune response. This initial delay has been postulated to be an important virulence mechanism of the organism (2, 3, 39, 40). An absence of this initial immune response is thought to aid rapid multiplication of bacteria, followed by dissemination of the bacteria to systemic organs, resulting in bacteremia. This causes widespread upregulation of multiple cytokines and chemokines that reflects contributions from both the host and the pathogen to an inappropriate inflammatory response (40, 59, 64). This kind of unbridled host response to a pathogen is now broadly accepted as the cause of host death in infectious diseases like malaria, influenza, and sepsis (6). In light of the absence of any known endo- or exotoxin activity of any virulence factor of Francisella, this hyperimmune response seems to be the cause of the mortality associated with respiratory tularemia (54).Adaptive immune responses following vaccination, as well as during sublethal infections, have highlighted the contributions of both B and T lymphocytes (8, 16, 44, 53). Most of the studies have been carried out with type B-infected humans, as well as mice (65). Both humans and mice develop antigen-specific antibodies, as well as CD4⁺ and CD8⁺ T cells, during sublethal infections (15, 17, 26, 57). The effector T-cell mechanisms that control the infections involve mainly gamma interferon (IFN-γ) and/or tumor necrosis factor alpha (TNF-α) (9, 66), but bacterial killing is partially mediated by NO produced by IFN-γ-activated macrophages (4, 14). However, a comprehensive study of the mechanisms triggering rapid death following systemic dissemination of bacteria before the onset of acquired immunity and the factors involved in bacterial clearance and host protection from lethal respiratory infection in the same experimental setting has not been done.Analysis of the genome sequence of Francisella revealed a family of five hypothetical proteins unique to this organism (38). One of these factors, a protein encoded by the FTT_0918 gene, has been shown to be a virulence factor, as mutants of type A strains lacking this gene are attenuated for infection in vitro and in vivo. In addition, intradermal inoculation with this mutant protects mice from intranasal challenge with virulent type A strains (63, 65). Our in vivo studies with the murine model organism F. novicida have shown that a transposon mutant (Mut) lacking a homolog of this 58-kDa protein is equally attenuated (54). In the current study we tested this mutant to determine whether it protects against murine respiratory tularemia and determined the host immune responses associated with protection. Intranasal immunization of C57BL/6 mice with Mut protected the mice from a subsequent challenge with an otherwise lethal dose of the wild-type (WT) bacteria. Importantly, the severe sepsis characterized by hypercytokinemia and bacteremia observed in nonvaccinated mice was not present in lungs of mice vaccinated with the mutant. Instead, a protective Th1 type of cytokine and antibody response was upregulated. Our results show that in the apparent absence of any endotoxins or exotoxins that could account for the lethality associated with respiratory tularensis, severe sepsis coupled with a lack of adaptive responses due to T-cell depletion is likely the major contributor to the severity of the disease and associated mortality, and an effective Th1 type of response coupled with the absence of severe sepsis and bacteremia is key for protection against this deadly infection.     

Francisella tularensis Phagosomal Escape Does Not Require Acidification of the Phagosome

Following uptake, Francisella tularensis enters a phagosome that acquires limited amounts of lysosome-associated membrane glycoproteins and does not acquire cathepsin D or markers of secondary lysosomes. With additional time after uptake, F. tularensis disrupts its phagosomal membrane and escapes into the cytoplasm. To assess the role of phagosome acidification in phagosome escape, we followed acidification using the vital stain LysoTracker red and acquisition of the proton vacuolar ATPase (vATPase) using immunofluorescence within the first 3 h after uptake of live or killed F. tularensis subsp. holarctica live vaccine strain (LVS) by human macrophages. Whereas 90% of the phagosomes containing killed LVS stained intensely for the vATPase and were acidified, only 20 to 30% of phagosomes containing live LVS stained intensely for the vATPase and were acidified. To determine whether transient acidification might be required for phagosome escape, we assessed the impact on phagosome permeabilization of the proton pump inhibitor bafilomycin A. Using electron microscopy and an adenylate cyclase reporter system, we found that bafilomycin A did not prevent phagosomal permeabilization by F. tularensis LVS or virulent type A strains (F. tularensis subsp. tularensis strain Schu S4 and a recent clinical isolate) or by “F. tularensis subsp. novicida,” indicating that F. tularensis disrupts its phagosomal membrane by a mechanism that does not require acidification.Francisella tularensis is a gram-negative facultative intracellular bacterium that causes a zoonosis in animals and a potentially fatal infection, tularemia, in humans. F. tularensis consists of four subspecies, F. tularensis subsp. tularensis, F. tularensis subsp. holarctica, F. tularensis subsp. mediasiatica and “F. tularensis subsp. novicida,” whose geographic distributions and virulence in humans differ (12, 25). F. tularensis subsp. tularensis (type A), found almost exclusively in North America, is highly virulent for humans. As few as 10 organisms received subcutaneously or 25 organisms received by inhalation can lead to a severe infection (32, 33). F. tularensis subsp. holarctica (type B, found in North America and in Europe) and F. tularensis subsp. mediasiatica (found in Asia) are less virulent. F. tularensis subsp. novicida, found in North America and Australia, is virulent in mice and has occasionally been reported to cause a mild disease, compared with type A infections, in humans (38). Because of its high infectivity and capacity to cause severe morbidity and mortality, F. tularensis subsp. tularensis is considered a potential agent of bioterrorism and is classified as a category A select agent.In animal models of tularemia, macrophages are important host cells for F. tularensis, and the virulence of the bacterium correlates with its capacity to grow in macrophages (2, 20). We have shown previously that efficient uptake of F. tularensis subsp. tularensis and F. tularensis subsp. holarctica live vaccine strain (LVS) by human macrophages requires complement and that it is mediated by a unique process involving spacious, asymmetric pseudopod loops (10). The mannose receptor (34) and class A scavenger receptors (26) have also been reported to play a role in uptake of F. tularensis LVS. We have demonstrated that following uptake, the bacterium enters a membrane-bound vacuole that acquires limited amounts of endosomal markers, including limited amounts of the late endosomal-lysosomal markers CD63, LAMP1, and LAMP2, but that the vacuole does not acquire the acid hydrolase cathepsin D, does not fuse with lysosomes, and is only minimally acidified to a pH of 6.7 at 3 h postinfection (11). With additional time after uptake, F. tularensis disrupts the phagosomal membrane and the bacterium escapes and replicates in the host cell cytosol (9, 11, 16). Celli and coworkers have studied the interaction of mouse bone marrow-derived macrophages with F. tularensis LVS (6) and F. tularensis Schu S4 (7) and also reported a transient interaction with the host endocytic pathway prior to escape with a more rapid kinetic profile than we have observed in human monocyte-derived macrophages (MDM). In addition, Chercoun et al. (6) have reported that at late times after infection (20 h) in mouse macrophages, a large proportion of F. tularensis cells enter an autophagosomal compartment. The Francisella pathogenicity island has been shown to be essential for the altered intracellular trafficking and escape of F. tularensis subsp. holarctica LVS (22) and F. tularensis subsp. novicida (31) into the cytoplasm.Some degree of acidification has been shown to be required for the escape of certain intracellular pathogens that replicate in the cytosol. For example, acidification of the vacuole occupied by Listeria monocytogenes is required for activation of listeriolysin O for permeablization of the vacuole (1), and acidification of either early or late endosomes is required for pH-dependent changes in adenoviral proteins to mediate the translocation of adenovirus into the host cell cytoplasm (23). While we have reported previously that the F. tularensis phagosome is only minimally acidified to a pH of 6.7 at 3 h postinfection, this finding does not preclude the possibility that some degree of acidification, even transient acidification, might be required for the bacterium to disrupt its phagosome and escape into the cytoplasm. Indeed, Santic et al. recently reported that nearly 80 to 85% of F. tularensis subsp. novicida phagosomes are acidified at 15 to 30 min postinfection in human MDM and that inhibition of acidification with bafilomycin A completely blocks escape (30). In contrast to these results for human macrophages with F. tularensis subsp. novicida, Chong et al. (7) have recently reported that F. tularensis Schu S4 phagosomes in mouse bone marrow-derived macrophages are transiently acidified and that inhibition of acidification delays, but does not prevent, phagosome disruption. To explore the importance of phagosomal pH on subsequent intracellular trafficking events for F. tularensis in human macrophages, we have examined the time course of colocalization of F. tularensis with the proton vacuolar ATPase (vATPase) and with a vital stain for acidified compartments, and we have examined the effect of inhibitors of acidification on phagosomal disruption.     

Directed Screen of Francisella novicida Virulence Determinants Using Drosophila melanogaster

Francisella tularensis is a highly virulent, facultative intracellular human pathogen whose virulence mechanisms are not well understood. Occasional outbreaks of tularemia and the potential use of F. tularensis as a bioterrorist agent warrant better knowledge about the pathogenicity of this bacterium. Thus far, genome-wide in vivo screens for virulence factors have been performed in mice, all however restricted by the necessity to apply competition-based, negative-selection assays. We wanted to individually evaluate putative virulence determinants suggested by such assays and performed directed screening of 249 F. novicida transposon insertion mutants by using survival of infected fruit flies as a measure of bacterial virulence. Some 20% of the genes tested were required for normal virulence in flies; most of these had not previously been investigated in detail in vitro or in vivo. We further characterized their involvement in bacterial proliferation and pathogenicity in flies and in mouse macrophages. Hierarchical cluster analysis of mutant phenotypes indicated a functional linkage between clustered genes. One cluster grouped all but four genes of the Francisella pathogenicity island and other loci required for intracellular survival. We also identified genes involved in adaptation to oxidative stress and genes which might induce host energy wasting. Several genes related to type IV pilus formation demonstrated hypervirulent mutant phenotypes. Collectively, the data demonstrate that the bacteria in part use similar virulence mechanisms in mammals as in Drosophila melanogaster but that a considerable proportion of the virulence factors active in mammals are dispensable for pathogenicity in the insect model.Francisella tularensis is the causative agent of tularemia, a zoonotic disease affecting a wide variety of small vertebrates as well as humans (49). The severity and the clinical manifestations of the disease are highly dependent on the infecting strain and the route of entry. If inhaled, as few as 10 bacteria can cause infection in humans, and if untreated, the mortality rate can reach 60% (45). To date, two subspecies that cause disease in humans, F. tularensis subspecies holarctica and F. tularensis subspecies tularensis, have been identified. A closely related species, F. novicida, is an environmental pathogen and appears not to affect healthy humans, since F. novicida infections have been reported almost exclusively in immunocompromised individuals (8, 19, 58), but it has been recognized as relevant when Francisella virulence in various mouse infection models is investigated. Genome comparisons revealed high sequence similarities between the F. novicida isolate U112 and the clinically important F. tularensis subspecies tularensis strain SCHU S4. The former represents the evolutionarily oldest and most complete Francisella genome, supporting good metabolic competence, while human-pathogenic strains, in adaptation to an intracellular niche, have lost many genes to genetic drift and are much more fastidious (26, 43).The only tularemia treatment to date relies on antibiotics. To enable development of vaccines and new antimicrobial drugs, it is vital to understand the molecular mechanisms behind the interaction of this pathogen with humans. F. tularensis escapes from the host cell phagosome and propagates in the cytosol (16). Multiplication results in cell death and release of bacteria (25), allowing them to spread to regional lymph nodes and to colonize spleen, liver, and lung (52). A substantial proportion of the bacterial burden can persist extracellularly in the bloodstream (14, 59).Despite knowledge about the in vivo life cycle, genome sequence data, and techniques for mutant generation, we still know little about specific virulence determinants of F. tularensis. Factors that are known to play a role are involved in lipopolysaccharide biosynthesis or intracellular survival. A focus of attention has been a genomic region called the Francisella pathogenicity island (FPI), which is required for escape from the phagosome and proliferation inside the cytosol and which encodes a putative type VI secretion system (reviewed in references 2, 13, and 35).Lately, libraries of transposon insertion mutants of different Francisella reference strains were used to screen for virulence factors in various mammalian in vitro and in vivo infection models (23, 31, 38, 51, 53, 57). In vivo screens in mice applied a competition-based, negative-selection strategy to identify bacterial mutants that cannot survive in and/or colonize a target organ (23, 51, 57). Results from these studies suggested a large number of genes to be involved in Francisella pathogenicity. While this strategy is a sensitive and efficient way to screen the whole bacterial genome, it probably overestimates the number of virulence genes and their importance as well as providing limited information as to the specific roles of individual genes in pathogenesis.Thus, far, the mouse has been the preferred model host for in vivo studies of Francisella. Recently, however, analysis of various human pathogens in model organisms like Caenorhabditis elegans or Drosophila melanogaster have demonstrated that bacteria to a large extent rely on the same virulence strategies in invertebrates as in humans (24). Because of their simplicity and the genetic tools available, nonmammalian models offer a unique opportunity to unravel the basis of host-pathogen interactions in great detail. Our previous work, in which we introduced D. melanogaster as a model host for Francisella infections, suggested that the fruit fly might be valuable for the identification and characterization of virulence determinants (56). In addition, blood-feeding arthropods, like ticks, mosquitoes, and biting flies, have long been acknowledged as vectors of tularemia (34, 37), implying that the bacterium has evolved strategies for persistence and replication in such organisms. Drosophila melanogaster might not represent a natural host for Francisella tularensis, but it is a highly relevant model for immune mechanisms and basic physiology in arthropods.Here we individually analyzed nearly 250 genes that had previously been suggested as candidate virulence determinants by negative-selection screens in mice in order to confirm their importance for pathogenicity in a robust infection model. By using survival of infected flies as a measure of bacterial virulence, we identified 49 genes as being required for normal virulence in flies. These genes were further investigated for their role in bacterial proliferation in flies and in mouse macrophage-like cells. All mutant phenotypes were analyzed by hierarchical cluster analysis to provide new insights into functional relationships among the corresponding genes. Our collected data and comparison of bacterial mutants in two model systems also allowed for an evaluation of D. melanogaster as a screening model.     

Francisella tularensis Schu S4 O-Antigen and Capsule Biosynthesis Gene Mutants Induce Early Cell Death in Human Macrophages

Francisella tularensis is capable of rampant intracellular growth and causes a potentially fatal disease in humans. Whereas many mutational studies have been performed with avirulent strains of Francisella, relatively little has been done with strains that cause human disease. We generated a near-saturating transposon library in the virulent strain Schu S4, which was subjected to high-throughput screening by transposon site hybridization through primary human macrophages, negatively selecting 202 genes. Of special note were genes in a locus of the Francisella chromosome, FTT1236, FTT1237, and FTT1238. Mutants with mutations in these genes demonstrated significant sensitivity to complement-mediated lysis compared with wild-type Schu S4 and exhibited marked defects in O-antigen and capsular polysaccharide biosynthesis. In the absence of complement, these mutants were phagocytosed more efficiently by macrophages than wild-type Schu S4 and were capable of phagosomal escape but exhibited reduced intracellular growth. Microscopic and quantitative analyses of macrophages infected with mutant bacteria revealed that these macrophages exhibited signs of cell death much earlier than those infected with Schu S4. These data suggest that FTT1236, FTT1237, and FTT1238 are important for polysaccharide biosynthesis and that the Francisella O antigen, capsule, or both are important for avoiding the early induction of macrophage death and the destruction of the replicative niche.Much of the recent interest in Francisella tularensis, the etiological agent of tularemia, is due to concern about its potential use as an agent of bioterrorism coupled with an incomplete understanding of the molecular basis of its pathogenicity. F. tularensis is highly pathogenic by the pneumonic route, causing disease in humans with an inoculum as small as 10 organisms, and infection by this route carries a mortality rate of 30 to 60% if untreated (43, 67). Due to its extreme virulence and ease of aerosol dissemination, several nations have weaponized F. tularensis and the U.S. Centers for Disease Control and Prevention have classified this organism as a category A select agent (20). F. tularensis has a remarkably broad host range: it is capable of infecting over 250 known species from across the entire phylogenetic tree, including amoebae, insects, small mammals (such as rodents and lagomorphs), and primates (51). Tularemia is primarily a zoonosis, and humans are thought to be accidental hosts (23). The majority of human infections, the pneumonic infections reported on Martha''s Vineyard in 2000 (21) being an notable exception, are cutaneous, lead to ulceroglandular disease, and ensue following exposure to infected animals or animal products (47). The ability of F. tularensis to infect such a wide range of eukaryotes suggests that this organism either co-opts cellular mechanisms common to all hosts, has the requisite virulence genes to adapt to many different intraorganismal environments, or both.Despite the infectivity of Francisella for disparate hosts, relatively little is known about its virulence genetics. F. tularensis invades and replicates within many cell types: phagocytes, such as primary macrophages (human monocyte-derived macrophages [MDMs] and mouse bone marrow-derived macrophages) and macrophage-like cell lines (J774A.1 and THP-1), as well as in nonphagocytic cells such as bronchial airway epithelial cells, hepatocytes, human umbilical vein endothelial cells, and epithelium-derived tissue culture cell lines (i.e., HEp-2, A549, HBE, and HepG2) (17, 24, 31, 40, 61). Macrophages are known to bind and phagocytose F. tularensis using at least three receptors: complement receptor 3 (CR3), which binds to complement 3b (C3b) protein deposited upon the bacterium when exposed to fresh serum (14) (60); mannose receptor (MR) (60); and scavenger receptor A (53). Once internalized, F. tularensis organisms are able to alter intracellular trafficking of their phagosomes and prevent fusion with the lysosome, acquiring late endosomal markers such as lamp-1 transiently and altogether avoiding endosomes containing cathepsin D and S (15). Shortly thereafter, F. tularensis escapes from the phagosome and grows in the macrophage cytosol. The majority of genes known to be important for phagosomal escape and intracellular growth lie within the duplicated Francisella pathogenicity island (FPI), an ∼30-kb region of the chromosome carrying the igl and pdp operons (46). Mutants with mutations in many of the genes in the FPI, such as iglABCD, vgrG, and iglI, as well as pdpA and pdpD, are defective for intracellular growth in both primary and tissue culture cells in vitro, and these genes may encode a novel type VI secretion system (4). Mutational analysis implicates genes such as mglA, sspA, fevR, fslA, pmrA, and migR in regulation of FPI genes (5-8, 10, 38). However, knowledge of other virulence genes residing outside the FPI that play important roles in intracellular growth is limited.We undertook a high-throughput approach to identify genes important for the growth of virulent F. tularensis Schu S4 in primary human macrophages. F. tularensis has two major biovars: type B (F. tularensis subsp. holarctica), which is found throughout the Northern hemisphere, and type A (F. tularensis subsp. tularensis), which is found exclusively within North America and typically causes a more severe disease in humans than type B isolates. These biovars are further subdivided into clades that exhibit differences in virulence phenotypes (45). A significant research effort to date has focused on the virulence properties of the non-human pathogens Francisella novicida and the F. tularensis live vaccine strain (LVS), an attenuated F. tularensis subsp. holarctica variant generated in the Soviet Union in the 1950s. Although both of these strains are avirulent in healthy humans, they can grow in some human cells and cell lines in vitro and are virulent in the mouse model of infection, properties that make them attractive models for studying the pathogenesis of tularemia. Several random transposon mutant libraries generated in F. novicida and F. tularensis LVS (41, 50, 55, 66, 68, 70) have been screened using both in vitro tissue culture models and mice. In addition, Qin and Mann generated an ∼700-member Tn5 (EZ-TN) mutant library in the type A strain F. tularensis Schu S4 and screened each clone through the hepatocyte-like cell line HepG2 to detect mutants defective in intracellular growth. Among the genes identified in this screen is FTT1236 (55).Lipopolysaccharide (LPS) is a major component in the outer membranes of Gram-negative organisms, and O antigens (O Ags) are known virulence factors of many pathogenic bacteria that function in part to protect against damage by serum complement and antimicrobial peptides as well as mask bacterial surface antigens (16). LPS is composed of a lipid A moiety that secures it to the Gram-negative outer membrane on which a core polysaccharide and O Ag are assembled. Whereas the LPS of most bacterial species is recognized as a pathogen-associated molecular pattern (PAMP) through MD-2/TLR4 and is a potent activating signal for the innate immune system, the LPS of F. tularensis is nearly inert (27). Of note, the lipid A of F. tularensis has an atypical structure, does not bind to LPS-binding protein (LBP), and is therefore not recognized by TLR2 or TLR4 (3, 28, 52). In addition, the F. tularensis O Ag is structurally distinct from that of F. novicida (43). Although our understanding of the genetics of O-Ag biosynthesis in F. tularensis is incomplete, it is known that wbt operon mutants of F. tularensis LVS do not express an O Ag. These strains bind C3b more avidly and are more sensitive to bile salts and to complement-mediated lysis than wild-type strains (13, 39). These mutants are also defective for intracellular growth in J774A.1 cells and exhibit reduced virulence and dissemination in mice (41, 56, 62, 69). Of note, an LVS FTL0706 (designated FTT1238 in F. tularensis Schu S4) mutant lacks an O Ag and exhibits decreased replication within, and is cytotoxic to, J774A.1 cells (41). A corresponding Schu S4 FTT1238 mutant also lacks an O Ag, but its virulence properties have not yet been described.In many pathogenic bacteria, capsular polysaccharides also contribute to serum resistance yet, unlike O Ags, diminish phagocytosis (16). Francisella has long been thought to have an extracellular structure that resembles that of a capsular polysaccharide. Observed by Hood in 1976 (30), the nature of this capsule has remained elusive. Acridine orange treatment was used to produce an undefined LVS mutant that appears to lack an extracellular polysaccharide structure (58). This strain was designated Cap⁻ (alternately termed “rough” in more recent work) and is serum sensitive (13, 62). Furthermore, expression of the capsular polysaccharide was observed to be increased by repeated passage of LVS on Chamberlain''s defined medium, which in turn increased virulence in mice (12). Recently, our group has identified a capsular polysaccharide using a new monoclonal antibody that recognizes this structure as distinct from LPS O Ag. This capsule has immunological properties distinct from those of purified LPS and is also a potential vaccine (2).Advances in transposon delivery systems in our lab and others allowed us to generate a near-saturating random transposon mutant library in strain Schu S4 that is capable of high-throughput screening using the transposon site hybridization (TraSH) system previously used by Weiss et al. for F. novicida (70). Here we describe the utilization of this genetic system to identify and characterize new virulence genes important for F. tularensis Schu S4 entry and growth in human MDMs. Among the genes we identified is a locus required for LPS O-Ag and capsular polysaccharide biogenesis and for serum resistance and intracellular growth within MDMs. We further show that defects in intracellular growth are due to their induction of premature macrophage death, which deprives mutant organisms of their replicative niche.     

A tolC Mutant of Francisella tularensis Is Hypercytotoxic Compared to the Wild Type and Elicits Increased Proinflammatory Responses from Host Cells

The highly infectious bacterium Francisella tularensis is a facultative intracellular pathogen and the causative agent of tularemia. TolC, which is an outer membrane protein involved in drug efflux and type I protein secretion, is required for the virulence of the F. tularensis live vaccine strain (LVS) in mice. Here, we show that an LVS ΔtolC mutant colonizes livers, spleens, and lungs of mice infected intradermally or intranasally, but it is present at lower numbers in these organs than in those infected with the parental LVS. For both routes of infection, colonization by the ΔtolC mutant is most severely affected in the lungs, suggesting that TolC function is particularly important in this organ. The ΔtolC mutant is hypercytotoxic to murine and human macrophages compared to the wild-type LVS, and it elicits the increased secretion of proinflammatory chemokines from human macrophages and endothelial cells. Taken together, these data suggest that TolC function is required for F. tularensis to inhibit host cell death and dampen host immune responses. We propose that, in the absence of TolC, F. tularensis induces excessive host cell death, causing the bacterium to lose its intracellular replicative niche. This results in lower bacterial numbers, which then are cleared by the increased innate immune response of the host.Francisella tularensis is the etiological agent of tularemia. F. tularensis is classified as a category A agent of bioterrorism by the U.S. Centers for Disease Control and Prevention (http://emergency.cdc.gov/agent/agentlist-category.asp) due to its low infectious dose, ease of aerosol dissemination, and capacity to cause high morbidity and mortality (19). There are two clinically relevant subspecies of F. tularensis: subsp. tularensis, which is extremely pathogenic in humans, and subsp. holarctica, which causes a less severe clinical presentation (48). The most severe form of the disease is pneumonic tularemia caused by the inhalation of aerosolized F. tularensis subsp. tularensis (19). The F. tularensis subsp. holarctica-derived live vaccine strain (LVS) was used for many years as the vaccination against tularemia. However, the basis for its attenuation is unknown, and it is no longer in use as a vaccine (46). The LVS is highly virulent in mice, where it causes a disease closely resembling human tularemia (30). These features make the LVS an important model for the study of tularemia. An additional Francisella species, F. novicida, causes disease only in immunocompromised individuals. F. novicida, like the LVS, is highly virulent in mice and widely used as a model of tularemia (20).F. tularensis is a Gram-negative, facultative intracellular pathogen (50). Although factors important for the virulence of F. tularensis are beginning to be identified, the molecular mechanisms behind the extreme pathogenicity of this organism still are largely unknown. In vivo, F. tularensis is a stealth pathogen, evading host cell defenses and dampening host proinflammatory responses. F. tularensis produces an unusual lipopolysaccharide that has low toxicity and does not activate host cells in a TLR4-dependent manner (4, 22). A critical aspect of the pathogenesis of F. tularensis is its ability to escape the phagosome and replicate within the cytosol of a variety of host cells, including both murine and human macrophages and dendritic cells (2, 3, 16, 25, 49). Although F. tularensis does have an extracellular phase (24), it is thought that cytosolic replication allows the bacteria to grow to large numbers while avoiding detection by the host immune system.Host cells respond to F. tularensis invasion by inducing cell death pathways, including apoptosis and pyroptosis (32, 38). In the intrinsic apoptotic pathway, cytochrome c is released from mitochondria into the cytosol, leading to caspase-9 activation and ultimately to the activation of effector caspases such as caspase-3 and -7 (10). In pyroptosis, caspase-1 is activated through the inflammasome complex, resulting in the release of proinflammatory cytokines such as interleukin-1ß (IL-1ß) (6, 32). Lai and coworkers demonstrated that the infection of murine J774 macrophage-like cells with the LVS activated the intrinsic apoptotic pathway as early as 12 h postinfection. Activated caspase-3, but not caspase-1, was detected in the infected cells (38). In contrast, Mariathasan et al. found that the infection of preactivated murine peritoneal macrophages by either the LVS or strain U112 (F. novicida) triggered pyroptosis and the release of IL-1ß (42). In both studies, the induction of cell death was dependent upon the bacteria escaping the phagosome and initiating cytosolic replication. Weiss and colleagues isolated mutants of strain U112 that were attenuated in vivo and caused increased cell death in tissue culture compared to that caused by wild-type U112 (53). This suggests that although host cells initiate death pathways in response to F. tularensis infection, the bacteria has the ability to actively reduce cell death, and this is important for virulence.In addition to triggering death pathways, host cells respond to invading bacteria by mounting a proinflammatory response to alert neighboring cells of the impending bacterial threat (17). However, F. tularensis has been shown to actively suppress these innate host responses. Telepnev and coworkers showed that the LVS disrupted toll-like receptor signaling and blocked the secretion of the proinflammatory cytokines tumor necrosis factor alpha (TNF-α) and IL-1ß by murine and human macrophages (51, 52). Similarly, Bosio and colleagues showed that the LVS inhibited the innate immune response of murine pulmonary dendritic cells to bacterial ligands, and the infection of mice with the fully virulent Schu4 strain (F. tularensis subsp. tularensis) caused an overall state of immunosuppression in the lungs (8, 9).The genome analysis of F. tularensis identified only a few potential virulence factors, suggesting that the bacterium uses novel factors to achieve its high level of pathogenicity (40). Unique to F. tularensis is a 33.9-kb region of DNA termed the Francisella pathogenicity island (FPI) (29, 40, 45). The FPI encodes genes that are essential for intracellular survival and virulence, including iglABCD and pdpABCD (45). F. tularensis lacks type III and IV secretion systems, which is surprising considering its intracellular nature. These secretion systems commonly are used by intracellular pathogens to deliver effector proteins inside host cells to manipulate host cell responses (14, 26). F. tularensis does contain genes encoding a type IV pilus biogenesis system that also functions in the secretion of soluble proteins by a type II-like mechanism and that are important for virulence (12, 31, 54). Finally, F. tularensis appears to contain a functioning type I secretion system that is critical for pathogenesis (28).Type I secretion systems function in the secretion of a variety of toxins and other virulence factors directly from the cytoplasm to the extracellular milieu in a single energized step (33, 37). The type I system consists of three separate components: an outer membrane channel-forming protein, a periplasmic adaptor or membrane fusion protein, and an inner membrane pump that typically belongs to the ATP-binding cassette family. The TolC protein of Escherichia coli, which functions in hemolysin secretion, is the prototypical outer membrane channel component (37). In addition to protein secretion, TolC functions in the efflux of small noxious molecules, conferring multidrug resistance (37). F. tularensis contains three TolC paralogs, TolC, FtlC, and SilC, with TolC and FtlC exhibiting significant homology to the E. coli TolC protein (28, 35). In a previous study we created tolC and ftlC deletion mutants in the F. tularensis LVS (28). We found that both TolC and FtlC participate in multidrug resistance in F. tularensis, but only the ΔtolC mutant was attenuated for virulence in mice by the intradermal route. Thus, tolC is a critical virulence factor of F. tularensis and likely functions in type I secretion in addition to multidrug efflux.Here, we delineate the molecular mechanisms behind the attenuation of the LVS ΔtolC mutant in mice infected by both the intradermal and intranasal routes. In vivo organ burden assays revealed that the ΔtolC strain is decreased for the bacterial colonization of liver, spleen, and most prominently, lungs. In vitro experiments revealed that the ΔtolC mutant is hypercytotoxic to murine macrophages, causing increased apoptosis via a mechanism involving caspase-3 but not caspase-1. In addition, the LVS ΔtolC mutant was hypercytotoxic toward human macrophages and elicited the significantly increased secretion of the proinflammatory chemokines CXCL8 (also known as IL-8) and CCL2 (also known as monocyte chemoattractant protein [MCP-1]). Taken together, these data demonstrate a critical role for TolC, likely via a TolC-secreted toxin(s), in the successful intracellular lifestyle of F. tularensis, its ability to evade host innate immune responses, and its overall virulence.     

Indoleamine 2,3-Dioxygenase 1 Is a Lung-Specific Innate Immune Defense Mechanism That Inhibits Growth of Francisella tularensis Tryptophan Auxotrophs

Upon microbial challenge, organs at various anatomic sites of the body employ different innate immune mechanisms to defend against potential infections. Accordingly, microbial pathogens evolved to subvert these organ-specific host immune mechanisms to survive and grow in infected organs. Francisella tularensis is a bacterium capable of infecting multiple organs and thus encounters a myriad of organ-specific defense mechanisms. This suggests that F. tularensis may possess specific factors that aid in evasion of these innate immune defenses. We carried out a microarray-based, negative-selection screen in an intranasal model of Francisella novicida infection to identify Francisella genes that contribute to bacterial growth specifically in the lungs of mice. Genes in the bacterial tryptophan biosynthetic pathway were identified as being important for F. novicida growth specifically in the lungs. In addition, a host tryptophan-catabolizing enzyme, indoleamine 2,3-dioxygenase 1 (IDO1), is induced specifically in the lungs of mice infected with F. novicida or Streptococcus pneumoniae. Furthermore, the attenuation of F. novicida tryptophan mutant bacteria was rescued in the lungs of IDO1^−/− mice. IDO1 is a lung-specific innate immune mechanism that controls pulmonary Francisella infections.Organs at different anatomic sites of the body have different physiological functions and are exposed to vastly different microbial and environmental challenges on a daily basis (32). As a result, depending on its relative sterility, each organ senses impending danger of infection differently and employs unique innate immune responses in an organ-specific homeostatic manner to effectively fight off any potential infection without compromising organ physiology and function. For instance, a sterile organ like the spleen is likely to induce a strong proinflammatory and bactericidal defense to maintain organ sterility, while such a defense would not be induced in a nonsterile organ like the colon. Instead, innate immune responses in the colon support a peaceful coexistence with the gut microflora rather than achieving sterility. To gain a better understanding of tissue-specific innate immune defense mechanisms, we have used Francisella tularensis as a model bacterial pathogen that is capable of entering and surviving in many different host tissues and organs.Francisella tularensis is a facultative, intracellular Gram-negative bacterium that causes the highly debilitating zoonotic disease tularemia (28). There are currently 4 known subspecies of F. tularensis that cause disease of different severities. The most virulent subspecies of Francisella is F. tularensis subsp. tularensis, which is found predominantly in North America and has an infectious dose of less than 10 CFU in humans (33, 34, 37). It is also associated with lethal pulmonary infections. Francisella novicida is also found primarily in North America but rarely causes disease in immunocompetent individuals (16). However, F. novicida shares the same families of virulence genes as F. tularensis, causes a similar disease in mice (27), and is more genetically amenable than F. tularensis, thus making F. novicida infection of mice a good experimental model for the study of Francisella pathogenesis.Francisella infects mammalian hosts via extremely diverse routes of entry, colonizing and replicating in different organs. Tularemia occurs in several forms, depending on the initial route of infection. The most common form of tularemia is ulceroglandular tularemia, which occurs when the bacterium enters the skin subcutaneously (9). Other infection routes include inoculation via the conjunctiva, which leads to oculoglandular tularemia (9, 38), and the ingestion of contaminated food and water, which leads to oropharyngeal or gastrointestinal tularemia (9). The most acute and fatal form of tularemia is pneumonic tularemia, which is caused by inhalation of the bacterium into the lungs. Pneumonic tularemia can also occur as a result of complications from the above forms of tularemia (9, 12), when the bacteria spread systemically from the initial peripheral site of infection to the lungs.From the perspective of the fitness of a microbe, it is advantageous for a microbe to infect its host via multiple entry routes and infect multiple organs. Thus, it is plausible that F. tularensis, a microbe that infects a range of different organs, has to deal with a diverse repertoire of different innate immune responses launched in these various organs during infection. We utilized a genome-wide genetic screen in F. novicida as a tool to identify tissue-specific interactions between the host and pathogen.Genetic screens are very effective tools that have been successfully used for the large-scale identification of virulence factors of many bacterial pathogens in vivo (6, 19, 44). Here, we utilize a microarray-based, negative-selection technique called transposon site hybridization (TraSH) (44) in an intranasal (i.n.) model of Francisella infection to identify Francisella factors important for bacterial growth and/or survival in the lungs. The screen identified almost all of the known Francisella virulence genes, including the Francisella pathogenicity island (FPI) genes. We also identified novel genes not previously known to be important for bacterial growth and/or survival in the lungs.By comparing the genes that are important for Francisella growth and/or survival in the lungs with the genes identified previously for spleen colonization (44), we identified the Francisella tryptophan biosynthetic pathway as being important for bacterial survival and/or growth specifically in the lungs. We demonstrated that the host enzyme that is responsible for catalyzing the first rate-limiting step of tryptophan degradation, indoleamine 2,3-dioxygenase 1 (IDO1), is induced specifically in the lungs during Francisella infection, suggesting that the host innate immune response to F. novicida restricts the availability of this essential amino acid. Indeed, the F. novicida tryptophan mutant bacteria survived better in the lungs of mice that lack IDO1 compared to wild-type mice. These findings suggest that IDO1 acts as an organ-specific, host innate immune mechanism that defends against a highly virulent bacterial pathogen.     

Francisella tularensis Induces Extensive Caspase-3 Activation and Apoptotic Cell Death in the Tissues of Infected Mice

Although Francisella tularensis subsp. tularensis is known to cause extensive tissue necrosis, the pathogenesis of tissue injury has not been elucidated. To characterize cell death in tularemia, C57BL/6 mice were challenged by the intranasal route with type A F. tularensis, and the pathological changes in infected tissues were characterized over the next 4 days. At 3 days postinfection, well-organized inflammatory infiltrates developed in the spleen and liver following the spread of infection from the lungs. By the next day, extensive cell death, characterized by the presence of pyknotic cells containing double-strand DNA breaks, was apparent throughout these inflammatory foci. Cell death was not mediated by activated caspase-1, as has been reported for cells infected with other Francisella subspecies. Mouse macrophages and dendritic cells that had been stimulated with type A F. tularensis did not release interleukin-18 in vitro, a response that requires the activation of procaspase-1. Dying cells within type A F. tularensis-infected tissues expressed activated caspase-3 but very little activated caspase-1. When caspase-1-deficient mice were challenged with type A F. tularensis, pathological changes, including extensive cell death, were similar to those seen in infected wild-type mice. In contrast, type A F. tularensis-infected caspase-3-deficient mice showed much less death among their F4/80⁺ spleen cells than did infected wild-type mice, and they retained the ability to express tumor necrosis factor alpha and inducible NO synthase. These findings suggest that type A F. tularensis induces caspase-3-dependent macrophage apoptosis, resulting in the loss of potentially important innate immune responses to the pathogen.Tularemia is a zoonotic infectious disease that is caused by the facultative intracellular bacterium Francisella tularensis. Little is known about the virulence properties of this pathogen or the pathogenic mechanisms responsible for the diseases it causes. Two subspecies of F. tularensis account for infections in humans. F. tularensis subsp. tularensis (type A) is by far the more virulent of the two and causes most of the lethal cases of tularemia in North America. The minimum infectious dose for type A F. tularensis strains in humans challenged by aerosol inhalation has been estimated to be less than 15 CFU (24). In the mouse, the most lethal form of exposure to type A strains is through the inhalation of viable organisms (14, 29), and infection disseminates to the spleen and liver after only a few days (11). In contrast, F. tularensis subsp. holarctica (type B) is a frequent cause of nonfatal tularemia in Europe and Asia.The pathological features of human tularemia have been reported by Lamps et al. (22), who examined autopsy samples from confirmed cases of naturally acquired disease. These authors noted the presence of granulomas and irregular microabscesses with coagulation necrosis in the liver, spleen, kidney, and lymph nodes. Most patients also showed diffuse necrotizing pneumonia ranging in appearance from abundant fibrin and cellular debris in the alveolar walls and airways to confluent necrosis and hemorrhage. Thus, the hallmark features of end-stage tularemia in humans include foci of necrosis in the lungs, liver, and spleen indicative of extensive cell death. Similar findings have been reported for mice infected with type A F. tularensis (11), indicating that extensive in situ cell death is a defining characteristic of disseminated disease. Despite this information, little is known about the microbial factors responsible for inducing cell injury or the intracellular signaling pathways of programmed cell death that are activated during type A F. tularensis infections.Infection of mice with the live vaccine strain (LVS) of F. tularensis subsp. holarctica continues to be one of the most frequently used animal models for studying the pathogenesis of tularemia. The minimum lethal dose for LVS in C57BL/6 mice injected by the intranasal (i.n.) route is ∼10³ CFU (3, 23), and under these conditions viable organisms disseminate from the lungs to the liver and spleen by the second day postinfection (p.i.) (3, 12). LVS causes pneumonia and systemic disease in mice that is similar in some respects to what is seen in infected human beings (2, 3, 12, 27). The host response to primary infection is characterized by a high level of expression of proinflammatory genes, especially in the spleen and liver (10, 16). Cells that accumulate within infected tissues include CD11b⁺ macrophages (Mφ), CD3⁺ T cells, and Ly-6G⁺ immature myeloid cells that are capable of further differentiation into granulocytes and dendritic cells (DC) (3, 12, 27). In the liver, microgranulomas containing Mφ, T cells, and myeloid precursors form early in LVS infections and continue to grow in size until the death of the host. However, only sporadic cell death occurs within hepatic granulomas and splenic pyogranulomatous infiltrates in infected mice (3). Both granuloma formation and cell death in the liver are highly dependent on gamma interferon (IFN-γ) production (3, 9), which is primarily produced during primary infections by activated NK cells and T cells within the infected organs (3, 13, 23). Thus, in contrast to type A F. tularensis, which causes significant pathological changes indicative of extensive cell death in situ, the tissue response to LVS is more inflammatory and results in less overt cytotoxic tissue damage.Several mechanisms of programmed cell death have been associated with Francisella infections. Lai et al. (20, 21) first reported that mouse J774 Mφ-like cells underwent apoptotic death when challenged in vitro with LVS at high multiplicities of infection. Because infected cells showed cytochrome c release from their mitochondria, the degradation of poly-ADP-ribose polymerase, and the cleavage of procaspase-9 and procaspase-3, the authors concluded that death signaling involved the intrinsic apoptosis pathway. The morphological features of apoptosis, which include cytoplasmic condensation, cell membrane blebbing, and chromatin condensation (pyknosis) (3, 9, 27), as well as the cleavage of procaspase-3 (3, 27), have also been observed in LVS-infected tissues.A caspase-3-independent form of programmed cell death, termed pyroptosis, is initiated when procaspase-1 is activated after its recruitment to multiprotein cytosolic complexes known as inflammasomes. Activated caspase-1 catalyzes the processing of the interleukin-1β (IL-1β) and IL-18 precursor polypeptides and coordinates their release with the induction of cell death. Cells dying by pyroptosis show impaired membrane integrity, which leads to osmotic cell lysis and the release of additional proinflammatory cellular contents (15, 18, 28). Monack and coworkers (17, 18, 25) showed that procaspase-1 was activated in mouse bone marrow-derived Mφ after their infection with either LVS or F. novicida strain U112. In their earlier studies, Lai et al. (21) had not observed the cleavage of procaspase-1 in LVS-infected J774 Mφ-like cells, but this may reflect their use of a cell line that fails to activate the pyroptosis pathway (18).The objective of the present study was to understand the basis for tissue injury caused by highly virulent type A F. tularensis strains in a mouse respiratory challenge model of tularemia. Specifically, we wanted to determine whether any of the mechanisms of cell death described for the less-virulent subspecies contribute to tissue injury caused by the tularensis subspecies. The results indicate that type A F. tularensis causes a caspase-3-dependent destruction of phagocytic cells early in infection, which results in the loss of potentially important innate immune responses to the pathogen.     

Reintroduction of Two Deleted Virulence Loci Restores Full Virulence to the Live Vaccine Strain of Francisella tularensis

A disadvantage of several old vaccines is that the genetic events resulting in the attenuation are often largely unknown and reversion to virulence cannot be excluded. In the 1950s, a live vaccine strain, LVS, was developed from a type B strain of Francisella tularensis, the causative agent of tularemia. LVS, which is highly attenuated for humans but still virulent for mice by some infection routes, has been extensively studied and found to protect staff from laboratory-acquired tularemia. The efforts to improve biopreparedness have identified a demand for a vaccine against tularemia. Recently the rapid progress in genomics of different Francisella strains has led to identification of several regions of differences (RDs). Two genes carried within RDs, pilA, encoding a putative type IV pilin, and FTT0918, encoding an outer membrane protein, have been linked to virulence. Interestingly, LVS has lost these two genes via direct repeat-mediated deletions. Here we show that reintroduction of the two deleted regions restores virulence of LVS in a mouse infection model to a level indistinguishable from that of virulent type B strains. The identification of the two attenuating deletion events could facilitate the licensing of LVS for use in humans.Francisella tularensis is the causative agent of tularemia, and natural infections have been reported in a range of vertebrates and invertebrates (22). Infections can occur by many different routes, via ingestion of contaminated food or water, contact with infected animals, bites by infected arthropods including mosquitoes or ticks, or via inhalation (36). F. tularensis is further divided into three subspecies and one proposed subspecies, where F. tularensis subsp. holarctica (type B) is found in most of Europe, Asia, and North America and F. tularensis subsp. tularensis (type A) is found in North America. F. tularensis subsp. mediasiatica has been identified only in Central Asia, and “Francisella tularensensis subsp. novicida” has been isolated in several locations in North America and Australia (25, 39). Human infections are mainly caused by type A and type B strains, where type A strains cause severe infections and are significantly more virulent than type B strains. In mouse infection models, F. tularensis subsp. mediasiatica is as virulent as, or even slightly more virulent than, type B strains (unpublished results). However, infections with F. tularensis subsp. mediasiatica appear to be rare in humans (30). F. tularensis subsp. novicida is significantly less pathogenic than the other subspecies in humans and is only known to cause infection in immunocompromised persons. Due to the high infectivity and potential for airborne transmission, F. tularensis has been designated a category A agent of bioterrorism (4, 14). Still, relatively little is known about the virulence determinants of F. tularensis. The recent development of genomics and genetic tools (9) has been the key to increasing the understanding of the molecular mechanisms of F. tularensis infections.In a recent study, comparisons of different strains revealed the presence of regions of differences (RDs) (35). These RDs are flanked by direct-repeat sequences that are assumed to have facilitated deletion events in certain strains. Two RDs are particularly interesting, since they have been linked to virulence. One region, denoted RD19, encodes a putative type IV pilin (PilA) that has been shown to contribute to virulence in a type B strain, FSC354 (7). Type IV pili (Tfp) are complex adhesins involved in important host cell interactions and are required for virulence in many human pathogens, such as Neisseria spp., Pseudomonas aeruginosa, and Vibrio cholerae (8, 18, 37). Tfp have also been shown to be involved in twitching motility, biofilm formation, and cell signaling (13, 17, 23). The second region, RD18, has been shown to be essential for virulence of a highly virulent type A strain, SCHU S4 (38). One spontaneous avirulent variant of SCHU S4, FSC043, was found to lack RD18 (35, 38). Two genes, FTT0918 and FTT0919 (SCHU S4 nomenclature), were identified as defective in FSC043, and DNA sequencing showed that the deletion event resulted in an in-frame fusion consisting of the N terminus of the FTT0918 protein and the C terminus of the FTT0919 protein (35, 38). Both the FTT0918 and FTT0919 proteins belong to a novel protein family that is unique to Francisella and without any known function so far (38). Importantly, Twine and colleagues were able to establish the attenuation of strain FSC043 as being directly linked to FTT0918, since mutation of this gene in the highly virulent strain SCHU S4 resulted in attenuation (38). In contrast, mutation of FTT0919 had no effect on virulence.Even though these two RDs have been studied in two different subspecies (FTT0918 in type A and pilA in type B), it is reasonable to assume that these two loci are of significance for virulence of both type A and type B strains. Indeed, here we verify that FTT0918 is important for virulence of type B strains, and in another study, PilA has been demonstrated to be required for full virulence of a type A strain (unpublished results). A striking observation is that the extensively studied live vaccine strain of type B origin, LVS, lacks both the FTT0918 gene and pilA. Interestingly, in a genomic study with the aim of identifying mutations in the genome of LVS, Rohmer and colleagues found seven proteins with either an altered or lost function as likely candidates for the attenuation of LVS (27). Among the genes encoding these seven proteins, the two deletions described above, pilA and FTT0918, were included, as well as a third deletion of 93 bp in the part of the FTT0086 gene encoding the C terminus (SCHU S4 nomenclature) (FTL_1773 in LVS) (27). FTT0086 encodes a protein showing homology to numerous proteins denoted dyp-type peroxidases, known to be important to counter oxidative stress.There has been a significant interest in vaccine development in recent years, and LVS is frequently used in comparative studies. LVS appears to provide some protection as judged from animal infection experiments and from experience with vaccination of laboratory staff, but the protection may be more limited against airborne type A strains (2, 5, 12). However, in the absence of new vaccines, LVS could be an attractive alternative. One major argument against licensing LVS is that the genetic determinants causing the attenuation have not been identified. In this work, we set out to determine the contributions of the three different gene deletion events to the attenuation of LVS. To avoid any influence of expression from nonnative promoters or effects of plasmid copy number, we used a strategy whereby each deleted region was restored by complementation in cis. Complementation of the gene FTT0086 had no impact on mouse virulence, while reintroduction of either the FTT0918 or pilA gene resulted in increased virulence. Complementation of LVS with both FTT0918 and pilA restored virulence to a level indistinguishable from that of a recently isolated virulent clinical type B isolate.     

Francisella tularensis T-Cell Antigen Identification Using Humanized HLA-DR4 Transgenic Mice

There is no licensed vaccine against the intracellular pathogen Francisella tularensis. The use of conventional mouse strains to screen protective vaccine antigens may be problematic, given the differences in the major histocompatibility complex (MHC) binding properties between murine and human antigen-presenting cells. We used engineered humanized mice that lack endogenous MHC class II alleles but that express a human HLA allele (HLA-DR4 transgenic [tg] mice) to identify potential subunit vaccine candidates. Specifically, we applied a biochemical and immunological screening approach with bioinformatics to select putative F. tularensis subsp. novicida T-cell-reactive antigens using humanized HLA-DR4 tg mice. Cell wall- and membrane-associated proteins were extracted with Triton X-114 detergent and were separated by fractionation with a Rotofor apparatus and whole-gel elution. A series of proteins were identified from fractions that stimulated antigen-specific gamma interferon (IFN-γ) production, and these were further downselected by the use of bioinformatics and HLA-DR4 binding algorithms. We further examined the validity of this combinatorial approach with one of the identified proteins, a 19-kDa Francisella tularensis outer membrane protein (designated Francisella outer membrane protein B [FopB]; FTN_0119). FopB was shown to be a T-cell antigen by a specific IFN-γ recall assay with purified CD4⁺ T cells from F. tularensis subsp. novicida ΔiglC-primed HLA-DR4 tg mice and cells of a human B-cell line expressing HLA-DR4 (DRB1*0401) functioning as antigen-presenting cells. Intranasal immunization of HLA-DR4 tg mice with the single antigen FopB conferred significant protection against lethal pulmonary challenge with an F. tularensis subsp. holarctica live vaccine strain. These results demonstrate the value of combining functional biochemical and immunological screening with humanized HLA-DR4 tg mice to map HLA-DR4-restricted Francisella CD4⁺ T-cell epitopes.Francisella tularensis is a Gram-negative bacterium and the etiological agent of the zoonotic disease tularemia. F. tularensis is classified into four subspecies, namely, F. tularensis subsp. tularensis, F. tularensis subsp. holarctica, F. tularensis subsp. mediasiatica, and F. tularensis subsp. novicida (F. novicida) on the basis of their biochemical and genetic profiles, virulence properties, and geographical origins (51). To this end, F. tularensis subsp. tularensis (type A) is the most virulent subspecies, with the inhalation of as few as 10 organisms causing disease and mortality rates of between 30 and 60% in untreated cases of pneumonic tularemia (53). The live vaccine strain (LVS) derived from F. tularensis subsp. holarctica has been used as a prophylactic vaccine against tularemia (48). Millions of individuals in the Soviet Union were immunized with live vaccine strains between 1946 and 1960 (52). However, LVS has not been licensed for use in the United States due to a lack of understanding of the genetic mutations that are responsible for attenuation of this strain, although it is used as an investigational new drug (IND) to immunize at-risk workers, primarily tularemia researchers. F. novicida, which causes disease only in immunocompromised humans but which is highly virulent for mice, has been used as a comparative model organism due to the high degree of genetic similarity with type A strains (98.1% homology between sequences common to strains U112 and SCHU S4 [45]). We recently reported that a defined vaccine strain (ΔiglB) generated in strain U112 was effective in inducing heterologous protection against various Francisella strains in a mouse model of pulmonary tularemia, suggesting the conservation of protective antigens (12).Cell-mediated immunity has been documented to play an important role in protection against tularemia (2, 18, 19, 49, 56). The role of antibodies, via neutralization and Fc receptor-mediated clearance (43, 44) in response to infection, has also gained significant attention. Therefore, the availability of a combination of multiple Francisella antigens containing T-cell and/or B-cell epitopes would be desirable for formulating an effective multivalent vaccine against this organism. However, the use of conventional mouse strains to identify protective antigens may not be feasible, given the differences in the major histocompatibility complex (MHC) binding properties between murine and human MHCs. These constraints can be overcome with the use of engineered humanized mice, such as the HLA-DR4 transgenic (tg) mouse. This mouse was generated to express the extracellular human α1 and β1 domains of the HLA-DRA and HLA-DRB1*0401 haplotypes, which form the peptide binding sites for antigen presentation, in conjunction with the murine α2 and β2 domains (29). These chimeric molecules have been shown to exhibit the same antigen-binding specificity as HLA-DRB1*0401 and to be functional in presenting antigens to T cells (29). The frequency of the HLA-DR allele in humans is 29% in Caucasian individuals, 10% in African American individuals, and 34% in other individuals (38), underscoring the translational value of the epitopes identified in these mice for humans. We recently demonstrated the feasibility of using the HLA-DR4 tg mouse for the identification of vaccine antigens against genital Chlamydia infection (39), demonstrating the value of the use of these animals in the rational selection of vaccine candidates.In this study, we utilized a robust biochemical membrane protein fractionation method, cytokine recall assays, and humanized HLA-DR4 tg mice to identify putative CD4⁺ T-cell-reactive antigens from U112. Moreover, using bioinformatics tools, we further validated one of the identified antigens (FTN_0119), designated Francisella outer membrane protein B (FopB), as a potential subunit vaccine candidate against pneumonic tularemia in HLA-DR4 tg mice.     

Toll-Like Receptor 3 Agonist Protection against Experimental Francisella tularensis Respiratory Tract Infection

We investigated whether Toll-like receptor 3 (TLR3) stimulation would protect the host from inhaled Francisella tularensis. TLR3 is expressed by respiratory epithelial cells and macrophages and can be activated by a synthetic double-stranded RNA ligand called polyinosine-polycytosine [poly(I:C)]. Thus, we evaluated poly(I:C) as a novel treatment against inhaled F. tularensis. In vivo, BALB/c mice intranasally (i.n.) treated with poly(I:C) (100 μg/mouse) 1 h before or after Schu 4 or LVS (100 CFU) i.n. challenge showed that poly(I:C) treatment significantly reduced bacterial load in the lungs (P < 0.05). Bronchoalveolar lavage from poly(I:C)-treated mice alone or combined with F. tularensis infection significantly increased cytokine secretion and enhanced neutrophil influx to lung tissues. Poly(I:C) responses were transient but significantly prolonged the survival of treated mice after i.n. F. tularensis challenge relative to mock treated animals. This prolonged survival providing a longer window for initiation of levofloxacin (LEVO) treatment (40 mg/kg). Animals treated with poly(I:C), challenged with F. tularensis, and then treated with LEVO 5 days later had 100% survival relative to 0% survival in animals receiving LEVO alone. Mechanistically, poly(I:C) given to human monocyte-derived macrophages before or after Schu 4 or LVS challenge (multiplicity of infection, 20:1) had significantly reduced intracellular bacterial replication (P < 0.05). These data suggest that poly(I:C) may represent a potential therapeutic agent against inhaled F. tularensis that prolongs survival and the opportunity to initiate standard antibiotic therapy (i.e., LEVO).Inhalation is likely to be one of the primary routes by which a bioweapon will be delivered to a target population. Francisella tularensis is a potential bioweapon because it can be aerosolized due to its inherently hardy nature, and less than 20 inhaled organisms can be detrimental to the host (8, 10, 28). F. tularensis, the etiologic agent of tularemia, is a small, Gram-negative nonmotile coccus and a facultative intracellular bacterium (16, 36). There are two major subspecies of F. tularensis; one is designated type A and includes strains that induce aggressive pathologies in the host and can result in pulmonary tularemia causing death if not treated (18, 37). The commonly studied virulent type A strain, Schu 4, was isolated originally from a human case of tularemia (22, 31, 38). The type B subspecies of F. tularensis causes a milder disease in humans than do type A strains. The only vaccine against tularemia known at this time was derived from subspecies type B and is called the live vaccine strain (LVS) (3, 8, 19, 31). Interestingly, most animal modeling of F. tularensis infection has used LVS-infected mice because it mimics the human disease caused by type A strains (6, 7, 9, 12). The immunological efficacy of LVS in humans is not known; vaccination with LVS does not provide complete protection against the virulent type A strains of F. tularensis (7, 19). As a result, alternative intervention strategies and vaccines need to be developed.Ideally, vaccines against bioweapons will be established to protect the general population limiting the impact of such terroristic acts. Until such vaccines are available and widely distributed, alternate methods of broad range protection must be investigated. One interesting strategy is to engender innate immune resistance against mucosal pathogens (13, 17, 24). We have investigated the potential of Toll-like receptor (TLR) agonists recognized by TLRs highly expressed by respiratory epithelial cells. Specifically, polyinosine-polycytosine [poly(I:C)] is a synthetic double-stranded RNA analog that stimulates TLR3 triggering the induction of the host innate immune response including as RANTES, gamma interferon (IFN-γ), interleukin-8 (IL-8), and IL-6 (11, 17, 23, 26).Poly(I:C) can be delivered easily by a nose spray, is cheap to manufacture, and could be offered as an over-the-counter product unlike antibiotics. Importantly, the kinetics of cytokine secretion after poly(I:C) administration showed a transient response and offered no indication of toxicity, even with repeated use in our previous study (17). We therefore examined poly(I:C) as a topical treatment for a potential F. tularensis aerosol release. Theoretically, intranasal (i.n.) poly(I:C) could engender an innate immune response against F. tularensis, providing an extended period of resistance before an antibiotic, such as levofloxacin (LEVO), can be administered. LEVO belongs to the group of antibiotics known as fluoroquinolones and has been used to treat respiratory infections such as tularemia (1, 21, 25).Recently, we established that genital application of poly(I:C) protected against lethal HSV-2 challenge in mice (17). We have extended these findings by applying poly(I:C) to the respiratory mucosa testing the hypothesis that nucleic acid-based TLR agonists may prove to be useful prophylactic and possibly therapeutic measures against select agent respiratory infections including F. tularensis. Because F. tularensis suppresses the innate immune response (2, 4, 29, 39), the host does not detect and/or respond to the organism for approximately 48 to 72 h after F. tularensis infection (2; T. D. Eaves-Pyles, unpublished data). This large gap between the time of infection and host detection of the organism limits the development of an adequate immune response against F. tularensis. As such, we hypothesized that poly(I:C) would enhance the host''s response prior to or soon after F. tularensis exposure. Our in vivo and in vitro studies show that mice treated 1 h before or 1 h after the administration of poly(I:C) had significantly less bacteria in their lungs, increased neutrophil infiltration to the lung, and extended survival after LVS or Schu 4 infection. Moreover, mice treated with poly(I:C) (1 h after Schu 4 infection), followed by LEVO administration 5 days later, were fully protected from lethal outcomes. Corresponding to these in vivo studies, we show that poly(I:C)-treated human monocyte-derived macrophages (MDM) secreted high levels of specific cytokines and engendered enhanced intracellular bacterial killing after LVS and Schu 4 exposure compared to untreated animals.     

Francisella tularensis ΔpyrF Mutants Show that Replication in Nonmacrophages Is Sufficient for Pathogenesis In Vivo

The pathogenesis of Francisella tularensis has been associated with this bacterium''s ability to replicate within macrophages. F. tularensis can also invade and replicate in a variety of nonphagocytic host cells, including lung and kidney epithelial cells and hepatocytes. As uracil biosynthesis is a central metabolic pathway usually necessary for pathogens, we characterized ΔpyrF mutants of both F. tularensis LVS and Schu S4 to investigate the role of these mutants in intracellular growth. As expected, these mutant strains were deficient in de novo pyrimidine biosynthesis and were resistant to 5-fluoroorotic acid, which is converted to a toxic product by functional PyrF. The F. tularensis ΔpyrF mutants could not replicate in primary human macrophages. The inability to replicate in macrophages suggested that the F. tularensis ΔpyrF strains would be attenuated in animal infection models. Surprisingly, these mutants retained virulence during infection of chicken embryos and in the murine model of pneumonic tularemia. We hypothesized that the F. tularensis ΔpyrF strains may replicate in cells other than macrophages to account for their virulence. In support of this, F. tularensis ΔpyrF mutants replicated in HEK-293 cells and normal human fibroblasts in vitro. Moreover, immunofluorescence microscopy showed abundant staining of wild-type and mutant bacteria in nonmacrophage cells in the lungs of infected mice. These findings indicate that replication in nonmacrophages contributes to the pathogenesis of F. tularensis.Francisella tularensis causes the acute illness known as tularemia and is classified by the Centers for Disease Control and Prevention as a category A biodefense agent. This organism is highly infectious, as a single bacterium can lead to disease that may be fatal if untreated (16, 39, 53). The virulence of F. tularensis has been associated with this organism''s ability to replicate within phagocytic cells of the innate immune system, such as macrophages (4). In the murine model of respiratory tularemia, both airway macrophages and dendritic cells are infected within 1 h following inhalation of F. tularensis (7). As this disease progresses, macrophage numbers decline (26, 63), likely due to the induction of caspase-3-dependent apoptosis induced by the virulent type A F. tularensis strain (63). During interactions with host macrophages, F. tularensis can block the activation of these cells, as evidenced by the inhibition of proinflammatory cytokine production (9, 60, 61). In addition to macrophages and dendritic cells, F. tularensis can invade and replicate in nonphagocytic host cells, such as alveolar epithelial cells, kidney epithelial cells, hepatocytes, and fibroblasts (14, 21, 25-27, 47). Although there has been recent progress on understanding the bacterial molecules that contribute to intramacrophage growth, less has been done to investigate F. tularensis growth in other cell types. And in the current paradigm of tularemia pathogenesis, F. tularensis mutants deficient in intramacrophage replication should be attenuated for virulence during animal infection.The de novo synthesis of pyrimidines is a central metabolic pathway, as these molecules are precursors of RNA, DNA, cell membranes, and glycosylation substrates (18, 57). This pathway comprises the activity of six enzymatic reactions that culminate in the decarboxylation of orotidine-5′-phosphate to uridine-5′-monophosphate, a step mediated by the protein product of pyrF (2). Prior reports have shown that F. tularensis genes encoding homologs to enzymes mediating initial steps of pyrimidine biosynthesis, such as carAB and pyrB, are essential for intramacrophage growth (47, 56). Although F. tularensis mutants of these genes did not replicate in primary macrophages in vitro, the in vivo virulence of these strains was not detailed fully (56).The gene encoding the ultimate enzyme of pyrimidine biosynthesis, pyrF, has not been characterized for Francisella. pyrF mutations in other bacteria lead to uracil auxotrophy and resistance to 5-fluoroorotic acid (FOA), which provides a nonantibiotic counterselectable marker useful in applied bacterial genetics (30, 45, 54, 55). A counterselectable marker would be especially beneficial when working with a category A biodefense agent, such as F. tularensis, where the choice of antibiotic selection and the cognate resistance markers are limited. An F. tularensis strain containing a deletion of pyrF may be attenuated for virulence and would also be a potential vaccine candidate.In this report, we characterize pyrF mutant strains of both F. tularensis LVS and the fully virulent F. tularensis strain Schu S4. Using a genetic approach, we show that this gene encodes a functional orotidine-5′-phosphate decarboxylase and that its activity is critical for replication in macrophages in vitro. Although ΔpyrF mutants of both LVS and Schu S4 had similar phenotypes, we present evidence suggesting that F. tularensis Schu S4 possesses more mechanisms than LVS for silencing macrophage activation. Surprisingly, the ΔpyrF mutants were not attenuated in vivo. Furthermore, we show that intramacrophage replication is expendable during F. tularensis infection, provided that this bacterium can still replicate in nonmacrophage cells. These findings delineate the contribution of F. tularensis replication in the host''s nonmacrophage cells in vivo and provide novel insight into the pathogenesis of this bacterium.     

Variation in Susceptibility of Bloodstream Isolates of Candida glabrata to Fluconazole According to Patient Age and Geographic Location in the United States in 2001 to 2007

We examined the susceptibilities to fluconazole of 642 bloodstream infection (BSI) isolates of Candida glabrata and grouped the isolates by patient age and geographic location within the United States. Susceptibility of C. glabrata to fluconazole was lowest in the northeast region (46%) and was highest in the west (76%). The frequencies of isolation and of fluconazole resistance among C. glabrata BSI isolates were higher in the present study (years 2001 to 2007) than in a previous study conducted from 1992 to 2001. Whereas the frequency of C. glabrata increased with patient age, the rate of fluconazole resistance declined. The oldest age group (≥80 years) had the highest proportion of BSI isolates that were C. glabrata (32%) and the lowest rate of fluconazole resistance (5%).Candidemia is without question the most important of the invasive mycoses (6, 33, 35, 61, 65, 68, 78, 86, 88). Treatment of candidemia over the past 20 years has been enhanced considerably by the introduction of fluconazole in 1990 (7, 10, 15, 28, 29, 31, 40, 56-58, 61, 86, 90). Because of its widespread usage, concern about the development of fluconazole resistance among Candida spp. abounds (2, 6, 14, 32, 47, 53, 55, 56, 59, 60, 62, 80, 86). Despite these concerns, fluconazole resistance is relatively uncommon among most species of Candida causing bloodstream infections (BSI) (5, 6, 22, 24, 33, 42, 54, 56, 65, 68, 71, 86). The exception to this statement is Candida glabrata, of which more than 10% of BSI isolates may be highly resistant (MIC ≥ 64 μg/ml) to fluconazole (6, 9, 15, 23, 30, 32, 36, 63-65, 71, 87, 91). Suboptimal fluconazole dosing practices (low dose [<400 mg/day] and poor indications) may lead to an increased frequency of isolation of C. glabrata as an etiological agent of candidemia in hospitalized patients (6, 17, 29, 32, 35, 41, 47, 55, 60, 68, 85) and to increased fluconazole (and other azole) resistance secondary to induction of CDR efflux pumps (2, 11, 13, 16, 43, 47, 50, 55, 69, 77, 83, 84) and may adversely affect the survival of treated patients (7, 10, 29, 40, 59, 90). Among the various Candida species, C. glabrata alone has increased as a cause of BSI in U.S. intensive care units since 1993 (89). Within the United States, the proportion of fungemias due to C. glabrata has been shown to vary from 11% to 37% across the different regions (west, midwest, northeast, and south) of the country (63, 65) and from <10% to >30% within single institutions over the course of several years (9, 48). It has been shown that the prevalence of C. glabrata as a cause of BSI is potentially related to many disparate factors in addition to fluconazole exposure, including geographic characteristics (3, 6, 63-65, 71, 88), patient age (5, 6, 25, 35, 41, 42, 48, 63, 82, 92), and other characteristics of the patient population studied (1, 32, 35, 51). Because C. glabrata is relatively resistant to fluconazole, the frequency with which it causes BSI has important implications for therapy (21, 29, 32, 40, 41, 45, 56, 57, 59, 80, 81, 86, 90).Previously, we examined the susceptibilities to fluconazole of 559 BSI isolates of C. glabrata and grouped the isolates by patient age and geographic location within the United States over the time period from 1992 to 2001 (63). In the present study we build upon this experience and report the fluconazole susceptibilities of 642 BSI isolates of C. glabrata collected from sentinel surveillance sites throughout the United States for the time period from 2001 through 2007 and stratify the results by geographic region and patient age. The activities of voriconazole and the echinocandins against this contemporary collection of C. glabrata isolates are also reported.     

Antibodies contribute to effective vaccination against respiratory infection by type A Francisella tularensis strains

Pneumonic tularemia is a life-threatening disease caused by inhalation of the highly infectious intracellular bacterium Francisella tularensis. The most serious form of the disease associated with the type A strains can be prevented in experimental animals through vaccination with the attenuated live vaccine strain (LVS). The protection is largely cell mediated, but the contribution of antibodies remains controversial. We addressed this issue in a series of passive immunization studies in Fischer 344 (F344) rats. Subcutaneous LVS vaccination induced a robust serum antibody response dominated by IgM, IgG2a, and IgG2b antibodies. Prophylactic administration of LVS immune serum or purified immune IgG reduced the severity and duration of disease in naïve rats challenged intratracheally with a lethal dose of the virulent type A strain SCHU S4. The level of resistance increased with the volume of immune serum given, but the maximum survivable SCHU S4 challenge dose was at least 100-fold lower than that shown for LVS-vaccinated rats. Protection correlated with reduced systemic bacterial growth, less severe histopathology in the liver and spleen during the early phase of infection, and bacterial clearance by a T cell-dependent mechanism. Our results suggest that treatment with immune serum limited the sequelae associated with infection, thereby enabling a sterilizing T cell response to develop and resolve the infection. Thus, antibodies induced by LVS vaccination may contribute to the defense of F344 rats against respiratory infection by type A strains of F. tularensis.Pneumonic tularemia is a highly debilitating disease caused by the Gram-negative coccobacillus Francisella tularensis. Strains classified under subspecies tularensis (type A) are the most virulent and pose the biggest challenge from a clinical perspective (28), with a mortality rate estimated to exceed 30% in untreated patients (11). Prophylactic vaccination is the best countermeasure, and there is good historical evidence that pneumonic tularemia can be prevented by vaccination with the attenuated F. tularensis live vaccine strain (LVS) (37). However, LVS is unlikely to be licensed for mass vaccination because the mechanism of attenuation has not been defined. Due to the potential of a major public health threat, there is an urgent need to understand the protective mechanisms associated with an effective immune response so that novel vaccines can be developed.Protective immunity against F. tularensis infection is usually attributed to an effective T cell response. However, F. tularensis has a significant extracellular phase, which makes it accessible to humoral immune responses (18). Indeed, there is ample evidence that B cells and antibodies are necessary for mice to develop their natural resistance to primary and secondary LVS infections. Purified lipopolysaccharide (LPS) from LVS induced a population of B1-a cells within 2 to 3 days of administration that protected mice against intraperitoneal (i.p.) LVS challenge (6, 7, 14). Consistent with these results, μMT mice lacking mature B cells exhibited increased susceptibility to primary intradermal (i.d.) LVS infection and delayed bacterial clearance (15, 40). μMT mice were also more susceptible to secondary i.p. LVS infection, and this defect was corrected by reconstitution with LVS-primed B cells (15). The contribution of antibodies has been addressed repeatedly in passive immunization experiments, which showed that immune serum from humans and mice vaccinated with live or inactivated LVS protected naïve mice against challenges with LVS or other low virulence strains given by a variety of routes (13, 19, 26, 29, 33, 36, 40). The dominant antibody response was directed at LPS, but antibodies against protein antigens have also been found (17, 23, 31, 41, 43). Monoclonal antibodies specific for LPS or the outer membrane protein FopA provided significant protection against LVS challenge when given either prophylactically (38) or therapeutically (30, 38). Together, these results suggest that antibodies contribute toward effective control of attenuated or low-virulence F. tularensis strains.It has been much more difficult to demonstrate antibody-mediated protection against type A strains in mice (1, 20, 21, 38), even though they express many antigens recognized by LVS immune serum (13, 30). This is not surprising given the historical difficulties in generating protective immunity against type A strains in this animal model (5). However, Ray et al. recently showed that oral LVS vaccination protected mice against a pulmonary SCHU S4 challenge in an antibody-dependent manner (35). Klimpel et al. also reported a similar finding using immune serum from mice cured of a lethal intranasal (i.n.) SCHU S4 infection with levofloxacin in a passive immunization model (27). Thus, the protective effects of antibodies appear not to be restricted only to low-virulence strains but may also contribute to the protection against highly virulent type A strains.To further characterize the mechanism of antibody-mediated protection, we utilized the recently characterized Fischer 344 (F344) rat model (45). Since F344 rats developed much stronger resistance to respiratory SCHU S4 challenge after LVS vaccination than previously observed in mice, we speculated that antibodies may provide better protection in this model and allow us to define their protective mechanism more thoroughly. We now show in a passive immunization model that serum antibodies from LVS-vaccinated rats conferred protection against a lethal intratracheal (i.t.) SCHU S4 challenge. Protection correlated with reduced systemic bacterial growth and less severe histopathology during the early phase of infection and bacterial clearance by a T cell-dependent mechanism. Thus, antibodies contribute to but are not sufficient for the effective control of respiratory infections by fully virulent type A strains. Our studies provide valuable insights into the protective mechanisms of antibodies that will guide future development of tularemia vaccine candidates.     

Identification of Three Novel Superantigen-Encoding Genes in Streptococcus equi subsp. zooepidemicus,szeF, szeN,and szeP

The acquisition of superantigen-encoding genes by Streptococcus pyogenes has been associated with increased morbidity and mortality in humans, and the gain of four superantigens by Streptococcus equi is linked to the evolution of this host-restricted pathogen from an ancestral strain of the opportunistic pathogen Streptococcus equi subsp. zooepidemicus. A recent study determined that the culture supernatants of several S. equi subsp. zooepidemicus strains possessed mitogenic activity but lacked known superantigen-encoding genes. Here, we report the identification and activities of three novel superantigen-encoding genes. The products of szeF, szeN, and szeP share 59%, 49%, and 34% amino acid sequence identity with SPEH, SPEM, and SPEL, respectively. Recombinant SzeF, SzeN, and SzeP stimulated the proliferation of equine peripheral blood mononuclear cells, and tumor necrosis factor alpha (TNF-α) and gamma interferon (IFN-γ) production, in vitro. Although none of these superantigen genes were encoded within functional prophage elements, szeN and szeP were located next to a prophage remnant, suggesting that they were acquired by horizontal transfer. Eighty-one of 165 diverse S. equi subsp. zooepidemicus strains screened, including 7 out of 15 isolates from cases of disease in humans, contained at least one of these new superantigen-encoding genes. The presence of szeN or szeP, but not szeF, was significantly associated with mitogenic activity in the S. equi subsp. zooepidemicus population (P < 0.000001, P < 0.000001, and P = 0.104, respectively). We conclude that horizontal transfer of these novel superantigens from and within the diverse S. equi subsp. zooepidemicus population is likely to have implications for veterinary and human disease.Gene gain via the horizontal acquisition of mobile genetic elements is a key factor in the emergence of new pathogenic strains of streptococci. An immediate selective advantage may be conferred on recipient bacteria through acquisition of antibiotic resistance mechanisms or the production of new virulence functions. Streptococcus equi subsp. equi is the causative agent of equine strangles, characterized by abscessation of the lymph nodes of the head and neck. S. equi subsp. equi is believed to have evolved from an ancestral strain of Streptococcus equi subsp. zooepidemicus (23, 49), which is associated with a wide variety of diseases in horses and other animals, including humans, through a process of gene loss and gain (20). These pathogens share approximately 80% DNA identity with the important human pathogen Streptococcus pyogenes (20).During the 1980s, strains of S. pyogenes emerged that caused streptococcal toxic shock syndrome (STSS), which was associated with particularly high morbidity and mortality in humans and the production of superantigens (sAgs) (13, 22). sAgs bypass the conventional mechanism of major histocompatibility complex (MHC)-restricted antigen presentation (15) and interfere with the development of a protective immune response through the generation of an overzealous proinflammatory response (32), disruption of antigen-specific T-cell responses, and inhibition of specific antibody production (27, 43).A total of 11 S. pyogenes sAgs have been described: SPEA, SPEC, SPEG, SPEH, SPEI, SPEJ, SPEK, SPEL, SPEM, SSA, and SMEZ (18, 43). The genes encoding SPEG, SPEJ, and SMEZ are chromosomally located, while the remaining sAg genes are located on mobile genetic elements. SPEA, SPEH, SPEI, and SSA share sequence similarities with the staphylococcal enterotoxins SEB, SEC, and SEG (18, 43). The S. equi subsp. equi strain 4047 genome contains four genes: seeH, seeI, seeL, and seeM (20), also known as sepe-H, sepe-I, speL_Se, and speM_Se, respectively, that encode homologues of S. pyogenes sAgs (4, 36). These genes are carried on two prophages, seeL and seeM on φSeq3 and seeH and seeI on φSeq4. S. equi subsp. equi sAgs stimulate equine T-cell proliferation in vitro and likely play an important role in S. equi subsp. equi pathogenicity (2, 4, 33). Interestingly, S. equi subsp. equi strain CF32 contains these sAg genes and predates SPEL- and SPEM-producing strains of S. pyogenes (20, 22), suggesting that cross-species transfer may have been responsible for the emergence of invasive M3/T3 strains of S. pyogenes. Comparison of S. equi subsp. equi strain 4047 prophage sequences to those in the public databases revealed that they share extensive similarity with prophage from S. pyogenes, so much so that clustering analysis demonstrated that the individual S. equi subsp. equi prophage are more closely related to phage in the sequenced S. pyogenes genomes than they are to each other, suggesting that the exchange of converting phage continues to influence the virulence of these important pathogens (20).S. equi subsp. zooepidemicus is the most frequently isolated opportunistic pathogen of horses; it is associated with inflammatory airway disease in Thoroughbred racehorses (50, 51), uterine infections in mares (21, 41), and ulcerative keratitis (8). It is also associated with disease in a wide range of other animal hosts, including cattle (39), sheep (25, 44), pigs (38, 42), monkeys (38, 42), dogs (12, 34), and humans (7, 17, 19). The broad range of hosts and tissues infected by S. equi subsp. zooepidemicus is reflected in its population diversity as determined by multilocus sequence typing (MLST) (49). Screening of a diverse population of 140 S. equi subsp. zooepidemicus isolates identified 25 strains of S. equi subsp. zooepidemicus that elicited potent mitogenic activity but did not test positive for the presence of seeH, seeI, seeL, or seeM (20), raising the possibility that these strains produce as-yet-unidentified sAgs. Several of these strains were related by MLST (49) and clustered into three groups around sequence type 123 (ST-123), ST-7, and ST-8 (20).In this study, we sequenced the genome of S. equi subsp. zooepidemicus strain BHS5 (ST-123), isolated from a dog with acute fatal hemorrhagic pneumonia in the United Kingdom in 1999 (49). Using comparative genomic analysis, we report the identification, activity, and prevalence of three novel sAgs and provide further evidence that the S. equi subsp. zooepidemicus population harbors virulence loci of importance to cross-species pathogen evolution.     

T Cells from Lungs and Livers of Francisella tularensis-Immune Mice Control the Growth of Intracellular Bacteria

Parenteral and respiratory vaccinations with the intracellular bacterium Francisella tularensis have been studied using the live vaccine strain (LVS) in a mouse model, and spleen cells from immune mice are often used for immunological studies. However, mechanisms of host immunological responses may be different in nonlymphoid organs that are important sites of infection, such as lung and liver. Using parenteral (intradermal) or respiratory (cloud aerosol) vaccination, here we examine the functions of resulting LVS-immune liver or lung cells, respectively. Surprisingly, LVS was considerably more virulent when administered by cloud aerosol than by intranasal instillation, suggesting method-dependent differences in initial localization and/or dissemination patterns. Only low doses were sublethal, and resolution of sublethal cloud aerosol infection was dependent on gamma interferon (IFN-γ), tumor necrosis factor alpha, and inducible nitric oxide synthase. Nonetheless, survival of cloud aerosol or parenteral infection resulted in the development of a protective immune response against lethal LVS intraperitoneal or aerosol challenge, reflecting development of systemic secondary immunity in both cases. Such immunity was further detected by directly examining the functions of LVS-immune lung or liver lymphocytes in vitro. Lung lymphocytes primed by respiratory infection, as well as liver lymphocytes primed by parenteral infection, clearly controlled in vitro intracellular bacterial growth primarily via mechanisms that were not dependent on IFN-γ activity. Thus, our results indicate functional similarities between immune T cells residing in spleens, livers, and lungs of LVS-immune mice.Francisella tularensis is a small gram-negative intracellular pathogen that can infect a variety of mammalian and arthropod hosts and cause tularemia, a disease that can be rapidly fatal. Infection can be initiated by a variety of routes, including via wounds, ingestion of contaminated food or water, insect bites, or by inhalation exposure (17). Aerogenic infection with as few as 10 organisms of type A F. tularensis subsp. tularensis may cause a fulminant and even fatal pulmonary disease. The traits of low infectious dose, airborne transmission, and high mortality rates have led to categorization of F. tularensis as a potential biowarfare agent (10).An attenuated live vaccine strain (LVS⁴), derived from virulent type B F. tularensis subsp. holarctica in Russia in the 1940s, appears to provide some protection against tularemia in humans (10, 38). Moreover, LVS has been used as a convenient and safe experimental model, since it exhibits attenuated virulence in humans but can cause a fatal disease in mice that is similar to human type A infection (15). In mice, the outcome of LVS infection is critically dependent on the route of inoculation. For BALB/cByJ male mice, the intrapertioneal (i.p.) 50% lethal dose (LD₅₀) approaches 1 single bacterium, whereas the intradermal (i.d.) LD₅₀ is approximately 10⁶ bacteria (15). The LD₅₀ of respiratory LVS infection via intranasal (i.n.) instillation or nose-only aerosol apparatus has been reported to be intermediate, about 1,000 to 3,000 CFU (7, 42).The spectrum of effector functions provided by T cells during adaptive immune responses to intracellular pathogens, which clearly include T-cell production of gamma interferon (IFN-γ) and induction of nitric oxide, is only partially understood. For Francisella, the majority of the studies to date have focused on mechanisms of protection following parenteral LVS vaccination. Similar to other intracellular bacteria, a strong innate immune response to LVS develops in mice; this response involves the production of early IFN-γ and tumor necrosis factor alpha (TNF-α) (15). During adaptive immunity generated by infection with this live attenuated strain, T cells are absolutely required for host survival of LVS infection, whereas B cells appear to play a minor role (15). In murine spleens, both CD4⁺ and CD8⁺ as well as an unusual population of CD4⁻ CD8⁻ T cells appear to be important and effect control of intracellular bacterial growth in vitro that depends only partially on IFN-γ but more heavily on TNF-α and ultimately production of nitric oxide (8, 9, 15).However, for Francisella infections as well as many other intracellular infections, knowledge of T-cell mechanisms has been mostly derived from studies of splenic T cells, which are readily available in numbers sufficient for detailed study. Little is known about immunological effector mechanisms that control infection in nonlymphoid organs of liver and lung, the other major sites of Francisella infection. Further, little is known about the quality of T-cell effector mechanisms engendered by vaccination via parenteral compared to respiratory routes. Different methods of experimental aerosol infection, which include intranasal administration, intratracheal or intrabronchial instillation, nose-only inhalation exposure, or whole-body aerosol exposure, may also qualitatively influence immune mechanisms. Notably, i.d. LVS immunization protects BALB/c mice against i.d. challenge with virulent type A F. tularensis but not against respiratory challenge; respiratory LVS vaccination, in contrast, provides protection against virulent respiratory challenge (7, 42). Thus, the responding cell populations, frequencies, or immune effector functions may be different when elicited by parenteral or respiratory vaccination. Here, we used i.d. or respiratory vaccination via a whole-body inhalation exposure system to examine T-cell function. We show that, similar to LVS-immune splenic T cells, LVS-immune lung and liver lymphocytes both controlled the intramacrophage growth of Francisella LVS during secondary immune responses by largely IFN-γ-independent mechanisms.     

Efficient Differentiation of Mycobacterium avium Complex Species and Subspecies by Use of Five-Target Multiplex PCR

Infections caused by the Mycobacterium avium complex (MAC) are on the rise in both human and veterinary medicine. A means of effectively discriminating among closely related yet pathogenetically diverse members of the MAC would enable better diagnosis and treatment as well as further our understanding of the epidemiology of these pathogens. In this study, a five-target multiplex PCR designed to discriminate MAC organisms isolated from liquid culture media was developed. This MAC multiplex was designed to amplify a 16S rRNA gene target common to all Mycobacterium species, a chromosomal target called DT1 that is unique to M. avium subsp. avium serotypes 2 and 3, to M. avium subsp. silvaticum, and to M. intracellulare, and three insertion sequences, IS900, IS901, and IS1311. The pattern of amplification results allowed determination of whether isolates were mycobacteria, whether they were members of the MAC, and whether they belonged to one of three major MAC subspecies, M. avium subsp. paratuberculosis, M. avium subsp. avium, and M. avium subsp. hominissuis. Analytical sensitivity was 10 fg of M. avium subsp. paratuberculosis genomic DNA, 5 to 10 fg of M. avium subsp. avium genomic DNA, and 2 to 5 fg of DNA from other mycobacterial species. Identification accuracy of the MAC multiplex was evaluated by testing 53 bacterial reference strains consisting of 28 different mycobacterial species and 12 nonmycobacterial species. Identification accuracy in a clinical setting was evaluated for 223 clinical MAC isolates independently identified by other methods. Isolate identification agreement between the MAC multiplex and these comparison assays was 100%. The novel MAC multiplex is a rapid, reliable, and simple assay for discrimination of MAC species and subspecies in liquid culture media.Since the early 1980s, there has been an increase in disease caused by organisms broadly categorized as nontuberculous mycobacteria (NTM), a generic term for mycobacteria not in the Mycobacterium tuberculosis complex and other than M. leprae (32). Of these NTM, Mycobacterium avium complex (MAC) species are the most common cause of human and animal disease globally (6, 14, 16, 24). The clinical relevance of the MAC in humans has been amplified in recent decades with the increasing population of immunocompromised individuals resulting from longer life expectancy, immunosuppressive chemotherapy, and the AIDS pandemic (27). The MAC is divided into two main species: M. avium and M. intracellulare. M. avium is further subdivided (per Turenne et al.) into four subspecies: M. avium subsp. avium, M. avium subsp. hominissuis, M. avium subsp. paratuberculosis, and M. avium subsp. silvaticum (39).Members of the family Mycobacteriaceae, comprising the MAC, differ in virulence and ecology. Those designated M. avium subsp. hominissuis are genomically diverse, low-virulence, opportunistic pathogens for both animals and humans. The majority of human M. avium subsp. hominissuis infections occur in HIV-immunocompromised people, immunocompetent persons with underling pulmonary disease, and children with cystic fibrosis (2, 12, 17). Considered ubiquitous in the environment (the most likely source of infection for humans), M. avium subsp. hominissuis has been isolated from water, soil, and dust (9). Domestic water distribution systems have been reported as possible sources of M. avium subsp. hominissuis infections in hospitals, homes, and commercial buildings (26, 27). In animals, M. avium subsp. hominissuis is found as a cause of lymphadenitis of the head and mesenteric lymph nodes of swine recognized at slaughter.Mycobacterium avium subsp. avium has long been recognized as a primary pathogen causing avian tuberculosis in wild and domestic birds (37, 38). Members of this subspecies also sporadically cause disease in other animals (6, 15, 30).For veterinarians, the MAC member of greatest importance is M. avium subsp. paratuberculosis. This MAC member causes a chronic granulomatous enteritis called Johne''s disease or paratuberculosis, most often in ruminants (16, 22, 31). Mycobacterium avium subsp. paratuberculosis is capable of infecting and causing disease a wide array of animal species, including nonhuman primates, without need of immunosuppressive coinfections. The herd-level prevalence of M. avium subsp. paratuberculosis infections in dairy cattle exceeds 50% in most major dairy product-producing countries (29, 31). Two systematic reviews and meta-analyses report a consistent association of M. avium subsp. paratuberculosis with Crohn''s disease, and the zoonotic potential of M. avium subsp. paratuberculosis continues to be a controversial subject discussed in the literature (1, 11). Unlike for most other M. avium subspecies, isolation of M. avium subsp. paratuberculosis requires the addition of the siderophore mycobactin to culture media and prolonged culture incubation for successful isolation from a tissue, soil, or fecal samples (43). After this lengthy incubation period with special media, resultant acid-fast organisms then need to be accurately identified.Unlike the M. avium subspecies, whose type strains were obtained from nonhuman hosts, the type strain of M. intracellulare (ATCC 13950) was isolated from a human, specifically a child who died from disseminated disease. Recently, numerous isolates considered to be M. intracellulare were reclassified as M. chimaera sp. nov. as part of the MAC (35). Few of these isolates were found to be clinically relevant, suggesting that this MAC species has low pathogenicity, and this factor is crucial to therapeutic decision making. Mycobacterium intracellulare appears to have a distinct environmental niche, more prevalent in biofilms and at significantly higher CFU numbers than M. avium (10, 36). It accounts for more documented human infections than M. avium subsp. hominissuis in several countries, including South Korea and Japan (19, 20, 23).Contemporary methods for MAC identification, e.g., high-performance liquid chromatography (HPLC) of cell wall mycolic acids, and genetic probes based on rRNA targets, e.g., AccuProbe, cannot discriminate among M. avium subspecies (2, 9). Given the differences in pathogenicity among M. avium subspecies and the implications regarding the infection source, a practical and accurate method of simply identifying M. avium subspecies is needed (13, 25, 35). In this study, we describe the specificity, discrimination capacity, and sensitivity of a novel five-target PCR, called the MAC multiplex, using a wide array of reference and clinical MAC isolates and numerous nonmycobacterial organisms.