Phagocytic receptors dictate phagosomal escape and intracellular proliferation of Francisella tularensis

Francisella tularensis, the causative agent of tularemia, survives and proliferates within macrophages of the infected host as part of its pathogenic strategy, through an intracellular life cycle that includes phagosomal escape and extensive proliferation within the macrophage cytosol. Various in vitro models of Francisella-macrophage interactions have been developed, using either opsonic or nonopsonic phagocytosis, and have generated discrepant results on the timing and extent of Francisella phagosomal escape. Here we have investigated whether either complement or antibody opsonization of the virulent prototypical type A strain Francisella tularensis subsp. tularensis Schu S4 affects its intracellular cycle within primary murine bone marrow-derived macrophages. Opsonization of Schu S4 with either human serum or purified IgG enhanced phagocytosis but restricted phagosomal escape and intracellular proliferation. Opsonization of Schu S4 with either fresh serum or purified antibodies redirected bacteria from the mannose receptor (MR) to the complement receptor CR3, the scavenger receptor A (SRA), and the Fcγ receptor (FcγR), respectively. CR3-mediated uptake delayed maturation of the early Francisella-containing phagosome (FCP) and restricted phagosomal escape, while FcγR-dependent phagocytosis was associated with superoxide production in the early FCP and restricted phagosomal escape and intracellular growth in an NADPH oxidase-dependent manner. Taken together, these results demonstrate that opsonophagocytic receptors alter the intracellular fate of Francisella by delivering bacteria through phagocytic pathways that restrict phagosomal escape and intracellular proliferation.     

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.     

IglE Is an Outer Membrane-Associated Lipoprotein Essential for Intracellular Survival and Murine Virulence of Type A Francisella tularensis

IglE is a small, hypothetical protein encoded by the duplicated Francisella pathogenicity island (FPI). Inactivation of both copies of iglE rendered Francisella tularensis subsp. tularensis Schu S4 avirulent and incapable of intracellular replication, owing to an inability to escape the phagosome. This defect was fully reversed following single-copy expression of iglE in trans from attTn7 under the control of the Francisella rpsL promoter, thereby establishing that the loss of iglE, and not polar effects on downstream vgrG gene expression, was responsible for the defect. IglE is exported to the Francisella outer membrane as an ∼13.9-kDa lipoprotein, determined on the basis of a combination of selective Triton X-114 solubilization, radiolabeling with [³H]palmitic acid, and sucrose density gradient membrane partitioning studies. Lastly, a genetic screen using the iglE-null live vaccine strain resulted in the identification of key regions in the carboxyl terminus of IglE that are required for intracellular replication of Francisella tularensis in J774A.1 macrophages. Thus, IglE is essential for Francisella tularensis virulence. Our data support a model that likely includes protein-protein interactions at or near the bacterial cell surface that are unknown at present.     

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.     

Disruption of Francisella tularensis Schu S4 iglI,iglJ, and pdpC Genes Results in Attenuation for Growth in Human Macrophages and In Vivo Virulence in Mice and Reveals a Unique Phenotype for pdpC

Francisella tularensis is a facultative intracellular bacterial pathogen and the causative agent of tularemia. After infection of macrophages, the organism escapes from its phagosome and replicates to high density in the cytosol, but the bacterial factors required for these aspects of virulence are incompletely defined. Here, we describe the isolation and characterization of Francisella tularensis subsp. tularensis strain Schu S4 mutants that lack functional iglI, iglJ, or pdpC, three genes of the Francisella pathogenicity island. Our data demonstrate that these mutants were defective for replication in primary human monocyte-derived macrophages and murine J774 cells yet exhibited two distinct phenotypes. The iglI and iglJ mutants were similar to one another, exhibited profound defects in phagosome escape and intracellular growth, and appeared to be trapped in cathepsin D-positive phagolysosomes. Conversely, the pdpC mutant avoided trafficking to lysosomes, phagosome escape was diminished but not ablated, and these organisms replicated in a small subset of infected macrophages. The phenotype of each mutant strain was reversed by trans complementation. In vivo virulence was assessed by intranasal infection of BALB/c mice. The mutants appeared avirulent, as all mice survived infection with 10⁸ CFU iglJ- or pdpC-deficient bacteria. Nevertheless, the pdpC mutant disseminated to the liver and spleen before being eliminated, whereas the iglJ mutant did not. Taken together, our data demonstrate that the pathogenicity island genes tested are essential for F. tularensis Schu S4 virulence and further suggest that pdpC may play a unique role in this process, as indicated by its distinct intermediate phenotype.     

Importance of Branched-Chain Amino Acid Utilization in Francisella Intracellular Adaptation

Intracellular bacterial pathogens have adapted their metabolism to optimally utilize the nutrients available in infected host cells. We recently reported the identification of an asparagine transporter required specifically for cytosolic multiplication of Francisella. In the present work, we characterized a new member of the major super family (MSF) of transporters, involved in isoleucine uptake. We show that this transporter (here designated IleP) plays a critical role in intracellular metabolic adaptation of Francisella. Inactivation of IleP severely impaired intracellular F. tularensis subsp. novicida multiplication in all cell types tested and reduced bacterial virulence in the mouse model. To further establish the importance of the ileP gene in F. tularensis pathogenesis, we constructed a chromosomal deletion mutant of ileP (ΔFTL_1803) in the F. tularensis subsp. holarctica live vaccine strain (LVS). Inactivation of IleP in the F. tularensis LVS provoked comparable intracellular growth defects, confirming the critical role of this transporter in isoleucine uptake. The data presented establish, for the first time, the importance of isoleucine utilization for efficient phagosomal escape and cytosolic multiplication of Francisella and suggest that virulent F. tularensis subspecies have lost their branched-chain amino acid biosynthetic pathways and rely exclusively on dedicated uptake systems. This loss of function is likely to reflect an evolution toward a predominantly intracellular life style of the pathogen. Amino acid transporters should be thus considered major players in the adaptation of intracellular pathogens.     

Combined deletion of four Francisella novicida acid phosphatases attenuates virulence and macrophage vacuolar escape

Francisella tularensis is a facultative intracellular pathogen and the etiologic agent of tularemia. It is capable of escape from macrophage phagosomes and replicates in the host cell cytosol. Bacterial acid phosphatases are thought to play a major role in the virulence and intracellular survival of a number of intracellular pathogens. The goal of this study was to delete the four primary acid phosphatases (Acps) from Francisella novicida and examine the interactions of mutant strains with macrophages, as well as the virulence of these strains in mice. We constructed F. novicida mutants with various combinations of acp deletions and showed that loss of the four Acps (AcpA, AcpB, AcpC, and histidine acid phosphatase [Hap]) in an F. novicida strain (ΔABCH) resulted in a 90% reduction in acid phosphatase activity. The ΔABCH mutant was defective for survival/growth within human and murine macrophage cell lines and was unable to escape from phagosome vacuoles. With accumulation of Acp deletions, a progressive loss of virulence in the mouse model was observed. The ΔABCH strain was dramatically attenuated and was an effective single-dose vaccine against homologous challenge. Furthermore, both acpA and hap were induced when the bacteria were within host macrophages. Thus, the Francisella acid phosphatases cumulatively play an important role in intracellular trafficking and virulence.     

Identifying Francisella tularensis Genes Required for Growth in Host Cells

Francisella tularensis is a highly virulent Gram-negative intracellular pathogen capable of infecting a vast diversity of hosts, ranging from amoebae to humans. A hallmark of F. tularensis virulence is its ability to quickly grow to high densities within a diverse set of host cells, including, but not limited to, macrophages and epithelial cells. We developed a luminescence reporter system to facilitate a large-scale transposon mutagenesis screen to identify genes required for growth in macrophage and epithelial cell lines. We screened 7,454 individual mutants, 269 of which exhibited reduced intracellular growth. Transposon insertions in the 269 growth-defective strains mapped to 68 different genes. FTT_0924, a gene of unknown function but highly conserved among Francisella species, was identified in this screen to be defective for intracellular growth within both macrophage and epithelial cell lines. FTT_0924 was required for full Schu S4 virulence in a murine pulmonary infection model. The ΔFTT_0924 mutant bacterial membrane is permeable when replicating in hypotonic solution and within macrophages, resulting in strongly reduced viability. The permeability and reduced viability were rescued when the mutant was grown in a hypertonic solution, indicating that FTT_0924 is required for resisting osmotic stress. The ΔFTT_0924 mutant was also significantly more sensitive to β-lactam antibiotics than Schu S4. Taken together, the data strongly suggest that FTT_0924 is required for maintaining peptidoglycan integrity and virulence.     

Identification of Early Interactions between Francisella and the Host

The adaptive immune response to Francisella tularensis is dependent on the route of inoculation. Intradermal inoculation with the F. tularensis live vaccine strain (LVS) results in a robust Th1 response in the lungs, whereas intranasal inoculation produces fewer Th1 cells and instead many Th17 cells. Interestingly, bacterial loads in the lungs are similar early after inoculation by these two routes. We hypothesize that the adaptive immune response is influenced by local events in the lungs, such as the type of cells that are first infected with Francisella. Using fluorescence-activated cell sorting, we identified alveolar macrophages as the first cell type infected in the lungs of mice intranasally inoculated with F. novicida U112, LVS, or F. tularensis Schu S4. Following bacterial dissemination from the skin to the lung, interstitial macrophages or neutrophils are infected. Overall, we identified the early interactions between Francisella and the host following two different routes of inoculation.     

MglA and Igl proteins contribute to the modulation of Francisella tularensis live vaccine strain-containing phagosomes in murine macrophages

The Francisella tularensis live vaccine strain (LVS), in contrast to its iglC mutant, replicates in the cytoplasm of macrophages. We studied the outcome of infection of the murine macrophagelike cell line J774A.1 with LVS and with iglC, iglD, and mglA mutants, the latter of which is deficient in a global regulator. Compared to LVS, all of the mutants showed impaired intracellular replication up to 72 h, and the number of the mglA mutant bacteria even decreased. Colocalization with LAMP-1 was significantly increased for all mutants compared to LVS, indicating an impaired ability to escape into the cytoplasm. A lysosomal acidity-dependent dye accumulated in approximately 40% of the vacuoles containing mutant bacteria but not at all in vacuoles containing LVS. Preactivation of the macrophages with gamma interferon inhibited the intracellular growth of all strains and significantly increased acidification of phagosomes containing the mutants, but it only slightly increased the LAMP-1 colocalization. The intracellular replication and phagosomal escape of the iglC and iglD mutants were restored by complementation in trans. In conclusion, the IglC, IglD, and MglA proteins each directly or indirectly critically contribute to the virulence of F. tularensis LVS, including its intracellular replication, cytoplasmic escape, and inhibition of acidification of the phagosomes.     

Adaptation of Francisella tularensis to the mammalian environment is governed by cues which can be mimicked in vitro

The intracellular bacterium Francisella tularensis survives in mammals, arthropods, and freshwater amoeba. It was previously established that the conventional media used for in vitro propagation of this microbe do not yield bacteria that mimic those harvested from infected mammals; whether these in vitro-cultivated bacteria resemble arthropod- or amoeba-adapted Francisella is unknown. As a foundation for our goal of identifying F. tularensis outer membrane proteins which are expressed during mammalian infection, we first sought to identify in vitro cultivation conditions that induce the bacterium's infection-derived phenotype. We compared Francisella LVS grown in brain heart infusion broth (BHI; a standard microbiological medium rarely used in Francisella research) to that grown in Mueller-Hinton broth (MHB; the most widely used F. tularensis medium, used here as a negative control) and macrophages (a natural host cell, used here as a positive control). BHI- and macrophage-grown F. tularensis cells showed similar expression of MglA-dependent and MglA-independent proteins; expression of the MglA-dependent proteins was repressed by the supraphysiological levels of free amino acids present in MHB. We observed that during macrophage infection, protein expression by intracellular bacteria differed from that by extracellular bacteria; BHI-grown bacteria mirrored the latter, while MHB-grown bacteria resembled neither. Naïve macrophages responding to BHI- and macrophage-grown bacteria produced markedly lower levels of proinflammatory mediators than those in cells exposed to MHB-grown bacteria. In contrast to MHB-grown bacteria, BHI-grown bacteria showed minimal delay during intracellular replication. Cumulatively, our findings provide compelling evidence that growth in BHI yields bacteria which recapitulate the phenotype of Francisella organisms that have emerged from macrophages.     

Staphylococcus aureus Strain USA300 Perturbs Acquisition of Lysosomal Enzymes and Requires Phagosomal Acidification for Survival inside Macrophages

Methicillin-resistant Staphylococcus aureus (MRSA) causes invasive, drug-resistant skin and soft tissue infections. Reports that S. aureus bacteria survive inside macrophages suggest that the intramacrophage environment may be a niche for persistent infection; however, mechanisms by which the bacteria might evade macrophage phagosomal defenses are unclear. We examined the fate of the S. aureus-containing phagosome in THP-1 macrophages by evaluating bacterial intracellular survival and phagosomal acidification and maturation and by testing the impact of phagosomal conditions on bacterial viability. Multiple strains of S. aureus survived inside macrophages, and in studies using the MRSA USA300 clone, the USA300-containing phagosome acidified rapidly and acquired the late endosome and lysosome protein LAMP1. However, fewer phagosomes containing live USA300 bacteria than those containing dead bacteria associated with the lysosomal hydrolases cathepsin D and β-glucuronidase. Inhibiting lysosomal hydrolase activity had no impact on intracellular survival of USA300 or other S. aureus strains, suggesting that S. aureus perturbs acquisition of lysosomal enzymes. We examined the impact of acidification on S. aureus intramacrophage viability and found that inhibitors of phagosomal acidification significantly impaired USA300 intracellular survival. Inhibition of macrophage phagosomal acidification resulted in a 30-fold reduction in USA300 expression of the staphylococcal virulence regulator agr but had little effect on expression of sarA, saeR, or sigB. Bacterial exposure to acidic pH in vitro increased agr expression. Together, these results suggest that S. aureus survives inside macrophages by perturbing normal phagolysosome formation and that USA300 may sense phagosomal conditions and upregulate expression of a key virulence regulator that enables its intracellular survival.     

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.     

Importance of PdpC,IglC, IglI,and IglG for Modulation of a Host Cell Death Pathway Induced by Francisella tularensis

Modulation of host cell death pathways appears to be a prerequisite for the successful lifestyles of many intracellular pathogens. The facultative intracellular bacterium Francisella tularensis is highly pathogenic, and effective proliferation in the macrophage cytosol leading to host cell death is a requirement for its virulence. To better understand the prerequisites of this cell death, macrophages were infected with the F. tularensis live vaccine strain (LVS), and the effects were compared to those resulting from infections with deletion mutants lacking expression of either of the pdpC, iglC, iglG, or iglI genes, which encode components of the Francisella pathogenicity island (FPI), a type VI secretion system. Within 12 h, a majority of the J774 cells infected with the LVS strain showed production of mitochondrial superoxide and, after 24 h, marked signs of mitochondrial damage, caspase-9 and caspase-3 activation, phosphatidylserine expression, nucleosome formation, and membrane leakage. In contrast, neither of these events occurred after infection with the ΔiglI or ΔiglC mutants, although the former strain replicated. The ΔiglG mutant replicated effectively but induced only marginal cytopathogenic effects after 24 h and intermediate effects after 48 h. In contrast, the ΔpdpC mutant showed no replication but induced marked mitochondrial superoxide production and mitochondrial damage, caspase-3 activation, nucleosome formation, and phosphatidylserine expression, although the effects were delayed compared to those obtained with LVS. The unique phenotypes of the mutants provide insights regarding the roles of individual FPI components for the modulation of the cytopathogenic effects resulting from the F. tularensis infection.     

RipA, a cytoplasmic membrane protein conserved among Francisella species, is required for intracellular survival

Francisella tularensis is a highly virulent bacterial pathogen that invades and replicates within numerous host cell types, including macrophages and epithelial cells. In an effort to better understand this process, we screened a transposon insertion library of the F. tularensis live vaccine strain (LVS) for mutant strains that invaded but failed to replicate within alveolar epithelial cell lines. One such strain isolated from this screen contained an insertion in the gene FTL_1914, which is conserved among all sequenced Francisella species yet lacks significant homology to any gene with known function. A deletion strain lacking FTL_1914 was constructed. This strain did not replicate in either epithelial or macrophage-like cells, and intracellular replication was restored by the wild-type allele in trans. Based on the deletion mutant phenotype, FTL_1914 was termed ripA (required for intracellular proliferation, factor A). Following uptake by J774.A1 cells, F. tularensis LVS ΔripA colocalized with LAMP-1 then escaped the phagosome at the same rate and frequency as wild-type LVS-infected cells. Electron micrographs of the F. tularensis LVS ΔripA mutant demonstrated the reentry of the mutant bacteria into double membrane vacuoles characteristic of autophagosomes in a process that was not dependent on replication. The F. tularensis LVS ΔripA mutant was significantly impaired in its ability to persist in the lung and in its capacity to disseminate and colonize the liver and spleen in a mouse model of pulmonary tularemia. The RipA protein was expressed during growth in laboratory media and localized to the cytoplasmic membrane. Thus, RipA is a cytoplasmic membrane protein conserved among Francisella species that is required for intracellular replication within the host cell cytoplasm as well as disease progression, dissemination, and virulence.     

Rab14 Regulates Maturation of Macrophage Phagosomes Containing the Fungal Pathogen Candida albicans and Outcome of the Host-Pathogen Interaction

Avoidance of innate immune defense is an important mechanism contributing to the pathogenicity of microorganisms. The fungal pathogen Candida albicans undergoes morphogenetic switching from the yeast to the filamentous hyphal form following phagocytosis by macrophages, facilitating its escape from the phagosome, which can result in host cell lysis. We show that the intracellular host trafficking GTPase Rab14 plays an important role in protecting macrophages from lysis mediated by C. albicans hyphae. Live-cell imaging of macrophages expressing green fluorescent protein (GFP)-tagged Rab14 or dominant negative Rab14, or with small interfering RNA (siRNA)-mediated knockdown of Rab14, revealed the temporal dynamics of this protein and its influence on the maturation of macrophage phagosomes following the engulfment of C. albicans cells. Phagosomes containing live C. albicans cells became transiently Rab14 positive within 2 min following engulfment. The duration of Rab14 retention on phagosomes was prolonged for hyphal cargo and was directly proportional to hyphal length. Interference with endogenous Rab14 did not affect the migration of macrophages toward C. albicans cells, the rate of engulfment, the overall uptake of fungal cells, or early phagosome processing. However, Rab14 depletion delayed the acquisition of the late phagosome maturation markers LAMP1 and lysosomal cathepsin, indicating delayed formation of a fully bioactive lysosome. This was associated with a significant increase in the level of macrophage killing by C. albicans. Therefore, Rab14 activity promotes phagosome maturation during C. albicans infection but is dysregulated on the phagosome in the presence of the invasive hyphal form, which favors fungal survival and escape.     

Novel Catanionic Surfactant Vesicle Vaccines Protect against Francisella tularensis LVS and Confer Significant Partial Protection against F. tularensis Schu S4 Strain

Francisella tularensis is a Gram-negative immune-evasive coccobacillus that causes tularemia in humans and animals. A safe and efficacious vaccine that is protective against multiple F. tularensis strains has yet to be developed. In this study, we tested a novel vaccine approach using artificial pathogens, synthetic nanoparticles made from catanionic surfactant vesicles that are functionalized by the incorporation of either F. tularensis type B live vaccine strain (F. tularensis LVS [LVS-V]) or F. tularensis type A Schu S4 strain (F. tularensis Schu S4 [Schu S4-V]) components. The immunization of C57BL/6 mice with “bare” vesicles, which did not express F. tularensis components, partially protected against F. tularensis LVS, presumably through activation of the innate immune response, and yet it failed to protect against the F. tularensis Schu S4 strain. In contrast, immunization with LVS-V fully protected mice against intraperitoneal (i.p.) F. tularensis LVS challenge, while immunization of mice with either LVS-V or Schu S4-V partially protected C57BL/6 mice against an intranasal (i.n.) F. tularensis Schu S4 challenge and significantly increased the mean time to death for nonsurvivors, particularly following the i.n. and heterologous (i.e., i.p./i.n.) routes of immunization. LVS-V immunization, but not immunization with empty vesicles, elicited high levels of IgG against nonlipopolysaccharide (non-LPS) epitopes that were increased after F. tularensis LVS challenge and significantly increased early cytokine production. Antisera from LVS-V-immunized mice conferred passive protection against challenge with F. tularensis LVS. Together, these data indicate that functionalized catanionic surfactant vesicles represent an important and novel tool for the development of a safe and effective F. tularensis subunit vaccine and may be applicable for use with other pathogens.     

Infected-host-cell repertoire and cellular response in the lung following inhalation of Francisella tularensis Schu S4, LVS, or U112

Francisella tularensis causes systemic disease in humans and other mammals, with high morbidity and mortality associated with inhalation-acquired infection. F. tularensis is a facultative intracellular pathogen, but the scope and significance of cell types infected during disease is unknown. Using flow cytometry, we identified and quantified infected-cell types and assessed the impact of infection on cell populations following inhalation of F. tularensis strains U112, LVS, and Schu S4. Initially, alveolar macrophages comprised over 70% of Schu S4- and LVS-infected cells, whereas approximately 51% and 27% of U112-infected cells were alveolar macrophages and neutrophils, respectively. After 3 days, roughly half of Schu S4- and LVS- and nearly 80% of U112-infected cells were neutrophils. All strains infected CD11b^high macrophages, dendritic cells, monocytes, and alveolar type II cells throughout infection. Macrophage, monocyte, and dendritic-cell populations were reduced during U112 infection but not Schu S4 or LVS infection. These results demonstrate directly that F. tularensis is a promiscuous intracellular pathogen in the lung that invades and replicates within cell types ranging from migratory immune cells to structural tissue cells. However, the proportions of cell types infected and the cellular immune response evoked by the human pathogenic strain Schu S4 differ from those of the human avirulent U112.     

Molecular method for discrimination between Francisella tularensis and Francisella-like endosymbionts

Environmental studies on the distribution of Francisella spp. are hampered by the frequency of Francisella-like endosymbionts that can produce a misleading positive result. A new, efficient molecular method for detection of Francisella tularensis and its discrimination from Francisella-like endosymbionts, as well as two variants associated with human disease (unusual F. tularensis strain FnSp1 and F. tularensis subsp. novicida-like strain 3523), is described. The method is highly specific and sensitive, detecting up to one plasmid copy or 10 genome equivalents.