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.     

Acanthamoeba culbertsoni Elicits Soluble Factors That Exert Anti-Microglial Cell Activity

Acanthamoeba culbertsoni is an opportunistic pathogen that causes granulomatous amoebic encephalitis (GAE), a chronic and often fatal disease of the central nervous system (CNS). A hallmark of GAE is the formation of granulomas around the amoebae. These cellular aggregates consist of microglia, macrophages, lymphocytes, and neutrophils, which produce a myriad of proinflammatory soluble factors. In the present study, it is demonstrated that A. culbertsoni secretes serine peptidases that degrade chemokines and cytokines produced by a mouse microglial cell line (BV-2 cells). Furthermore, soluble factors present in cocultures of A. culbertsoni and BV-2 cells, as well as in cocultures of A. culbertsoni and primary neonatal rat cerebral cortex microglia, induced apoptosis of these macrophage-like cells. Collectively, the results indicate that A. culbertsoni can apply a multiplicity of cell contact-independent modes to target macrophage-like cells that exert antiamoeba activities in the CNS.Acanthamoeba culbertsoni belongs to a group of free-living amoebae, such as Balamuthia mandrillaris, Naegleria fowleri, and Sappinia pedata, that can cause disease in humans (46, 56). Acanthamoeba spp. are found worldwide and have been isolated from a variety of environmental sources, including air, soil, dust, tap water, freshwater, seawater, swimming pools, air conditioning units, and contaminated contact lenses (30). Trophozoites feed on bacteria and algae and represent the infective form (47, 56). However, under unfavorable environmental conditions, such as extreme changes in temperature or pH, trophozoites transform into a double-walled, round cyst (22, 45).Acanthamoeba spp. cause an infection of the eye known as amoebic keratitis (AK), an infection of the skin referred to as cutaneous acanthamoebiasis, and a chronic and slowly progressing disease of the central nervous system (CNS) known as granulomatous amoebic encephalitis (GAE) (22, 23, 30, 56). GAE is most prevalent in humans who are immunocompromised (30, 33, 40) and has been reported to occur among individuals infected with the human immunodeficiency virus (HIV) (28). It has been proposed that Acanthamoeba trophozoites access the CNS by passage through the olfactory neuroepithelium (32) or by hematogenous spread from a primary nonneuronal site of infection (23, 24, 33, 53).In immune-competent individuals, GAE is characterized by the formation of granulomas. These cellular aggregates consist of microglia, macrophages, polymorphonuclear cells, T lymphocytes, and B lymphocytes (24, 30). The concerted action of these immune cells results in sequestration of amoebae and is instrumental in slowing the progression of GAE. This outcome is consistent with the observation that granulomas are rarely observed in immunocompromised individuals (34) and in mice with experimentally induced immune suppression following treatment with the cannabinoid delta-9-tetrahydrocannabinol (Δ⁹-THC) (8).Microglia are a resident population of macrophages in the CNS. These cells, along with CNS-invading peripheral macrophages, appear to play a critical early effector role in the control of Acanthamoeba spread during GAE (4, 5, 29, 31). In vitro, microglia have been shown to produce an array of chemokines and cytokines in response to Acanthamoeba (31, 51). However, these factors appear not to have a deleterious effect on these amoebae (29).Acanthamoeba spp. produce serine peptidases, cysteine peptidases, and metallopeptidases (1, 2, 9, 10, 14, 16, 18, 19, 21, 25, 26, 37, 38, 41, 42, 52). In the present study, it is demonstrated that serine peptidases secreted by A. culbertsoni degrade chemokines and cytokines that are produced by immortalized mouse BV-2 microglia-like cells. In addition, soluble factors present in cocultures of A. culbertsoni and BV-2 cells induced apoptosis of the BV-2 cells. Collectively, these results suggest a mode through which A. culbertsoni can evade immune responsiveness in the CNS.     

Hag Mediates Adherence of Moraxella catarrhalis to Ciliated Human Airway Cells

Moraxella catarrhalis is a human pathogen causing otitis media in infants and respiratory infections in adults, particularly patients with chronic obstructive pulmonary disease. The surface protein Hag (also designated MID) has previously been shown to be a key adherence factor for several epithelial cell lines relevant to pathogenesis by M. catarrhalis, including NCIH292 lung cells, middle ear cells, and A549 type II pneumocytes. In this study, we demonstrate that Hag mediates adherence to air-liquid interface cultures of normal human bronchial epithelium (NHBE) exhibiting mucociliary activity. Immunofluorescent staining and laser scanning confocal microscopy experiments demonstrated that the M. catarrhalis wild-type isolates O35E, O12E, TTA37, V1171, and McGHS1 bind principally to ciliated NHBE cells and that their corresponding hag mutant strains no longer associate with cilia. The hag gene product of M. catarrhalis isolate O35E was expressed in the heterologous genetic background of a nonadherent Haemophilus influenzae strain, and quantitative assays revealed that the adherence of these recombinant bacteria to NHBE cultures was increased 27-fold. These experiments conclusively demonstrate that the hag gene product is responsible for the previously unidentified tropism of M. catarrhalis for ciliated NHBE cells.Moraxella catarrhalis is a gram-negative pathogen of the middle ear and lower respiratory tract (29, 40, 51, 52, 69, 78). The organism is responsible for ∼15% of bacterial otitis media cases in children and up to 10% of infectious exacerbations in patients with chronic obstructive pulmonary disease (COPD). The cost of treating these ailments places a large financial burden on the health care system, adding up to well over $10 billion per annum in the United States alone (29, 40, 52, 95, 97). In recent years, M. catarrhalis has also been increasingly associated with infections such as bronchitis, conjunctivitis, sinusitis, bacteremia, pneumonia, meningitis, pericarditis, and endocarditis (3, 12, 13, 17-19, 24, 25, 27, 51, 67, 70, 72, 92, 99, 102-104). Therefore, the organism is emerging as an important health problem.M. catarrhalis infections are a matter of concern due to high carriage rates in children, the lack of a preventative vaccine, and the rapid emergence of antibiotic resistance in clinical isolates. Virtually all M. catarrhalis strains are resistant to β-lactams (34, 47, 48, 50, 53, 65, 81, 84). The genes specifying this resistance appear to be gram positive in origin (14, 15), suggesting that the organism could acquire genes conferring resistance to other antibiotics via horizontal transfer. Carriage rates as high as 81.6% have been reported for children (39, 104). In one study, Faden and colleagues analyzed the nasopharynx of 120 children over a 2-year period and showed that 77.5% of these patients became colonized by M. catarrhalis (35). These investigators also observed a direct relationship between the development of otitis media and the frequency of colonization. This high carriage rate, coupled with the emergence of antibiotic resistance, suggests that M. catarrhalis infections may become more prevalent and difficult to treat. This emphasizes the need to study pathogenesis by this bacterium in order to identify vaccine candidates and new targets for therapeutic approaches.One key aspect of pathogenesis by most infectious agents is adherence to mucosal surfaces, because it leads to colonization of the host (11, 16, 83, 93). Crucial to this process are surface proteins termed adhesins, which mediate the binding of microorganisms to human cells and are potential targets for vaccine development. M. catarrhalis has been shown to express several adhesins, namely UspA1 (20, 21, 59, 60, 77, 98), UspA2H (59, 75), Hag (also designated MID) (22, 23, 37, 42, 66), OMPCD (4, 41), McaP (61, 100), and a type 4 pilus (63, 64), as well as the filamentous hemagglutinin-like proteins MhaB1, MhaB2, MchA1, and MchA2 (7, 79). Each of these adhesins was characterized by demonstrating a decrease in the adherence of mutant strains to a variety of human-derived epithelial cell lines, including A549 type II pneumocytes and Chang conjunctival, NCIH292 lung mucoepidermoid, HEp2 laryngeal, and 16HBE14o-polarized bronchial cells. Although all of these cell types are relevant to the diseases caused by M. catarrhalis, they lack important aspects of the pathogen-targeted mucosa, such as the features of cilia and mucociliary activity. The ciliated cells of the respiratory tract and other mucosal membranes keep secretions moving out of the body so as to assist in preventing colonization by invading microbial pathogens (10, 26, 71, 91). Given this critical role in host defense, it is interesting to note that a few bacterial pathogens target ciliated cells for adherence, including Actinobacillus pleuropneumoniae (32), Pseudomonas aeruginosa (38, 108), Mycoplasma pneumoniae (58), Mycoplasma hyopneumoniae (44, 45), and Bordetella species (5, 62, 85, 101).In the present study, M. catarrhalis is shown to specifically bind to ciliated cells of a normal human bronchial epithelium (NHBE) culture exhibiting mucociliary activity. This tropism was found to be conserved among isolates, and analysis of mutants revealed a direct role for the adhesin Hag in binding to ciliated airway cells.     

Genetics and Virulence Association of the Shigella flexneri Sit Iron Transport System

The sit-encoded iron transport system is present within pathogenicity islands in all Shigella spp. and some pathogenic Escherichia coli strains. The islands contain numerous insertion elements and sequences with homology to bacteriophage genes. The Shigella flexneri sit genes can be lost as a result of deletion within the island. The formation of deletions was dependent upon RecA and occurred at relatively high frequency. This suggests that the sit region is inherently unstable, yet sit genes are maintained in all of the clinical isolates tested. Characterization of the sitABCD genes in S. flexneri indicates that they encode a ferrous iron transport system, although the genes are induced aerobically. The sit genes provide a competitive advantage to S. flexneri growing within epithelial cells, and a sitA mutant is outcompeted by the wild type in cultured epithelial cells. The Sit system is also required for virulence in a mouse lung model. The sitA mutant was able to infect the mice and induce a protective immune response but was avirulent compared to its wild-type parent strain.The Shigella species are invasive enteric pathogens that cause dysentery in their human host. Following ingestion, the bacteria invade colonic epithelial cells and multiply within the cytoplasm. They spread from cell to cell, eventually causing death of the host cells and provoking an intense inflammatory response (39, 51).Shigella spp. are closely related to and considered part of the same species as Escherichia coli (40, 41). The patterns that have emerged from analysis of the E. coli and Shigella genomes are that functional genes that are present in two or more members of this enteric group show a high degree of sequence conservation, but the overall organization of the genomes is different. Compared with the E. coli K-12 genome, each of the Shigella genomes contains a significant number of rearrangements, as well as insertions and deletions. Some of the insertions, termed pathogenicity islands (PAI), are quite large and include genes that increase virulence or fitness. Deletions in the chromosome may also affect pathogenicity; for example, the deletion of lysine decarboxylase genes in S. flexneri is associated with increased virulence (25, 26).One class of genes that shows significant variability among Shigella and E. coli strains are those encoding high-affinity transport systems for iron. Iron is an essential element for Shigella, but the acquisition of iron is complicated by its insolubility in aerobic environments at neutral pH. Bacteria often have multiple pathways for importing iron, allowing the utilization of Fe⁺⁺, Fe⁺⁺⁺, and iron bound to a variety of carriers.Ferric iron is efficiently transported into E. coli K-12 by the siderophore enterobactin (Ent) (11). This low-molecular-weight iron chelator is synthesized and secreted into the environment, where it binds ferric iron with high affinity (32), and the ferri-siderophore complex is transported back into the cell via a specific transporter system (Fep). Most E. coli, Shigella dysenteriae type 1, and Shigella sonnei strains synthesize and transport enterobactin (11, 34, 36, 38). However, many Shigella boydii and Shigella flexneri strains are Ent⁻ due to deletions and point mutations within the ent/fep operons (36, 52). Shigella strains that fail to produce enterobactin produce a different siderophore, aerobactin, and some strains produce both siderophores (22, 36). Aerobactin is a secondary hydroxamate, and genes for its synthesis (iucABCD) and receptor (iutA) are located within a single operon (4, 22). These genes may be on either the chromosome (22, 24) or a plasmid (4, 8). The chromosomal genes are located within PAI in S. flexneri and S. boydii (31, 42, 57).The Shigella species have additional iron transport systems that are not found in E. coli K-12. A transport system with homology to the Salmonella enteritidis Sit system is found in all of the Shigella species. Some strains have heme transport systems, the best characterized of which are the S. dysenteriae Shu heme transporter (30, 61) and a nearly identical system in E. coli O157:H7 (54). S. dysenteriae type 1 strains also have the iro genes (43) for the biosynthesis of salmochelin, a modified form of enterobactin first described in Salmonella (13). Additional transport systems for iron are present in enteric pathogens, and some of these are completely uncharacterized.The only iron transport system that appears to be common to all members of the E. coli/Shigella group is Feo. This is a ferrous iron cytoplasmic membrane transporter encoded by the feoABC genes (5, 19). FeoB is a cytoplasmic membrane protein with GTPase activity (23), but the mechanism of transport and the functions of FeoA and FeoC have not been fully determined.Despite the apparent variation in the iron transport systems found in Shigella, there are consistent patterns. All express at least one siderophore and have the Feo and Sit transporters. S. flexneri serotype 2a strains SA100 and 2457T have only these three systems and thus were chosen for analysis of iron transport in vitro and within the host cell cytoplasm.Analysis of isogenic strains lacking one or more of the iron transporters showed that no single mutation eliminated intracellular growth, as the single iucD, feoB, and sitA mutants all produced plaques in cultured cells (48). The double mutants produced smaller plaques, but only the triple mutant was completely defective in growth and plaque formation (48).Additional information about the roles of these three systems in iron acquisition comes from our previous studies of their regulation. All of the iron transport systems are negatively regulated by Fur (1, 19), an iron-binding repressor protein. However, analysis of the expression of iron transport genes by wild-type S. flexneri growing in the intracellular environment indicated that only the sit genes were highly expressed in this environment (47). The aerobactin genes were downregulated in the intracellular environment (14), and feo expression appeared unchanged. The fact that, of the three iron transport systems, only sit is normally induced intracellularly suggests that it plays an important role when the bacteria inhabit the host cell cytoplasm. The failure of a sit mutant to show a defect in plaque formation may indicate that iron starvation induced by loss of the Sit system resulted in upregulation of the feo and iuc genes when they would not normally have been expressed. Further, since all of these genes appear to be regulated similarly by Fur in vitro, this differential expression of the iron transport genes in the intracellular environment suggested that factors other than iron were contributing to their regulation.An additional environmental factor that controls the expression of these genes is oxygen (3). As previously noted in E. coli K-12 (19), expression of the feo ferrous iron transport operon was induced under anaerobic conditions, where ferrous iron should predominate over ferric iron. In contrast, the aerobactin genes were induced aerobically, consistent with their role in ferric iron uptake (3). Surprisingly, the S. flexneri sit genes were repressed when the cells were grown anaerobically (3), although the homologous sitABCD genes in Salmonella encode a ferrous iron uptake system (20, 63). Consistent with aerobic induction, the Sit system was sufficient to support plaque formation by S. flexneri under aerobic, but not anaerobic, conditions (3).Because the Sit iron transport system was found in all of the Shigella species and was induced when the bacteria were growing intracellularly, we undertook a characterization of its genetics and role in the virulence of S. flexneri.     

Plasminogen Acquisition and Activation at the Surface of Leptospira Species Lead to Fibronectin Degradation

Pathogenic Leptospira species are the etiological agents of leptospirosis, a widespread disease of human and veterinary concern. In this study, we report that Leptospira species are capable of binding plasminogen (PLG) in vitro. The binding to the leptospiral surface was demonstrated by indirect immunofluorescence confocal microscopy with living bacteria. The PLG binding to the bacteria seems to occur via lysine residues because the ligation is inhibited by addition of the lysine analog 6-aminocaproic acid. Exogenously provided urokinase-type PLG activator (uPA) converts surface-bound PLG into enzymatically active plasmin, as evaluated by the reaction with the chromogenic plasmin substrate d-Val-Leu-Lys 4-nitroanilide dihydrochloridein. The PLG activation system on the surface of Leptospira is PLG dose dependent and does not cause injury to the organism, as cellular growth in culture was not impaired. The generation of active plasmin within Leptospira was observed with several nonvirulent high-passage strains and with the nonpathogenic saprophytic organism Leptospira biflexa. Statistically significant higher activation of plasmin was detected with a low-passage infectious strain of Leptospira. Plasmin-coated virulent Leptospira interrogans bacteria were capable of degrading purified extracellular matrix fibronectin. The breakdown of fibronectin was not observed with untreated bacteria. Our data provide for the first time in vitro evidence for the generation of active plasmin on the surface of Leptospira, a step that may contribute to leptospiral invasiveness.The spirochete Leptospira interrogans is a highly invasive pathogen and the causal agent of leptospirosis, one of the most widespread zoonoses of human and veterinary concern (7, 20, 25, 39, 76). The disease occurs mainly in peripheral metropolitan regions lacking adequate sanitary conditions during activities that involve direct contact with contaminated water, soil, or animals (25, 36, 76). Humans are accidental and terminal hosts in the transmission process of leptospirosis (20, 65). The leptospires enter the body via abrasions on skin or actively through mucosa, spreading to any tissue, but particularly colonizing kidneys and liver (39).Despite its importance and the genomic sequencing of five strains of Leptospira, four pathogenic (9, 57, 66) and one saprophytic (64), molecular aspects of the pathogenesis, virulence, and invasion processes by which the leptospires infect the hosts and initiate tissue colonization are poorly characterized. To date, few virulence factors contributing to the pathogenesis of the disease have been identified (3, 48, 67).It is known that one characteristic of leptospiral infection is the rapid dissemination within the host and colonization of renal tubules that constitute immunologically safe environments (20). The ability of the leptospires to adhere to extracellular matrix (ECM) macromolecules has been shown (4), and to date a few adhesins, ECM-binding proteins, have been identified (4, 11, 29, 30, 72). After adherence, the next step must be to overcome the barriers imposed by epithelial tissues and ECMs. For this, the proteolytic activity achieved by subversion of host proteases by pathogens, such as plasmin, has been demonstrated to be important during several bacterial infections (37).Plasmin is a broad-spectrum serine protease component of the fibrinolytic system, which has plasminogen (PLG) as the main component. It has been shown that several pathogens, including the spirochete Borrelia burgdorferi, bind PLG on the surface and convert it to plasmin by host activators (6, 13, 16, 19, 22, 31, 34, 37, 38, 60, 68, 73); this binding promotes degradation of ECM components and is essential for dissemination of the bacteria through the host tissues, suggesting its role during infection and pathogenesis (12, 14, 15, 28, 37, 58).Based on these assertions, we were prompted to investigate the ability of pathogenic L. interrogans to bind PLG. We show in this work by in vitro assays that leptospires are capable of capturing PLG in its outer surface, that the conversion to enzymatically active plasmin could be achieved by an exogenous source, and that the active plasmin generated on the surface of Leptospira can degrade the fibronectin ECM component. Outer membrane proteins (OMPs) are involved with PLG acquisition, but aqueous soluble proteins also contribute to the binding. Neither temperature shift to the mammalian body, under normal and febrile conditions, nor physiologic osmolarity affected plasmin generation by leptospires. We also demonstrate a significant difference in the plasminogen activation system (PAS) between infectious and noninfectious leptospires, suggesting that this feature might have a role in leptospiral virulence.     

Tracking the Emerging Human Pathogen Pseudallescheria boydii by Using Highly Specific Monoclonal Antibodies

Pseudallescheria boydii has long been known to cause white grain mycetoma in immunocompetent humans, but it has recently emerged as an opportunistic pathogen of humans, causing potentially fatal invasive infections in immunocompromised individuals and evacuees of natural disasters, such as tsunamis and hurricanes. The diagnosis of P. boydii is problematic since it exhibits morphological characteristics similar to those of other hyaline fungi that cause infectious diseases, such as Aspergillus fumigatus and Scedosporium prolificans. This paper describes the development of immunoglobulin M (IgM) and IgG1 κ-light chain monoclonal antibodies (MAbs) specific to P. boydii and certain closely related fungi. The MAbs bind to an immunodominant carbohydrate epitope on an extracellular 120-kDa antigen present in the spore and hyphal cell walls of P. boydii and Scedosporium apiospermum. The MAbs do not react with S. prolificans, Scedosporium dehoogii, or a large number of clinically relevant fungi, including A. fumigatus, Candida albicans, Cryptococcus neoformans, Fusarium solani, and Rhizopus oryzae. The MAbs were used in immunofluorescence and double-antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISAs) to accurately differentiate P. boydii from other infectious fungi and to track the pathogen in environmental samples. Specificity of the DAS-ELISA was confirmed by sequencing of the internally transcribed spacer 1 (ITS1)-5.8S-ITS2 rRNA-encoding regions of environmental isolates.Pseudallescheria boydii is an infectious fungal pathogen of humans (7, 16, 40, 58, 59). It is the etiologic agent of white grain mycetoma in immunocompetent humans (7) and has emerged over recent years as the cause of fatal disseminated infections in individuals with neutropenia, AIDS, diabetes, renal failure, bone marrow or solid organ transplants, systemic lupus erythematous, and Crohn''s disease; in those undergoing corticosteroid treatment; and in leukemia and lymphoma patients (1, 2, 3, 18, 27, 31, 32, 34, 36, 37, 38, 47, 49, 52). The fungus is the most prevalent species after Aspergillus fumigatus in the lungs of cystic fibrosis patients (8), where it causes allergic bronchopulmonary disease (5) and chronic lung lesions simulating aspergillosis (24). Near-drowning incidents and recent natural disasters, such as the Indonesian tsunami in 2004, have shown P. boydii and the related species Scedosporium apiospermum and Scedosporium aurantiacum to be the causes of fatal central nervous system infections and pneumonia in immunocompetent victims who have aspirated polluted water (4, 11, 12, 21, 22, 25, 30, 33, 57). Its significance as a potential pathogen of disaster evacuees has led to its recent inclusion in the Centers for Disease Control and Prevention list of infectious etiologies in persons with altered mental statuses, central nervous system syndromes, or respiratory illness.P. boydii is thought to be an underdiagnosed fungus (60), and misidentification is one of the reasons that the mortality rate due to invasive pseudallescheriasis is high. Detection of invasive P. boydii infections, based on cytopathology and histopathology, is problematic since it can occur in tissue and bronchoalveolar and bronchial washing specimens with other hyaline septated fungi, such as Aspergillus and Fusarium spp. (7, 23, 53, 60), which exhibit similar morphological characteristics upon microscopic examination (2, 23, 24, 28, 37, 44, 53, 60). Early diagnosis of infection by P. boydii and differentiation from other agents of hyalohyphomycosis is imperative, since it is refractory to antifungal compounds, such as amphotericin B, that are commonly administered for the control of fungal infections (10, 39, 58).The immunological diagnosis of Pseudallescheria infections has focused on the detection of antigens by counterimmunoelectrophoresis, and by immunohistological techniques using polyclonal fluorescent antibodies, but cross-reactions with antigens from other fungi, such as Aspergillus species, occurs (7, 19, 23). Pinto and coworkers (41, 42) isolated a peptidorhamnomannan from hyphae of P. boydii and proposed the antigen as a diagnostic marker for the pathogen. Cross-reactivity with Sporothrix schenckii and with Aspergillus have, however, been noted (23, 41). Furthermore, it is uncertain whether a similar antigen is present in the related pathogenic species S. prolificans, an important consideration in patient groups susceptible to mixed Scedosporium infections (6, 18).Hybridoma technology allows the production of highly specific MAbs that are able to differentiate between closely related species of fungi (54, 55, 56). The purpose of this paper is to report the development of MAbs specific to P. boydii and certain closely related species and their use to accurately discriminate among P. boydii, A. fumigatus, and other human pathogenic fungi by using immunofluorescence and double-antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISAs).Currently, the natural environmental habitat of P. boydii is unknown, but nutrient-rich, brackish waters, such as estuaries, have been suggested (9, 17). In combination with a semiselective isolation procedure, I show how the DAS-ELISA can be used to rapidly and accurately track the pathogen in naturally infested estuarine muds, and in doing so illustrate the potential of the DAS-ELISA as a diagnostic platform for detection of P. boydii and related species within the Pseudallescheria complex.     

Characterization of a Campylobacter jejuni VirK Protein Homolog as a Novel Virulence Determinant

Campylobacter jejuni is a leading cause of food-borne illness in the United States. Despite significant recent advances, its mechanisms of pathogenesis are poorly understood. A unique feature of this pathogen is that, with some exceptions, it lacks homologs of known virulence factors from other pathogens. Through a genetic screen, we have identified a C. jejuni homolog of the VirK family of virulence factors, which is essential for antimicrobial peptide resistance and mouse virulence.Campylobacter jejuni is a leading cause of infectious diarrhea in industrialized and developing countries (2, 67). Although most often self-limiting, C. jejuni infections can also lead to severe disease and harmful sequelae, such as Guillain-Barré syndrome (4, 55). Despite the significant progress made during the past few years, the mechanisms of C. jejuni pathogenesis remain poorly understood. A number of potential virulence factors have been identified, and in some cases, their role in virulence and/or colonization has been demonstrated in animal models of infection. For example, motility has been shown to be crucial in order for C. jejuni to colonize or cause disease in several animal models of infection (1, 15, 30, 54). A variety of surface structures, such as adhesins (34, 40, 64) and polysaccharides (5, 6), and glycosylation systems (38, 74), which presumably modify some of these surface structures, have also been shown to be important for infection. Additional studies have revealed the importance of specific metabolic pathways in C. jejuni growth both in vitro and within animals (16, 25, 31, 60, 76). The ability of C. jejuni to invade and survive within nonphagocytic cells has also been proposed to be an important virulence determinant (21, 41, 57, 58, 68, 75, 80).The available genome sequences of several C. jejuni strains have provided significant insight into C. jejuni physiology and metabolism (22, 32, 62, 63, 65). Remarkably, however, analysis of these C. jejuni genome sequences has revealed very few homologs of common virulence factors from other pathogens. A notable exception is the toxin CDT (cytolethal distending toxin), which is also encoded by several other important bacterial pathogens (36, 44, 45). In this paper we describe the identification of a transposon insertion mutant in C. jejuni 81-176, which results in increased susceptibility to antimicrobial peptides and a significant defect in the ability of the organism to cause disease in an animal model of infection. The insertion mutant was mapped to the CJJ81176_1087 open reading frame (Cj1069 in the C. jejuni NCT 11168 reference strain), which encodes a protein with very significant amino acid sequence similarity to the VirK (DUF535) family of virulence factors (13, 20, 56).     

Molecular Identification and Susceptibility of Trichosporon Species Isolated from Clinical Specimens in Qatar: Isolation of Trichosporon dohaense Taj-Aldeen,Meis & Boekhout sp. nov.

Trichosporon species have been reported as emerging pathogens and usually occur in severely immunocompromised patients. In the present work, 27 clinical isolates of Trichosporon species were recovered from 27 patients. The patients were not immunocompromised, except for one with acute myeloid leukemia. Sequence analysis revealed the isolation of Trichosporon dohaense Taj-Aldeen, Meis & Boekhout sp. nov., with CBS 10761^T as the holotype strain, belonging to the Ovoides clade. In the D1-D2 large-subunit rRNA gene analysis, T. dohaense is a sister species to T. coremiiforme, and in the internal transcribed spacer analysis, the species is basal to the other species of this clade. Molecular identification of the strains yielded 17 T. asahii, 3 T. inkin, 2 T. japonicum, 2 T. faecale, and 3 T. dohaense isolates. The former four species exhibited low MICs for five antifungal azoles but showed high MICs for amphotericin B. T. dohaense demonstrated the lowest amphotericin B MIC (1 mg/liter). For the majority of T. asahii isolates, amphotericin B MICs were high (MIC at which 90% of isolates were inhibited [MIC₉₀], ≥16 mg/liter), and except for fluconazole (MIC₉₀, 8 mg/liter), the azole MICs were low: MIC₉₀s were 0.5 mg/liter for itraconazole, 0.25 mg/liter for voriconazole, 0.25 mg/liter for posaconazole, and 0.125 mg/liter for isavuconazole. The echinocandins, caspofungin and anidulafungin, demonstrated no activity against Trichosporon species.Trichosporon species are yeast-like fungi, widely distributed in nature and commonly isolated from soil and other environmental sources, which have been involved in a variety of opportunistic infections and have been recognized as emerging fungal pathogens in immunocompromised hosts (19, 79, 80). Disseminated Trichosporon infections are potentially life-threatening and are often fatal in neutropenic patients (7, 22). Although uncommon, pathogenic species of this genus have been reported increasingly, mostly in patients with malignant diseases (3, 6, 9, 10, 11, 20, 32, 44, 47, 48, 63, 77), neonates (18, 56, 84), a bone marrow transplant recipient (22), a solid organ transplant recipient (50), and patients with human immunodeficiency virus (34, 35, 46). Trichosporon has also been reported to cause fungemia (5, 9, 25, 29, 30, 33, 53, 62). Members of the genus Trichosporon have occasionally been implicated as nail pathogens (16, 28, 74) and in subcutaneous infections (66). Trichosporon is considered an opportunistic agent, and therefore, recovery of Trichosporon species capable of growing at 37°C, especially from immunocompromised patients, should be regarded as potentially significant. Several reports have addressed the difficulty of identifying Trichosporon to the species level by physiological and biochemical characteristics (2, 64); therefore, molecular methods based on the sequencing of the internal transcribed spacer (ITS) have been developed (15, 69, 71, 72).In the present paper, we report the isolation of Trichosporon species from clinical specimens over a 4-year period in Qatar, the poor performance of biochemical identification methods, the significance of molecular identification, and the antifungal susceptibility data for the isolates. While investigating the molecular identification of Trichosporon species, we found three strains that do not match any of the published strains in the literature. We describe this organism as Trichosporon dohaense Taj-Aldeen, Meis & Boekhout, sp. nov., the name proposed for this species.     

Requirement for Formin-Induced Actin Polymerization during Spread of Shigella flexneri

Actin polymerization in the cytosol and at the plasma membrane is locally regulated by actin nucleators. Several microbial pathogens exploit cellular actin polymerization to spread through tissue. The movement of the enteric pathogen Shigella flexneri, both within the cell body and from cell to cell, depends on actin polymerization. During intercellular spread, actin polymerization at the bacterial surface generates protrusions of the plasma membrane, which are engulfed by adjacent cells. In the cell body, polymerization of actin by Shigella spp. is dependent on N-WASP activation of the Arp2/Arp3 complex. Here we demonstrate that, in contrast, efficient protrusion formation and intercellular spread depend on actin polymerization that involves activation of the Diaphanous formin Dia. While the Shigella virulence protein IpgB2 can bind and activate Dia1 (N. M. Alto et al., Cell 124:133-145, 2006), its absence does not result in a detectable defect in Dia-dependent protrusion formation or spread. The dependence on the activation of Dia during S. flexneri infection contrasts with the inhibition of this pathway observed during vaccinia virus infection.During infection, several human bacterial pathogens enter host cells and spread through host tissues by moving directly from one cell into adjacent cells. These microorganisms, including Shigella spp., Listeria monocytogenes (44), Rickettsia spp. (43), Burkholderia spp. (21), and Mycobacterium marinum (42), induce the polymerization of host actin into tails that propel them through the cell cytoplasm to the cell periphery. Actin tail assembly in the cell body involves local activation of actin polymerization through the Arp2/Arp3 (Arp2/3) complex (6, 11, 14, 19, 27, 49). The Arp2/3 complex initiates new filament assembly and cross-links those filaments at 70° angles (28). At the cell periphery, Shigella spp. push outwardly against the plasma membrane, creating a membrane-bound cell extension (“protrusion”) that extends tens of micrometers from the cell surface and contains a bacterium at its tip (5). Contact of a protrusion tip with the membrane of an adjacent cell is followed by its uptake into the adjacent cell by a process that resembles macropinocytosis (20), leading to the spread of the infection into adjacent cells.Although it is clear that actin assembly is required for the formation of protrusions by Shigella spp., the specific molecular mechanisms involved are poorly understood. Shigella spp. frequently form protrusions in tissue culture cells at sites of focal adhesions (30). The actin network at the base of protrusions contains filaments that are oriented in parallel arrays, in contrast to the angled arrays of actin filaments that predominate in actin tails associated with bacteria in the cell body (15), suggesting that actin nucleation processes independent of the Arp2/3 complex may be involved in protrusion formation.Formins are ubiquitously expressed proteins that, like the Arp2/3 complex, initiate de novo polymerization of actin (31, 36). In contrast to Arp2/3 complex-mediated actin polymerization, formin-mediated actin polymerization leads to cross-linking of actin polymers in parallel arrays (31, 36). Formins play critical roles in a variety of cytoskeletal processes in different cell types, including cytokinesis, cell polarity, cell migration and adhesion, and intracellular trafficking (13). At the cell membrane, the mammalian Diaphanous-related formins Dia1 and Dia2 function as effectors of the small GTPase RhoA (1, 39, 48). RhoA plays a critical role in the generation of actin stress fibers that attach at adherens junctions and focal adhesions. The localization of Dia1 and Dia2 at sites of potential Shigella flexneri exit from the cell makes them ideal candidates as mediators of protrusion formation. Their potential role in this process is examined here.     

Mannheimia haemolytica and Its Leukotoxin Cause Neutrophil Extracellular Trap Formation by Bovine Neutrophils

Mannheimia haemolytica is an important member of the bovine respiratory disease complex, which is characterized by abundant neutrophil infiltration into the alveoli and fibrin deposition. Recently several authors have reported that human neutrophils release neutrophil extracellular traps (NETs), which are protein-studded DNA matrices capable of trapping and killing pathogens. Here, we demonstrate that the leukotoxin (LKT) of M. haemolytica causes NET formation by bovine neutrophils in a CD18-dependent manner. Using an unacylated, noncytotoxic pro-LKT produced by an ΔlktC mutant of M. haemolytica, we show that binding of unacylated pro-LKT stimulates NET formation despite a lack of cytotoxicity. Inhibition of LKT binding to the CD18 chain of lymphocyte function-associated antigen 1 (LFA-1) on bovine neutrophils reduced NET formation in response to LKT or M. haemolytica cells. Further investigation revealed that NETs formed in response to M. haemolytica are capable of trapping and killing a portion of the bacterial cells. NET formation was confirmed by confocal microscopy and by scanning and transmission electron microscopy. Prior exposure of bovine neutrophils to LKT enhanced subsequent trapping and killing of M. haemolytica cells in bovine NETs. Understanding NET formation in response to M. haemolytica and its LKT provides a new perspective on how neutrophils contribute to the pathogenesis of bovine respiratory disease.Mannheimia haemolytica is a member of the bovine respiratory disease complex (BRD), causing a severe fibrinous pleuropneumonia sometimes referred to as shipping fever. The pneumonia is characterized by intense neutrophil infiltration in alveoli, intra-alveolar hemorrhage, fibrin deposition, and consolidation of the lungs (42, 56). The importance of neutrophils in the production of inflammatory mediators, recruitment of other leukocytes, and lung damage (17, 56, 67, 74) was demonstrated in calves that were depleted of neutrophils before challenge with M. haemolytica (10, 56). Neutrophil-depleted calves displayed less lung pathology than did control calves infected with M. haemolytica (10, 56). From these data, it is clear that neutrophils are a key player in the pathology of bovine pleuropneumonia; however, the mechanisms by which they contribute to host defense and tissue destruction are not clearly defined.The most important virulence factor for M. haemolytica is its leukotoxin (LKT), a 104-kDa exotoxin produced during logarithmic-phase growth (18, 32). LKT is a member of the repeats-in-toxin (RTX) toxin family of exoproteins produced by a wide variety of Gram-negative bacteria, including Escherichia coli, Actinobacillus pleuoropneumoniae, and Aggregatibacter actinomycetemcomitans (70). RTX toxins are characterized by a C-terminal glycine-rich nonapeptide repeat region (-G-G-X-G-X-D-X-U-X, where U is a hydrophobic residue) that binds calcium (Ca²⁺). The latter is required for membrane binding and cytotoxicity (30, 70). RTX toxins can insert into the plasma membrane of target cells, causing lysis and necrotic cell death (30, 70). The N-terminal domain contains amphipathic and hydrophobic domains believed to be required for pore stabilization and formation, respectively (70). More recently, it was shown that LKT also causes apoptosis via a caspase 9-dependent pathway and that LKT is internalized and transported via the cytoskeleton to mitochondria (4-6).The leukotoxin operon contains the genes lktC, lktA, lktB, and lktD (36, 37, 58). lktA encodes the inactive pro-LKT protein that is not cytotoxic until acylated (62) by the transacylase encoded by lktC. lktB and lktD encode proteins responsible for leader sequence-independent secretion of LKT from the bacterial cell (36, 37, 58). The acylated LKT then binds the CD18 chain of the β₂-integrin lymphocyte function-associated antigen 1 (LFA-1) (3, 21-26, 33, 40, 41, 44, 55, 63) on ruminant leukocytes. LKT binding to amino acids 5 to 17 of the signal sequence of CD18 is required for cell death and restricts cytotoxicity to ruminant leukocytes, because the signal sequence for CD18 is not present on mature leukocytes from other mammalian species (55). Other investigators have shown that both the pro- form and mature LKT are capable of binding CD18, although the pro-LKT does not cause cytotoxicity (62). No biological role has been assigned to the pro- form of LKT.Recently, several authors have shown that human neutrophils are able to undergo a form of cell death, called NETosis, that is distinct from apoptosis and necrosis (12, 13, 31, 51, 69). NETosis is defined as the release of nuclear DNA from an activated neutrophil into the extracellular environment, with little concomitant release of lactate dehydrogenase (LDH) (12). The extracellular DNA and associated proteins (e.g., histones) released by activated neutrophils have been termed neutrophil extracellular traps (NETs) (12). There are four steps leading to NET formation. These are neutrophil activation, nuclear envelope degradation, mixing of nuclear DNA with cytosolic proteins, and extrusion of the DNA-protein mixture from the cell (31). Treatment of human neutrophils with interleukin-8 (IL-8), phorbol 12-myristate 13-acetate (PMA), or lipopolysaccharide (LPS) causes NET formation (12, 31, 69). NET formation also occurs in response to prokaryotic and eukaryotic pathogens (12, 35, 64). To date, no bacterial exotoxin has been shown to cause NET formation.NETs are composed of extracellular DNA that is studded with antimicrobial proteins. The latter include nuclear histones and primary, secondary, and tertiary granular components such as neutrophil elastase, myeloperoxidase, lactoferrin, and gelatinase (51, 69). When neutrophils become activated and commit to NET formation, they also are capable of trapping and killing pathogens. To date, NETs have been shown to kill a variety of Gram-negative and Gram-positive bacteria, fungi, and protozoans (2, 7-9, 12, 13, 15, 19, 20, 27, 28, 31, 34, 35, 43, 50-53, 59, 64, 67, 70). Here, we examine if M. haemolytica and its LKT cause NET formation by bovine neutrophils and whether NETs are capable of trapping and killing M. haemolytica cells in vitro.     

Virulence and Cellular Interactions of Burkholderia multivorans in Chronic Granulomatous Disease

Chronic granulomatous disease (CGD) patients are susceptible to life-threatening infections by the Burkholderia cepacia complex. We used leukocytes from CGD and healthy donors and compared cell association, invasion, and cytokine induction by Burkholderia multivorans strains. A CGD isolate, CGD1, showed higher cell association than that of an environmental isolate, Env1, which correlated with cell entry. All B. multivorans strains associated significantly more with cells from CGD patients than with those from healthy donors. Similar findings were observed with another CGD pathogen, Serratia marcescens, but not with Escherichia coli. In a mouse model of CGD, strain CGD1 was virulent while Env1 was avirulent. B. multivorans organisms were found in the spleens of CGD1-infected mice at levels that were 1,000 times higher than those found in Env1-infected mice, which was coincident with higher levels of the proinflammatory cytokine interleukin-1β. Taken together, these results may shed light on the unique susceptibility of CGD patients to specific pathogens.Chronic granulomatous disease (CGD) is a rare primary immunodeficiency resulting from genetic defects in the phagocyte NAPDH oxidase. It is characterized by life-threatening infections caused by specific bacteria and fungi, leading to pneumonias, tissue abscesses, and exuberant granuloma formation (38). The Burkholderia cepacia complex (Bcc) includes at least 10 distinct species and is a leading cause of bacterial infections in CGD (44). Patients with cystic fibrosis (CF) also develop Bcc infections with various outcomes, ranging from no change in clinical course to a more rapid deterioration of lung function to the dreadful cepacia syndrome, which is characterized by necrotizing pneumonia and sepsis (25, 45). Interestingly, Bcc rarely causes infection in healthy individuals, but it can infect patients undergoing bronchoscopies and other procedures (4).Within the Bcc, Burkholderia cenocepacia and Burkholderia multivorans are commonly isolated from CF and non-CF patients (4, 32); the rate of B. multivorans infection now exceeds that of B. cenocepacia at several CF centers (15). In contrast to the high transmissibility of some CF B. cenocepacia strains (i.e., the epidemic lineage ET12) (24, 25), CF B. multivorans infections likely reflect independent acquisitions from unrelated sources (24). Curiously, unlike B. cenocepacia, B. multivorans has been recovered from environmental samples only rarely (1, 24), and it is the most frequently found species among CGD patients (16, 17).The mechanisms by which the Bcc causes disease specifically in CF are not known. Bcc isolates can survive within macrophages (28, 33) and respiratory epithelial cells (5, 21) and can invade epithelial cells in vivo (8, 10) and persist in the lung (9, 10). Cell infection assays using monocytes, macrophages, and epithelial cells (10, 11, 29, 46) show great variability among individual Bcc strains, with no clear correlation between those isolated from CF patients and those isolated from the environment (22). For the most part, these studies have been carried out using tissue culture models (28, 29, 43) and, in some cases, CF human or CF mouse cell systems (34, 35).Much less is known about the interaction between the Bcc and CGD despite the availability of animal models for the disease (20, 31). B. cenocepacia induced the necrosis of human CGD neutrophils but not normal controls (6). Similarly to healthy people, normal mice are resistant to the Bcc and usually show only transient infections upon inoculation (8, 37). On the other hand, CGD mice are highly susceptible to Bcc infection and show clinical signs that are similar to those of the human disease (20, 31, 37).To address why B. multivorans is a pathogen in CGD, we initiated studies with strains isolated from CGD patients and CGD cells. Strains of B. multivorans differed in cell association and cell entry. We found a preferential association of bacteria with CGD instead of normal leukocytes as shown by microscopy and culture techniques. This preferential association is shared by another CGD pathogen, Serratia marcescens, but not by Escherichia coli. Finally, we demonstrate dramatic differences in virulence in B. multivorans strains in a mouse model of CGD.     

Extracellular Vesicles from Cryptococcus neoformans Modulate Macrophage Functions

Cryptococcus neoformans and distantly related fungal species release extracellular vesicles that traverse the cell wall and contain a varied assortment of components, some of which have been associated with virulence. Previous studies have suggested that these extracellular vesicles are produced in vitro and during animal infection, but the role of vesicular secretion during the interaction of fungi with host cells remains unknown. In this report, we demonstrate by fluorescence microscopy that mammalian macrophages can incorporate extracellular vesicles produced by C. neoformans. Incubation of cryptococcal vesicles with murine macrophages resulted in increased levels of extracellular tumor necrosis factor alpha (TNF-α), interleukin-10 (IL-10), and transforming growth factor β (TGF-β). Vesicle preparations also resulted in a dose-dependent stimulation of nitric oxide production by phagocytes, suggesting that vesicle components stimulate macrophages to produce antimicrobial compounds. Treated macrophages were more effective at killing C. neoformans yeast. Our results indicate that the extracellular vesicles of C. neoformans can stimulate macrophage function, apparently activating these phagocytic cells to enhance their antimicrobial activity. These results establish that cryptococcal vesicles are biologically active.Cryptococcus neoformans is an encapsulated yeast that causes disease in diverse species, including humans. Infection is most commonly acquired by inhalation of environmental propagules. C. neoformans rarely causes disease in immunocompetent individuals, but patients with immunological disorders can develop disseminated and neural cryptococcosis (63).Extracellular microbial products have been amply demonstrated to modulate the interaction between host cells and pathogens. Many virulence factors and immunogens are released in their soluble forms by fungal cells to the extracellular space (4, 9, 16, 19, 37, 49, 53, 60, 62, 65, 67). C. neoformans, for instance, constitutively secretes large amounts of its capsular polysaccharide glucuronoxylomannan (GXM) (61). Disease progress is associated with detection of GXM, which is a potent modulator of the immune response (reviewed in reference 81). Other secreted virulence-related factors include galactoxylomannan (GalXM) (14), phospholipases (16), and urease (12, 62). In addition to acting as virulence factors, culture supernatant components are immunogenic, conferring protection against C. neoformans infection (51, 53).Phagocytes are particularly important effector cells in the control of systemic mycoses (54). The interaction of C. neoformans with phagocytes, including macrophages, monocytes, dendritic cells, and neutrophils, has been widely studied (23, 32, 43, 46, 50, 59, 68, 77). Cryptococcal GXM is antiphagocytic (34) and a powerful immunomodulator (45, 79). C. neoformans capsule size directly correlates with the efficacy of phagocytosis in vitro (6, 15, 82). Phagocytosis of C. neoformans can result in either fungal killing (24, 30) or survival (2, 3, 39-41, 71, 80). Killing of C. neoformans apparently involves the production of oxidative species (24), while the mechanisms of fungal escape include phagosome extrusion, cell-to-cell spread, and phagosomal permeabilization (2, 3, 40, 41, 71). Capsular polysaccharides and melanin are known to modulate the interaction of C. neoformans with phagocytes in favor of the fungus (27, 39, 47, 48, 71, 72, 74, 76), but the role of other structures in the outcome of yeast phagocytosis is virtually unknown.A number of recent studies have shown that GXM, GalXM, pigments, proteins, and lipids are trafficked in vesicles that traverse the cell wall (7, 14, 20, 56, 57, 62, 64, 65). Extracellular vesicles are also produced by the pathogens Candida albicans, C. parapsilosis, Sporothrix schenckii, and Histoplasma capsulatum, as well as by the model yeast Saccharomyces cerevisiae (1), suggesting that extracellular vesicle secretion is a general property of fungal cells. Secreted vesicles are heterogeneous. For instance, vesicles secreted by C. neoformans were classified into four different groups based on morphology and electron density (64). Additionally, vesicle diameter ranges from 30 to 400 nm, with the majority having dimensions of 100 to 150 nm (20, 64, 65). The combined use of serology, biochemistry, proteomics, and lipidomics led to the identification of 2 polysaccharides, phospholipids, 4 neutral lipids, and 76 proteins as extracellular vesicle components secreted by C. neoformans, which means that at least 81 different molecules are released to the extracellular milieu by vesicular secretion (14, 57, 64). It is likely that this number is an underestimate resulting from the difficulty of proteomic studies in vesicles from highly encapsulated cryptococcal cells, since a higher number of vesicular proteins were characterized in other fungi. For example, in H. capsulatum, proteomics and lipidomics of extracellular vesicles revealed an even more complex composition, including 283 proteins and 17 different phospholipids (1).In this study, we evaluated the influence of extracellular vesicles on the fate of C. neoformans after phagocytosis by mouse macrophages. Our results show that fungal vesicles are biologically active and stimulate macrophages. Moreover, our results demonstrate that vesicles from an acapsular mutant strain were more effective in eliciting macrophage activation and augmenting fungal killing than vesicles from encapsulated strains. Taken together, our findings suggest that fungal secretory vesicles have the potential to influence the interaction of C. neoformans with host cells.     

Borrelia burgdorferi Infection-Associated Surface Proteins ErpP,ErpA, and ErpC Bind Human Plasminogen

Host-derived plasmin plays a critical role in mammalian infection by Borrelia burgdorferi. The Lyme disease spirochete expresses several plasminogen-binding proteins. Bound plasminogen is converted to the serine protease plasmin and thereby may facilitate the bacterium''s dissemination throughout the host by degrading extracellular matrix. In this work, we demonstrate plasminogen binding by three highly similar borrelial outer surface proteins, ErpP, ErpA, and ErpC, all of which are expressed during mammalian infection. Extensive characterization of ErpP demonstrated that this protein bound in a dose-dependent manner to lysine binding site I of plasminogen. Removal of three lysine residues from the carboxy terminus of ErpP significantly reduced binding of plasminogen, and the presence of a lysine analog, ɛ-aminocaproic acid, inhibited the ErpP-plasminogen interaction, thus strongly pointing to a primary role for lysine residues in plasminogen binding. Ionic interactions are not required in ErpP binding of plasminogen, as addition of excess NaCl or the polyanion heparin did not have any significant effect on binding. Plasminogen bound to ErpP could be converted to the active enzyme, plasmin. The three plasminogen-binding Erp proteins can also bind the host complement regulator factor H. Plasminogen and factor H bound simultaneously and did not compete for binding to ErpP, indicating separate binding sites for both host ligands and the ability of the borrelial surface proteins to bind both host proteins.Lyme disease is the most commonly reported arthropod-borne disease in the United States (8). Borrelia burgdorferi, the causative agent of Lyme disease, is transmitted to its hosts through the bites of infected Ixodes ticks. In the earliest stage of Lyme disease, a bull''s-eye-shaped rash, erythema migrans, occurs as the spirochete spreads outward from the site of the tick bite. If left untreated, serious clinical outcomes can occur, including arthritis, neuropathies, and carditis (48).The bacterium disseminates from the bite site to other host tissues. B. burgdorferi can traverse the epithelium and invade vascular walls but is rarely abundant in blood (1). In addition, B. burgdorferi can pass through the blood-brain barrier to enter the central nervous system (58). The spirochete, unlike many invasive pathogens, lacks surface protease activities (12, 26). Therefore, binding of host proteases to the surface of the bacterium may aid in the spirochete''s dissemination. Indeed, B. burgdorferi binds plasminogen, a component of the host''s fibrinolytic system (12, 19). Plasminogen circulates in the plasma as an inactive proenzyme and is activated by tissue-type plasminogen activator and urokinase-type plasminogen activator (uPA) to plasmin (55). Plasminogen binding is an important virulence factor for invasive pathogens such as group A streptococci and Staphylococcus, as well as Borrelia species (10, 43, 55). The binding of plasminogen to bacteria and its subsequent activation allow bacteria to degrade the host''s extracellular matrix and basement membranes either through the direct protease activity of plasmin or by plasmin''s activation of host matrix metalloproteases (MMPs). B. burgdorferi has previously been shown to bind plasminogen, which is rapidly converted to active plasmin in the presence of host plasminogen activator (11). In vitro, plasmin-coated B. burgdorferi is able to penetrate endothelial cell monolayers (12). Surface-associated plasmin on B. burgdorferi can directly degrade fibronectin, a major component of the extracellular matrix, as well as laminin and vitronectin (11, 19). B. burgdorferi induces the release of MMP-9 (gelatinase) and MMP-1 (collagenase) from human cells, and plasmin-coated B. burgdorferi activates pro-MMP-9 (20), initiating a cascade that leads to degradation of basement membranes. Plasminogen has previously been shown to be important in B. burgdorferi pathogenesis. Although not strictly required for infection, plasminogen was required for efficient dissemination in ticks, and its absence decreased spirochetemia in plasminogen-deficient mice (10).Plasminogen-binding proteins of B. burgdorferi have previously been identified, including the outer-surface lipoprotein OspA (19). A role for OspC in plasminogen binding has also been suggested (31). However, OspA is generally not expressed during human infection, and OspC production ceases within the first few days of mammalian infection (13, 24, 25, 34, 42). Other, unidentified plasminogen-binding proteins have been observed in B. burgdorferi, including a protein(s) with an approximate molecular mass of 20 kDa, which is close to the size of several Erp proteins (12, 19). The members of the Erp family of outer-surface lipoproteins are expressed at high levels during mammalian infection (15, 23, 38-41).Lyme disease spirochetes contain numerous DNA elements, including the main chromosome as well as linear and circular plasmids (6). Infectious isolates carry several distinct yet homologous elements called cp32s, circular prophages of approximately 32 kb (54). All cp32 elements encode one or two Erp proteins, which can vary widely in amino acid sequence (50). However, all erp loci are preceded by nearly identical promoter regions (36, 53). Hence, most of the erp genes analyzed follow the same pattern of expression, being repressed in the tick vector but synthesized during mammalian infection (15, 21, 23, 35, 37-41). Roles for most of the Erp proteins have yet to be defined. ErpX has been demonstrated to bind host laminin (our unpublished results and reference 3). Three Erp proteins bind the host complement regulator factor H and factor H-related protein 1: ErpP, ErpC, and ErpA (22, 28, 29). Some factor H binding proteins of other human pathogens have been demonstrated to bind multiple ligands, including plasminogen (30, 47). These data, and the presence of unidentified plasminogen-binding proteins in B. burgdorferi, prompted us to examine if Erp proteins are able to bind plasminogen.     

Growth in a Biofilm Induces a Hyperinfectious Phenotype in Vibrio cholerae

Biofilm formation plays a multifaceted role in the life cycles of a wide variety of microorganisms. In the case of pathogenic Vibrio cholerae, biofilm formation in its native aquatic habitats is thought to aid in persistence during interepidemic seasons and to enhance infectivity upon oral ingestion. The structure of V. cholerae biofilms has been hypothesized to protect the bacteria during passage through the stomach. Here, we directly test the role of biofilm architecture in the infectivity of V. cholerae by comparing the abilities of intact biofilms, dispersed biofilms, and planktonic cells to colonize the mouse small intestine. Not only were V. cholerae biofilms better able to colonize than planktonic cells, but the structure of the biofilm was also found to be dispensable: intact and dispersed biofilms colonized equally, and both vastly out-colonized planktonic cells. The infectious dose for biofilm-derived V. cholerae was orders of magnitude lower than that of planktonic cells. This biofilm-induced hyperinfectivity may be due in part to a higher growth rate of biofilm-derived cells during infection. These results suggest that the infectious dose of naturally occurring biofilms of V. cholerae may be much lower than previously estimated using cells grown planktonically in vitro. Furthermore, this work implies the existence of factors specifically induced during growth in a biofilm that augment infection by V. cholerae.Bacteria are often found in biofilms, surface-attached aggregates of microorganisms encased in an extracellular polysaccharide or protein matrix (14). Mature bacterial biofilms often assume a three-dimensional structure composed of pillars of bacteria separated by fluid-filled channels (15). Compared to their free-living, planktonic (PL) counterparts, biofilm-associated bacteria have been shown to be recalcitrant to a variety of stresses and antimicrobial agents, including chlorine, low pH, UV irradiation, antibiotics, host defenses, and more (4, 16, 21, 22, 39, 40, 44, 63, 72). The structure of the biofilm itself has been thought to physically protect the bacteria within. The decreased susceptibility of biofilms to antibiotics, for example, is understood to be due at least in part to decreased permeability of the biofilm to the antibiotic (20, 30, 60). However, growth rate and metabolic state have also been proposed to contribute to biofilm-related protection from certain antimicrobials and other stresses (5, 25, 59, 68). In addition, distinct genetic mechanisms of antibiotic resistance employed by biofilm microorganisms have been described (34, 41, 42).The ability to form biofilms is a virulence determinant of many microorganisms. Classic examples of biofilm infections include chronic infections of the cystic fibrosis lung by Pseudomonas aeruginosa, Haemophilus influenzae and Streptococcus pneumoniae in chronic otitis media, uropathogenic Escherichia coli in recurrent urinary tract infections, and disease caused by microbial biofilms on a variety of indwelling medical devices (27, 35, 36, 52, 56). The reduced susceptibility of in vivo biofilms to antimicrobials has a huge impact on human health due to the difficulty involved in their eradication.In other instances, biofilm formation contributes to a microorganism''s survival in an environmental niche, such as in water system piping or on other solid surfaces, and consequently affects the likelihood of contact with a host (24, 58, 69). Vibrio cholerae is the causative agent of cholera and is a natural inhabitant of freshwater, marine, and estuarine environments. In the aquatic environment, V. cholerae has been observed to form biofilms on abiotic and biotic surfaces, including those of zooplankton, phytoplankton, algae, and crustaceans (23, 33, 62). This surface-attached state is thought to be the primary means of persistence of V. cholerae in the environment, providing protection from a variety of stresses and, when the bacteria are attached to chitin, a source of nutrients and a forum for acquiring new genetic material (43, 45, 46, 50, 67, 71). Moreover, biofilms are likely a form in which pathogenic (toxigenic) V. cholerae is consumed by humans, and they provide a means by which humans can obtain a concentrated infective dose (13, 28, 32). Because chitin- and biofilm-associated V. cholerae is better able to survive acid exposure (50, 72), it has been hypothesized that V. cholerae biofilms are protected during transit through the gastric acid barrier of the stomach, thus allowing more bacteria to reach the small intestine colonization site. Yet the role of stomach acid in susceptibility to cholera is unclear in humans (26), and the infant mouse, with neutral pH in the stomach, may be an irrelevant model of this phenomenon.In this work, we test the hypothesis that the biofilm structure itself enhances colonization of the small intestine by V. cholerae. Instead, we found that V. cholerae bacteria dispersed from a biofilm are as infectious as those in an intact biofilm and that both are dramatically more infectious than free-living, planktonic cells in the infant mouse model. This work suggests that the physiological state of V. cholerae in biofilms, and not the biofilm structure, is the primary contributor to hyperinfectivity.     

Physical and Chemical Characterization and Immunologic Properties of Salmonella enterica Serovar Typhi Capsular Polysaccharide-Diphtheria Toxoid Conjugates

Typhoid fever remains a serious public health problem in developing countries, especially among young children. Recent studies showed more than 50% of typhoid cases are in children under 5 years old. Licensed vaccines, such as Salmonella enterica serovar Typhi capsular Vi, did not confer protection against typhoid fever for this age group. Vi conjugate, prepared by binding Vi to Pseudomonas aeruginosa recombinant exoprotein A (rEPA), induces protective levels of antibody at as young as 2 years old. Because of the lack of regulatory precedent for rEPA in licensing vaccines, we employed diphtheria toxoid (DT) as the carrier protein to accommodate accessibility in developing countries. Five lots of Vi-DT conjugates were prepared using adipic acid dihydrazide (ADH) as the linker. All 5 lots showed consistency in their physical and chemical characteristics and final yields. These Vi-DT conjugates elicited levels of IgG anti-Vi in young mice significantly higher than those in mice injected with Vi alone and induced a booster response upon reinjection. This booster effect was absent if the Vi replaced one of the two conjugate injections. Vi-DT was stable under repeated freeze-thaw (20 cycles). We plan to perform clinical evaluation of the safety and immunogenicity of Vi-DT when added to the infant combination vaccines.Typhoid fever, a serious systemic infection caused by Salmonella enterica serovar Typhi, remains a major public health problem in Central Asia, Southeast Asia, Africa, and Latin America (11, 52, 53). It was estimated that more than 21 million cases of typhoid fever and >200,000 deaths occurred in 2000 (10). The treatment of patients and management of asymptomatic carriers are becoming more difficult due to the worldwide emergence of multidrug-resistant (MDR) strains (2, 15, 29, 42, 43). Vaccination is considered the most promising strategy for the control of typhoid fever in developing countries (11, 19, 52, 53).Typhoid fever in children younger than 5 years old has often been unrecognized due to atypical clinical symptoms, difficulties in the number and volume of blood drawings, and use of less than optimal culture media (35, 46). Several studies have shown that the incidence of typhoid fever among children less than 5 years old is similar to that in school age children and young adults (14, 27, 34, 50, 51).The 3 licensed typhoid vaccines have limited efficacy, and none are suitable for young children under 5 years old. The use of heat-inactivated whole-cell vaccine was suspended in many countries because of its reactogenicity. The parenteral Vi polysaccharide and the live attenuated oral Ty21a vaccine were introduced in the late 1980s; both vaccines are well accepted and confer moderate protection (50 to 70%) in older children and adults. However, neither vaccine is licensed for routine immunization of infants (52).The Vi capsular polysaccharide is both an essential virulence factor and a protective antigen for S. Typhi (36, 38, 39). The concentration of serum IgG anti-Vi is correlated with immunity to the pathogen (22, 25, 26, 28, 36, 38, 49). However, Vi is not suitable for routine immunization of infants and young children because of its age-related immunogenicity and T-cell independence. As was shown for other capsular polysaccharides, such as Haemophilus influenzae type b (8, 37); meningococcus groups A, C, and W135; and Streptococcus pneumoniae (12, 20), Vi covalently bound with protein conferred T-cell dependence and increased immunogenicity (48-50). To date, diphtheria toxoid (DT), tetanus toxoid (TT), cholera toxins (CT), the B subunit of the heat-labile toxin (LT-B) of Escherichia coli, recombinant outer membrane protein of Klebsiella pneumoniae (rP40), and iron-regulated outer-membrane proteins (IROMPs) of S. Typhi have served as carriers for Vi polysaccharide in laboratory studies (16, 17, 32, 48-50; personal communications). An improved method was developed (24), utilizing adipic acid dihydrazide (ADH) as the linker and Pseudomonas aeruginosa recombinant exoprotein A (rEPA) as the carrier. Clinical trials of Vi-rEPA conjugates conferred 89% protection in Vietnamese children 2 to 5 years old for 46 months (23, 26, 28). The level of serum IgG anti-Vi induced by Vi-rEPA conjugates was correlated with prevention of typhoid fever in these studies (7, 21-23, 26, 28).One limitation of using rEPA as the carrier protein is the lack of regulatory precedent in licensing vaccines. In this report, five lots of Vi conjugates using DT manufactured by pharmaceutical companies in China and India were prepared (24, 48, 49). Modifications of conjugation procedures were made for the purposes of easy adoption and scale up by manufacturers. The stability of Vi-DT was studied for the feasibility of stockpiling in disaster relief.Another important aspect of conjugate vaccine implementation is the optimum immunization formulation and schedule using alternating injections of polysaccharide and conjugate. Priming or boosting effects of polysaccharide on its conjugate vaccine have been observed in infants injected with pneumococcal and meningococcal vaccines (3, 4, 31, 40). There was no consistent conclusion about various types of polysaccharides studied (6, 9, 31, 40, 41). Here, we compared the immune response of Vi polysaccharide injected before or after the administration of Vi-DT with the responses of those receiving 2 injections of Vi-DT. We also investigated the dosage effect for the purpose of better formulation.     

Plasmid-Borne Virulence-Associated Genes Have a Conserved Organization in Virulent Strains of Avian Pathogenic Escherichia coli

Avian pathogenic Escherichia coli (APEC) is an important respiratory pathogen of poultry. Various virulence factors are responsible for determining the pathogenicity of these strains, and it is commonly believed they are encoded on large plasmids the strains carry. This study examined a series of strains, the pathogenicity of which had previously been determined by aerosol exposure, for possession of large plasmids and found all isolates carried at least one large plasmid, regardless of the level of virulence. Virulence-associated genes carried on these plasmids were also examined, and it was shown that highly virulent strains carried at least four virulence-associated genes on their largest plasmid. Two of the virulence-associated genes were shown to be chromosomally located in a strain of intermediate virulence, while no virulence-associated genes were carried by the low-virulence strain. The organization of the virulence-associated genes was shown to be highly conserved among APEC isolates of high virulence, supporting the concept of a conserved portion of the putative virulence region that contributes to the pathogenicity of APEC strains.Avian pathogenic Escherichia coli (APEC) strains cause respiratory disease and septicemia in poultry and are economically important worldwide, causing significant mortality (13). The carriage of large plasmids is considered characteristic of APEC isolates (8), and pathogenicity is thought to be determined by virulence-associated factors encoded by them (15). These factors include serum resistance, encoded by the iss gene (14), temperature-sensitive hemagglutination, encoded by tsh (10), adhesins, the production of colicin V (ColV) and the possession of iron-scavenging mechanisms, such as aerobactin production (encoded by the iucABCD operon), and the more recently identified putative iron transport system encoded by the etsABC operon (18).Another iron acquisition system found in APEC utilizes salmochelin, a catecholate siderophore. The chromosomal iroA gene cluster that encodes this system was first found in Salmonella enterica (2) and is absent from the corresponding region of the E. coli chromosome (32), although it has been found on a transmissible plasmid from a uropathogenic E. coli isolate (34). The iroA gene cluster has been found on multiple APEC virulence plasmids (9, 17, 18, 37), and deletion studies have shown that the iroA gene cluster is required for full virulence (9).A further iron transport system, designated the sitABCD system, was first identified on a pathogenicity island in Salmonella enterica serovar Typhimurium (39), and it has been shown that sitABCD is required for full virulence of Salmonella serovar Typhimurium (16). Genomic subtraction identified the plasmid-located sitA gene from the sitABCD operon as unique to an APEC strain (32), and the sitA gene was found to be more prevalent in APEC than in commensal E. coli (18, 29, 32).The sitABCD operon occurs on APEC virulence plasmids (17, 18, 30, 37), but a sitABCD deletion mutant was still pathogenic for birds, suggesting that other iron transport systems are able to compensate for the loss of sitABCD (30).The carriage of ColV plasmids has previously been thought to be essential for virulence (3, 33, 38). However, other studies have suggested it is not the presence of the ColV gene itself but other genes that these plasmids carry that are responsible for virulence (28, 35). The well-characterized APEC virulence plasmids pAPEC-O2-ColV (18) and pAPEC-1 (9) encode ColV, while carriage of the Australian APEC virulence plasmid pVM01 does not confer production of ColV (12). Despite various ColV statuses, all three of these virulence plasmids are F-type plasmids, and hence this is potentially another way to characterize APEC virulence plasmids.SopA and SopB, which have similarity to the ParA and ParB proteins of the P1 plasmid, are thought to be essential for F-plasmid partitioning (22, 24). Detection of the genes of the sopABC locus could thus indicate the presence of a putative virulence plasmid.Strain E3 is an O-nontypeable:H28 APEC field isolate (11) that carries the 151-kb virulence plasmid pVM01 (12), which contains a virulence region with the virulence-associated genes iucA, tsh, iss, iroN, and sitA, as well as hlyF, ompT, and the etsABC operon (37). The arrangement of the virulence-associated genes around pVM01 (37) is similar to that in the plasmids pAPEC-O2-ColV from APEC strain O2 (18), pAPEC-O1-ColBM from APEC strain O1 (17), and pAPEC-1 from APEC strain χ7122 (23). Identifying a specific region that is conserved in highly virulent APEC strains will facilitate diagnosis of colibacillosis by differentiation of pathogenic strains from commensal E. coli and will also enable surveillance for pathogenic isolates in the environment of poultry.This study examined six E. coli strains, some of which were isolated from diseased birds and some of which were recovered from healthy birds (11, 36). The pathogenicity of these strains has been determined using aerosol exposure (11, 36), making this the largest known collection of APEC strains fulfilling Koch''s postulates. The series of strains includes the highly virulent strains E3, E30, and E956 and the less-virulent strains E133, E1043, and E1292. The presence of the virulence-associated genes iucA, tsh, and iss in these strains has previously been elucidated by PCR amplification (36). However, while previous studies have found many of these virulence factors to be encoded by APEC strains associated with disease (29) and have suggested that they are encoded on virulence plasmids (18), they have not conclusively determined whether they are encoded on virulence plasmids or are chromosomally encoded. Similarly, although previous studies suggest that these virulence-associated genes are consistently present in isolates from diseased birds (1, 6, 18, 21, 26, 29), no study has yet determined if these genes are consistently associated with each other.The aim of this study was to examine a series of strains of known pathogenicities for the possession of large plasmids and to determine if known virulence-associated genes from the putative virulence region were carried on them. The second objective was to investigate any association between the virulence-associated genes.     

Borrelia burgdorferi BmpA Is a Laminin-Binding Protein

The Borrelia burgdorferi BmpA outer surface protein plays a significant role in mammalian infection by the Lyme disease spirochete and is an important antigen for the serodiagnosis of human infection. B. burgdorferi adheres to host extracellular matrix components, including laminin. The results of our studies indicate that BmpA and its three paralogous proteins, BmpB, BmpC, and BmpD, all bind to mammalian laminin. BmpA did not bind mammalian type I or type IV collagens or fibronectin. BmpA-directed antibodies significantly inhibited the adherence of live B. burgdorferi to laminin. The laminin-binding domain of BmpA was mapped to the carboxy-terminal 80 amino acids. Solubilized collagen inhibited BmpA-laminin binding, suggesting interactions through the collagen-binding domains of laminin. These results, together with previous data, indicate that BmpA and its paralogs are targets for the development of preventative and curative therapies for Lyme disease.Early during the course of Lyme disease, humans frequently produce antibodies directed against a Borrelia burgdorferi antigen originally described as “P39” (66). Antibodies recognizing P39 are considered to be specific and diagnostic for Lyme disease spirochete infection (5, 18, 30, 62, 64). The antigenic protein was subsequently identified as BmpA (Borrelia membrane protein A) (65). The bmpA gene is located on the main borrelial chromosome, adjacent to three paralogous genes named bmpB, bmpC, and bmpD, which together form a complex operon (3, 4, 28, 32, 55, 56, 65). These other Bmp proteins are also often antigenic in infected humans (14). In addition to the serological data described above, examination of B. burgdorferi within skin and joint tissues confirmed the production of BmpA protein during mammalian infection (21, 49). BmpA is located in the borrelial outer membrane (46), where it is exposed to the external environment and can be a target of bactericidal antibodies (49, 63; F. Cabello, personal communication). BmpA and its paralogs have been implicated as playing roles in some symptoms of Lyme disease (49, 72). B. burgdorferi mutants in which bmpA or bmpB is specifically deleted are unable to persist in mouse joint tissues (49), indicating an important role for these proteins in the maintenance of mammalian infection. Despite the extensive research conducted on these important antigens, functions for the Bmp proteins had not been determined previously.B. burgdorferi is an extracellular organism, frequently found associated with its hosts'' connective tissues (6-9, 16, 17, 24, 26, 31, 36, 39, 48). In the laboratory, B. burgdorferi shows affinity for various host extracellular matrix (ECM) components, such as type I collagen, fibronectin, and decorin (16, 33, 34, 50, 74). We recently determined that B. burgdorferi also adheres to mammalian laminin, an important component of many mammalian ECMs (13). Ligand affinity blot analyses of a B. burgdorferi cell fraction enriched for outer membrane components revealed that the type strain, B31, can produce several distinct laminin-binding proteins, one of which we previously identified as being the surface-exposed outer membrane lipoprotein ErpX (11, 13, 69). We now present data indicating that BmpA and its paralogs are also laminin-binding proteins.