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.     

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.     

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.     

Ail Binding to Fibronectin Facilitates Yersinia pestis Binding to Host Cells and Yop Delivery

Yersinia pestis, the causative agent of plague, evades host immune responses and rapidly causes disease. The Y. pestis adhesin Ail mediates host cell binding and is critical for Yop delivery. To identify the Ail receptor(s), Ail was purified following overexpression in Escherichia coli. Ail bound specifically to fibronectin, an extracellular matrix protein with the potential to act as a bridge between Ail and host cells. Ail expressed by E. coli also mediated binding to purified fibronectin, and Ail-mediated E. coli adhesion to host cells was dependent on fibronectin. Ail expressed by Y. pestis bound purified fibronectin, as did the Y. pestis adhesin plasminogen activator (Pla). However, a KIM5 Δail mutant had decreased binding to host cells, while a KIM5 Δpla mutant had no significant defect in adhesion. Furthermore, treatment with antifibronectin antibodies decreased Ail-mediated adhesion by KIM5 and the KIM5 Δpla mutant, indicating that the Ail-fibronectin interaction was important for cell binding. Finally, antifibronectin antibodies inhibited the KIM5-mediated cytotoxicity of host cells in an Ail-dependent fashion. These data indicate that Ail is a key adhesin that mediates binding to host cells through interaction with fibronectin on the surface of host cells, and this interaction is important for Yop delivery by Y. pestis.The three species of Yersinia pathogenic for humans, Yersinia enterocolitica, Y. pseudotuberculosis, and Y. pestis, cause distinct diseases. Y. pseudotuberculosis and Y. enterocolitica typically cause acute gastroenteritis and mesenteric lymphadenitis. On the other hand, Y. pestis, the causative agent of the plague, is one of the most deadly human infectious diseases (8). Y. pestis is a close relative of Y. pseudotuberculosis, diverging only 1,500 to 20,000 years ago (1). To accommodate flea-borne transmission, Y. pestis has acquired two unique plasmids not harbored by enteropathogenic Yersinia species. All three pathogenic Yersinia species inject cytotoxic Yersinia outer proteins (Yops) into host cells via the Ysc type III secretion system (TTSS) to establish an infection (11). Host cell contact is essential for engagement of the TTSS and secretion of Yops (9, 54). Within the host cell, Yops effect actin rearrangements, inhibit phagocytosis, and block proinflammatory signals (4, 40, 42). Both Y. enterocolitica and Y. pseudotuberculosis express the well-studied adhesin molecules invasin (Inv) and YadA, capable of mediating Yop delivery (9, 54). However, Y. pestis does not express either adhesin due to an IS1541 element insertion within inv (58) and a frameshift mutation in yadA (44, 55). Y. pestis has a number of other adhesins capable of mediating host cell interaction. Both the pH 6 antigen (Psa [29, 63]) and plasminogen activator (Pla [28]) of Y. pestis have been shown to be adhesins. Psa is a tightly regulated pilus expressed at a pH of <6.7 and 37°C (52, 67) and is known to bind to β-linked galactosylated glycosphingolipids (46), low-density lipoprotein (31), and human IgG (69). Pla, expressed at 26°C but further induced at 37°C (49), is known to bind to several extracellular matrix components (23, 28, 30). The putative autotransporter, YapC, is also capable of mediating cell adhesion when it is expressed in Escherichia coli (15), as is the pilus encoded by the chaperone/usher system locus y0561-0563 (16), but neither yapC nor y0561-0563 results in significantly decreased adhesion when they are deleted from Y. pestis (15, 16).Recently, an additional adhesin of Y. pestis, Ail (adherence and invasion locus), was determined to facilitate cell binding (14, 25). Ail (encoded by y1324) is a 21.5-kDa outer membrane protein of the OmpX family that is predicted to have eight transmembrane domains and four extracellular loops extending above the surface of the bacterium (17, 65). Ail homologues include OmpX of Escherichia coli (32) and Enterobacter cloacae (61), PagC in Salmonella (53), and Opa proteins from Neisseria (10). Ail from Y. enterocolitica has been studied previously and shown to have three activities: cell adhesion, cell invasion (36), and the ability to confer serum resistance (5, 51) by binding to complement regulatory proteins (24). The residues for all three activities have been mapped to particular amino acids in the surface-exposed loops (35). Y. pseudotuberculosis Ail also confers adhesion and invasion functions (T. M. Tsang and E. S. Krukonis, unpublished data) and serum resistance (68), although the two amino acid changes between Y. pseudotuberculosis Ail and Y. pestis Ail result in decreased adhesion and invasion mediated by the former (Tsang and Krukonis, unpublished). More recently, Y. pestis Ail was also shown to mediate cell adhesion (14, 25), autoaggregation (25), and serum resistance (3, 24, 25) and to facilitate Yop delivery to host cells (14). Furthermore, Y. pestis Ail is required for virulence, as a Y. pestis Δail mutant has a >3,000-fold increase in the 50% lethal dose (14). A Y. pestis Δail mutant shows reduced binding to both epithelial and phagocytic human-derived cell lines, and in a mouse model of infection, a Y. pestis KIM5 Δail mutant colonizes host tissue to much lower levels than the parental KIM5 strain (14). Over the course of 7 days, the Δail mutant is cleared from the host (14). Together, these data demonstrate that Ail is an important adhesin that contributes to colonization and virulence.Cell adhesion is important for the establishment of a successful infection. Adhesion is also significant in Y. pestis pathogenesis because host cell contact is required for the production and translocation of the Yop effector proteins (48, 54). Bacteria can bind directly to host cell receptors (21) or use molecules like extracellular matrix (ECM) components to mediate attachment to host cells (12, 22, 30, 45, 57, 64). Common components of the cellular matrix that facilitate bacterial binding include fibronectin (22, 28, 64), collagen (23, 45), and laminin (28, 30, 45). Interactions between bacteria and ECM can lead to bridge-like attachments to host cells.Fibronectin is a large glycoprotein that is a key structural component in many tissues. This ∼220-kDa protein is commonly found as a dimer that is linked by two disulfide bonds located near the C terminus. Fibronectin is a complex molecule made up of three types of modular repeating units (43, 47). Fibronectin can bind to many substrates, including collagen (13), integrin receptors on host cells (50, 56), and heparin (60). Additionally, fibronectin contains a binding site for several bacterial pathogens at the N-terminal end of the molecule (39, 59).A number of fibronectin binding proteins on bacterial pathogens have been identified and studied, including SigB from Staphylococcus aureus (34), protein F from Streptococcus pyogenes (41), and YadA from Y. pseudotuberculosis (12, 19) and Y. enterocolitica (64). Binding of Y. pseudotuberculosis YadA to fibronectin allows Y. pseudotuberculosis to utilize β₁ integrins on the surface of host cells for invasion (12). Given the key role of Y. pestis Ail in cell adhesion, Yop delivery, and virulence, we sought to determine the component on host cells to which Ail binds.Although Ail has been studied extensively in other Yersinia species, the substrate on host cells with which Ail interacts is not known. In this study, we used a purified Y. pestis Ail to identify the extracellular matrix component, fibronectin, as a protein bound by Ail. Furthermore, Ail-mediated binding to host cells through fibronectin is important for the delivery of Yop effector proteins.     

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.     

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).     

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.     

Development of a Bead Immunoassay To Measure Vi Polysaccharide-Specific Serum IgG after Vaccination with the Salmonella enterica Serovar Typhi Vi Polysaccharide

Vi polysaccharide from Salmonella enterica serotype Typhi is used as one of the available vaccines to prevent typhoid fever. Measurement of Vi-specific serum antibodies after vaccination with Vi polysaccharide by enzyme-linked immunosorbent assay (ELISA) may be complicated due to poor binding of the Vi polysaccharide to ELISA plates resulting in poor reproducibility of measured antibody responses. We chemically conjugated Vi polysaccharide to fluorescent beads and performed studies to determine if a bead-based immunoassay provided a reproducible method to measure vaccine-induced anti-Vi serum IgG antibodies. Compared to ELISA, the Vi bead immunoassay had a lower background and therefore a greater signal-to-noise ratio. The Vi bead immunoassay was used to evaluate serum anti-Vi IgG in 996 subjects from the city of Kolkata, India, before and after vaccination. Due to the location being one where Salmonella serotype Typhi is endemic, approximately 45% of the subjects had protective levels of anti-Vi serum IgG (i.e., 1 μg/ml anti-Vi IgG) before vaccination, and nearly 98% of the subjects had protective levels of anti-Vi serum IgG after vaccination. Our results demonstrate that a bead-based immunoassay provides an effective, reproducible method to measure serum anti-Vi IgG responses before and after vaccination with the Vi polysaccharide vaccine.Typhoid fever is caused by Salmonella enterica serotype Typhi (32). Humans are the only natural host and reservoir of S. enterica serotype Typhi (32, 41). Typhoid fever represents a spectrum of diseases ranging from an acute uncomplicated disease—including fever, headache, malaise, and disturbances of bowel function (constipation in adults and diarrhea in children)—to a more severe, complicated form of disease in 10 to 20% of infected patients that includes bleeding in the gastrointestinal tract, intestinal perforation (in 1 to 3% of hospital typhoid fever cases) and an altered mental state (32, 41). The case fatality rate is highly variable, depending on the medical treatment available and geographic location. For example, the average fatality rate is less than 1% overall but may range between 2% fatality in hospitalized patients in Pakistan and Vietnam and 50% fatality in hospitalized patients in some parts of Indonesia and Papua New Guinea (32, 41). Worldwide, typhoid fever remains a significant public health problem, with an estimated 17,000,000 cases of typhoid fever each year and up to 600,000 deaths (2, 10, 32, 41).Typhoid vaccines currently available are composed of purified Vi polysaccharide or live attenuated S. enterica serotype Typhi (Ty21a) organisms (10, 39). The Vi polysaccharide vaccine induces protective serum antibody responses that reach a maximum at 28 days after a single intramuscular vaccination with 25 μg purified Vi polysaccharide (39), a capsular polysaccharide (Vi for virulence) that increases the virulence of S. enterica serotype Typhi (32). Protective antibody levels have been estimated to be 1 μg/ml anti-Vi IgG antibody in the serum (20). Protective efficacy of the Vi polysaccharide vaccine as determined by protection against disease is modest, with only 55 to 72% of subjects protected against disease through 3 years postvaccination (1, 20, 39). The live attenuated Ty21a vaccine is administered orally as three or four doses of enteric capsules (39). Due to its use as an oral, mucosally administered vaccine, the Ty21a vaccine induces protection against typhoid fever by induction of mucosal IgA and serum IgG antibodies specific for lipopolysaccharide antigens (39). The protective efficacy of the Ty21a vaccine at 3 years postvaccination was reported to range from 42 to 67% when using three doses of Ty21a enteric capsules (11, 39). Next-generation vaccines that utilize Vi conjugated to protein carriers that provide superior induction of anti-Vi antibodies are currently in development (14, 21, 25, 36).Despite its ability to induce protective immune responses when used alone or conjugated to protein carriers, the use of Vi polysaccharide as a coating antigen in enzyme-linked immunosorbent assay (ELISA) to measure vaccine-induced anti-Vi antibody responses has been reported to be problematic. The use of polysaccharides (lipopolysaccharide [LPS], Haemophilus influenzae type b capsular polysaccharide, Vi polysaccharide) as coating antigens for immunoassays is plagued by problems such as a poor binding of polysaccharides to ELISA plates and inconsistent results (3, 15, 16, 26, 33). To increase binding of Vi antigen to ELISA plates and produce more-robust assays, others have biotinylated Vi and then added it to streptavidin-coated plates (12) or conjugated Vi to tyramine (22, 26). However, some reports indicate that Vi was used without any additional treatment as an ELISA coating antigen (7, 19, 21) although a Vi ELISA performed on plates was less sensitive than a radioimmunoassay procedure (19).Immunoassays based on the use of fluorescent beads as the solid surface have recently been developed and compared to ELISA for the measurement of antigen-specific antibodies for polysaccharides from Streptococcus pneumoniae, Neisseria meningitidis, or Haemophilus influenzae type b (HiB) (5, 8, 23, 27, 34, 35). The fluorescent bead assays were comparable to ELISA and sometimes were noted as having enhanced dynamic ranges or increased sensitivity (5, 8, 27, 35). An additional benefit of fluorescent bead immunoassays is their ability to be multiplexed to permit the simultaneous measurement of antibodies specific for different antigens (8, 23, 27, 34, 35). This study was performed to evaluate a fluorescent bead immunoassay for its ability to measure vaccine-induced antibodies specific for Salmonella serotype Typhi Vi polysaccharide. The performance of the fluorescent bead assay was compared to that of ELISA.     

Glyceraldehyde-3-Phosphate Dehydrogenase Is a Surface-Associated,Fibronectin-Binding Protein of Trichomonas vaginalis

Trichomonas vaginalis colonizes the urogenital tract of humans and causes trichomonosis, the most prevalent nonviral sexually transmitted disease. We have shown an association of T. vaginalis with basement membrane extracellular matrix components, a property which we hypothesize is important for colonization and persistence. In this study, we identify a fibronectin (FN)-binding protein of T. vaginalis. A monoclonal antibody (MAb) from a library of hybridomas that inhibited the binding of T. vaginalis organisms to immobilized FN was identified. The MAb (called ws1) recognized a 39-kDa protein and was used to screen a cDNA expression library of T. vaginalis. A 1,086-bp reactive cDNA clone that encoded a protein of 362 amino acids with identity to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was obtained. The gapdh gene was cloned, and recombinant GAPDH (rGAPDH) was expressed in Escherichia coli cells. Natural GAPDH and rGAPDH bound to immobilized FN and to plasminogen and collagen but not to laminin. MAb ws1 inhibited binding to FN. GAPDH was detected on the surface of trichomonads and was upregulated in synthesis and surface expression by iron. Higher levels of binding to FN were seen for organisms grown in iron-replete medium than for organisms grown in iron-depleted medium. In addition, decreased synthesis of GAPDH by antisense transfection of T. vaginalis gave lower levels of organisms bound to FN and had no adverse effect on growth kinetics. Finally, GAPDH did not associate with immortalized vaginal epithelial cells (VECs), and neither GAPDH nor MAb ws1 inhibited the adherence of trichomonads to VECs. These results indicate that GAPDH is a surface-associated protein of T. vaginalis with alternative functions.Trichomonas vaginalis, an extracellular protozoan parasite, is the cause of trichomonosis, the most prevalent nonviral sexually transmitted disease (47). In women, vaginitis due to T. vaginalis clinically manifests with symptoms of vaginal itching, odor, and discharge. Adverse health outcomes for women with this sexually transmitted disease include cervical cancer (46) and preterm delivery and low-birth-weight infants (25). There is a relationship between seropositivity to T. vaginalis and prostate cancer (43). This disease is significant due to its association with human immunodeficiency virus (33, 45). More recently, persistent, undetected T. vaginalis infections associated with asymptomatic carriage were found among women (40).T. vaginalis penetration of the mucous layer (28), followed by adherence to vaginal epithelial cells (VECs), is preparatory for colonization (9, 10). VEC adherence by parasites is mediated by numerous distinct trichomonad surface adhesins (5, 10, 18). Brief contact of T. vaginalis with VECs and fibronectin (FN) elicited dramatic changes in parasite morphology, suggesting a host-specific signaling of parasites (8, 9). Importantly, iron and cell contact by parasites each upregulated the expression of adhesins in a coordinated fashion via distinct mechanisms (2, 4, 6, 21, 29). Genetic approaches using antisense (AS) inhibition of synthesis (36, 37) and heterologous expression in Tritrichomonas foetus (26, 36) have reaffirmed the role of these T. vaginalis proteins as adhesins. T. vaginalis organisms secrete or release numerous metabolic enzymes, including adhesin AP65 (decarboxylating malic enzyme), α-enolase, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) during growth and multiplication (27). AP65 and α-enolase were found to reassociate with the parasite surface for the expression of adhesin function (19) and binding to plasminogen (35), respectively.There is an increased awareness of the existence of metabolic enzymes on the surfaces of bacterial pathogens, yeast, and parasites (12, 24, 35). These surface-associated enzymes appear to be novel virulence factors (17, 22, 38, 39). The anchorless glycolytic enzymes GAPDH (13, 31, 38) and α-enolase (39) are present on the surface of group A streptococcus. The surface-associated GAPDH of Candida albicans binds with strong affinity to FN and laminin (22). In enterohemorrhagic Escherichia coli and enteropathogenic E. coli, GAPDH is an extracellular protein that binds human plasminogen and fibrinogen and also interacts with intestinal epithelial cells (17).We demonstrate that GAPDH is another enzyme on the surface of T. vaginalis. A monoclonal antibody (MAb) that inhibited parasite associations with FN was immunoreactive with GAPDH. Importantly, iron was found to regulate gene expression and synthesis and surface placement of GAPDH. Both low-iron-grown trichomonads and AS-transfected parasites with decreased amounts of GAPDH had smaller amounts of surface GAPDH and corresponding lower levels of binding to FN. GAPDH was not involved in adherence of trichomonads to immortalized VECs. Interestingly, as with other microbial pathogens, T. vaginalis GAPDH also bound plasminogen and collagen but not laminin (17, 22).     

Identification of a Variety of Staphylococcus Species by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry

Whole-cell fingerprinting by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) in combination with a dedicated bioinformatic software tool (MALDI Biotyper 2.0) was used to identify 152 staphylococcal strains corresponding to 22 staphylococcal species. Spectra of the 152 isolates, previously identified at the species level using a sodA gene-based oligonucleotide array, were analyzed against the main spectra of 3,030 microorganisms. A total of 151 strains out of 152 (99.3%) were correctly identified at the species level; only one strain was identified at the genus level. The MALDI-TOF MS method revealed different clonal lineages of Staphylococcus epidermidis that were of either human or environmental origin, which suggests that the MALDI-TOF MS method could be useful in the profiling of staphylococcal strains. The topology of the dendrogram generated by the MALDI Biotyper 2.0 software from the spectra of 120 Staphylococcus reference strains (representing 36 species) was in general agreement with that inferred from the 16S rRNA gene-based analysis. Our findings indicate that the MALDI-TOF MS technology, associated with a broad-spectrum reference database, is an effective tool for the swift and reliable identification of Staphylococci.Most staphylococci are harmless and reside normally on the skin and mucous membranes of humans and other organisms (16, 22, 34). Staphylococcal strains are isolated from various food products in which they are involved in fermentation (18, 29). Staphylococcus species can cause a wide variety of diseases in humans and other animals (2, 22, 30-32, 35). S. aureus is a major pathogen in human infections (31). Several other Staphylococcus species have also been implicated in human infections, notably S. saprophyticus, S. epidermidis, S. lugdunensis, and S. schleiferi (4, 16, 31, 34). Coagulase-negative staphylococci (CoNS) have emerged as predominant pathogens in hospital-acquired infections (4, 16, 31, 34). One of the major challenges of daily diagnostic work is therefore to identify Staphylococcus species.Several manual and automated methods based on phenotypic characteristics have been developed for the identification of Staphylococci (12, 24). Unfortunately, these systems have their limitations, mostly due to phenotypic differences between strains from the same species (6, 10, 19, 21). Over the last 10 years, many genotypic methods based on the analysis of selected DNA targets have been designed for species-level identification of most common isolated CoNS (20, 26, 33). The sequence polymorphism of the sodA gene has significant discriminatory power (20) and allows the development of assays based on DNA chip technologies (“Staph array”) (8). Recently, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) using protein “fingerprints” was used for the identification of microorganisms (1, 3, 5, 9, 11, 14, 25, 36). In the present study, we assessed the ability of the MALDI Biotyper system (Bruker Daltonique, Wissembourg, France) to identify Staphylococcus species of clinical and environmental origins previously identified by sodA gene-based oligonucleotide array (8).     

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.     

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.     

Protection of Sheep against Rift Valley Fever Virus and Sheep Poxvirus with a Recombinant Capripoxvirus Vaccine

Rift Valley fever (RVF) is an epizootic viral disease of sheep that can be transmitted from sheep to humans, particularly by contact with aborted fetuses. A capripoxvirus (CPV) recombinant virus (rKS1/RVFV) was developed, which expressed the Rift Valley fever virus (RVFV) Gn and Gc glycoproteins. These expressed glycoproteins had the correct size and reacted with monoclonal antibodies (MAb) to native glycoproteins. Mice vaccinated with rKS1/RVFV were protected against RVFV challenge. Sheep vaccinated with rKS1/RVFV twice developed neutralizing antibodies and were significantly protected against RVFV and sheep poxvirus challenge. These findings further document the value of CPV recombinants as ruminant vaccine vectors and support the inclusion of RVFV genes encoding glycoproteins in multivalent recombinant vaccines to be used where RVF occurs.Rift Valley fever (RFV) virus (RVFV) is a mosquito-borne member of the genus Phlebovirus, family Bunyaviridae. It is widely distributed in Africa, causing endemic and epidemic disease in both humans and livestock, including sheep, cattle, and goats. RVF was first described in Kenya and was shown to be caused by a filterable virus transmissible via blood (9). Acute RVF in lambs is characterized by fever and death within 24 to 48 h of being detected (43). Signs in adult sheep include fever, mucopurulent nasal discharge, hemorrhagic diarrhea, and abortion in pregnant ewes (43). RVFV can be transmitted from infected sheep to humans, particularly when humans are exposed to aborted sheep fetuses and blood.Attenuated live RVFV vaccines are available for use in livestock. A mutagen-attenuated RVFV vaccine induces protective immune responses in lambs and appears to be safe (25); however, other studies documented teratogenic effects on lambs from vaccinated pregnant ewes similar to those caused by the attenuated RVFV strain Smithburn (18). An inactivated RVFV vaccine induces neutralizing antibody responses in humans (33), and its use in sheep would not induce teratogenic effects or abortions. However, the inactivated vaccine requires 3 doses (33) and is expensive to produce. Efforts to make RVFV vaccines without these disadvantages include an attenuated RVFV developed by reverse genetics and lacking the NSs and NSm genes (4) and other new-generation RVFV vaccines (reviewed in reference 19) that protect mice against virus challenge (7, 16, 24, 27).The middle (M) RNA segment of the RVFV genome encodes the viral glycoproteins Gn and Gc (8, 20), and recombinant vaccinia virus expressing these glycoproteins induces neutralizing antibody and protective immunity to RVFV in mice (7). Vaccinia virus is safe for animals, but there is some risk to humans, as it was reported previously to spread from human vaccinees to contacts (28, 55) and to cause serious clinical disease in human immunodeficiency virus-infected patients (36). Although modified vaccinia virus Ankara is a safer alternative for humans (6, 57), there are animal poxviruses with naturally restricted host ranges for vaccine vectors in animals (1, 13, 30, 31, 40, 46, 47, 52, 53).For ruminants, the genus Capripoxvirus (CPV) of the family Poxviridae has been an effective recombinant vector to induce protective immunity against several other viruses (3, 17, 29, 32, 40, 41, 51). This genus has three closely related species causing sheep pox, goat pox, and lumpy skin disease (LSD) of cattle. A recombinant LSD vaccine expressing the Gn and Gc glycoproteins of RVFV induced protection against RVFV challenge in mice (52, 53) and sheep (52). The three species of CPV have 96 to 97% nucleotide identity (49) and are restricted to ruminants, with no evidence of human infections (10, 11). Furthermore, attenuated CPV vaccines are in use in Africa and the Middle East to control ruminant poxvirus disease (11, 21). The use of a CPV vector to deliver virus vaccines to ruminants also induces immunity to the CPV vector, thus increasing the valence of the vaccine (3, 17, 39, 40). We report here the construction of a recombinant CPV that expresses the RVFV Gn and Gc glycoproteins and induces protective immunity against RVFV and sheep poxvirus (SPV) challenge in sheep.     

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.     

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.     

Evaluation of Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Identification of Clinically Important Yeast Species

We evaluated the use of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for the rapid identification of yeast species. Using Bruker Daltonics MALDI BioTyper software, we created a spectral database library with m/z ratios of 2,000 to 20,000 Da for 109 type and reference strains of yeast (44 species in 8 genera). The database was tested for accuracy by use of 194 clinical isolates (23 species in 6 genera). A total of 192 (99.0%) of the clinical isolates were identified accurately by MALDI-TOF MS. The MALDI-TOF MS-based method was found to be reproducible and accurate, with low consumable costs and minimal preparation time.Invasive fungal infections due to opportunistic pathogens are a significant cause of morbidity and mortality (2, 5, 8). The current rise in fungal infections correlates with the widespread use of broad-spectrum antibacterial agents, prolonged hospitalization of critically ill patients, and the increased number of immunocompromised patients. Candida species comprise the fourth most common cause of nosocomial bloodstream infections, and Cryptococcus neoformans is the most common cause of fungus-related mortality in HIV-infected patients (15, 19). While Candida albicans is still involved in more than half of all Candida-related bloodstream infections, an increase in recovery of non-C. albicans Candida spp., Rhodotorula spp., Trichosporon spp., and Malassezia spp. has occurred (2, 29). Treatment with amphotericin B may be useful for these organisms and inefficient for those belonging to other genera (5, 8). While many Candida species remain susceptible to fluconazole, it is important to differentiate the more resistant organisms, namely, Candida glabrata, Candida krusei, Rhodotorula spp., and some members of the genus Trichosporon. Additionally, Rhodotorula spp. have an innate resistance to voriconazole, and Trichosporon, Cryptococcus, and Rhodotorula are intrinsically resistant to the echinocandins (1, 15). These organisms present new challenges not only to treatment but also to standard identification methods used in the clinical laboratory (4, 8, 28).Commercially available biochemical test systems identify most of the commonly isolated species of yeast accurately but may result in no identification or misidentification of more-unusual isolates (4, 21, 28). Additionally, samples for these tests must be incubated for 1 to 3 days before results are obtained. To overcome the inaccuracies of biochemical identification methods, nucleic acid-based tests have been developed. These tests amplify and then sequence a target gene, such as the rRNA genes or the internal transcribed spacer (ITS) region (9, 10, 14, 17). While these assays are highly accurate, they require considerable processing time and costly reagents.As an alternative to biochemical and genome-based identification schemes, proteomic profiling by mass spectral analysis was recently evaluated for use in species differentiation of a variety of microorganisms. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) is emerging as a rapid and accurate tool for identifying pathogens, including Gram-positive and Gram-negative bacteria, mycobacteria, molds, and yeast species (3, 6, 11-13, 16, 18, 22, 23, 27). The technique can be performed rapidly, with minimal consumable expenses, and produces reproducible, species-specific spectral patterns that are not dependent upon the age of culture, growth conditions, or medium selection (7, 13, 20, 26).In this work, we present the development of a yeast database library consisting of 109 type and reference strains (44 species in 8 genera), and we tested the robustness and accuracy of this library by using 194 well-characterized clinical isolates (23 species in 6 genera).     

Salmonella-Containing Vacuoles Display Centrifugal Movement Associated with Cell-to-Cell Transfer in Epithelial Cells

Intracellular Salmonella enterica serovar Typhimurium (serovar Typhimurium) occupies a Salmonella-containing vacuole (SCV) where bacterial effector proteins are secreted into the host cell using type III secretion systems (T3SS). Cytoskeletal motor proteins and T3SS-delivered effector proteins facilitate SCV positioning to juxtanuclear positions where bacterial replication occurs. Here, we show that this characteristic SCV positioning is not maintained by all SCVs during infection of HeLa cells. Notably, juxtanuclear SCV localization that occurs by 8 to 14 h postinfection is followed by significant centrifugal displacement of a subset of SCVs toward the host cell periphery by 24 h postinfection. This novel phenotype requires bacterial protein synthesis, a functional Salmonella pathogenicity island 2 (SPI-2)-encoded T3SS, intact microtubules, and kinesin-1 motor protein. Bacteria lacking PipB2, a kinesin-recruiting T3SS effector, did not exhibit centrifugal displacement and remained at juxtanuclear positions throughout 24 h of infection. While levels of the SPI-2 effectors PipB2 and SifA increased during 24 h postinfection, a corresponding decrease in levels of the SPI-1 T3SS effectors SipA and SopB, both known to mediate juxtanuclear SCV positioning, was observed. A fluorescence-based assay indicated that wild-type serovar Typhimurium transferred from infected to uninfected epithelial cells while strains deficient in SPI-2 T3SS secretion or PipB2 did not. Our results reveal a novel SCV phenotype implicated in the cell-to-cell spread of serovar Typhimurium during infection.Salmonella enterica serovar Typhimurium (serovar Typhimurium) is a cause of gastroenteritis in humans and typhoid-like disease in certain strains of mice (55). Serovar Typhimurium is a facultative intracellular pathogen that can actively invade nonphagocytic cells through the delivery of bacterial proteins, termed effectors, into the host cell cytosol using the type III secretion system (T3SS) encoded by Salmonella pathogenicity island 1 (SPI-1) (19). Following entry, serovar Typhimurium typically resides in a membrane-bound compartment termed the Salmonella-containing vacuole (SCV) (16, 50, 54). From here, additional effector proteins required for intracellular replication and virulence are delivered into the host cytosol using a second T3SS encoded by another pathogenicity island, SPI-2 (10, 28, 40, 48, 58). These effectors modulate various host cell activities, including endosomal trafficking, actin assembly dynamics, and microtubule-based transport (5, 8, 24, 33, 50, 51, 58).One characteristic trait of SCVs is their localization to a juxtanuclear, Golgi apparatus-associated region of the host cell several hours postinfection (1, 5, 41, 45). In general, with some variation likely due to differing experimental methods, this distinct localization is observed as early as 4 h postinfection (hpi) and is maintained at 8 to 16 hpi (2, 5, 13, 24, 26, 41, 42, 45). A recent study has shown that serovar Typhimurium can redirect host secretory traffic, resulting in an accumulation of post-Golgi vesicles around the SCV (36). It has been proposed that serovar Typhimurium targets the Golgi apparatus to acquire nutrients and/or membrane materials for maintenance of a replicative niche within the SCV (23, 45).Several different effectors secreted by the SPI-2 T3SS appear to mediate this centralized positioning of SCVs, including SseF, SseG, and SifA (2, 5, 13, 41, 42, 45). Deletion of sseF or sseG results in SCVs that are displaced from their usual juxtanuclear, Golgi compartment-associated position (1, 13, 45). SseF and SseG have been shown to interact with each other (13) and appear to promote the recruitment of the minus-end-directed microtubule motor dynein to SCVs to permit their juxtanuclear localization (2). SseG has also been proposed to act by tethering SCVs to the Golgi region (42).Deletion of sifA also results in SCVs that are displaced from the nucleus and located toward the host cell periphery (5). SifA was shown to interact with a host SifA- and kinesin-interacting protein that negatively regulates the recruitment of plus-end-directed kinesin-1 motors to the SCV, thus favoring the inward migration and maintenance of the SCV around the nucleus (5). In apparent opposition to SifA, the SPI-2 effector PipB2 has been shown to recruit kinesin-1 to the SCV (26). However, the characteristic positioning of SCVs to juxtanuclear regions suggests that the kinesin-inhibitory action of SifA may be dominant over the effects of PipB2 (26), at least at 8 to 14 hpi.Interestingly, some effectors secreted by the SPI-1 T3SS that is traditionally associated with Salmonella invasion appear to persist in host cells (6, 14) and are also implicated in modulating intracellular SCV positioning (6, 57). We recently demonstrated a role for the SPI-1 T3SS effector SopB in maintaining the juxtanuclear positioning of SCVs through the action of nonmuscle myosin II actin motors (57). Another SPI-1 effector, SipA, has also been shown to persist in host cells after bacterial entry and appears to act with SifA to ensure perinuclear positioning of SCVs (6). Hence, it appears that stringent control of microtubule and actin motor activity on the SCV by both SPI-1 and SPI-2 T3SS effectors is an important facet of SCV intracellular positioning (26).Overall, much remains to be resolved regarding the mediators and implications of intracellular SCV positioning. By remaining at the juxtanuclear region, the bacteria likely modify their compartments into a replicative niche where nutrient acquisition and SCV maintenance can occur (23, 36, 45). As a result of replication, high numbers of intracellular bacteria would presumably lead to host cell lysis, resulting in bacterial release; however, little is known about any mechanism(s) of Salmonella escape from host cells.The present study was conducted to examine the intracellular positioning of SCVs over the course of a 24-h infection. We show that at later stages of epithelial cell infection, the positioning of a significant proportion of SCVs is not maintained at a juxtanuclear region but is situated closer to the host cell periphery. This outward displacement of SCVs was dependent upon the SPI-2 T3SS, host microtubules and kinesin, and the SPI-2 effector PipB2. Furthermore, the dynamic positioning of SCVs is associated with a decrease in protein levels of SPI-1 effectors previously shown to mediate juxtanuclear positioning. Results from a cell-to-cell infection assay indicate that serovar Typhimurium strains that did not exhibit peripheral displacement at later stages of infection were also impaired in their ability to infect newly introduced host cells. Our results provide new insight into the nature of SCV positioning and demonstrate that intracellular SCV positioning is a dynamic process, with implications for bacterial cell-to-cell transfer.     

Staphylococcus aureus Induces Microglial Inflammation via a Glycogen Synthase Kinase 3β-Regulated Pathway

A proinflammatory role for glycogen synthase kinase 3β (GSK-3β) has been demonstrated. Here, we addressed its roles on heat-inactivated Staphylococcus aureus-induced microglial inflammation. Heat-inactivated S. aureus induced tumor necrosis factor alpha (TNF-α) and nitric oxide (NO) production, at least in part, via a Toll-like receptor 2-regulated pathway. Neutralization of TNF-α largely blocked heat-inactivated S. aureus-induced NO. Heat-inactivated S. aureus activated GSK-3β, and inhibiting GSK-3β reduced TNF-α production as well as inducible NO synthase (iNOS)/NO biosynthesis. While activation of NF-κB was essential for heat-inactivated S. aureus-induced TNF-α and NO, inhibiting GSK-3β blocked heat-inactivated S. aureus-induced NF-κB p65 nuclear translocation. Additionally, inhibiting GSK-3β enhanced heat-inactivated S. aureus-induced interleukin-10 (IL-10) production (IL-10 is an anti-inflammatory cytokine which inhibits TNF-α production). Neutralization of IL-10 reduced TNF-α downregulation caused by GSK-3β inhibition. These results suggest that GSK-3β regulates heat-inactivated S. aureus-induced TNF-α and NO production in microglia mainly by activating NF-κB and probably by inhibiting IL-10.Staphylococcus aureus, a gram-positive bacterium, causes a variety of diseases, such as bacteremia, peritonitis, subcutaneous and brain abscess, and life-threatening staphylococcal septic shock (15). The mechanisms that lead to staphylococcal septic shock are multifactorial but involve especially immunogenic and toxic injuries (10, 40). Cell wall components and secreted virulence factors, including enterotoxins and exotoxins, have been shown to be inflammatory and cytotoxic to the host. Pathogen-associated molecular patterns are recognized by the innate immune system through a family of pattern recognition receptors, such as Toll-like receptors (TLRs) (2, 6, 26). Microglia, the resident macrophages in the brain, express TLR2 to recognize S. aureus peptidoglycan and play a critical role in neuroinflammation (7, 35, 37). Induction of neuroinflammation by S. aureus is partially mediated by TLR2- and nuclear factor-κB (NF-κB)-regulated pathways (23, 26, 36, 51).Infection of S. aureus causes the deregulated production of inflammatory cytokines, including tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and IL-10, and chemokines, including monocyte chemoattractant protein 1 (MCP-1) and RANTES (regulated on activation, normal T cell expressed and secreted protein) (24, 25, 32, 45). TNF-α, a potent proinflammatory cytokine, causes severe inflammatory responses, including cytokine and chemokine production and inducible nitric oxide (NO) synthase (iNOS)/NO biosynthesis in S. aureus infection (49). The deregulated generation of NO contributes to S. aureus-induced circulatory failure and liver injury (34). IL-10, a potent anti-inflammatory cytokine, inhibits the synthesis of the proinflammatory cytokines (TNF-α, IL-1, IL-6, IL-12, IL-18, and IL-10 itself), chemokines (IL-8, MCP-1, and RANTES), and iNOS/NO (4, 30, 43). IL-10 knockout mice display high mortality and are more susceptible to S. aureus-induced brain abscess (48). Exogenous IL-10 inhibits lethal sepsis, hepatic injury, and TNF-α production induced by staphylococcal enterotoxin B in mice (46, 48).Inhibiting glycogen synthase kinase 3β (GSK-3β) downregulates TLR-mediated inflammatory responses but increases IL-10 production (41, 53). Since NF-κB is important for inflammatory activation, GSK-3β is also involved in activating NF-κB in response to inflammatory stimuli (17-21, 29, 44, 50, 52). Therefore, GSK-3β inhibitors have been used to confer anti-inflammation against TNF-α administration, endotoxemia, experimental colitis, type II collagen-induced arthritis, ovalbumin-induced asthma, and experimental autoimmune encephalomyelitis (5, 12-14, 18, 20, 31, 41, 50, 52). Notably, current studies also show the effects of GSK-3β inhibition in reducing gram-negative coccobacillus Francisella-induced inflammation (55). GSK-3β inhibitors have also been widely used to reduce microglial inflammation and neurotoxicity (31, 54). In search of strategies against S. aureus-induced microglial inflammation, we investigated the possible effects of GSK-3β inhibition. In the present study, we report that inhibiting GSK-3β blocks NF-κB activation, TNF-α production, and iNOS/NO biosynthesis, but increases IL-10 production in heat-inactivated S. aureus-stimulated microglia.