A Burkholderia pseudomallei colony variant necessary for gastric colonization.

UNLABELLED: Diverse colony morphologies are a hallmark of Burkholderia pseudomallei recovered from infected patients. We observed that stresses that inhibit aerobic respiration shifted populations of B. pseudomallei from the canonical white colony morphotype toward two distinct, reversible, yet relatively stable yellow colony variants (YA and YB). As accumulating evidence supports the importance of B. pseudomallei enteric infection and gastric colonization, we tested the response of yellow variants to hypoxia, acidity, and stomach colonization. Yellow variants exhibited a competitive advantage under hypoxic and acidic conditions and alkalized culture media. The YB variant, although highly attenuated in acute virulence, was the only form capable of colonization and persistence in the murine stomach. The accumulation of extracellular DNA (eDNA) was a characteristic of YB as observed by 4',6-diamidino-2-phenylindole (DAPI) staining of gastric tissues, as well as in an in vitro stomach model where large amounts of eDNA were produced without cell lysis. Transposon mutagenesis identified a transcriptional regulator (BPSL1887, designated YelR) that when overexpressed produced the yellow phenotype. Deletion of yelR blocked a shift from white to the yellow forms. These data demonstrate that YB is a unique B. pseudomallei pathovariant controlled by YelR that is specifically adapted to the harsh gastric environment and necessary for persistent stomach colonization. IMPORTANCE: Seemingly uniform populations of bacteria often contain subpopulations that are genetically identical but display unique characteristics which offer advantages when the population is faced with infrequent but predictable stresses. The pathogen Burkholderia pseudomallei is capable of forming several reversible colony types, and it interconverted between one white type and two yellow types under certain environmental stresses. The two yellow forms exhibited distinct advantages in low-oxygen and acidic environments. One yellow colony variant was the only form capable of chronic stomach colonization. Areas of gastric infection were marked by bacteria encased in a DNA matrix, and the yellow forms were able to produce large amounts of extracellular DNA in vitro. We also identified the regulator in control of yellow colony variant formation. These findings demonstrate a role in infection for colony variation and provide a mechanism for chronic stomach colonization-a frequently overlooked niche in melioidosis.

In vitro and in vivo assessment of Salmonella enterica serovar Typhimurium DT104 virulence.

Multidrug-resistant Salmonella enterica serovar Typhimurium phage type DT104 has become a widespread cause of human and other animal infection worldwide. The severity of clinical illness in S. enterica serovar Typhimurium DT104 outbreaks has led to the suggestion that this strain possesses enhanced virulence. In the present study, in vitro and in vivo virulence-associated phenotypes of several clinical isolates of S. enterica serovar Typhimurium DT104 were examined and compared to S. enterica serovar Typhimurium ATCC 14028s. The ability of these DT104 isolates to survive within murine peritoneal macrophages, invade cultured epithelial cells, resist antimicrobial actions of reactive oxygen and nitrogen compounds, and cause lethal infection in mice were assessed. Our results failed to demonstrate that S. enterica serovar Typhimurium DT104 isolates are more virulent than S. enterica serovar Typhimurium ATCC 14028s.

Defective localization of the NADPH phagocyte oxidase to Salmonella-containing phagosomes in tumor necrosis factor p55 receptor-deficient macrophages.

Tumor necrosis factor receptor (TNFR) p55-knockout (KO) mice are susceptible profoundly to Salmonella infection. One day after peritoneal inoculation, TNFR-KO mice harbor 1,000-fold more bacteria in liver and spleen than wild-type mice despite the formation of well organized granulomas. Macrophages from TNFR-KO mice produce abundant quantities of reactive oxygen and nitrogen species in response to Salmonella but nevertheless exhibit poor bactericidal activity. Treatment with IFN-gamma enhances killing by wild-type macrophages but does not restore the killing defect of TNFR-KO cells. Bactericidal activity of macrophages can be abrogated by a deletion in the gene encoding TNFalpha but not by saturating concentrations of TNF-soluble receptor, suggesting that intracellular TNFalpha can regulate killing of Salmonella by macrophages. Peritoneal macrophages from TNFR-KO mice fail to localize NADPH oxidase-containing vesicles to Salmonella-containing vacuoles. A TNFR-KO mutation substantially restores virulence to an attenuated mutant bacterial strain lacking the type III secretory system encoded by Salmonella pathogenicity island 2 (SPI2), suggesting that TNFalpha and SPI2 have opposing actions on a common pathway of vesicular trafficking. TNFalpha-TNFRp55 signaling plays a critical role in the immediate innate immune response to an intracellular pathogen by optimizing the delivery of toxic reactive oxygen species to the phagosome.

Oxygen-dependent anti-Salmonella activity of macrophages.

Numerous observations have established a crucial role for phagocytic cells in host resistance to Salmonella. Activated macrophages rely on a complex array of oxygen-dependent antimicrobial molecules to inhibit or kill intracellular Salmonella. An initial oxidative bactericidal phase, which is dependent on the respiratory burst phagocyte oxidase (phox) is succeeded by a prolonged nitrosative bacteriostatic phase, which is dependent on inducible nitric oxide synthase (iNOS). The sequential contribution of phox and iNOS to anti-Salmonella innate immunity has been demonstrated both in vitro and in vivo. The temporal progression from the predominant production of reactive oxygen species to the production of nitrogen oxides could optimize the initial reduction in microbial burden while minimizing the immunopathological consequences of the host inflammatory response.

Salmonella evasion of the NADPH phagocyte oxidase.

The bacteria-phagocyte interaction is of central importance in Salmonella pathogenesis. Immediately following phagocytosis, the NADPH phagocyte oxidase complex assembles in vesicles and produces highly toxic reactive oxygen species that play a major role in initial Salmonella killing by phagocytes. However, Salmonella has evolved a number of strategies to reduce the efficacy of oxygen-dependent phagocyte antimicrobial systems. Some of these strategies, such as superoxide dismutases, hydroperoxidases, oxidoreductases, scavengers and repair systems are common to most aerobic bacteria. In addition, Salmonella has acquired, by horizontal gene transfer, a type III secretory system encoded by Salmonella pathogenicity island 2 that interferes with the trafficking of vesicles containing functional NADPH phagocyte oxidase to the phagosome, thereby enhancing the survival of Salmonella within macrophages.

Antimicrobial actions of the NADPH phagocyte oxidase and inducible nitric oxide synthase in experimental salmonellosis. I. Effects on microbial killing by activated peritoneal macrophages in vitro.

The contribution of the NADPH phagocyte oxidase (phox) and inducible nitric oxide (NO) synthase (iNOS) to the antimicrobial activity of macrophages for Salmonella typhimurium was studied by using peritoneal phagocytes from C57BL/6, congenic gp91phox(-/)-, iNOS(-/)-, and doubly immunodeficient phox(-/)-iNOS(-/)- mice. The respiratory burst and NO radical (NO.) made distinct contributions to the anti-Salmonella activity of macrophages. NADPH oxidase-dependent killing is confined to the first few hours after phagocytosis, whereas iNOS contributes to both early and late phases of antibacterial activity. NO-derived species initially synergize with oxyradicals to kill S. typhimurium, and subsequently exert prolonged oxidase-independent bacteriostatic effects. Biochemical analyses show that early killing of Salmonella by macrophages coincides with an oxidative chemistry characterized by superoxide anion (O(2).(-)), hydrogen peroxide (H(2)O(2)), and peroxynitrite (ONOO(-)) production. However, immunofluorescence microscopy and killing assays using the scavenger uric acid suggest that peroxynitrite is not responsible for macrophage killing of wild-type S. typhimurium. Rapid oxidative bacterial killing is followed by a sustained period of nitrosative chemistry that limits bacterial growth. Interferon gamma appears to augment antibacterial activity predominantly by enhancing NO. production, although a small iNOS-independent effect was also observed. These findings demonstrate that macrophages kill Salmonella in a dynamic process that changes over time and requires the generation of both reactive oxidative and nitrosative species.

Antimicrobial actions of the NADPH phagocyte oxidase and inducible nitric oxide synthase in experimental salmonellosis. II. Effects on microbial proliferation and host survival in vivo.

The roles of the NADPH phagocyte oxidase (phox) and inducible nitric oxide synthase (iNOS) in host resistance to virulent Salmonella typhimurium were investigated in gp91phox(-/)-, iNOS(-/)-, and congenic wild-type mice. Although both gp91phox(-/)- and iNOS(-/)- mice demonstrated increased susceptibility to infection with S. typhimurium compared with wild-type mice, the kinetics of bacterial replication were dramatically different in the gp91phox(-/)- and iNOS(-/)- mouse strains. Greater bacterial numbers were present in the spleens and livers of gp91phox(-/)- mice compared with C57BL/6 controls as early as day 1 of infection, and all of the gp91phox(-/)- mice succumbed to infection within 5 d. In contrast, an increased bacterial burden was detected within reticuloendothelial organs of iNOS(-/)- mice only beyond the first week of infection. Influx of inflammatory CD11b(+) cells, granuloma formation, and serum interferon gamma levels were unimpaired in iNOS(-/)- mice, but the iNOS-deficient granulomas were unable to limit bacterial replication. The NADPH phagocye oxidase and iNOS are both required for host resistance to wild-type Salmonella, but appear to operate principally at different stages of infection.

Salmonella pathogenicity island 2-dependent evasion of the phagocyte NADPH oxidase.

A type III protein secretion system encoded by Salmonella pathogenicity island 2 (SPI2) has been found to be required for virulence and survival within macrophages. Here, SPI2 was shown to allow Salmonella typhimurium to avoid NADPH oxidase-dependent killing by macrophages. The ability of SPI2-mutant bacteria to survive in macrophages and to cause lethal infection in mice was restored by abrogation of the NADPH oxidase-dependent respiratory burst. Ultrastructural and immunofluorescence microscopy demonstrated efficient localization of the NADPH oxidase in the proximity of vacuoles containing SPI2-mutant but not wild-type bacteria, suggesting that SPI2 interferes with trafficking of oxidase-containing vesicles to the phagosome.

Disparate requirement for T cells in resistance to mucosal and acute systemic candidiasis.

Although highly susceptible to orogastric candidiasis, T-cell receptor delta- and alpha-chain knockout mice, deficient in gammadelta and alphabeta T cells, respectively, were found to be resistant to disseminated candidiasis of endogenous origin and to acute systemic candidiasis (resulting from intravenous injection).

Cellular routes of invasion by enteropathogens.

The cellular pathways of infection utilized by pathogenic enteric bacteria have important implications for their clinical manifestations. Yersinia reaches Peyer's patches via M cells and uses plasmid-encoded factors to resist phagocytic cells. Shigella also translocates via M cells and incapacitates phagocytes, but subsequently re-enters the epithelium basolaterally to elicit an acute inflammatory response. Salmonella has recently been shown to both colonize Peyer's patches via M cells and independently disseminate to extraintestinal sites via CD18-expressing phagocytes. M cell-mediated entry can lead to gastroenteritis and mucosal antibody production, while systemic dissemination can result in septicemia and elicitation of systemic immune responses.

Effects of probiotic bacteria on humoral immunity to Candida albicans in immunodeficient bg/bg-nu/nu and bg/bg-nu/+ mice.

Germfree beige-nude ( bg/bg-nu/nu) and beige-heterozygous ( bg/bg-nu/+) mice were colonized with a pure culture of Candida albicans or with a probiotic bacterium (Lactobacillus acidophilus, Lactobacillus reuteri, Lactobacillus casei, or Bifidobacterium infantis). Probiotic-colonized mice were subsequently challenged orally with C. albicans. The effect of prior colonization with probiotic bacteria on the antibody responses of the immunodeficient mice to alimentary tract colonization with C. albicans was compared to the antibody responses of the gnotobiotic mice colonized only with C. albicans. This study demonstrated that, although the probiotic bacteria did not induce a vigorous antibody response to their own antigens, they altered the antibody responses of mice to C. albicans. In T cell competent bg/bg-nu/+mice, B. infantis enhanced and focused IgG1, IgG2A, and IgA responses to C. albicans antigens. Some of the probiotic bacteria also enhanced the IgG1 and IgG2A antibody responses of bg/bg-nu/nu mice to C. albicans antigens. This study not only shows the value of gnotobiotic animal models in demonstrating that probiotic bacteria can affect the capacity of mice to form antibodies to C. albicans, but it also points out their usefulness in comparing the capacity of different probiotic bacteria to produce beneficial health effects in mice.

Extraintestinal dissemination of Salmonella by CD18-expressing phagocytes.

Specialized epithelia known as M cells overlying the lymphoid follicles of Peyer's patches are important in the mucosal immune system, but also provide a portal of entry for pathogens such as Salmonella typhimurium, Mycobacterium bovis, Shigella flexneri, Yersinia enterocolitica and reoviruses. Penetration of intestinal M cells and epithelial cells by Salmonella typhimurium requires the invasion genes of Salmonella Pathogenicity Island 1 (SPI1). SPI1-deficient S. typhimurium strains gain access to the spleen following oral administration and cause lethal infection in mice without invading M cells or localizing in Peyer's patches, which indicates that Salmonella uses an alternative strategy to disseminate from the gastrointestinal tract. Here we report that Salmonella is transported from the gastrointestinal tract to the bloodstream by CD18-expressing phagocytes, and that CD18-deficient mice are resistant to dissemination of Salmonella to the liver and spleen after oral administration. This CD18-dependent pathway of extraintestinal dissemination may be important for the development of systemic immunity to gastrointestinal pathogens, because oral challenge with SPI1-deficient S. typhimurium elicits a specific systemic IgG humoral immune response, despite an inability to stimulate production of specific mucosal IgA.

Mucosal and systemic candidiasis in IL-8Rh-/- BALB/c mice.

Germ-free BALB/c mice, genetically engineered to be deficient for interleukin-8 (IL-8) receptor homolog (IL-8Rh-/-), were more susceptible to gastric candidiasis after oral challenge and to acute systemic candidiasis after intravenous challenge than IL-8Rh+/+ controls. In comparison to IL-8Rh+/+ mice, the IL-8Rh-/- mice had slower influx of polymorphonuclear neutrophils (PMN) into Candida albicans-infected tissues and a lower percentage of PMN in peritoneal exudate cells (PEC) elicited with heat-killed C. albicans. PEC from IL-8Rh-/- mice exhibited less luminol-dependent chemiluminescence in response to C. albicans and did not kill C. albicans hyphae as well as PEC from IL-8Rh+/+ mice. C. albicans-colonized IL-8Rh-/- mice showed no histological evidence of systemic candidiasis. These results suggest a role for the IL-8Rh in murine resistance to gastric and acute systemic candidiasis, but not in resistance to systemic candidiasis of endogenous origin.

Early resistance of interleukin-10 knockout mice to acute systemic candidiasis.

In contrast to immunocompetent controls, interleukin-10 (IL-10) knockout (KO) mice eliminated an experimental intravenous inoculation with Candida albicans from their kidneys. Improved clearance of C. albicans from the kidneys of IL-10 KO mice was evident at 24 h after intravenous challenge with the fungus. Conversely, mice with a deletion of the IL-4 cytokine gene were more susceptible to systemic candidiasis than were immunocompetent controls. The hyperresistance of IL-10 KO mice to acute systemic candidiasis did not seem to correlate with nitric oxide-mediated immunity, but rather, it appeared to be associated with more efficient effector function of innate cells, possibly neutrophils. In support of the latter hypothesis, we observed that neutrophils from IL-10 KO mice were more efficient at killing C. albicans blastoconidia and hyphae than were neutrophils from immunocompetent control mice. Neither IL-10 KO nor IL-4 KO mice that were monoassociated with C. albicans for 4 weeks showed any histologic evidence of systemic candidiasis of endogenous origin. In contrast to systemic candidiasis, we observed no significant (P < 0.05) differences in susceptibility among IL-10 KO, IL-4 KO, and wild-type (immunocompetent) mice to orogastric candidiasis. Our results suggest that IL-10 exerts a negative effect on the early, innate response to acute systemic candidiasis; however, in comparison to immunocompetent control (wild-type) mice, neither IL-10 nor IL-4 deficiency enhanced susceptibility to orogastric candidiasis.

Genes encoding putative effector proteins of the type III secretion system of Salmonella pathogenicity island 2 are required for bacterial virulence and proliferation in macrophages.

The type III secretion system of Salmonella pathogenicity island 2 (SPI-2) is required for systemic infection of this pathogen in mice. Cloning and sequencing of a central region of SPI-2 revealed the presence of genes encoding putative chaperones and effector proteins of the secretion system. The predicted products of the sseB, sseC and sseD genes display weak but significant similarity to amino acid sequences of EspA, EspD and EspB, which are secreted by the type III secretion system encoded by the locus of enterocyte effacement of enteropathogenic Escherichia coli. The transcriptional activity of an sseA::luc fusion gene was shown to be dependent on ssrA, which is required for the expression of genes encoding components of the secretion system apparatus. Strains carrying nonpolar mutations in sseA, sseB or sseC were severely attenuated in virulence, strains carrying mutations in sseF or sseG were weakly attenuated, and a strain with a mutation in sseE had no detectable virulence defect. These phenotypes were reflected in the ability of mutant strains to grow within a variety of macrophage cell types: strains carrying mutations in sseA, sseB or sseC failed to accumulate, whereas the growth rates of strains carrying mutations in sseE, sseF or sseG were only modestly reduced. These data suggest that, in vivo, one of the functions of the SPI-2 secretion system is to enable intracellular bacterial proliferation.

Candidiasis in interferon-gamma knockout (IFN-gamma-/-) mice.

Germ-free C57BL/6 x 129 interferon-gamma knockout (IFN-gamma(-/-)) mice and their immunocompetent (+/-, +/+) counterparts were colonized with a pure culture of Candida albicans to assess their natural susceptibility to mucosal and systemic candidiasis of endogenous origin. Colonization with a pure culture of C. albicans was not lethal for adult or neonatal IFN-gamma(-/-) gnotobiotic mice over the 15-week study. The IFN-gamma(-/-) mice were more susceptible to gastric (cardia-antrum section), anorectal, and acute systemic (intravenous challenge) candidiasis than immunocompetent controls, and some IFN-gamma(-/-) mice developed intestinal adenomas after colonization with C. albicans. The enhanced susceptibility of IFN-gamma(-/-) mice, compared with immunocompetent controls, may be associated with a poor proliferative response of spleen cells to C. albicans antigens and a T helper 2 (IgG1) serum antibody response to C. albicans antigens. Thus, IFN-gamma is important for murine resistance to gastric, anorectal, and acute systemic candidiasis.

The transcriptional regulator SoxS is required for resistance of Salmonella typhimurium to paraquat but not for virulence in mice.

In Escherichia coli, the SoxRS regulon is required for resistance to redox-cycling agents which elevate cytosolic superoxide levels, as well as for resistance to nitric oxide-dependent macrophage killing. In Salmonella typhimurium, SoxS is also required for enhanced expression of Mn-superoxide dismutase and resistance to paraquat, but not for resistance to nitric oxide donor compounds in vitro, resistance to macrophage killing, or virulence in mice. Differences in other antioxidant defense systems or compensation by homologous regulons may account for species-specific differences in the role of SoxS.

Macrophages in resistance to candidiasis.

Candida albicans, an increasingly common opportunistic pathogenic fungus, frequently causes disease in immunodeficient but not immunocompetent hosts. Clarifying the role of the phagocytic cells that participate in resistance to candidiasis not only is basic to understanding how the host copes with this dimorphic pathogen but also will expedite the development of innovative prophylactic and therapeutic approaches for treating the multiple clinical presentations that candidiasis encompasses. In this review, we present evidence that a diverse population of mononuclear phagocytes, in different states of activation and differentiation and from a variety of host species, can phagocytize C. albicans blastoconidia via an array of opsonic and nonopsonic mechanisms and can kill C. albicans blastoconidia and hyphae by means of oxygen-dependent and -independent mechanisms. Reactive nitrogen intermediates should now be added to the well-established candidacidal reactive oxygen intermediates of macrophages. Furthermore, what were thought to be two independent pathways, i.e., nitric oxide and superoxide anion, have now been shown to combine to form a potent macrophage candidacidal molecule, peroxynitrite. In contrast to monocytes and neutrophils, which are important in resistance to early stages of C. albicans infections, more differentiated macrophages activated by cytokines such as gamma interferon participate in the acquired resistance of hosts with C. albicans-specific, cell-mediated immunity. Evidence presented in this review demonstrates that mononuclear phagocytes, in some instances in the absence of other professional phagocytes such as neutrophils, play an import role in resistance to systemic and mucosal candidiasis.

Periplasmic superoxide dismutase protects Salmonella from products of phagocyte NADPH-oxidase and nitric oxide synthase.

Superoxide dismutase (SOD) catalyzes the conversion of superoxide radical to hydrogen peroxide. Periplasmic localization of bacterial Cu,Zn-SOD has suggested a role of this enzyme in defense against extracellular phagocyte-derived reactive oxygen species. Sequence analysis of regions flanking the Salmonella typhimurium sodC gene encoding Cu,Zn-SOD demonstrates significant homology to lambda phage proteins, reflecting possible bacteriophage-mediated horizontal gene transfer of this determinant among pathogenic bacteria. Salmonella deficient in Cu,Zn-SOD has reduced survival in macrophages and attenuated virulence in mice, which can be restored by abrogation of either the phagocyte respiratory burst or inducible nitric oxide synthase. Moreover, a sodC mutant is extremely susceptible to the combination of superoxide and nitric oxide. These observations suggest that SOD protects periplasmic or inner membrane targets by diverting superoxide and limiting peroxynitrite formation, and they demonstrate the ability of the respiratory burst and nitric oxide synthase to synergistically kill microbial pathogens in vivo.