Johanna Rivera

Bacillus anthracis produces membrane-derived vesicles containing biologically active toxins.

Extracellular vesicle production is a ubiquitous process in Gram-negative bacteria, but little is known about such process in Gram-positive bacteria. We report the isolation of extracellular vesicles from the supernatants of Bacillus anthracis, a Gram-positive bacillus that is a powerful agent for biological warfare. B. anthracis vesicles formed at the outer layer of the bacterial cell had double-membrane spheres and ranged from 50 to 150 nm in diameter. Immunoelectron microscopy with mAbs to protective antigen, lethal factor, edema toxin, and anthrolysin revealed toxin components and anthrolysin in vesicles, with some vesicles containing more than one toxin component. Toxin-containing vesicles were also visualized inside B. anthracis-infected macrophages. ELISA and immunoblot analysis of vesicle preparations confirmed the presence of B. anthracis toxin components. A mAb to protective antigen protected macrophages against vesicles from an anthrolysin-deficient strain, but not against vesicles from Sterne 34F2 and Sterne δT strains, consistent with the notion that vesicles delivered both toxin and anthrolysin to host cells. Vesicles were immunogenic in BALB/c mice, which produced a robust IgM response to toxin components. Furthermore, vesicle-immunized mice lived significantly longer than controls after B. anthracis challenge. Our results indicate that toxin secretion in B. anthracis is, at least, partially vesicle-associated, thus allowing concentrated delivery of toxin components to target host cells, a mechanism that may increase toxin potency. Our observations may have important implications for the design of vaccines, for passive antibody strategies, and provide a previously unexplored system for studying secretory pathways in Gram-positive bacteria.

More Is Not Necessarily Better: Prozone-Like Effects in Passive Immunization with IgG

Despite a century of study, the relationship between Ag-specific Ig concentration and protection remains poorly understood for the majority of pathogens. In certain conditions, administration of high Ab doses before challenge with an infectious agent can be less effective than smaller Ab doses, a phenomenon which is consistent with a prozone-like effect. In this study, the relationship between IgG1, IgG2a, IgG2b, and IgG3 dose, infective inocula, and protection was investigated in a mouse model of Cryptococcus neoformans infection. The activity of each IgG subclass ranged from protective to disease-enhancing depending on both the Ab dose and infective inocula used. Enhanced dissemination to the brain was observed in mice given a high IgG2a dose and a relatively low inoculum. Ab administration had immunomodulatory effects, with cytokine expression in lung, brain, and spleen varying as a function of the infective inoculum Ab dose and IgG subclass. In vitro studies did not predict or explain the mechanism of in vivo prozone-like effects, because all isotypes were opsonic and elicited NO release from macrophages. IgG2a was most efficient in inducing a macrophage oxidative burst. These results reveal that an individual Ab can be protective, nonprotective, or disease-enhancing depending on its concentration relative to a challenge inoculum. Our findings have implications for the potential contribution of Ab responses to defense against microbial diseases because Ab-mediated immunity may be protective, nonprotective, or even deleterious to the host.

Neutropenia alters lung cytokine production in mice and reduces their susceptibility to pulmonary cryptococcosis

Neutrophils are generally considered to contribute to host defense through their potent microbicidal activity. However, there is accumulating evidence that neutrophils also have an important regulatory role in establishing the balance of Th1 and Th2 responses. This study investigated the role of neutrophils in defense against pulmonary Cryptococcus neoformans infection using neutrophil‐depleted BALB/c mice generated by administering mAb RB6–8C5. Neutropenic mice with pulmonary infection survived significantly longer than control mice, but there was no difference between groups infected intravenously. On day 1 of infection, neutropenic mice had significantly smaller fungal burdens than control mice. On day 7, neutropenic mice had significantly higher lung concentrations of IL‐10, TNF‐α, IL‐4, and IL‐12 than control mice, but there was no difference in IFN‐γ and MCP‐1 levels. Neutrophils influenced the outcome of cryptococcal infection in mice through mechanisms that did not involve a reduction in early fungal burden. The absence of neutrophils in lung tissue during the initial stages of infection appeared to alter the inflammatory response in a manner thatwas subsequently beneficial to the host. Higher levels of Th1‐ and Th2‐associated cytokines in neutropenic mice could have simultaneously promoted a strong cellular response while reducing inflammatory damage to the lung. Our results support the emerging concept that neutrophils play an important function in modulating the development of the immune response.

Identification and characterization of the Cryptococcus neoformans phosphomannose isomerase-encoding gene, MAN1, and its impact on pathogenicity

The polysaccharide capsule surrounding Cryptococcus neoformans comprises manose, xylose and glucuronic acid, of which mannose is the major constituent. The GDP‐mannose biosynthesis pathway is highly conserved in fungi and consists of three key enzymes: phosphomannose isomerase (PMI), phosphomannomutase (PMM) and GDP‐mannose pyrophosphorylase (GMP). The MAN1 gene, encoding for the PMI enzyme, was isolated and sequenced from C. neoformans, and a disruption of the MAN1 gene was generated. One MAN1 disruption mutant, man1, which showed poor capsule formation, reduced polysaccharide secretion and morphological abnormalities, was chosen for virulence studies. In both the rabbit and the mouse models of invasive cryptococcosis, man1 was shown to be severely impaired in its virulence, with complete elimination of the yeast from the host. A reconstituted strain of man1 was constructed using gene replacement at the native locus. The wild‐type and reconstituted strains were significantly more virulent than the knock‐out mutant in both animal models. Our findings reveal that PMI activity is essential for the survival of C. neoformans in the host. The fact that the man1 mutant was not pathogenic suggests that blocking mannose synthesis could be fungicidal in the mammalian host and thus an excellent target for antifungal drug development.

Antibody Efficacy in Murine Pulmonary Cryptococcus neoformans Infection: A Role for Nitric Oxide

We investigated the pathogenesis of pulmonary Cryptococcus neoformans infection and passive Ab efficacy in mice deficient in inducible NO synthase (NOS2−/−) and the parental strain. Parental mice lived significantly longer than NOS2−/− mice after intratracheal infection, despite having a higher lung fungal burden. Administration of Ab reduced lung CFU in both NOS2−/− and parental mice, but prolonged survival and increased the inflammatory response only in parental mice. Ab administration was associated with increased serum nitrite and reduced polysaccharide levels in parental mice. Eosinophils were present in greater numbers in the lung of infected NOS2−/− mice than parental mice, irrespective of Ab administration. C. neoformans infection in NOS2−/− mice resulted in significantly higher levels of IFN-γ, monocyte chemoattractant protein-1, and macrophage-inflammatory protein-1α than parental mice. Ab administration had different effects on infected NOS2−/− and parental mice with respect to IFN-γ, monocoyte chemoattractant protein-1, and macrophage-inflammatory protein-1α levels. Ab administration increased lung levels of IFN-γ in parental mice and reduced levels in NOS2−/− mice. The results indicate that NO is involved in the regulation of cytokine expression in response to cryptococcal pneumonia and is necessary for Ab efficacy against C. neoformans in mice. Our findings indicate a complex relationship between Ab efficacy against C. neoformans and cytokine expression, underscoring the interdependency of cellular and humoral defense mechanisms.

The Relative Susceptibility of Mouse Strains to Pulmonary Cryptococcus neoformans Infection Is Associated with Pleiotropic Differences in the Immune Response

ABSTRACT CBA/J mice were highly susceptible to intratracheal (i.t.) Cryptococcus neoformans infection relative to BALB/c mice, while both strains were equally susceptible to intravenous (i.v.) infection. Increased susceptibility in i.t. infection was associated with higher brain CFU, lower serum immunoglobulin M (IgM) and IgG responses to glucuronoxylomannan (GXM), lack of IgE regulation during infection, and alveolar macrophage permissiveness to intracellular replication in vitro. In contrast, for BALB/c mice, relative resistance was associated with increased interleukin-12 (IL-12) and decreased IL-10 pulmonary levels. In CBA/J mice, relative susceptibility was associated with a decreased proportion of CD4+ and CD8+ T cells and an increase in macrophage percentage in pulmonary infiltrates. In contrast, no significant differences in these cytokines or cell recruitment were observed in the i.v. model, consistent with no differences in the survival rate. Passive antibody (Ab) protection experiments revealed a prozone effect in the BALB/c mice with i.v. infection, such that Ab efficacy decreased at higher doses. In the i.t. model using CBA/J mice, low Ab doses were disease enhancing and protection was observed only at high doses. Our results show (i) that differences in mouse strain susceptibility are a function of the infection model, (ii) that susceptibility to pulmonary infection was associated with macrophage permissiveness for intracellular replication, and (iii) that the efficacy of passive Ab in pulmonary infection is a function of dose and mouse strain. The results highlight significant differences in the pathogenesis of cryptococcal infection among inbred mice and associate their relative susceptibility with differences in numerous components of the innate and adaptive immune responses.

A Monoclonal Antibody to Bacillus anthracis Protective Antigen Defines a Neutralizing Epitope in Domain 1

ABSTRACT Antibody (Ab) responses to Bacillus anthracis toxins are protective, but relatively few protective monoclonal antibodies (MAbs) have been reported. Protective antigen (PA) is essential for the action of B. anthracis lethal toxin (LeTx) and edema toxin. In this study, we generated two MAbs to PA, MAbs 7.5G and 10F4. These MAbs did not compete for binding to PA, consistent with specificities for different epitopes. The MAbs were tested for their ability to protect a monolayer of cultured macrophages against toxin-mediated cytotoxicity. MAb 7.5G, the most-neutralizing MAb, bound to domain 1 of PA and reduced LeTx toxicity in BALB/c mice. Remarkably, MAb 7.5G provided protection without blocking the binding of PA or lethal factor or the formation of the PA heptamer complex. However, MAb 7.5G slowed the proteolytic digestion of PA by furin in vitro, suggesting a potential mechanism for Ab-mediated protection. These observations indicate that some Abs to domain 1 can contribute to host protection.

Antibody-Mediated Protection against Cryptococcus neoformans Pulmonary Infection Is Dependent on B Cells

ABSTRACT The pathogenesis of pulmonary Cryptococcus neoformans infection and the efficacy of passive immunoglobulin G1 (IgG1) administration were investigated in B-cell-deficient and C57BL/6J mice. C57BL/6J mice lived longer than B-cell-deficient mice after both intratracheal and intravenous infections. Administration of IgG1 prior to infection prolonged the survival of C57BL/6J mice but had no effect on the survival or numbers of CFU in the lungs of B-cell-deficient mice. C. neoformans infection in B-cell-deficient mice resulted in significantly higher levels of gamma interferon (IFN-γ), monocyte chemoattractant protein-1 (MCP-1), and macrophage inflammatory protein-1α (MIP-1α) than in C57BL/6J mice. IgG1 administration reduced IFN-γ and MCP-1 levels in C57BL/6J mice but not in B-cell-deficient mice. In addition, compared to its effect in C57BL/6J mice, C. neoformans infection in FcRγIII-deficient, athymic, and SCID mice significantly increased IFN-γ and MCP-1 levels. IgG1 administration was associated with reduced IFN-γ levels in C57BL/6J mice but not in FcRγIII-deficient, athymic, and SCID mice. These observations suggest that IgG1-mediated protection in this system is a consequence of alterations in the inflammatory response that translate into less damage to the host without directly reducing the fungal burden. For hosts with impaired immunities, the ineffectiveness of passive antibody (Ab) may reflect an inability to down-regulate inflammation and avoid self-damage. The results indicate an important role for B cells in host defense against C. neoformans infection and demonstrate a surprising dependence of Ab-mediated protection on B cells in this system.

Mouse Genetic Background Is a Major Determinant of Isotype-Related Differences for Antibody-Mediated Protective Efficacy against Cryptococcus neoformans

The protective efficacy of mAbs to Cryptococcus neoformans glucuronoxylomannan depends on Ab isotype. Previous studies in A/JCr and C57BL/6J mice showed relative protective efficacy of IgG1, IgG2a ≫ IgG3. However, we now report that in C57BL/6J × 129/Sv mice, IgG3 is protective while IgG1 is not protective, with neither isotype being protective in 129/Sv mice. IgG1, IgG2a, and IgG3 had different effects on IFN-γ expression in infected C57BL/6J × 129/Sv mice. IgG1-treated C57BL/6J × 129/Sv mice had significantly more pulmonary eosinophilia than IgG2a- and IgG3-treated C57BL/6J × 129/Sv mice. C. neoformans infection and Ab administration had different effects on FcγRI, FcγRII, and FcγRIII expression in C57BL/6J, 129/Sv, and C57BL/6J × 129/Sv mice. Our results indicate that the relative efficacy of Ab isotype function against C. neoformans is a function of the genetic background of the host and that IgG3-mediated protection in C57BL/6J × 129/Sv mice was associated with lower levels of IFN-γ and fewer pulmonary eosinophils. The dependence of isotype efficacy on host genetics underscores a previously unsuspected complex relationship between the cellular and humoral arms of the adaptive immune response.

Antibody interactions with the capsule of Cryptococcus neoformans.

ABSTRACT Monoclonal antibodies to the encapsulated fungus Cryptococcus neoformans produce different immunofluorescence (IF) patterns after binding to the polysaccharide capsule. To explore the relationship between the IF pattern and the location of antibody binding, two immunoglobulin M (IgM) monoclonal antibodies (MAbs) (12A1 and 13F1) that differ in protective efficacy and IF pattern and one protective IgG1 MAb (2H1) were studied by IF and electron microscopy (EM). Fixing C. neoformans cells in lung tissue for EM resulted in significantly better preservation of the capsule than fixing yeast cells in suspension. The localization of MAbs 12A1 and 13F1 by immunogold EM differed depending on whether the MAb was bound to cells in cut tissue sections embedded in plastic or to cells in solution. In cut tissue sections, MAbs 12A1 and 13F1 bound throughout the capsule, whereas in solution both MAbs bound near the capsule surface. To investigate whether antibody binding to the C. neoformans capsule affected the binding of other primary or secondary reagents, various combinations of MAbs 12A1, 13F1, and 2H1 were studied by direct and indirect IF. The IF pattern and location of binding for MAbs 12A1, 13F1, and 2H1 varied depending on the presence of other capsule-binding MAbs and the method of detection. The results show that (i) binding of MAbs to the C. neoformanspolysaccharide capsule can modify the binding of subsequent primary or secondary antibodies; (ii) the IgM MAbs bind primarily to the outer capsule regions despite the occurrence of their epitopes throughout the capsule; and (iii) MAb 2H1 staining of newly formed buds is reduced, suggesting quantitative or qualitative differences in bud capsule.