RESUMEN
During infection, Bacillus anthracis bacilli encounter potent antimicrobial peptides (AMPs) such as defensins. We examined the role that B. anthracis capsule plays in protecting bacilli from defensins and other cationic AMPs by comparing their effects on a fully virulent encapsulated wild type (WT) strain and an isogenic capsule-deficient capA mutant strain. We identified several human defensins and non-human AMPs that were capable of killing B. anthracis. The human alpha defensins 1-6 (HNP-1-4, HD-5-6), the human beta defensins 1-4 (HBD-1-4), and the non-human AMPs, protegrin, gramicidin D, polymyxin B, nisin, and melittin were all capable of killing both encapsulated WT and non-encapsulated capA mutant B. anthracis. However, non-encapsulated capA mutant bacilli were significantly more susceptible than encapsulated WT bacilli to killing by nearly all of the AMPs tested. We demonstrated that purified capsule bound HBD-2, HBD-3, and HNP-1 in an electrophoretic mobility shift assay. Furthermore, we determined that the capsule layer enveloping WT bacilli bound and trapped HBD-3, substantially reducing the amount reaching the cell wall. To assess whether released capsule might also play a protective role, we pre-incubated HBD-2, HBD-3, or HNP-1 with purified capsule before their addition to non-encapsulated capA mutant bacilli. We found that free capsule completely rescued the capA mutant bacilli from killing by HBD-2 and -3 while killing by HNP-1 was reduced to the level observed with WT bacilli. Together, these results suggest an immune evasion mechanism by which the capsule, both that enveloping the bacilli and released fragments, contributes to virulence by binding to and inhibiting the antimicrobial activity of cationic AMPs.
Asunto(s)
Bacillus anthracis , Nisina , alfa-Defensinas , beta-Defensinas , Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Péptidos Antimicrobianos , Defensinas/genética , Defensinas/farmacología , Gramicidina , Humanos , Meliteno , Polimixina B , alfa-Defensinas/farmacologíaRESUMEN
BACKGROUND: Inhalational anthrax is rare and clinical experience limited. Expert guidelines recommend treatment with combination antibiotics including protein synthesis-inhibitors to decrease toxin production and increase survival, although evidence is lacking. METHODS: Rhesus macaques exposed to an aerosol of Bacillus anthracis spores were treated with ciprofloxacin, clindamycin, or ciprofloxacin + clindamycin after becoming bacteremic. Circulating anthrax lethal factor and protective antigen were quantitated pretreatment and 1.5 and 12 hours after beginning antibiotics. RESULTS: In the clindamycin group, 8 of 11 (73%) survived demonstrating its efficacy for the first time in inhalational anthrax, compared to 9 of 9 (100%) with ciprofloxacin, and 8 of 11 (73%) with ciprofloxacin + clindamycin. These differences were not statistically significant. There were no significant differences between groups in lethal factor or protective antigen levels from pretreatment to 12 hours after starting antibiotics. Animals that died after clindamycin had a greater incidence of meningitis compared to those given ciprofloxacin or ciprofloxacin + clindamycin, but numbers of animals were very low and no definitive conclusion could be reached. CONCLUSION: Treatment of inhalational anthrax with clindamycin was as effective as ciprofloxacin in the nonhuman primate. Addition of clindamycin to ciprofloxacin did not enhance reduction of circulating toxin levels.
Asunto(s)
Carbunco/sangre , Carbunco/prevención & control , Antígenos Bacterianos/sangre , Bacillus anthracis/efectos de los fármacos , Bacillus anthracis/fisiología , Toxinas Bacterianas/sangre , Ciprofloxacina/uso terapéutico , Clindamicina/uso terapéutico , Infecciones del Sistema Respiratorio/sangre , Infecciones del Sistema Respiratorio/prevención & control , Animales , Carbunco/microbiología , Carbunco/mortalidad , Antibacterianos/uso terapéutico , Biomarcadores , Ciprofloxacina/farmacología , Clindamicina/farmacología , Modelos Animales de Enfermedad , Quimioterapia Combinada , Macaca mulatta , Pronóstico , Infecciones del Sistema Respiratorio/microbiología , Infecciones del Sistema Respiratorio/mortalidad , Resultado del TratamientoRESUMEN
For safety, designated Select Agents in tissues must be inactivated and viability tested before the tissue undergoes further processing and analysis. In response to the shipping of samples of "inactivated" Bacillus anthracis that inadvertently contained live spores to nonregulated entities and partners worldwide, the Federal Register now mandates in-house validation of inactivation procedures and standardization of viability testing to detect live organisms in samples containing Select Agents that have undergone an inactivation process. We tested and validated formaldehyde and glutaraldehyde inactivation procedures for animal tissues infected with virulent B. anthracis, Burkholderia pseudomallei, Francisella tularensis, and Yersinia pestis. We confirmed that our fixation procedures for tissues containing these Tier 1 Select Agents resulted in complete inactivation and that our validated viability testing methods do not interfere with detection of live organisms. Institutions may use this work as a guide to develop and conduct their own testing to comply with the policy.
Asunto(s)
Bacterias/efectos de los fármacos , Desinfectantes/farmacología , Formaldehído/farmacología , Glutaral/farmacología , Viabilidad Microbiana/efectos de los fármacos , Animales , Cobayas , Especificidad de Órganos , Esporas Bacterianas/efectos de los fármacos , Factores de TiempoRESUMEN
The antiphagocytic capsule of Bacillus anthracis is a major virulence factor. We hypothesized that it may also mediate virulence through inhibition of the host's immune responses. During an infection, the capsule exists attached to the bacterial surface but also free in the host tissues. We sought to examine the impact of free capsule by assessing its effects on human monocytes and immature dendritic cells (iDCs). Human monocytes were differentiated into iDCs by interleukin-4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) over 7 days in the presence of capsule derived from wild-type encapsulated B. anthracis Ames (WT) or a control preparation from an isogenic B. anthracis Ames strain that produces only 2% of the capsule of the WT (capA mutant). WT capsule consistently induced release of IL-8 and IL-6 while the capA mutant control preparation elicited either no response or only a minimal release of IL-8. iDCs that were differentiated in the presence of WT capsule had increased side scatter (SSC), a measure of cellular complexity, when assessed by flow cytometry. iDCs differentiated in the presence of WT capsule also matured less well in response to subsequent B. anthracis peptidoglycan (Ba PGN) exposure, with reduced upregulation of the chemokine receptor CCR7, reduced CCR7-dependent chemotaxis, and reduced release of certain cytokines. Exposure of naive differentiated control iDCs to WT capsule did not alter cell surface marker expression but did elicit IL-8. These results indicate that free capsule may contribute to the pathogenesis of anthrax by suppressing the responses of immune cells and interfering with the maturation of iDCs.
Asunto(s)
Bacillus anthracis/inmunología , Cápsulas Bacterianas/inmunología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Tolerancia Inmunológica , Células Cultivadas , Humanos , Interleucina-6/metabolismo , Interleucina-8/metabolismoRESUMEN
The capsule of Bacillus anthracis is composed of a d isomer poly-γ-glutamic acid polymer, which is especially nonstimulatory to dendritic cells, even more so than similar mixed d, l isomer polymers from nonpathogenic Bacillus species. Capsule is an essential virulence factor for B. anthracis, protecting the bacilli from phagocytosis by innate immune cells. In this study, we demonstrate that encapsulation provides a further pathogenic advantage by shielding more inflammatory Ags on the bacillus surface, thereby reducing dendritic cell responses. We exposed human immature dendritic cells (DCs) to increasing multiplicities of infection (MOIs) of killed B. anthracis bacilli from the fully encapsulated wild-type Ames strain (WT) and an isogenic capsule-deficient strain (capA mutant). Both strains elicited robust cytokine responses, but IL-23, TNF-α, and IL-10 were significantly reduced in response to the encapsulated WT compared with capA mutant up to an MOI of 15. capA mutant bacilli could induce phenotypic maturation of immature DCs with upregulation of MHC classes I and II, CD83, and CCR7 at an MOI of 3.75, whereas encapsulated WT bacilli still did not induce significant upregulation of MHC classes I and II at an MOI of 15. DCs exposed to capA mutant bacilli (MOI 3.75) exhibited CCR7-dependent chemotaxis that was comparable to that of LPS-stimulated controls, whereas DCs exposed to encapsulated WT bacilli exhibited significantly less chemotaxis. We conclude that capsule shields more inflammatory surface Ags, delaying development of an adaptive immune response by reducing TNF-α, thereby inhibiting DC maturation.
Asunto(s)
Bacillus anthracis/inmunología , Cápsulas Bacterianas/inmunología , Células Dendríticas/inmunología , Macrófagos/inmunología , Ácido Poliglutámico/análogos & derivados , Citocinas/metabolismo , Humanos , Inmunidad Innata , Fagocitosis , Ácido Poliglutámico/inmunología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Capsule depolymerase (CapD) is a gamma-glutamyl transpeptidase and a product of the Bacillus anthracis capsule biosynthesis operon. In this study, we examined the effect of modulating capD expression on B. anthracis capsule phenotype, interaction with phagocytic cells and virulence in guinea pigs. Transcriptional fusions of capD were made to the genes encoding heat-shock protein 60 (hsp60) and elongation factor Tu (EFTu), and to capA, a B. anthracis capsule biosynthesis gene. Translation signals were altered to improve expression of capD, including replacing the putative ribosome-binding site with a consensus sequence and the TTG start codon with ATG. CapD was not detected by immunoblotting in lysates from wild-type B. anthracis Ames but was detected in strains engineered with a consensus ribosome-binding site for capD. Strains overexpressing capD at amounts detected by immunoblotting were found to have less surface-associated capsule and released primarily lower-molecular-mass capsule into culture supernatants. Overexpression of capD increased susceptibility to neutrophil phagocytic killing and adherence to macrophages and resulted in reduced fitness in a guinea pig model of infection. These data suggest that B. anthracis may have evolved weak capD expression resulting in optimized capsule-mediated virulence.
Asunto(s)
Bacillus anthracis/enzimología , Bacillus anthracis/patogenicidad , Cápsulas Bacterianas/metabolismo , Glicósido Hidrolasas/metabolismo , Animales , Bacillus anthracis/genética , Chaperonina 60/genética , Clonación Molecular , Evolución Molecular , Glicósido Hidrolasas/biosíntesis , Glicósido Hidrolasas/genética , Cobayas , Macrófagos/citología , Macrófagos/metabolismo , Operón , Fenotipo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Virulencia/genéticaRESUMEN
The opsono-adherence assay is a functional assay that enumerates the attachment of bacterial pathogens to professional phagocytes. Because adherence is requisite to phagocytosis and killing, the assay is an alternative method to opsono-phagocytic killing assays. An advantage of the opsono-adherence assay is the option of using inactivated pathogens and mammalian cell lines, which allows standardization across multiple experiments. The use of an inactivated pathogen in the assay also facilitates work with biosafety level 3 infectious agents and other virulent pathogens. In our work, the opsono-adherence assay was used to assess the functional ability of antibodies, from sera of animals immunized with an anthrax capsule-based vaccine, to induce adherence of fixed Bacillus anthracis to a mouse macrophage cell line, RAW 264.7. Automated fluorescence microscopy was used to capture images of bacilli adhering to macrophages. Increased adherence was correlated with the presence of anti-capsule antibodies in the serum. Non-human primates that exhibited high serum anti-capsule antibody concentrations were protected from anthrax challenge. Thus, the opsono-adherence assay can be used to elucidate the biological functions of antigen specific antibodies in sera, to evaluate the efficacy of vaccine candidates and other therapeutics, and to serve as a possible correlate of immunity.
Asunto(s)
Vacunas contra el Carbunco/inmunología , Carbunco/inmunología , Anticuerpos Antibacterianos/inmunología , Bacillus anthracis/inmunología , Adhesión Bacteriana , Proteínas Opsoninas/inmunología , Animales , Carbunco/microbiología , Carbunco/prevención & control , Antígenos Bacterianos/inmunología , Fluoresceína-5-Isotiocianato/metabolismo , Fluorescencia , Humanos , Macrófagos/inmunología , Ratones , Primates/inmunología , Primates/microbiología , Células RAW 264.7RESUMEN
The efficacy of currently licensed anthrax vaccines is largely attributable to a single Bacillus anthracis immunogen, protective antigen. To broaden protection against possible strains resistant to protective antigen-based vaccines, we previously developed a vaccine in which the anthrax polyglutamic acid capsule was covalently conjugated to the outer membrane protein complex of Neisseria meningitidis serotype B and demonstrated that two doses of 2.5µg of this vaccine conferred partial protection of rhesus macaques against inhalational anthrax . Here, we demonstrate complete protection of rhesus macaques against inhalational anthrax with a higher 50µg dose of the same capsule conjugate vaccine. These results indicate that B. anthracis capsule is a highly effective vaccine component that should be considered for incorporation in future generation anthrax vaccines.
Asunto(s)
Vacunas contra el Carbunco/inmunología , Carbunco/prevención & control , Cápsulas Bacterianas/inmunología , Ácido Poliglutámico/inmunología , Infecciones del Sistema Respiratorio/prevención & control , Animales , Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/inmunología , Toxinas Bacterianas/inmunología , Femenino , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Macaca mulatta , Masculino , Conejos , Vacunas Conjugadas/inmunologíaRESUMEN
Bacillus anthracis, the causative agent of anthrax, is recognized as one of the most serious bioterrorism threats. The current human vaccines are based on the protective antigen component of the anthrax toxins. Concern about possible vaccine resistant strains and reliance on a single antigen has prompted the search for additional immunogens. Bacterial capsules, as surface-expressed virulence factors, are well-established components of several licensed vaccines. In a previous study we showed that an anthrax vaccine consisting of the B. anthracis poly-γ-D-glutamic acid capsule covalently conjugated to the outer membrane protein complex of Neisseria meningitidis serotype B protected mice against parenteral B. anthracis challenge. Here we tested this vaccine in rabbits and monkeys against an aerosol spore challenge. The vaccine induced anti-capsule antibody responses in both species, measured by ELISA and a macrophage opsono-adherence assay. While rabbits were not protected against a high aerosol challenge dose, significant protection was observed in monkeys receiving the capsule conjugate vaccine. The results confirm that the capsule is a protective immunogen against anthrax, being the first non-toxin antigen shown to be efficacious in monkeys and suggest that addition of capsule may broaden and enhance the protection afforded by protective antigen-based vaccines.
Asunto(s)
Vacunas contra el Carbunco/inmunología , Carbunco/prevención & control , Cápsulas Bacterianas/inmunología , Proteínas de la Membrana Bacteriana Externa/química , Animales , Carbunco/inmunología , Vacunas contra el Carbunco/administración & dosificación , Anticuerpos Antibacterianos/sangre , Cápsulas Bacterianas/química , Proteínas de la Membrana Bacteriana Externa/aislamiento & purificación , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Femenino , Macaca mulatta , Macrófagos/inmunología , Masculino , Neisseria meningitidis/química , Proteínas Opsoninas/sangre , Fagocitosis , Conejos , Análisis de Supervivencia , Vacunas Conjugadas/administración & dosificación , Vacunas Conjugadas/inmunologíaRESUMEN
The poly-gamma-d-glutamic acid capsule confers antiphagocytic properties on Bacillus anthracis and is essential for virulence. In this study, we showed that CapD, a gamma-polyglutamic acid depolymerase encoded on the B. anthracis capsule plasmid, degraded purified capsule and removed the capsule from the surface of anthrax bacilli. Treatment with CapD induced macrophage phagocytosis of encapsulated B. anthracis and enabled human neutrophils to kill encapsulated organisms. A second glutamylase, PghP, a gamma-polyglutamic acid hydrolase encoded by Bacillus subtilis bacteriophage PhiNIT1, had minimal activity in degrading B. anthracis capsule, no effect on macrophage phagocytosis, and only minimal enhancement of neutrophil killing. Thus, the levels of both phagocytosis and killing corresponded to the degree of enzyme-mediated capsule degradation. The use of enzymes to degrade the capsule and enable phagocytic killing of B. anthracis offers a new approach to the therapy of anthrax.
Asunto(s)
Bacillus anthracis/metabolismo , Cápsulas Bacterianas/metabolismo , Ácido Poliglutámico/metabolismo , gamma-Glutamiltransferasa/metabolismo , Animales , Antígenos Bacterianos/genética , Antígenos Bacterianos/metabolismo , Bacillus anthracis/efectos de los fármacos , Bacillus anthracis/genética , Bacillus subtilis/efectos de los fármacos , Bacillus subtilis/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/farmacología , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/farmacología , Células Cultivadas , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Fagocitosis/efectos de los fármacos , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , gamma-Glutamil Hidrolasa/metabolismo , gamma-Glutamiltransferasa/genética , gamma-Glutamiltransferasa/farmacologíaRESUMEN
Efficacy of a poly-gamma-D-glutamic acid anthrax capsule vaccine was assessed in a mouse model of infection. Capsule by itself was protective against lethal challenge with a toxin(-), capsule(+) Bacillus anthracis strain. Conjugation of capsule to bovine serum albumin resulted in enhanced IgG anti-capsule antibodies measured by ELISA, but completely abrogated the protection. The protective unconjugated capsule vaccine elicited significantly higher IgM titers and opsonic activity than did the non-protective capsule conjugate. When tested against a fully virulent toxin(+), capsule(+) B. anthracis strain, neither capsule nor protective antigen alone was protective. However, the combination of the two protected against a lethal challenge. These results suggest that capsule may enhance the protection afforded by protective antigen vaccines against anthrax if opsonizing antibodies are produced. Surprisingly, some protection was also observed when protective antigen was conjugated to itself.