Infections with the Gram-negative bacterium Burkholderia pseudomallei (melioidosis) are associated with high mortality, and there is currently no approved vaccine to prevent the development of melioidosis in humans. Infected patients also do not develop protective immunity to reinfection, and some individuals will develop chronic, subclinical infections with B. pseudomallei. At present, our understanding of what constitutes effective protective immunity against B. pseudomallei infection remains incomplete. Therefore, we conducted a study to elucidate immune correlates of vaccine-induced protective immunity against acute B. pseudomallei infection. BALB/c and C57BL/6 mice were immunized subcutaneously with a highly attenuated, Select Agent-excluded purM deletion mutant of B. pseudomallei (strain Bp82) and then subjected to intranasal challenge with virulent B. pseudomallei strain 1026b. Immunization with Bp82 generated significant protection from challenge with B. pseudomallei, and protection was associated with a significant reduction in bacterial burden in lungs, liver, and spleen of immunized mice. Humoral immunity was critically important for vaccine-induced protection, as mice lacking B cells were not protected by immunization and serum from Bp82-vaccinated mice could transfer partial protection to nonvaccinated animals. In contrast, vaccineinduced protective immunity was found to be independent of both CD4 and CD8 T cells. Tracking studies demonstrated uptake of the Bp82 vaccine strain predominately by neutrophils in vaccine-draining lymph nodes and by smaller numbers of dendritic cells (DC) and monocytes. We concluded that protection following cutaneous immunization with a live attenuated Burkholderia vaccine strain was dependent primarily on generation of effective humoral immune responses.

Bacillus anthracis is surrounded by an antiphagocytic capsule composed of poly-␥-D-glutamic acid (␥DPGA).Bacterial and fungal capsular polysaccharides are shed into body fluids in large amounts during infection. The goal of our study was to examine the in vivo fate and distribution of the ␥DPGA capsular polypeptide. Mice were injected via the intravenous route with various amounts of purified ␥DPGA. Blood, urine, and various organs were harvested at different times after treatment. Sites of ␥DPGA accumulation were determined by immunoassay using monoclonal antibodies specific for ␥DPGA. The results showed that the liver and spleen were the primary sites for the accumulation of ␥DPGA. As found in previous studies of capsular polysaccharides, the Kupffer cells of the liver and splenic macrophages were sites for the cellular accumulation of ␥DPGA. Unlike capsular polysaccharides, the hepatic sinusoidal endothelial cells were also sites for ␥DPGA accumulation. ␥DPGA was rapidly cleared from serum and was excreted into the urine. ␥DPGA in the urine showed a reduced molecular size relative to native ␥DPGA. The results indicate that in vivo clearance of the polypeptide capsular antigen of B. anthracis shares several features with the clearance of capsular polysaccharides. Key differences between the in vivo behaviors of ␥DPGA and capsular polysaccharides include the accumulation of ␥DPGA in hepatic sinusoidal endothelial cells and a ␥DPGA clearance rate that was more rapid than the clearance reported for capsular polysaccharides.Most bacterial capsules are composed of polysaccharides. These capsules are essential to the virulence of many pathogenic bacteria, such as Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria meningitidis as well as the opportunistic yeast Cryptococcus neoformans. The capsules are characteristically antiphagocytic, and antibodies to capsular polysaccharides are protective. Capsular polysaccharides display a repeating epitope structure, have a high molecular weight, and resist degradation in vivo. These properties are characteristic of antigens classified as thymus-independent type 2 (22).Unlike the capsular polysaccharides that surround most bacteria, the capsule of Bacillus anthracis is a homopolymer of D-glutamic acid residues that are linked by the gamma carboxyl group (poly-␥-D-glutamic acid [␥DPGA]) (11). The ␥DPGA capsule is essential for virulence (5,14,30). Like capsular polysaccharides, ␥DPGA is poorly immunogenic, and the coupling of ␥DPGA to immunogenic protein carriers greatly enhances immunogenicity (15,24,25,29). Finally, as with capsular polysaccharides, antibodies to ␥DPGA are protective in murine models of pulmonary anthrax (2,15,18).Studies of the in vivo behaviors of capsular polysaccharides of S. pneumoniae, H. influenzae type b, and C. neoformans found that capsular polysaccharides accumulate in cells of the reticuloendothelial system and persist for weeks in tissues and serum. Despite the essential role played by ␥DPGA in the virulence of B. anthracis, little i...

e Francisella tularensis is a highly infectious intracellular bacterium that causes the zoonotic infection tularemia. While much literature exists on the host response to F. tularensis infection, the vast majority of work has been conducted using attenuated strains of Francisella that do not cause disease in humans. However, emerging data indicate that the protective immune response against attenuated F. tularensis versus F. tularensis type A differs. Several groups have recently reported that interleukin-17 (IL-17) confers protection against the live vaccine strain (LVS) of Francisella. While we too have found that IL-17R␣ ؊/؊ mice are more susceptible to F. tularensis LVS infection, our studies, using a virulent type A strain of F. tularensis (SchuS4), indicate that IL-17R␣ ؊/؊ mice display organ burdens and pulmonary gamma interferon (IFN-␥) responses similar to those of wild-type mice following infection. In addition, oral LVS vaccination conferred equivalent protection against pulmonary challenge with SchuS4 in both IL-17R␣؊/؊ and wild-type mice. While IFN-␥ was found to be critically important for survival in a convalescent model of SchuS4 infection, IL-17 neutralization from either wild-type or IFN-␥ ؊/؊ mice had no effect on morbidity or mortality in this model. IL-17 protein levels were also higher in the lungs of mice infected with the LVS rather than F. tularensis type A, while IL23p19 mRNA expression was found to be caspase-1 dependent in macrophages infected with LVS but not SchuS4. Collectively, these results demonstrate that IL-17 is dispensable for host immunity to type A F. tularensis infection, and that induced and protective immunity differs between attenuated and virulent strains of F. tularensis.

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.

Successful treatment of pneumonic infection with Francisella tularensis, the causative agent of tularemia, requires rapid initiation of antibiotic therapy, yet even then treatment failures may occur. Consequently, new treatments are needed to enhance the effectiveness of antimicrobial therapy for acute pneumonic tularemia. In a prior study, immunization with F. tularensis membrane protein fraction (MPF) antigens 3 days prior to challenge was reported to induce significant protection from inhalational challenge. We therefore hypothesized that MPF immunization might also be effective in enhancing infection control if combined with antibiotic therapy and administered after infection as post-exposure immunotherapy. To address this question, a 24 h post-exposure treatment model of acute pulmonary Schu S4 strain of F. tularensis infection in BALB/c mice was used. Following exposure, mice were immunized with MPF and treated with low-dose gentamicin, alone or in combination and the effects on survival, bacterial burden and dissemination were assessed. We found that immunization with MPF significantly increased the effectiveness of subtherapeutic gentamicin therapy for post-exposure treatment of pneumonic tularemia, with 100% of combination-treated mice surviving long-term. Bacterial burdens in the liver and spleen were significantly reduced in combination MPF-gentamicin treated mice at 7 days after challenge. Passively transferred antibodies against MPF antigens also increased the effectiveness of gentamicin therapy. Thus, we concluded that post-exposure immunization with MPF antigens was an effective means of enhancing conventional antimicrobial therapy for pneumonic tularemia.