Tod J. Merkel

Cytokine Response to Infection with Bacillus anthracis Spores

ABSTRACT Bacillus anthracis, the etiological agent of anthrax, is a gram-positive, spore-forming bacterium. The inhalational form of anthrax is the most severe and is associated with rapid progression of the disease and the outcome is frequently fatal. Transfer from the respiratory epithelium to regional lymph nodes appears to be an essential early step in the establishment of infection. This transfer is believed to occur by means of carriage within alveolar macrophages following phagocytosis. Therefore, the ability of B. anthracis to transit through the host macrophage or dendritic cell appears to be an early and critical step in B. anthracis pathogenesis. In this work, we examined the cytokine responses to spore infection in mouse primary peritoneal macrophages, in primary human dendritic cells, and during a spore aerosol infection model utilizing the susceptible A/J mouse strain. We demonstrated that both mouse peritoneal macrophages and human dendritic cells exhibited significant intracellular bactericidal activity during the first hours following uptake, providing the necessary time to mount a cytokine response prior to cell lysis. Strong tumor necrosis factor (TNF-α) and interleukin-6 (IL-6) responses were seen in mouse peritoneal macrophages. In addition to TNF-α and IL-6, human dendritic cells produced the cytokines IL-1β, IL-8, and IL-12. A mixture of Th1 and Th2 cytokines were detected in sera obtained from infected animals. In this study, we provide further evidence of an acute cytokine response when cells in culture and mice are infected with B. anthracis spores.

CHARACTERIZATION OF THE ACTIVATING REGION OF ESCHERICHIA COLI CATABOLITE GENE ACTIVATOR PROTEIN (CAP). II: ROLE AT CLASS I AND CLASS II CAP-DEPENDENT PROMOTERS

CAP-dependent promoters can be divided into classes based on the position of the DNA site for CAP. In class I CAP-dependent promoters, the DNA site for CAP is located upstream of the DNA site for polymerase; the DNA site for CAP can be located at various distances from the transcription start point, provided that the DNS site for CAP and the DNA site for RNA polymerase are on the same face of the DNA helix. In class II CAP-dependent promoters, the DNA site for CAP overlaps the DNA site for RNA polymerase, replacing the -35 determinants for binding of RNA polymerase. In previous work, we have shown that a surface loop consisting of amino acid residues 152 to 166 of CAP is essential for transcription activation at the best-characterized class I CAP-dependent promoter, the lac promoter, and we proposed that this surface loop makes direct protein-protein contact with RNA polymerase in the ternary complex of lac promoter, CAP, and RNA polymerase. Here, we show that the surface loop consisting of amino acid residues 152 to 166 is essential for transcription activation at other class I CAP-dependent promoters and at a class II CAP-dependent promoter. We show further that the effects of alanine substitutions of residues 152 to 166 are qualitatively identical at the lac promoter and other class I CAP-dependent promoters, but are different at a class II CAP-dependent promoter. We propose that the surface loop consisting of residues 152 to 166 makes identical molecular interactions in transcription activation at all class I CAP-dependent promoters, irrespective of distance between the DNA site for CAP and the transcription start point, but makes a different set of molecular interactions in transcription activation at class II CAP-dependent promoters.

Role of Anthrax Toxins in Dissemination, Disease Progression, and Induction of Protective Adaptive Immunity in the Mouse Aerosol Challenge Model

ABSTRACT Anthrax toxins significantly contribute to anthrax disease pathogenesis, and mechanisms by which the toxins affect host cellular responses have been identified with purified toxins. However, the contribution of anthrax toxin proteins to dissemination, disease progression, and subsequent immunity after aerosol infection with spores has not been clearly elucidated. To better understand the role of anthrax toxins in pathogenesis in vivo and to investigate the contribution of antibody to toxin proteins in protection, we completed a series of in vivo experiments using a murine aerosol challenge model and a collection of in-frame deletion mutants lacking toxin components. Our data show that after aerosol exposure to Bacillus anthracis spores, anthrax lethal toxin was required for outgrowth of bacilli in the draining lymph nodes and subsequent progression of infection beyond the lymph nodes to establish disseminated disease. After pulmonary exposure to anthrax spores, toxin expression was required for the development of protective immunity to a subsequent lethal challenge. However, immunoglobulin (immunoglobulin G) titers to toxin proteins, prior to secondary challenge, did not correlate with the protection observed upon secondary challenge with wild-type spores. A correlation was observed between survival after secondary challenge and rapid anamnestic responses directed against toxin proteins. Taken together, these studies indicate that anthrax toxins are required for dissemination of bacteria beyond the draining lymphoid tissue, leading to full virulence in the mouse aerosol challenge model, and that primary and anamnestic immune responses to toxin proteins provide protection against subsequent lethal challenge. These results provide support for the utility of the mouse aerosol challenge model for the study of inhalational anthrax.

Anthrax Protective Antigen Delivered by Salmonella enterica Serovar Typhi Ty21a Protects Mice from a Lethal Anthrax Spore Challenge

ABSTRACT Bacillus anthracis, the etiological agent of anthrax disease, is a proven weapon of bioterrorism. Currently, the only licensed vaccine against anthrax in the United States is AVA Biothrax, which, although efficacious, suffers from several limitations. This vaccine requires six injectable doses over 18 months to stimulate protective immunity, requires a cold chain for storage, and in many cases has been associated with adverse effects. In this study, we modified the B. anthracis protective antigen (PA) gene for optimal expression and stability, linked it to an inducible promoter for maximal expression in the host, and fused it to the secretion signal of the Escherichia coli alpha-hemolysin protein (HlyA) on a low-copy-number plasmid. This plasmid was introduced into the licensed typhoid vaccine strain, Salmonella enterica serovar Typhi strain Ty21a, and was found to be genetically stable. Immunization of mice with three vaccine doses elicited a strong PA-specific serum immunoglobulin G response with a geometric mean titer of 30,000 (range, 5,800 to 157,000) and lethal-toxin-neutralizing titers greater than 16,000. Vaccinated mice demonstrated 100% protection against a lethal intranasal challenge with aerosolized spores of B. anthracis 7702. The ultimate goal is a temperature-stable, safe, oral human vaccine against anthrax infection that can be self-administered in a few doses over a short period of time.

Role of Purine Biosynthesis in Bacillus anthracis Pathogenesis and Virulence

ABSTRACT Bacillus anthracis, the etiological agent of anthrax, is a spore-forming, Gram-positive bacterium and a category A biothreat agent. Screening of a library of transposon-mutagenized B. anthracis spores identified a mutant displaying an altered phenotype that harbored a mutated gene encoding the purine biosynthetic enzyme PurH. PurH is a bifunctional protein that catalyzes the final steps in the biosynthesis of the purine IMP. We constructed and characterized defined purH mutants of the virulent B. anthracis Ames strain. The virulence of the purH mutants was assessed in guinea pigs, mice, and rabbits. The spores of the purH mutants were as virulent as wild-type spores in mouse intranasal and rabbit subcutaneous infection models but were partially attenuated in a mouse intraperitoneal model. In contrast, the purH mutant spores were highly attenuated in guinea pigs regardless of the administration route. The reduced virulence in guinea pigs was not due solely to a germination defect, since both bacilli and toxins were detected in vivo, suggesting that the significant attenuation was associated with a growth defect in vivo. We hypothesize that an intact purine biosynthetic pathway is required for the virulence of B. anthracis in guinea pigs.

Epicutaneous Model of Community-Acquired Staphylococcus aureus Skin Infections

ABSTRACT Staphylococcus aureus is one of the most common etiological agents of community-acquired skin and soft tissue infection (SSTI). Although the majority of S. aureus community-acquired SSTIs are uncomplicated and self-clearing in nature, some percentage of these cases progress into life-threatening invasive infections. Current animal models of S. aureus SSTI suffer from two drawbacks: these models are a better representation of hospital-acquired SSTI than community-acquired SSTI, and they involve methods that are difficult to replicate. For these reasons, we sought to develop a murine model of community-acquired methicillin-resistant S. aureus SSTI (CA-MRSA SSTI) that can be consistently reproduced with a high degree of precision. We utilized this model to begin to characterize the host immune response to this type of infection. We infected mice via epicutaneous challenge of the skin on the outer ear pinna using Morrow-Brown allergy test needles coated in S. aureus USA300. When mice were challenged in this model, they developed small, purulent, self-clearing lesions with predictable areas of inflammation that mimicked a human infection. CFU in the ear pinna peaked at day 7 before dropping by day 14. The Th1 and Th17 cytokines gamma interferon (IFN-γ), interleukin-12 (IL-12) p70, tumor necrosis factor alpha (TNF-α), IL-17A, IL-6, and IL-21 were all significantly increased in the draining lymph node of infected mice, and there was neutrophil recruitment to the infection site. In vivo neutrophil depletion demonstrated that neutrophils play a protective role in preventing bacterial dissemination and fatal invasive infection.

Activation of the vrg6 Promoter of Bordetella pertussis by RisA

The BvgAS two-component system positively regulates the expression of the virulence genes of Bordetella pertussis and negatively regulates a second set of genes whose function is unknown. The BvgAS-mediated regulation of the bvg-repressed genes is accomplished through the activation of expression of the negative regulator, BvgR. A second two-component regulatory system, RisAS, is required for expression of the bvg-repressed surface antigens VraA and VraB. We examined the roles of BvgR and RisA in the regulation of four bvg-repressed genes in B. pertussis. Our analyses demonstrated that all four genes are repressed by the product of the bvgR locus and are activated by the product of the risA locus. Deletion analysis of the vrg6 promoter identified the upstream and downstream boundaries of the promoter and, in contrast to previously published results, demonstrated that sequences downstream of the start of transcription are not required for the regulation of expression of vrg6. Gel mobility-shift experiments demonstrated sequence-specific binding of RisA to the vrg6 and vrg18 promoters, and led to the identification of two putative RisA binding sites. Finally, transcriptional analysis and Western blot analysis demonstrated that BvgR regulates neither the expression nor the stability of RisA.

The multifaceted RisA regulon of Bordetella pertussis.

The whooping cough agent Bordetella pertussis regulates the production of its virulence factors by the BvgA/S system. Phosphorylated BvgA activates the virulence-activated genes (vags) and represses the expression of the virulence-repressed genes (vrgs) via the activation of the bvgR gene. In modulating conditions, with MgSO4, the BvgA/S system is inactive, and the vrgs are expressed. Here, we show that the expression of almost all vrgs depends on RisA, another transcriptional regulator. We also show that some vags are surprisingly no longer modulated by MgSO4 in the risA− background. RisA also regulates the expression of other genes, including chemotaxis and flagellar operons, iron-regulated genes, and genes of unknown function, which may or may not be controlled by BvgA/S. We identified RisK as the likely cognate RisA kinase and found that it is important for expression of most, but not all RisA-regulated genes. This was confirmed using the phosphoablative RisAD60N and the phosphomimetic RisAD60E analogues. Thus the RisA regulon adds a new layer of complexity to B. pertussis virulence gene regulation.

Multiple Roles of Myd88 in the Immune Response to the Plague F1-V Vaccine and in Protection against an Aerosol Challenge of Yersinia pestis CO92 in Mice

The current candidate vaccine against Yersinia pestis infection consists of two subunit proteins: the capsule protein or F1 protein and the low calcium response V protein or V-antigen. Little is known of the recognition of the vaccine by the hosts innate immune system and how it affects the acquired immune response to the vaccine. Thus, we vaccinated Toll-like receptor (Tlr) 2, 4, and 2/4-double deficient, as well as signal adaptor protein Myd88-deficient mice. We found that Tlr4 and Myd88 appeared to be required for an optimal immune response to the F1-V vaccine but not Tlr2 when compared to wild-type mice. However, there was a difference between the requirement for Tlr4 and MyD88 in vaccinated animals. When F1-V vaccinated Tlr4 mutant (lipopolysaccharide tolerant) and Myd88-deficient mice were challenged by aerosol with Y. pestis CO92, all but one Tlr4 mutant mice survived the challenge, but no vaccinated Myd88-deficient mice survived the challenge. Spleens from these latter nonsurviving mice showed that Y. pestis was not cleared from the infected mice. Our results suggest that MyD88 appears to be important for both an optimal immune response to F1-V and in protection against a lethal challenge of Y. pestis CO92 in F1-V vaccinated mice.