Scott Stibitz

Routine markerless gene replacement in Bacillus anthracis

ABSTRACT An improved genetic tool suitable for routine markerless allelic exchange in Bacillus anthracis has been constructed. Its utility was demonstrated by the introduction of insertions, deletions, and missense mutations on the chromosome and plasmid pXO1 of the Sterne strain of B. anthracis.

Four Superoxide Dismutases Contribute to Bacillus anthracis Virulence and Provide Spores with Redundant Protection from Oxidative Stress

ABSTRACT The Bacillus anthracis genome encodes four superoxide dismutases (SODs), enzymes capable of detoxifying oxygen radicals. That two of these SODs, SOD15 and SODA1, are present in the outermost layers of the B. anthracis spore is indicated by previous proteomic analyses of the exosporium. Given the requirement that spores must survive interactions with reactive oxygen species generated by cells such as macrophages during infection, we hypothesized that SOD15 and SODA1 protect the spore from oxidative stress and contribute to the pathogenicity of B. anthracis. To test these theories, we constructed a double-knockout (Δsod15 ΔsodA1) mutant of B. anthracis Sterne strain 34F2 and assessed its lethality in an A/J mouse intranasal infection model. The 50% lethal dose of the Δsod15 ΔsodA1 strain was similar to that of the wild type (34F2), but surprisingly, measurable whole-spore SOD activity was greater than that in 34F2. A quadruple-knockout strain (Δsod15 ΔsodA1 ΔsodC ΔsodA2) was then generated, and as anticipated, spore-associated SOD activity was diminished. Moreover, the quadruple-knockout strain, compared to the wild type, was attenuated more than 40-fold upon intranasal challenge of mice. Spore resistance to exogenously generated oxidative stress and to macrophage-mediated killing correlated with virulence in A/J mice. Allelic exchange that restored sod15 and sodA1 to their wild-type state restored wild-type characteristics. We conclude that SOD molecules within the spore afford B. anthracis protection against oxidative stress and enhance the pathogenicity of B. anthracis in the lung. We also surmise that the presence of four SOD alleles within the genome provides functional redundancy for this key enzyme.

The Response Regulator BvgA and RNA Polymerase α Subunit C-Terminal Domain Bind Simultaneously to Different Faces of the Same Segment of Promoter DNA

Examination of the binding of FeBABE-conjugated BvgA to the fha promoter of Bordetella pertussis has revealed that three dimers, formed by head-to-head association of monomers, bind one face of the DNA helix from the inverted-heptad primary binding site to the -35 region. The orientation of BvgA monomers within the dimers is the same as that recently demonstrated by X-ray crystallographic methods for a dimer of the C-terminal domain of NarL bound to DNA. Use of FeBABE conjugates of RNAP alpha subunit C-terminal domain showed that binding of this domain is linearly coincident with binding of the BvgA dimers, but to a different helical face. These results reveal a previously undescribed mode of interaction between RNAP alpha-CTD and a transcriptional activator.

Demonstration of differential virulence gene promoter activation in vivo in Bordetella pertussis using RIVET

Bordetella pertussis, the etiologic agent of whooping cough, causes disease by employing an array of virulence factors controlled by the BvgA–BvgS two‐component signal transduction system. Regulation by this system has been extensively characterized in vitro, where bvg‐activated genes are repressed in a process known as phenotypic modulation. Differential regulation of these genes by the response regulator BvgA results in promoters that are activated early, middle, or late after being released from modulation. However, the in vivo environmental signal and regulation pattern has not been described. In order to investigate BvgAS‐mediated regulation of B. pertussis virulence factors in vivo using the mouse aerosol challenge model, we have adapted the recombinase‐based in vivo technology (RIVET) system for use in B. pertussis. We have demonstrated that these strains show resolution during in vitro growth under non‐modulating conditions. In addition, we have demonstrated that modulating strains by growth on media containing MgSO4 does not affect virulence in the mouse aerosol challenge model. We have therefore used the RIVET system to reveal the time‐course of gene expression in vivo for selected B. pertussis virulence factors (cya, fha, prn and ptx). Our data indicate that this method can be effectively used to monitor and compare in vivo and in vitro gene expression in B. pertussis, and that temporal regulation patterns previously observed in vitro are mirrored in vivo.

O Antigen Protects Bordetella parapertussis from Complement

ABSTRACT Bordetella pertussis, a causative agent of whooping cough, expresses BrkA, which confers serum resistance, but the closely related human pathogen that also causes whooping cough, Bordetella parapertussis, does not. Interestingly, B. parapertussis, but not B. pertussis, produces an O antigen, a factor shown in other models to confer serum resistance. Using a murine model of infection, we determined that O antigen contributes to the ability of B. parapertussis to colonize the respiratory tract during the first week of infection, but not thereafter. Interestingly, an O antigen-deficient strain of B. parapertussis was not defective in colonizing mice lacking the complement cascade. O antigen prevented both complement component C3 deposition on the surface and complement-mediated killing of B. parapertussis. In addition, O antigen was required for B. parapertussis to systemically spread in complement-sufficient mice, but not complement-deficient mice. These data indicate that O antigen enables B. parapertussis to efficiently colonize the lower respiratory tract by protecting against complement-mediated control and clearance.

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.

Genetic and Biochemical Analyses of BvgA Interaction with the Secondary Binding Region of the fha Promoter of Bordetella pertussis

The BvgA-BvgS two-component signal transduction system regulates expression of virulence factors in Bordetella pertussis. The BvgA response regulator activates transcription by binding to target promoters, which include those for the genes encoding filamentous hemagglutinin (fha) and pertussis toxin (ptx). We have previously shown that at both promoters the phosphorylated form of BvgA binds multiple high- and low-affinity sites. Specifically, at the fha promoter, we proposed that there may be high- and a low-affinity binding sites for the BvgA dimer. In our present investigation, we used DNA binding analyses and in vitro and in vivo assays of promoters with substitutions and deletions to support and extend this hypothesis. Our observations indicate that (i) binding of BvgA approximately P to a primary (high-affinity) site and a secondary binding region (lower affinity) is cooperative, (ii) although both the primary binding site and the secondary binding region are required for full activity of the wild-type (undeleted) promoter, deletion of two helical turns within the secondary binding region can produce a fully active or hyperactive promoter, and (iii) BvgA binding to the secondary binding region shows limited DNA sequence specificity.

BvgA functions as both an activator and a repressor to control Bvgi phase expression of bipA in Bordetella pertussis

The Bordetella bipA gene is expressed maximally when the BvgAS phosphorelay is semi‐active, i.e. in the Bvg‐intermediate (Bvgi) phase. We used a BvgA‐FeBABE cleavage approach together with site‐directed mutagenesis and bipA–lacZ fusion analyses to determine precisely where BvgA‐phosphate (BvgA∼P) binds at the bipA promoter and how that binding contributes to the complex transcription pattern displayed by bipA. BvgA∼P bound with high affinity and cooperatively with RNAP to sequences at the bipA promoter immediately 5′ to and overlapping those bound by RNAP to activate transcription under Bvgi phase conditions. bipA therefore, like fhaB, appears to be similar to classical class‐II promoters with regard to the mechanism by which its transcription is activated. BvgA∼P bound with relatively low affinity to sequences immediately 3′ of those bound by RNAP at the bipA promoter and this binding mediated repression of bipA transcription under Bvg+ phase conditions. BvgA∼P binding to these sequences occurred simultaneously, if not cooperatively, with RNAP, indicating that BvgA∼P represses bipA expression by inhibiting transcription initiation and/or elongation, rather than by competing with RNAP for binding. As bipA is the first Bvgi phase gene to be characterized, and the first gene shown to be repressed by BvgA∼P directly, our results will provide a basis for comparison as additional Bvg‐regulated genes are identified and characterized.

In vivo phosphorylation dynamics of the Bordetella pertussis virulence-controlling response regulator BvgA

We have used protein electrophoresis through polyacrylamide gels derivatized with the proprietary ligand Phos‐tag™ to separate the response regulator BvgA from its phosphorylated counterpart BvgA∼P. This approach has allowed us to readily ascertain the degree of phosphorylation of BvgA in in vitro reactions, or in crude lysates of Bordetella pertussis grown under varying laboratory conditions. We have used this technique to examine the kinetics of BvgA phosphorylation after shift of B. pertussis cultures from non‐permissive to permissive conditions, or of its dephosphorylation following a shift from permissive to non‐permissive conditions. Our results provide the first direct evidence that levels of BvgA∼P in vivo correspond temporally to the expression of early and late BvgA‐regulated virulence genes. We have also examined a number of other aspects of BvgA function predicted from previous studies and by analogy with other two‐component response regulators. These include the site of BvgA phosphorylation, the exclusive role of the cognate BvgS sensor kinase in its phosphorylation in Bordetella pertussis, and the effect of the T194M mutation on phosphorylation. We also detected the phosphorylation of a small but consistent fraction of BvgA purified after expression in Escherichia coli.

Stably Luminescent Staphylococcus aureus Clinical Strains for Use in Bioluminescent Imaging

In vivo bioluminescent imaging permits the visualization of bacteria in live animals, allowing researchers to monitor, both temporally and spatially, the progression of infection in each animal. We sought to engineer stably luminescent clinical strains of Staphylococcus aureus, with the goal of using such strains in mouse models. The gram-positive shuttle vector pMAD was used as the backbone for an integration plasmid. A chloramphenicol resistance gene, a modified lux operon from Photorhabdus luminescens, and approximately 650 bp of homology to the chromosome of the USA300 S. aureus strain NRS384 were added, generating plasmid pRP1195. Electroporation into strain RN4220 followed by temperature shift led to integration of pRP1195 into the chromosome. The integrated plasmid was transferred to clinical strains by phage transduction. Luminescent strains displayed no in vitro growth defects. Moreover, luminescence was stable in vitro after three rounds of subculture over 48 hours of growth in the absence of antibiotics. Mice were infected with a luminescent strain of NRS384 in skin and intravenous models. In a mouse skin model, luminescent bacteria were present in lesions that formed and cleared over the course of several days, and in an intravenous model, bacteria inoculated in the mouse tail vein were observed spreading to multiple tissues. No statistically significant difference in virulence was observed between NRS384 and the luminescent strain in either infection model. These preliminary data suggest that this luminescent USA300 strain is suitable for use in mouse models. Similar strains were engineered using other sequenced clinical strains. Because these strains are stably luminescent, they should prove useful in animal models of infection.