Our research program is directed towards gaining an understanding of the basic biology of the host-pathogen interaction between two model respiratory pathogens; B. anthracis and B. pertussis. Molecular and genetic methods are employed in the identification of genes required for virulence of B. pertussis and B. anthracis in tissue culture and animal models of disease. Identification and regulation of virulence factors of B. anthracis. Principle Objectives of the Current Research: (1) Identification of Bacillus anthracis genes that are essential for survival in human macrophages (2) Development of an animal model of B. anthracis infection. Bacillus anthracis is a Gram (+) rod. The development of 1-2 micron endospores that are resistant to temperature and drying provide the bacterium with a dormant state in order to survive within the soil. Infection is thought to occur by inhalation, abrasion or ingestion. Most commonly, anthrax is diagnosed as a cutaneous infection in those with occupational contact with animals or animal products. Cutaneous anthrax is readily curable using antibiotics and rarely progresses to systemic infection. In contrast, a systemic infection (usually resulting from inhalation of anthrax spores) has a mortality rate approaching 100%. Following inhalation, spores are phagocytosed by alveolar macrophages, triggering germination to the vegetative form. Viable cells are released into the lymphatic system and later into the bloodstream, causing massive bacteraemia of between 107 to 108 cfu/ml. The vegetative form of B. anthracis produces a number of virulence factors including specific toxins. The production of an acute cytokine response contributes to hypotension, septic shock and death. After testing a number of transposon delivery systems used in a variety of Bacillus and Lactobacillus species, we identified a mini-Tn10 transposon system that we have used successfully to generate transposon insertion mutations in the B. anthracis chromosome. Using this transposon mutagenesis system, we have generated a library of mutagenized B. anthracis. In order to identify B. anthracis transposon mutants affected for their ability to survive within human macrophages, we developed in vitro screens for resistance to killing by human macrophages. To date we have identified five unlinked, chromosomal transposon insertions that result in the loss of resistance to killing by macrophages. The chromosomal location of each transposon insertion has been identified and confirmed by sequence analysis. Each of the open reading frames likely to be affected by each transposon insertion will be individually knocked out and the phenotype of the resulting strains will be assessed using in vitro human macrophage invasion assays. An important aspect of this work will be the generation of unmarked, in-frame deletions of each of the open reading frames of interest. The lack of a functional allele replacement system for B. anthracis has required researchers in the field to rely on Campbell-insertion of entire plasmids in order to generate knock-out mutations. We have successfully tested a conditionally-lethal genetic marker as well as a temperature-sensitive plasmid origin of replication in B. anthracis and we are in the process of combining these elements to construct an allele-replacement plasmid. This plasmid should allow us to generate unmarked, in-frame deletions of defined open reading frames in the B. anthracis. Although our current methods for screening mutants are labor intensive, they continue to yield new loci required for survival of B. anthracis within human macrophages. Until we begin to isolate insertions in previously identified loci, without identifying new loci, it is reasonable to conclude that our mutagenesis and screening system will continue to be productive. Therefore, in the upcoming year, we will continue to utilize our current screens to screen our existing library of B. anthracis transposon mutants for additional insertion mutants affected for their ability to resist killing by macrophages. In order to study the early steps of B. anthracis infection, it will be necessary to utilize an animal model of infection that mimics, as much as possible, the relevant infection in humans. The route of B. anthracis infection that is of most concern is the aerosol route. Therefore, we have undertaken the development of an animal aerosol-challenge model of B. anthracis infection. The injection of A/J mice with the Sterne I (pX01+, pX02-) strain of B. anthracis leads to a systemic infection resulting in death (14). Aerosol exposure of A/J mice with the Sterne I strain has been used successfully to demonstrate vaccine efficacy and therefore should provide a useful model of B. anthracis infection (3). The appropriate dose of B. anthracis spores to be used in these studies will be optimized during initial experiments. In addition, endpoints will be evaluated to determine which are most relevant for the determination of virulence. Those endpoints under consideration include percent survival, time to death, and the number of bacterial colony-forming units isolated from host tissues (lungs, lymph nodes, liver, spleen, and blood) at various timepoints post-infection. Molecular and Genetic Studies of Virulence in Bordetella pertussis. The Regulation of the bvg-Repressed Genes in B. pertussis: Our work is directed towards a better understanding of the interaction between Bordetella pertussis and it's human host. Specifically we are addressing the mechanism of regulation of the bvg-repressed genes and their role in the infectious life cycle. In previous work we identified the bvgR locus of B. pertussis and demonstrated that it is responsible for the repression of expression of the bvg-repressed genes. We (and others)also determined that expression of the bvg-repressed genes is activated by the transcriptional activator RisA. As a first step in the examination of regulation of the bvgR-repressed genes, we constructed, recombinant strains of Escherichia coli that overexpress the B. pertussis BvgR and RisA proteins and have undertaken a biochemical analysis of the interaction between BvgRand RisA and bvg-repressed promoters. We have demonstrated binding of the bvg-repressed promoters by RisA but not by BvgR. We have also undertaken a genetic analysis of the promoter of the bvg-repressed genes vrg6 and vrg18. We constructed a detailed collection of 5' and 3' nested deletions and linker substitutions of the vrg6 and vrg18 promoters and have made transcriptional fusions of these constructs to a lacZ reporter. We transferred these PbvgR-lacZ fusions onto the B. pertussis chromosome in single copy and assayed the activity of the recombinant promoters. This analysis, combined with the results of biochemical studies discussed above allowed us identify the elements of the vrg6 and vrg18 promoters required for transcriptional activation by RisA. Our recent results have led to the development of a new model of regulation of the bvg-Repressed genes by BvgR which we are currently testing. Therapeutic Development: Bordetella pertussis, the etiologic agent of whooping cough, is a highly infectious human pathogen with a strong capacity to infect the human respiratory tract. Pertussis in the United States and Canada is primarily seen in young adults with waning immunity and unprotected infants under 6 months of age. Infants with severe pertussis often require long hospitalizations. Although antibiotics and bronchodilators are often given, they do little to alter the severity or duration of the disease. The development of therapies that can reduce the severity and duration of the paroxysmal coughing is required. Compelling evidence has accumulated that suggests that anti-PT antibodies provide protection against disease. It has been demonstrated that both polyclonal and monoclonal anti-PT antibodies can reverse the severe systemic manifestations of pertussis infection in mice and two recent small, clinical studies indicated that the administration of high-titer immunoglobulin prepared from the plasma of donors immunized with purified pertussis toxin was well tolerated by infants and resulted in a decrease in the severity and duration of pyroxysmal coughing {518,519}. Because of the need for an effective therapy for severe pertussis and because of the recent encouraging clinical results with high titer anti-pertussis immunoglobulin in the treatment of pertussis in infants, we sought to develop human monoclonal antibodies and single-chain antibodies against pertussis toxin for use in pertussis therapy. The use of monoclonal antibodies ensures a homogenous preparation of high and well-characterized activity. Work conducted during the last year in our collaborator's laboratory at the University of Minnisota has resulted in the successful humanization of the murine monoclonal antibodies 1B7 and 11E6. These particularly potent anti-pertussis toxin antibodies have previously been shown to be protective in the mouse aerosol challenge model. Ongoing work in our collaborator's laboratory is directed towards the construction of cell lines capable of producing the humanized 1B7 and 11E6 antibodies at commercially viable levels. We will undertake a series of mouse aerosol challenge experiments designed to determine if the humanized monoclonal antibodies Mab 1B7 and murine Mab 11E6 can provide a therapeutic benefit to mice exposed to virulent B. pertussis in the mouse aerosol challenge model. This study will provide us with important insights into the mechanism by which anti-pertussis antibodies confer protection against B. pertussis infection and will provide the data required to support an Investigational New Drug application when these therapeutic agents move forward to clinical studies. This project incorporates FY2002 projects 1Z01BJ003011-02 and 1Z01BJ003013-02.
