Animal Models to Study Plague Infection and Immunity

National Institute Of Allergy And Infectious Diseases

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Abstract

The molecular pathogenesis of fully virulent, wild-type Y. pestis in relevant animal models has been relatively neglected because of the scarcity of secure BSL-3 facilities and trained personnel certified to work with this Class A select agent. The threat of bioterrorism and the emergence of multiply-antibiotic resistant strains of Y. pestis increases the urgency for a more detailed understanding of the host-pathogen relationship at the molecular level that may lead to the design of improved medical countermeasures and diagnostics. Y. pestis is one of the most invasive and virulent of bacterial pathogens. Bubonic plague pathogenesis is noted by an initial stealth phase, during which the bacteria multiply and spread to secondary lymphoid tissue without stimulating a strong innate immune response, followed soon thereafter by an aggression phase characterized by rapid systemic spread, hyperinflammation, and fatal septic shock. We have established rodent models of bubonic plague that incorporate flea-borne transmission to investigate the role of specific Y. pestis virulence factors and to characterize the host response to naturally acquired infection. We have characterized the kinetics, microbiology, and histopathology of bubonic plague following intradermal injection of Y. pestis; and used this model to characterize the gene expression profile of Y. pestis in the infected lymph node during bubonic plague. Our previous work has shown that three important Y. pestis virulence factors, Ail (an outer surface protein), the Type III secretion system encoded on the Yersinia virulence plasmid, and the plasminogen activator (Pla) encoded on a Y. pestis-specific plasmid all act to limit the polymorphonuclear leukocyte (PMN) response to bubonic plague infection in vivo. Thus, several lines of evidence indicate that the PMN response correlates with successful outcome to infection, and this aspect of host-pathogen interaction has become a focus of our lab. To facilitate studies of Y. pestis-neutrophil interactions in vitro we have established a system to generate immortalized murine neutrophil progenitor cells based on retroviral transduction of a Hoxb8-estrogen receptor construct into bone marrow cells (ref: Nat Methods. 2006 Apr;3(4):287-93). We have used this method to generate large numbers of murine neutrophils and macrophages suitable for a variety of in vitro assays. We have also used CRISPR-Cas9 technology to create targeted deletions of genes in the immortalized progenitor cell lines. Experiments with these Hoxb8ER-derived PMNs show they are almost indistinguishable from primary bone marrow neutrophils, in terms of morphology, physiology or cell surface marker expression. We have characterized the response of murine neutrophils to Y. pestis and examined the effects of antibody opsonization on uptake and killing of bacteria. We have also used these Hoxb8ER immortalized cells as a source of murine macrophages and have evaluated several Y. pestis mutants for intracellular growth deficiencies. During the past year, we continued to develop techniques for imaging Y. pestis in tissues, focusing on bacterium-host phagocyte interactions in the skin-draining lymph nodes in collaboration with the Biological Imaging Section of the Research Technologies Branch. We have focused on imaging fixed and sectioned lymph node tissue by immunofluorescence assay to characterize Y. pestis-host cell interactions early after dissemination of the bacteria to the lymph node. We used these methods to obtain high-resolution images of subcapsular sinus macrophages, neutrophils, and blood vessels in lymph nodes from Y. pestis-infected mice. However, the number of bacteria present in the lymph node early after infection is highly variable from mouse to mouse and is often very low. These issues have proven to be difficult to overcome. We continued to characterize murine neutrophils derived from bone marrow progenitors immortalized by transduction with Hoxb8-estrogen receptor construct. We have confirmed expression and colocalization of NADPH oxidase components, and quantified ROS production capacity in comparison to primary bone marrow neutrophils. We have found these cells to be good substitutes for primary murine neutrophils and very useful in examining Y. pestis-neutrophil interactions in vitro. During FY2022 we initiated a collaboration with Matthew Lawrenz (University of Louisville) that takes advantage of our ability to combine intravital microscopy techniques with a mouse model of bubonic plague. Specifically, this collaboration is examining the role of the inflammatory lipid mediator Ltb4 in neutrophil recruitment and swarming behavior in response to Y. pestis infection in the skin. During the last year we also continued a project to evaluate the role of the Y. pestis F1 capsule in promoting mammal-to-flea transmission. To be transmitted, Y. pestis must infect the small blood capillary vessels in the superficial layer of the skin, which are the source of blood for a feeding flea. The objective of this study is to test the hypothesis that one function of the Y. pestis capsule is to prevent the formation of bacterial aggregates that would be inaccessible to a feeding flea because they are too large to readily enter into and flow through the small capillaries. According to this hypothesis, although the capsule is not required for virulence per se, it is important to complete the transmission cycle.