Animal Models to Study Plague Infection and Immunity

National Institute Of Allergy And Infectious Diseases

Investigators

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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 RTB. We are expanding upon our earlier studies, which focused mainly on neutrophil responses to Y. pestis, to characterize monocyte, macrophage, and dendritic cell responses and interactions with Y. pestis in the lymph node as well. Both virulent and highly attenuated strains of Y. pestis can disseminate from the skin to the draining lymph node. The virulent strains replicate and can often eventually escape and spread systemically, whereas attenuated strains are contained and eventually killed. The containment mechanisms are not completely understood. We will determine how the cellular responses develop and change over the course of infection. During FY2021 we completed and published a study evaluating Y. pestis-phagocyte interactions in the presence of opsonizing anti-Y. pestis antibodies both in vitro and in vivo. We found minimal effect on macrophage or neutrophil interactions in vitro, but a dramatic effect of antibody opsonization on Y. pestis-neutrophil interactions in both the skin and draining lymph node of mice. We continued to examine the role of lymph node subcapsular sinus macrophages in the pathogenesis of bubonic plague. Jason Cyster (UCSF) provided CD169-DTR transgenic mice that allow for depletion of subcapsular sinus macrophages using diphtheria toxin treatment. Work in the coming year will examine how the presence or absence of subcapsular sinus macrophages affect the fate of Y. pestis within and immune cell recruitment to infected lymph nodes. We continue a collaboration with Kim Hasenkrug (LPVD), looking at the effects of Y. pestis infection on neutrophil and macrophage CD47 and Sirp-alpha expression. CD47 is expressed on most cell types and serves as a dont eat me signal by binding the receptor Sirp-alpha on phagocytic cells. Engagement of Sirp-alpha by CD47 inhibits phagocytosis and has a suppressive effect on several inflammatory processes involved in innate and adaptive immune responses. Our preliminary results show a marked increase in Sirp- alpha surface expression on neutrophils after infection with attenuated strains of Y. pestis in vitro, but fully virulent Y. pestis inhibits/dampens this increase. We are currently working to evaluate potential effects of this regulation on neutrophil physiology and bubonic plague pathogenesis. We have also begun a collaboration with Ferenc Scheeren (U. of Leiden) examining the potential role of a glutaminyl cyclase enzyme encoded by Y. pestis in bacterium-phagocyte interactions. The mammalian version of this enzyme is known to be important for Sirp-alpha-CD47 interactions, but what role, if any, the Y. pestis encoded enzyme is playing in pathogenesis is unknown. During FY2021 we also published a study that used an in vivo imaging system (IVIS) in conjunction with bioluminescent Y. pestis strains to monitor the incidence and dissemination patterns of infection in mice challenged by flea bite. Fleas were used within the first week after infection to evaluate what is known as early-phase transmission, and again 1 to 2 weeks after infection to evaluate a second transmission mechanism that is dependent on Y. pestis biofilm formation in the flea foregut. Results showed that flea-borne bubonic plague can follow an acute course, with dissemination from the flea bite site to the draining lymph node and systemic spread within a few days; or a prolonged course in which the bacteria multiply extensively in the intradermal flea bite site over several days, before either resolving or finally disseminating to cause systemic disease. This clinical picture differs from that seen following intradermal inoculation by needle, which invariably leads to acute disease. The chronic skin infection seen following flea bite challenge provides a new model to study immune responses in the dermis. During the last year we 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.