Search

Primary pneumonic plague occurs when Yersinia pestis is inhaled into the lower respiratory tract where it invades the alveoli and grows. Rapid bacterial growth eventually elicits a neutrophilic inflammatory response that is ineffective and damaging, leading to accelerated progression of disease. In the laboratory, modeling of primary pneumonic plague can be accomplished by instillation of bacterial culture in the nares of anesthetized mice and rats. Although primary pneumonic plague can develop from this method, variability in dosing and side effects of anesthesia can complicate data interpretation. In contrast, aerosol challenge models allow for well-controlled studies of pneumonic plague with minimal experimental bias and unwanted side effects. For these reasons, antibiotic testing and the licensing of new treatments depend on efficacy data generated from aerosol delivery of Y. pestis in order to more accurately model transmission and the early stages of human pneumonic plague. In order to meet this need, we have extensively characterized pneumonic plague in mice and rats challenged by nose-only exposure to Yersinia pestis. With this approach, simultaneous challenge of large cohorts of animals, gently restrained and not anesthetized, assures safe, well-controlled, unbiased, and uniform infection. In this chapter, we present a standardized method for reproducible aerosol delivery of wild-type Y. pestis to rodents for experimental models of primary pneumonic plague.

Unresolved experimental Lyme arthritis in C3H 5-lipoxygenase (5-LOX) -/- mice is associated with impaired macrophage phagocytosis of Borrelia burgdorferi In the present study, we further investigated the effects of the 5-LOX metabolite, leukotriene (LT)B 4 on phagocytosis of B. burgdorferi Bone marrow-derived macrophages (BMDMs) from 5-LOX -/- mice were defective in the uptake and killing of B. burgdorferi from the earliest stages of spirochete internalization. BMDMs from mice deficient for the LTB 4 high-affinity receptor (BLT1 -/- ) were also unable to efficiently phagocytose B. burgdorferi Addition of exogenous LTB 4 augmented the phagocytic capability of BMDMs from both 5-LOX -/- and BLT1 -/- mice, suggesting that the low-affinity LTB 4 receptor, BLT2, might be involved. Blocking BLT2 activity with the specific antagonist, LY255283, inhibited phagocytosis in LTB 4 -stimulated BLT1 -/- BMDMs, demonstrating a role for BLT2. However, the lack of a phagocytic defect in BLT2 -/- BMDMs suggested that this was a compensatory effect. In contrast, 12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid, a natural BLT2-specific high-affinity ligand, and resolvin E1, a BLT1 agonist, were both unable to boost phagocytosis in BMDMs from either 5-LOX -/- or BLT1 -/- mice, suggesting a specific role for LTB 4 in mediating phagocytosis in murine macrophages. This study demonstrates that LTB 4 promotes macrophage phagocytosis of bacteria via BLT1, and that BLT2 can fulfill this role in the absence of BLT1., (Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.)

Many Gram-negative bacterial pathogens use type III secretion systems to export proteins that act directly on the host and aid in the infectious process. Extracellular bacteria primarily rely upon the type III secretion system to insert or inject effector proteins into the cytosol of their host cell in order to perturb intracellular signaling events and aid in pathogenesis. Intracellular bacteria can also depend on the T3SS translocation of effector proteins from vacuolar compartments into the vacuolar membrane or host cell cytosol where they can modulate intracellular trafficking and/or signaling pathways necessary for their growth and survival. Biochemical fractionation of infected cells in vitro enables detection of these events, making it possible to identify relevant protein-protein interactions, characterize phenotypes of mutant strains and understand how these effector proteins impact host cells. In this chapter we provide methods for the analysis of translocated effector proteins using biochemical and mechanical fractionation procedures.