Adaptive immune cells and CCR2 are dispensable, while CXCR2 is necessary for Brucella-induced focal neutrophil recruitment and inflammation.

Molecular studies of bacterial virulence are enhanced by expression of recombinant DNA during infection to allow complementation of mutants and expression of reporter proteins in vivo. For highly pathogenic bacteria, such as Yersinia pestis, these studies are currently limited because deliberate introduction of antibiotic resistance is restricted to those few which are not human treatment options. In this work, we report the development of alternatives to antibiotics as tools for host-pathogen research during Yersinia pestis infections focusing on the diaminopimelic acid (DAP) pathway, a requirement for cell wall synthesis in eubacteria. We generated a mutation in the dapA-nlpB(dapX) operon of Yersinia pestis KIM D27 and CO92 which eliminated the expression of both genes. The resulting strains were auxotrophic for diaminopimelic acid and this phenotype was complemented in trans by expressing dapA in single and multi-copy. In vivo, we found that plasmids derived from the p15a replicon were cured without selection, while selection for DAP enhanced stability without detectable loss of any of the three resident virulence plasmids. The dapAX mutation rendered Y. pestis avirulent in mouse models of bubonic and septicemic plague which could be complemented when dapAX was inserted in single or multi-copy, restoring development of disease that was indistinguishable from the wild type parent strain. We further identified a high level, constitutive promoter in Y. pestis that could be used to drive expression of fluorescent reporters in dapAX strains that had minimal impact to virulence in mouse models while enabling sensitive detection of bacteria during infection. Thus, diaminopimelic acid selection for single or multi-copy genetic systems in Yersinia pestis offers an improved alternative to antibiotics for in vivo studies that causes minimal disruption to virulence.

No abstract

Osteoarticular complications are the most common focal manifestation of brucellosis in humans. Little is known about the pathology of osteoarticular brucellosis due to the lack of relevant models. However, we recently reported that ablation of IFN-γ in mice results in articular inflammation during infection with Brucella melitensis. Here, we show that adaptive immune cells are dispensable for Brucella-induced articular inflammation. Wild-type, µMT-/-, and Rag1-/- mice all developed articular inflammation at a similar rate and severity when infected with B. melitensis and depleted of IFN-γ. In addition, minimal levels of cytokines associated with T cell responses were detected in arthritic joints. However, we found increased numbers of neutrophils and macrophages in joints from mice with Brucella-induced arthritis. In addition, the concentration of CCR2 and CXCR2 ligands, was strikingly enhanced in Brucella-infected joints. Interestingly, CXCR2-/-, but not CCR2-/- mice, displayed markedly lower joint inflammation relative to wild-type mice when infected with B. melitensis and depleted of IFN-γ. Collectively, these results show that chemokines, such as CXCR2 ligands, are important mediators of Brucella-induced articular inflammation while adaptive immune cells are not required. Therefore, the targeting of CXCR2 and/or other chemokine receptors may have potential as a complementary antibiotic therapy for the resolution of Brucella-induced arthritis.