No abstract

Commensals are important for the proper functioning of multicellular organisms. How a commensal establishes persistent colonization of its host is little understood. Studies of this aspect of microbe-host interactions are impeded by the absence of an animal model. We have developed a natural small animal model for identifying host and commensal determinants of colonization and of the elusive process of persistence. Our system couples a commensal bacterium of wild mice, Neisseria musculi, with the laboratory mouse. The pairing of a mouse commensal with its natural host circumvents issues of host restriction. Studies are performed in the absence of antibiotics, hormones, invasive procedures, or genetic manipulation of the host. A single dose of N. musculi, administered orally, leads to long-term colonization of the oral cavity and gut. All mice are healthy. Susceptibility to colonization is determined by host genetics and innate immunity. For N. musculi, colonization requires the type IV pilus. Reagents and powerful tools are readily available for manipulating the laboratory mouse, allowing easy dissection of host determinants controlling colonization resistance. N. musculi is genetically related to human-dwelling commensal and pathogenic Neisseria and encodes host interaction factors and vaccine antigens of pathogenic Neisseria. Our system provides a natural approach for studying Neisseria-host interactions and is potentially useful for vaccine efficacy studies.

Neisseria musculi, isolated from the oral cavity of wild-caught mice, does not colonize most inbred mouse strains. N. musculi does weakly (50%) colonize C57BL/6J (B6) mice but readily colonizes CAST/EiJ (CAST) mice. In this study, we examined whether differences in the CAST and B6 host response could elucidate mechanisms governing N. musculi colonization. In vivo stimulation of B6 or CAST splenocytes with wild type (WT) Neisseria or Escherichia coli LPS showed that CAST mice had a blunted inflammatory response, producing significantly lower levels of IL-6 than B6 mice. The use of specific genetic knockouts highlighted a need for an intact innate immune system to prevent colonization. B6–RAG-1−/− mice were colonized at a similar rate as WT B6 mice, whereas B6-MyD88−/− and TLR4−/− mice were readily colonized like CAST (100%) mice. Sequence analysis revealed a unique point mutation in TLR4 in CAST mice. However, crosses to TLR4−/− mice and analysis of recombinant inbred Collaborative Cross mice showed that TLR4 from CAST mice was not sufficient to allow Neisseria colonization. In vitro stimulation of B6 bone marrow–derived macrophages or splenocytes with WT Neisseria yielded low levels of IL-6 compared with LPS stimulation. Surprisingly, UV-inactivated Neisseria induced high levels of IL-6, suggesting suppression of IL-6 production is an active bacterial process. Consistent with a critical role for IL-6 in preventing colonization, mice deficient for the IL-6 receptor were efficiently colonized, indicating host IL-6 production plays a critical role in determining host colonization susceptibility.

Heart outflow tract septation in mouse embryos carrying mutations in retinoic acid receptor genes fails with complete penetrance. In this mutant background, ectopic TGFβ signaling in the distal outflow tract is responsible for septation failure, but it was uncertain what tissue was responsive to ectopic TGFβ and why this response interfered with septation. By combining RAR gene mutation with tissue-specific Cre drivers and a conditional type II TGFβ receptor (Tgfbr2) allele, we determined that ectopic activation of TGFβ signaling in the endocardium is responsible for septation defects. Ectopic TGFβ signaling results in ectopic mesenchymal transformation of the endocardium and thereby in improperly constituted distal OFT cushions. Our analysis highlights the interactions between myocardium, endocardium, and neural crest cells in outflow tract morphogenesis, and demonstrates the requirement for proper TGFβ signaling in outflow tract cushion organization and septation.