A Natural Mouse Model for Neisseria Persistent Colonization.

We have developed a natural mouse model to study persistent colonization by commensal Neisseria. The system couples the ordinary lab mouse with Neisseria musculi (Nmus), a commensal in the oral cavity and gut of the wild mouse, Mus musculus. The pairing of Nmus with its natural reservoir circumvents host restriction barriers that have impeded previous studies of Neisseria in vivo behavior. The model allows, for the first time, for the dissection of host and neisserial determinants of asymptomatic colonization. Inoculation procedures are noninvasive and susceptibility to Nmus colonization varies with host genetic background. In colonized mice, bacterial burdens are detectable up to 1-year post inoculation, making it an ideal model for the study of persistence. As Nmus encodes several Neisseria gonorrhoeae (and Neisseria meningitidis) host interaction factors, the system can be used to query the in vivo functions of these commonly held genes and factors. Nmus also encodes many pathogenic Neisseria vaccine targets including a polysaccharide capsule, making the model potentially useful for vaccine development. The ease of genetic manipulation of Nmus enhances the feasibility of such studies.

RpoN and the Nps and Npa two-component regulatory system control pilE transcription in commensal Neisseria.

Over 20 genes are involved in the biogenesis and function of the Neisseria Type IV pilus (Tfp). In the pathogenic species, RpoD and the integration host factor (IHF) protein regulate expression of pilE, encoding the Tfp structural subunit. We previously reported that in commensal species, pilE transcription is regulated by RpoN, IHF, and activator Npa. Npa has many hallmarks of response regulators in two-component regulatory systems, leading us to search for its response regulator partner. We report that Npa partners with sensor kinase Nps to control pilE transcription. Among the genes involved in Tfp biogenesis and function, only pilE is controlled by RpoN and Npa/Nps. We summarize our findings in a model, and discuss the implications of the differential regulation of pilE the context of Neisseria Tfp biogenesis.

A Natural Mouse Model for Neisseria Colonization.

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.

The Commensal Neisseria musculi Modulates Host Innate Immunity To Promote Oral Colonization.

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.

Isolation and characterization of Neisseria musculi sp. nov., from the wild house mouse.

Members of the genus Neisseria have been isolated from or detected in a wide range of animals, from non-human primates and felids to a rodent, the guinea pig. By means of selective culture, biochemical testing, Gram staining and PCR screening for the Neisseria-specific internal transcribed spacer region of the rRNA operon, we isolated four strains of the genus Neisseria from the oral cavity of the wild house mouse, Mus musculus subsp. domesticus. The isolates are highly related and form a separate clade in the genus, as judged by tree analyses using either multi-locus sequence typing of ribosomal genes or core genes. One isolate, provisionally named Neisseria musculi sp. nov. (type strain AP2031T=DSM 101846T=CCUG 68283T=LMG 29261T), was studied further. Strain AP2031T/N. musculi grew well in vitro. It was naturally competent, taking up DNA in a DNA uptake sequence and pilT-dependent manner, and was amenable to genetic manipulation. These and other genomic attributes of N. musculi sp. nov. make it an ideal candidate for use in developing a mouse model for studying Neisseria-host interactions.

Dysregulated endocardial TGFß signaling and mesenchymal transformation result in heart outflow tract septation failure.

Heart outflow tract septation in mouse embryos carrying mutations in retinoic acid receptor genes fails with complete penetrance. In this mutant background, ectopic TGFß signaling in the distal outflow tract is responsible for septation failure, but it was uncertain what tissue was responsive to ectopic TGFß and why this response interfered with septation. By combining RAR gene mutation with tissue-specific Cre drivers and a conditional type II TGFß receptor (Tgfbr2) allele, we determined that ectopic activation of TGFß signaling in the endocardium is responsible for septation defects. Ectopic TGFß 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 TGFß signaling in outflow tract cushion organization and septation.