Although vertebrates harbor bacterial communities in their gastrointestinal tract whose composition is host-specific, little is known about the mechanisms by which bacterial lineages become selected. The goal of this study was to characterize the ecological processes that mediate host-specificity of the vertebrate gut symbiont Lactobacillus reuteri, and to systematically identify the bacterial factors that are involved. Experiments with monoassociated mice revealed that the ability of L. reuteri to form epithelial biofilms in the mouse forestomach is strictly dependent on the strain's host origin. To unravel the molecular basis for this host-specific biofilm formation, we applied a combination of transcriptome analysis and comparative genomics and identified eleven genes of L. reuteri 100-23 that were predicted to play a role. We then determined expression and importance of these genes during in vivo biofilm formation in monoassociated mice. This analysis revealed that six of the genes were upregulated in vivo, and that genes encoding for proteins involved in epithelial adherence, specialized protein transport, cell aggregation, environmental sensing, and cell lysis contributed to biofilm formation. Inactivation of a serine-rich surface adhesin with a devoted transport system (the SecA2-SecY2 pathway) completely abrogated biofilm formation, indicating that initial adhesion represented the most significant step in biofilm formation, likely conferring host specificity. In summary, this study established that the epithelial selection of bacterial symbionts in the vertebrate gut can be both specific and highly efficient, resulting in biofilms that are exclusively formed by the coevolved strains, and it allowed insight into the bacterial effectors of this process.

Escherichia coli is a facultative anaerobic symbiont found widely among mammalian gastrointestinal tracts. Several human studies have reported increased commensal E. coli abundance in the intestine during inflammation; however, host immunological responses toward commensal E. coli during inflammation are not well-defined. Here, we show that colonization of gnotobiotic mice with different genotypes of commensal E. coli isolated from healthy conventional microbiota mice and representing distinct populations of E. coli elicited strain-specific disease phenotypes and immunopathological changes following treatment with the inflammatory stimulus, dextran sulfate sodium (DSS). Production of the inflammatory cytokines GM-CSF, IL-6, and IFN-γ was a hallmark of the severe inflammation induced by E. coli strains of Sequence Type 129 (ST129) and ST375 following DSS administration. In contrast, colonization with E. coli strains ST150 and ST468 caused mild intestinal inflammation and triggered only low levels of pro-inflammatory cytokines, a response indistinguishable from that of E. coli-free control mice treated with DSS. The disease development observed with ST129 and ST375 colonization was not directly associated with their abundance in the GI tract as their levels did not change throughout DSS treatment, and no major differences in bacterial burden in the gut were observed among the strains tested. Data mining and in vivo neutralization identified IL-6 as a key cytokine responsible for the observed differential disease severity. Collectively, our results show that the capacity to exacerbate acute intestinal inflammation is a strain-specific trait that can potentially be overcome by blocking the pro-inflammatory immune responses that mediate intestinal tissue damage.

The eco-evolutionary interactions among members of the vertebrate gut microbiota that ultimately result in host-specific communities are poorly understood. Here we show that Lactobacillus reuteri coexists with  species that belong to the Lactobacillus johnsonii cluster (L. johnsonii, L. gasseri, and L taiwanensis) in a taxonomically wide range of rodents, suggesting cohabitation over evolutionary times. The two dominant Lactobacillus species found in wild mice establish a commensalistic relationship in gastric biofilms when introduced together into germ-free mice in which L. reuteri facilitates colonization of L. taiwanensis. Genomic analysis revealed allopatric diversification in strains of both species that originated from geographically separated locations (Scotland and France). Allopatry of the strains resulted in reduced formation of mixed biofilms in vitro, indicating that interspecies interactions in gastric Lactobacillus-biofilms are the result of an adaptive evolutionary process that occurred in a biogeographical context. In summary, these findings suggest that members within the vertebrate gut microbiota can evolve inter-dependencies through ecological facilitation, which could represent one mechanism by which host-specific bacterial communities assemble across vertebrate species and an explanation for their spatial and biogeographic patterns.

During Theiler's murine encephalomyelitis virus (TMEV) infection of macrophages, it is thought that high interleukin-6 (IL-6) levels contribute to the demyelinating disease found in chronically infected SJL/J mice but absent in B10.S mice capable of clearing the infection. Therefore, IL-6 expression was measured in TMEV-susceptible SJL/J and TMEV-resistant B10.S macrophages during their infection with TMEV DA strain or responses to lipopolysaccharide (LPS) or poly(I · C). Unexpectedly, IL-6 production was greater in B10.S macrophages than SJL/J macrophages during the first 24 h after stimulation with TMEV, LPS, or poly(I · C). Further experiments showed that in B10.S, SJL/J, and RAW264.7 macrophage cells, IL-6 expression was dependent on extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) and enhanced by exogenous IL-12. In SJL/J and RAW264.7 macrophages, exogenous IL-6 resulted in decreased TMEV replication, earlier activation of STAT1 and STAT3, production of nitric oxide, and earlier upregulation of several antiviral genes downstream of STAT1. However, neither inhibition of IL-6-induced nitric oxide nor knockdown of STAT1 diminished the early antiviral effect of exogenous IL-6. In addition, neutralization of endogenous IL-6 from SJL/J macrophages with Fab antibodies did not exacerbate early TMEV infection. Therefore, endogenous IL-6 expression after TMEV infection is dependent on ERK MAPK, enhanced by IL-12, but too slow to decrease viral replication during early infection. In contrast, exogenous IL-6 enhances macrophage control of TMEV infection through preemptive antiviral nitric oxide production and antiviral STAT1 activation. These results indicate that immediate-early production of IL-6 could protect macrophages from TMEV infection.

IRF3 is an innate anti-viral factor whose role in limiting Theiler’s Murine Encephalomyelitis Virus (TMEV) infection and preventing TMEV-induced disease is unclear. Acute disease and innate immune responses of macrophages were examined in IRF3 knockout mice compared with C57Bl/6 mice following in vitro or intracranial infection with either TMEV GDVII or DA. IRF3 deficiency augmented viral infection, as well as morbidity and mortality following intracranial infection with neurovirulent TMEV GDVII. In contrast, IRF3 deficiency prevented hippocampal injury following intracranial infection with persistent TMEV DA. The extent of TMEV infection in macrophages from C57Bl/6 mice was significantly less than that in IRF3 deficient macrophages, which was associated with poor IFN-γ and IL-6 expression in response to TMEV. Reestablishing IRF3 expression in IRF3 deficient macrophages increased control of TMEV replication and increased expression of IFN-γ and IL-6. In addition, IRF3 deficient macrophages failed to exhibit IL-6 antiviral effects, which was associated with inability to sustain IL-6-induced STAT1 activation compared with C57BL/6 macrophages. Altogether, IRF3 contributes to early control of TMEV replication through induction of IL-6 and IFN-γ and support of IL-6 antiviral effects, but contributes to TMEV-induced hippocampal injury.