Engineering E. coli strains using antibiotic-resistance-gene-free plasmids.

We created a generalizable pipeline for antibiotic-resistance-gene-free plasmid (ARGFP)-based cloning using a dual auxotrophic- and essential-gene-based selection strategy. We use auxotrophic selection to construct plasmids in engineered E. coli DH10B cloning strains and both auxotrophic- and essential-gene-based selection to (1) select for recombinant strains and (2) maintain a plasmid in E. coli Nissle 1917, a common chassis for engineered probiotic applications, and E. coli MG1655, the laboratory "wild-type" E. coli strain. We show that our approach has comparable efficiency to that of antibiotic-resistance-gene-based cloning. We also show that the double-knockout Nissle and MG1655 strains are simple to transform with plasmids of interest. Notably, we show that the engineered Nissle strains are amenable to long-term plasmid maintenance in repeated culturing as well as in the mouse gut, demonstrating the potential for broad applications while minimizing the risk of antibiotic resistance spread via horizontal gene transfer.

Altered IgA Response to Gut Bacteria Is Associated with Childhood Asthma in Peru.

Alterations in gut microbiota in early life have been associated with the development of asthma; however, the role of gut bacteria or the IgA response to gut bacteria in school-aged children with asthma is unclear. To address this question, we profiled the microbial populations in fecal and nasal swab samples by 16S rRNA sequencing from 40 asthma and 40 control children aged 9-17 y from Peru. Clinical history and laboratory evaluation of asthma and allergy were obtained. Fecal samples were analyzed by flow cytometry and sorted into IgA+ and IgA- subsets for 16S rRNA sequencing. We found that the fecal or nasal microbial 16S rRNA diversity and frequency of IgA+ fecal bacteria did not differ between children with or without asthma. However, the α diversity of fecal IgA+ bacteria was decreased in asthma compared with control. Machine learning analysis of fecal bacterial IgA-enrichment data revealed loss of IgA binding to the Blautia, Ruminococcus, and Lachnospiraceae taxa in children with asthma compared with controls. In addition, this loss of IgA binding was associated with worse asthma control (Asthma Control Test) and increased odds of severe as opposed to mild to moderate asthma. Thus, despite little to no change in the microbiota, children with asthma exhibit an altered host IgA response to gut bacteria compared with control participants. Notably, the signature of altered IgA responses is loss of IgA binding, in particular to members of Clostridia spp., which is associated with greater severity of asthma.

A Potential Role for Stress-Induced Microbial Alterations in IgA-Associated Irritable Bowel Syndrome with Diarrhea.

Stress is a known trigger for flares of inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS); however, this process is not well understood. Here, we find that restraint stress in mice leads to signs of diarrhea, fecal dysbiosis, and a barrier defect via the opening of goblet-cell associated passages. Notably, stress increases host immunity to gut bacteria as assessed by immunoglobulin A (IgA)-bound gut bacteria. Stress-induced microbial changes are necessary and sufficient to elicit these effects. Moreover, similar to mice, many diarrhea-predominant IBS (IBS-D) patients from two cohorts display increased antibacterial immunity as assessed by IgA-bound fecal bacteria. This antibacterial IgA response in IBS-D correlates with somatic symptom severity and was distinct from healthy controls or IBD patients. These findings suggest that stress may play an important role in patients with IgA-associated IBS-D by disrupting the intestinal microbial community that alters gastrointestinal function and host immunity to commensal bacteria.

Dynamic immunoglobulin responses to gut bacteria during inflammatory bowel disease.

Aberrant immune responses against gut microbiota are thought to be key drivers of inflammatory bowel disease (IBD) pathogenesis. However, the extent and targets of immunoglobulin (Ig) A versus IgG responses to gut bacteria in IBD and its association with IBD severity is not well understood. Here, we address this by analyzing fecal samples from Crohn's disease (CD), ulcerative colitis (UC), and Non-IBD patients by flow cytometry for the frequency of bacteria that were endogenously bound with IgA and/or IgG. Assessment of IBD patients from two geographically distinct cohorts revealed increased percentages of IgA- and IgG-bound fecal bacteria compared to non-IBD controls. Notably, the two major subsets of IBD showed distinct patterns of Ig-bound bacteria, with CD activity associated with increases in both IgA and IgG-bound bacteria, whereas UC activity correlated only with increases in IgG-bound bacteria. Analysis of the flow sorted Ig-bound bacterial repertoire by 16S rDNA sequencing revealed taxa that were Ig-bound specifically in IBD. Notably, this included bacteria that are also thought to reside in the oral pharynx, including Gemella, Peptostreptococcus, and Streptococcus species. These data show that the pattern of IgA and IgG binding to fecal bacteria is distinct in UC and CD. In addition, the frequency of Ig-bound fecal bacteria may have potential as a non-invasive biomarker for disease activity. Finally, our results support the hypothesis that immune responses to oral pharyngeal bacteria may play an important role in the pathogenesis of IBD.

Helicobacter species are potent drivers of colonic T cell responses in homeostasis and inflammation.

Specific gut commensal bacteria improve host health by eliciting mutualistic regulatory T (Treg) cell responses. However, the bacteria that induce effector T (Teff) cells during inflammation are unclear. We addressed this by analyzing bacterial-reactive T cell receptor (TCR) transgenic cells and TCR repertoires in a murine colitis model. Unexpectedly, we found that mucosal-associated Helicobacter species triggered both Treg cell responses during homeostasis and Teff cell responses during colitis, as suggested by an increased overlap between the Teff/Treg TCR repertoires with colitis. Four of six Treg TCRs tested recognized mucosal-associated Helicobacter species in vitro and in vivo. By contrast, the marked expansion of luminal Bacteroides species seen during colitis did not trigger a commensurate Teff cell response. Unlike other Treg cell-inducing bacteria, Helicobacter species are known pathobionts and cause disease in immunodeficient mice. Thus, our study suggests a model in which mucosal bacteria elicit context-dependent Treg or Teff cell responses to facilitate intestinal tolerance or inflammation.