Human microbiota modulation via QseC sensor kinase mediated in the Escherichia coli O104:H4 outbreak strain infection in microbiome model

Abstract

Background: The intestinal microbiota plays a crucial role in human health, adjusting its composition and the microbial metabolites protects the gut against invading microorganisms. Enteroaggregative E. coli (EAEC) is an important diarrheagenic pathogen, which may cause acute or persistent diarrhea (≥14 days). The outbreak strain has the potent Shiga toxin, forms a dense biofilm and communicate via QseBC two-component system regulating the expression of many important virulence factors.

Results: Herein, we investigated the QseC histidine sensor kinase role in the microbiota shift during O104:H4 C227-11 infection in the colonic model SHIME® (Simulator of the Human Intestinal Microbial Ecosystem) and in vivo mice model. The microbiota imbalance caused by C227-11 infection affected ỿ-Proteobacteria and Lactobacillus spp. predominance, with direct alteration in intestinal metabolites driven by microbiota change, such as Short-chain fatty acids (SCFA). However, in the absence of QseC sensor kinase, the microbiota recovery was delayed on day 3 p.i., with change in the intestinal production of SCFA, like an increase in acetate production. The higher predominance of Lactobacillus spp. in the microbiota and significant augmented qseC gene expression levels were also observed during C227-11 mice infection upon intestinal depletion. Novel insights during pathogenic bacteria infection with the intestinal microbiota were observed. The QseC kinase sensor seems to have a role in the microbiota shift during the infectious process by Shiga toxin-producing EAEC C227-11.

Conclusions: The QseC role in C227-11 infection helps to unravel the intestine microbiota modulation and its metabolites during SHIME® and in vivo models, besides they contribute to elucidate bacterial intestinal pathogenesis and the microbiota relationships.

Keywords: EAEC; Microbiota; QseC; SHIME®.

Fig. 1

SHIME® infection model overview employed with all controlled containers representing the gastrointestinal tract, with stomach and its feed, small intestine and its pancreatic juice and the ascending colon triplicates employed here. The arrows indicate the flow direction of the pumps, dashed lines for gas and solid lines for liquids (a). Experimental protocol with all period steps developed in 5 weeks during C227–11 infection (b). C227–11 and C227–11::qseC strains presence in output was verified by PCR amplification

Fig. 2

Microbiota predominance modulated via QseC during C227–11 infection in the SHIME® model. Relative microbiota abundance analysis via qRT-PCR of 16 s rRNA of phyla and genera. Microbiota composition from days 0 to 3 p.i with strain C227–11 infection, respectively, phyla and genera (a and b), and with strain C227–11::qseC infection, respectively, phyla and genera (c and d). ELISA Immunoassay capture to measure the Stx levels from the output collected during the SHIME® infection, day 1, ** p = 0.002 and 3 p.i., ** p = 0.009 (e). The statistical significance analyzes were performed on GraphPad Prism 7 via t-test

Fig. 3

Direct acetate, propionate and butyrate production analysis (mmol/L) from day 0 to day 3.p.i. via gas chromatography. SCFA composition from C227–11 infection period (a) (*** p = 0.0003) and C227–11::qseC (b). Analyzes were performed individually for each SCFA compared to day 0. The statistical significance analyzes were performed on GraphPad Prism 7 via one-way ANOVA and Tukey post hoc test (*p = 0.0371, *p = 0.0309, *** p = 0.0001)

Fig. 4

Percentile balance of acetate, propionate and butyrate occurrence from total SCFA production analysis via gas chromatography, daily kinetics during infection, from 0 to day 3 p.i. Differential occurrence in the C227–11(a) and C227–11::qseC strains (b) infection. Gradient from 0% (blue) to 100% (red) concentration of each SCFA

Fig. 5

Microbiota predominance during C57BL/6 mice infection, C227–11and C227–11::qseC strains (a). Expression levels of qseC during early and later infection (day 1-3p.i.) of C227–11, 042 and DH5α strains, p-values are respectively p =0.006 (**), p = 0.001 (**) and p =0.004 (**) (b). Relative expression levels were measured in vitro of stx2a gene from the C227–11, C227–11::qseC, and C227–11qseC⁺ (pBAD33 qseC), p =0.01 (**), p =0.001 (***) (c)