Analysis of Shigella flexneri Resistance, Biofilm Formation, and Transcriptional Profile in Response to Bile Salts

Abstract

The Shigella species cause millions of cases of watery or bloody diarrhea each year, mostly in children in developing countries. While many aspects of Shigella colonic cell invasion are known, crucial gaps in knowledge regarding how the bacteria survive, transit, and regulate gene expression prior to infection remain. In this study, we define mechanisms of resistance to bile salts and build on previous research highlighting induced virulence in Shigella flexneri strain 2457T following exposure to bile salts. Typical growth patterns were observed within the physiological range of bile salts; however, growth was inhibited at higher concentrations. Interestingly, extended periods of exposure to bile salts led to biofilm formation, a conserved phenotype that we observed among members of the Enterobacteriaceae. Characterization of S. flexneri 2457T biofilms determined that both bile salts and glucose were required for formation, dispersion was dependent upon bile salts depletion, and recovered bacteria displayed induced adherence to HT-29 cells. RNAsequencing analysis verified an important bile salt transcriptional profile in S. flexneri 2457T, including induced drug resistance and virulence gene expression. Finally, functional mutagenesis identified the importance of the AcrAB efflux pump and lipopolysaccharide O-antigen synthesis for bile salt resistance. Our data demonstrate that S. flexneri 2457T employs multiple mechanisms to survive exposure to bile salts, which may have important implications for multidrug resistance. Furthermore, our work confirms that bile salts are important physiological signals to activate S. flexneri 2457T virulence. This work provides insights into how exposure to bile likely regulates Shigella survival and virulence during host transit and subsequent colonic infection.

Shigella flexneri adherence factor expression in in vivo-like conditions

“…Furthermore, upon bacterial dispersion from the biofilm, recovered bacteria displayed induced adherence to colonic HT-29 cells. This analysis enabled us to expand the Shigella infection paradigm to incorporate biofilm formation during small intestinal passage, dispersion upon colonic transition following the loss of the bile salts signal, and induced infection rates due to the IVLCs encountered in the small intestine (31, 32). Since adherence factors are important components of biofilm formation (32, 33), we performed EM analysis of bacteria isolated from the IVLC-induced biofilm to visualize possible adherence factors.…”

“…Functional analyses of the mutants were next performed to evaluate the role of each factor in biofilm formation and adhesion to epithelial cells. First, given the importance of adherence in the initiation of biofilms (33, 51), we analyzed the mutants in the biofilm assay (31, 32). All mutants exhibited reduced biofilm formation at 3 hours (Figure 6), a timepoint used to examine the role of adherence factors in early biofilm formation (32).…”

“…Characterization of the three structural genes in this study demonstrates that S. flexneri 2457T utilizes “traditional” adherence factors to initiate biofilm formation and to facilitate contact to colonic epithelial cells. Several observations influenced the investigation, including the lack of an adherence null mutant in OspE1 and OspE2 analysis (30), the subsequent biofilm formation and induced adherence observed following IVLC exposure (31, 32), as well as the presence of the various adherence gene clusters in the S. flexneri 2457T genome. The literature on “traditional” Shigella adherence factors is contradictory.…”

“…Furthermore, we have previously demonstrated that tryptic soy broth media supplemented with bile salts induce the adherence of S. flexneri 2457T to colonic epithelial cells, which is facilitated at least in part by the type-III secretion system effector proteins OspE1 and OspE2 (30). Finally, our recent publication characterizes an adhesive biofilm phenotype following prolonged exposure to a combination of bile salts and glucose that represent aspects of the in vivo -like conditions (IVLCs) found in the small intestine (31). Given this literature and the fact that deletion of both ospE1 and ospE2 did not completely abrogate adherence (30), we sought to determine if additional adherence factors were produced by S. flexneri 2457T following IVLC exposure.…”

Complete genome sequence and annotation of the laboratory reference strainShigella flexneriserotype 5a M90T and genome-wide transcriptional start site determination

“…Transcriptomic analysis has been increasingly employed to dissect the molecular mechanisms of host-pathogen interactions for a wide range of bacteria [16][17][18][19]. However, only few studies employing RNA-seq have been carried out in Shigella [13,20,21].…”