Virulence and Stress Responses of Shigella flexneri Regulated by PhoP/PhoQ

Abstract

The two-component signal transduction system PhoP/PhoQ is an important regulator for stress responses and virulence in most Gram-negative bacteria, but characterization of PhoP/PhoQ in <i>Shigella</i> has not been thoroughly investigated. In the present study, we found that deletion of <i>phoPQ</i> (Δ<i>phoPQ</i>) from <i>Shigella flexneri</i> 2a 301 (<i>Sf</i>301) resulted in a significant decline (reduced by more than 15-fold) in invasion of HeLa cells and Caco-2 cells, and less inflammation (- or +) compared to <i>Sf</i>301 (+++) in the guinea pig Sereny test. In low Mg²⁺ (10 μM) medium or pH 5 medium, the Δ<i>phoPQ</i> strain exhibited a growth deficiency compared to <i>Sf</i>301. The Δ<i>phoPQ</i> strain was more sensitive than <i>Sf</i>301 to polymyxin B, an important antimicrobial agent for treating multi-resistant Gram-negative infections. By comparing the transcriptional profiles of Δ<i>phoPQ</i> and <i>Sf</i>301 using DNA microarrays, 117 differentially expressed genes (DEGs) were identified, which were involved in Mg²⁺ transport, lipopolysaccharide modification, acid resistance, bacterial virulence, respiratory, and energy metabolism. Based on the reported PhoP box motif [(T/G) GTTTA-5nt-(T/G) GTTTA], we screened 38 suspected PhoP target operons in <i>S. flexneri</i>, and 11 of them (<i>phoPQ, mgtA, slyB, yoaE, yrbL, icsA, yhiWX, rstA, hdeAB, pagP</i>, and <i>shf</i>-<i>rfbU</i>-<i>virK</i>-<i>msbB2</i>) were demonstrated to be PhoP-regulated genes based on electrophoretic mobility shift assays and β-galactosidase assays. One of these PhoP-regulated genes, <i>icsA</i>, is a well-known virulence factor in <i>S. flexneri</i>. In conclusion, our data suggest that the PhoP/PhoQ system modulates <i>S. flexneri</i> virulence (in an <i>icsA</i>-dependent manner) and stress responses of Mg²⁺, pH and antibacterial peptides.