QseC Signaling in the Outbreak O104:H4 Escherichia coli Strain Combines Multiple Factors during Infection

Abstract

Enteroaggregative Escherichia coli (EAEC) from the O104:H4 specific serotype caused a large outbreak of bloody diarrhea with some complicated cases of hemolytic-uremic syndrome (HUS) in Europe in 2011. The outbreak strain consisted in an EAEC capable to produce the Shiga toxin (Stx) subtype 2a, a characteristic from enterohemorrhagic E. coli QseBC two-component system detects AI-3/Epi/NE and mediates the chemical signaling between pathogen and mammalian host. This system coordinates a cascade of virulence genes expression in important human enteropathogens. The blocking of QseC of EAEC C227-11 (Stx⁺) strain by N-phenyl-4-{[(phenylamino) thioxomethyl]amino}-benzenesulfonamide (also known as LED209) in vivo demonstrated a lower efficiency of colonization. The periplasmic protein VisP, which is related to survival mechanisms in a colitis model of infection, bacterial membrane maintenance, and stress resistance, here presented high levels of expression during the initial infection within the host. Under acid stress conditions, visP expression levels were differentiated in an Stx-dependent way. Together, these results emphasize the important role of VisP and the histidine kinase sensor QseC in the C227-11 (Stx⁺) outbreak strain for the establishment of the infectious niche process in the C57BL/6 mouse model and of LED209 as a promising antivirulence drug strategy against these enteric pathogens.IMPORTANCE EAEC is a remarkable etiologic agent of acute and persistent diarrhea worldwide. The isolates harbor specific subsets of virulence genes and their pathogenesis needs to be better understood. Chemical signaling via histidine kinase sensor QseC has been shown as a potential target to elucidate the orchestration of the regulatory cascade of virulence factors.

Keywords: EAEC; Escherichia coli; O104:H4; QseC; Shiga toxin; VisP; chemical signaling.

FIG 1

Mouse colonization with 10¹⁰ CFU of O104:H4 strains after treatment with LED209. The CFU counts for WT and ::qseC (C227-11) strains (A) or the BA3826 strain (B) recovered days 1 to 14 after the administration of 58.8 mg/kg of LED209 or vehicle were determined. A Student t test was used to determine the statistical significance between treated and nontreated groups. Error bars indicate the SD of the mean (*, P < 0.01; **, P < 0.001; ***, P < 0.0001; ns, not significant).

FIG 2

LED209 blocking of QseC during in vivo infection decreases the expression levels of qseC gene in O104:H4 Stx⁺ and Stx^– strains. (A and B) qRT-PCR analyses of qseC from feces collected from days 1 to 10 p.i. of both C227-11 and BA3826 strains. (C) qRT-PCR of qseC with RNA extracted from cultures of EHEC 86-42, C227-11 (Stx⁺), BA3826 (Stx^–), 17-2, and 042 strains grown in LB medium until reaching an OD₆₀₀ of 1.0. A Student t test was used to determine the statistical significance between treated and nontreated groups, or ANOVA was used for comparisons to EHEC 86-24. Error bars indicate the SD of the mean (**, P < 0.001; ***, P < 0.0001; ns, not significant).

FIG 3

Stress conditions stimulate overexpression of visP in diarrheagenic E. coli strains. (A and B) qRT-PCR of visP from feces collected from days 1 to 10 p.i. of both C227-11 (Stx⁺) and BA3826 (Stx^–) strains after treatment with LED209. (C and D) qRT-PCR of visP from RNA collected from cultures of WT C227-11 (Stx⁺), ::qseC, qseC⁺, BA3826 (Stx^–), 042 and EHEC 86-24 strains after 1 h of static incubation at 37°C in pH 7.2 (C) and 3.0 (D). A Student t test was employed to determine statistical significance between treated and nontreated groups or ANOVA comparing to WT C227-11 (Stx⁺) strain. Error bars indicate the SD of the mean (*, P < 0.01; **, P < 0.001; ***, P < 0.0001; ns, not significant).

FIG 4

Gut microbiota changes in ampicillin mouse model of infection from days 5 (A) and 15 (B) p.i. with various EAEC strains. Proteobacteria, Bacteroidetes, and Firmicutes (more common intestinal phylogenetic groups) abundance analyses by qRT-PCR of 16S rRNA from feces were performed.

FIG 5

General phenotypic differences between EAEC strains during biofilm formation. (A) WT C227-11 (Stx⁺), ::qseC C227-11 (Stx⁺), qseC⁺ C227-11 (Stx⁺), and BA3826 (Stx^–) strains on 96-well polystyrene plate in the presence or absence of 1% α-methyl d-mannoside incubated for 24 h at 37°C. (B) Motility profile between E. coli strains compared to WT C227-11 (Stx⁺) plated on LB agar 0.3% and incubated at 37°C. Growth halos were measured 24 h after incubation. Statistical analysis was performed using a Student t test between each group or ANOVA. Error bars indicate the SD of the mean (**, P < 0.001; ***, P < 0.0001; ns, not significant).

FIG 6

Role of type I fimbriae in O104:H4 Stx⁺ and Stx^– strains during in vitro and in vivo essays. (A) qRT-PCR of fimH of RNA extracted from 042, C227-11 (Stx⁺), and BA3826 (Stx^–) cultivated strains in LB medium until reaching an OD₆₀₀ of 1.0. (B) fimH expression levels determined via qRT-PCR from RNAs of EAEC and DH5α strains extracted from feces on day 1 p.i. A Student t test or ANOVA was used to determine the statistical significance for each replicate. Error bars indicate the SD of the mean (**, P < 0.001; ***, P < 0.0001).

FIG 7

Proposed model for QseC and VisP role. Blocking of QseC phosphorylation by LED209 or knockout of qseC prevents the recognition of environmental signals and inhibits the transcription cascade of virulence genes in vivo. (A) Interruption of this pathway triggers other survival mechanisms, such as transcription of visP. (B) In vitro, the periplasmic protein VisP plays a role under different pH conditions between O104:H4 Stx⁺ and Stx^– strains, and TIF mediates attachment on the abiotic surface.