Chemical sensing in mammalian host-bacterial commensal associations

Abstract

The mammalian gastrointestinal (GI) tract is colonized by a complex consortium of bacterial species. Bacteria engage in chemical signaling to coordinate population-wide behavior. However, it is unclear if chemical sensing plays a role in establishing mammalian host-bacterial commensal relationships. Enterohemorrhagic Escherichia coli (EHEC) is a deadly human pathogen but is a member of the GI flora in cattle, its main reservoir. EHEC harbors SdiA, a regulator that senses acyl-homoserine lactones (AHLs) produced by other bacteria. Here, we show that SdiA is necessary for EHEC colonization of cattle and that AHLs are prominent within the bovine rumen but absent in other areas of the GI tract. We also assessed the rumen metagenome of heifers, and we show that it is dominated by Clostridia and/or Bacilli but also harbors Bacteroidetes. Of note, some members of the Bacteroidetes phyla have been previously reported to produce AHLs. SdiA-AHL chemical signaling aids EHEC in gauging these GI environments, and promotes adaptation to a commensal lifestyle. We show that chemical sensing in the mammalian GI tract determines the niche specificity for colonization by a commensal bacterium of its natural animal reservoir. Chemical sensing may be a general mechanism used by commensal bacteria to sense and adapt to their mammalian hosts. Additionally, because EHEC is largely prevalent in cattle herds, interference with SdiA-mediated cattle colonization is an exciting alternative to diminish contamination of meat products and cross-contamination of produce crops because of cattle shedding of this human pathogen.

Fig. 1.

SdiA-mediated AHL regulation of the EHEC LEE genes. (A) Schematic depiction of the LEE pathogenicity island. (B) Quantitative RT-PCR (qRT-PCR) of the LEE genes (ler/LEE1 and espA/LEE4) in WT EHEC (86-24), isogenic sdiA mutant, and complemented (Cpl) strain (ΔsdiA with pDH6; sdiA cloned in pACYC177) grown in DMEM to an OD₆₀₀ of 1.0 in the absence and presence of AHL (10 μM oxo-C6-homoserine lactone). (C) EMSAs of LEE1, LEE4, LEE5, gadW, ftsQ (positive control), and kanamycin-resistant gene, kan promoter, (negative control) with SdiA-AHL. (D) Western blots of whole-cell lysates of WT, sdiA mutant, and sdiA mutant complemented with pDH6 probed with antisera against EspA and RpoA (loading control). (E) Western blots of the secreted proteins of WT, sdiA mutant, and sdiA mutant complemented with pDH6 strains in the absence and presence of AHL (10 μM oxo-C6-HSL) probed with an antiserum against EspA. In conditions where no AHLs were added, the same amount of the ethyl-acetate solvent was added to ensure that the solvent had no effect in gene expression.

Fig. 2.

SdiA regulation of the gad acid-resistance system. (A) qRT-PCR of the gadW gene encoding the master regulator of the gad system in WT EHEC (86-24), sdiA mutant, sdiA mutant with pACYC177, and sdiA mutant complemented with pDH6 (sdiA in pACYC177) strains grown in DMEM to an OD₆₀₀ of 1.0 in the absence and presence of AHL (10 μM oxo-C8-HSL and 10 nM oxo-C8-HSL). (B) qRT-PCR of the gadX gene in WT EHEC (86-24), sdiA mutant, sdiA mutant with pACYC177, and sdiA mutant complemented with pDH6 (sdiA in pACYC177) strains grown in DMEM to an OD₆₀₀ of 1.0 in the absence and presence of AHL (10 μM oxo-C8-HSL). (C) Acid resistance (survival in acidic pH) through the gad system in the absence and presence of 10 μM oxo-C8-HSL. In conditions where no AHLs were added, the same amount of the ethyl-acetate solvent was added to ensure that the solvent had no effect in gene expression.

Fig. 3.

Rumen AHLs affect expression of EHEC genes. (A) TLC from AHLs extracted from 50 mL (evaporated to 5 μL that were added to the TLC) of rumen fluid (RU) collected from three different cows (RU1, RU2, and RU3). C6-HSL (402 pmol), C8-HSL (15.8 pmol), oxo-C6-HSL (400 pmol), and oxo-C8-HSL (15 pmol) were used as controls. (B) TLC from AHLs extracted from 50 mL (evaporated to 5 μL that were added to the TLC) of rumen fluid (R) that has been subjected to alkaline treatment (ALK; hydrolyses the homoserine lactone of AHLs) and acidification (AC; ALK+AC restores the homoserine lactone); oxo-C8-HSL (AHL) undergoing the same treatments was used as a control. (C) Preparative TLC from AHLs extracted from 50 mL (evaporated to 5 μL that were added to the TLC) of other portions of the GI of ruminants. The positive control turns blue. (D) qRT-PCR of ler (LEE1) from WT and sdiA mutant in the presence of rumen AHLs (5 μL extract). In conditions where no rumen extracts were added, the same amount of the dichloromethane solvent was added to ensure that the solvent had no effect in gene expression. (E) qRT-PCR of eae (LEE5) gene from WT and sdiA mutant in the presence of rumen AHLs (5 μL extract). (F) qRT-PCR of gadX from WT and sdiA mutant in the presence of rumen AHLs (5 μL extract). (G) qRT-PCR of gadX from WT and sdiA mutant containing either empty vector or the aiiA gene from B. cereus (encodes a lactonase that inactivates AHLs) cloned into pBADMYcHisA grown in filtered, nonconcentrated rumen fluid in the presence of arabinose. The presence of AHLs in this rumen fluid was previously confirmed (Fig. S3). (H) Graph depicting the bacterial composition of the rumen of eight heifers on a grain diet.

Fig. 4.

Competition of WT and sdiA mutant in the bovine rumen and RAJ. Eight 1.5-year-old fully ruminant and ruminally cannulated Charolais heifers were inoculated with equal cfus of WT and sdiA mutant. (A) As a control, equal cfus of WT and sdiA were grown in coculture in vitro, and their competitive indexes were determined throughout growth (early, mid, late, and stationary phases of growth). A competitive index of 1 means no difference, a competitive index <1 means that WT was in higher numbers than the sdiA mutant, and a competitive index >1 means that the mutant was in higher numbers than WT. The ratio of WT to mutant bacteria was determined in the (B) rumen and (C) RAJ. (D) Schematic model of SdiA-AHL–dependent EHEC gene expression in the bovine GI tract.