Autoinducer-2 Quorum Sensing Influences Viability of Escherichia coli O157:H7 under Osmotic and In Vitro Gastrointestinal Stress Conditions

Abstract

Bacteria use autoinducer molecules to communicate both at intra-species and inter-species levels by quorum sensing. One such cell density-dependent signaling system is the luxS-mediated universal quorum sensing using autoinducer-2 (AI-2). Virulence of several pathogens is determined by an AI-2 system and is related to colonization and infection of the host. From this concept, numerous papers have suggested that AI-2 inhibition is an important strategy toward designing of new antimicrobial agents. However, recent studies indicate that the AI-2 system is also involved in adaptation and survival under environmental stress conditions. Therefore, we hypothesized that interaction between quorum sensing and environmental conditions may be critical in influencing predicted results in a control and when combating of target pathogens. We investigated the growth of enterohemorrhagic Escherichia coli O157:H7 (EHEC) and its luxS-deficient (non AI-2 producing) mutant strain under various stress conditions, and found significant differences in the growth rate under osmotic stress. Moreover, we could also show the impact of the AI-2 molecule on viability in the gastrointestinal tract model representing a complex environmental condition. Differences in vital responses of the strains suggest that AI-2 quorum sensing has a significant influence on the viability of EHEC under environmental stress conditions.

Keywords: EHEC; autoinducer-2; bacterial survival; gastrointestinal stress; osmotic stress; quorum sensing.

FIGURE 1

Growth of EHEC wild-type strain (ATCC 43894) and the luxS-deficient mutant strain under different culture conditions. NaCl (A), bile (B), and pH (C) tests were performed using LB broth at 37°C for 24 h. The fold changes were calculated from optical density values measured at 600 nm, and compared to those for the wild-type strain. Error bars indicate the standard deviations. ^∗p ≤ 0.05.

FIGURE 2

Field Emission Scanning electron analysis (FE-SEM) analysis. EHEC wild-type (A) and luxS-deficient mutant (B) were cultured in 0.6M NaCl LB broth for 12 h.

FIGURE 3

Influence of AI-2 on growth of E. coli wild-type strain (ATCC 43894) and the luxS-deficient mutant strain under osmotic stress. EHEC wild-type (WT), luxS-deficient mutant (MT), and the mutant with 3 μM (as final concentration) of AI-2 (MT + AI-2) were cultured for 10 h under 0.6M NaCl LB broth. The growth rates were measured from optical density 600 nm values (A). Individual cell length-to-width ratio (B) was measured and calculated from at least 90 representative objects. Error bars indicate the standard deviations. Significance is indicated (^∗p ≤ 0.05; ^∗∗p ≤ 0.01; ^∗∗∗p ≤ 0.001).

FIGURE 4

Cluster analysis using a hierarchical clustering heatmap.

FIGURE 5

Influence of different pH-values of gastric juice on viability of EHEC wild-type strain (ATCC 43894) and the luxS-deficient mutant strain in an in vitro GIT model. ATP activity was determined and used as basis for comparison of viability of the EHEC wild-type and the luxS deficient mutant. The fold changes were calculated on the basis of the value of the wild-type strain. Error bars indicate the standard deviations. Significance is indicated (^∗p ≤ 0.05).

FIGURE 6

Comparison of the survival of the EHEC wild-type and the luxS-deficient mutant strains in the mouse gastrointestinal tract. The values were determined by FITC detection. Calculation of the fold changes was based on the value of the wild-type strain. Error bars indicate the standard deviations.