The role of quorum sensing in Escherichia coli (ETEC) virulence factors
Introduction
Enterotoxigenic Escherichia coli (ETEC) strains are a major cause of diarrheal disease in humans and animals (Zhu et al., 2011). ETEC is the most common cause of traveler’s diarrhea and can be fatal for children (Qadri et al., 2005). The major virulence factors are bacterial fimbriae that mediate bacterial attachment to host enterocytes allowing its colonization and proliferation, and the production of enterotoxins, which stimulate fluid and electrolyte secretion by intestinal cells leading to diarrhea (Fairbrother et al., 2005).
ETEC in pigs is responsible for much of the neonatal, and a majority of the post weaning diarrheal infections (Berberov et al., 2004). Stress caused by handling, isolation of litters from the sow, or by cold, has been reported to increase fecal excretion of ETEC in comparison to control piglets (Dowd et al., 2007). Stress induces the activation of the adrenal, leading to the release of stress-related catecholamine hormones that can impair immune response and activate growth and expression of virulence factors in bacteria (Jones et al., 2001). The catecholamines (epinephrine and norepinephrine) can be present in the gastrointestinal tract (Horger et al., 1998). Norepinephrine is produced within adrenergic neurons present in the enteric nervous system, and epinephrine is synthesized in both the central nervous system and the adrenal medulla and is involved in systemic responses (Purves et al., 2001). The levels of epinephrine in the intestine are in the micromolar range, similar to the level tested in the present study, however, during an ETEC infection, the altered electrolyte secretion may compromise the integrity of the enterocyte, leading to an increase in its concentration (Polotsky et al., 1994, Freestone et al., 2008).
E. coli strains regulate their virulence gene expression in response to a variety of environmental factors and can use quorum sensing (QS) to modulate gene expression. QS is a bacterial cell-to-cell communication mechanism, comprising the production and detection of autoinducers (AI). Report of a signaling molecule, AI-3, whose synthesis is not dependent on LuxS (Sperandio et al., 2003), suggests that this molecule is involved in Enterohemorrhagic E. coli (EHEC) cross talk with the epinephrine-norepinephrine host signaling system (Freestone et al., 2002). EHEC senses the host hormones epinephrine and norepinephrine through the membrane protein QseC (Walters et al., 2006). QseC senses both AI-3 and epinephrine and thus functions in interkingdom cross-signaling (Rasko et al., 2008, Moreira et al., 2010). QseC is part of a two-component system, QseB/C, in which QseC phosphorylate on binding of its ligand and transfer the phosphate moiety to QseB transcriptional regulators, by doing so modulating the expression of genes under their control (Sperandio et al., 2002). However, two-component sensory systems are known to integrate multiple signals and the extent to which such sensors influence virulence by sensing stress-related catecholamines is not totally understood.
Given that AI-3 production is not limited to EHEC strains, and that AI-3/epinephrine may be involved in interspecies/interkingdom signaling, one can suggest that this signaling pathway might also play a role in the pathogenesis of ETEC. The specific aim of this study was to determine the effect of conditioned medium (containing factors e.g., autoinducer molecules), and epinephrine in the growth, motility, fimbriae and heat-labile toxin gene expression in ETEC E68.
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Bacteria and culture media
The E. coli E68 (O141; K88ab; H4) (Gil Turnes et al., 1999), and E. coli DH5α (fhuA2 lac(del)U169 phoA glnV44 Φ80′ lacZ(del)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17, Invitrogen) strains used in this experiment were provided by bacteria collection of Cenbiot Bacteriology Laboratory – UFPel. The culture media used was BrainHeart Infusion broth (BHI, Difco), Luria Bertani (LB, Acumedia), and Minimum Essential Medium eagle (MEM, Sigma). Supplements of epinephrine (Hipolabor), and atenolol a β
Cell growth dynamics
The addition of conditioned medium to the cultures increased ETEC E68 growth significantly (p < 0.05) starting at the 3rd hour of culture and continuing at the 4th and 5th hour, however by the 6th hour the difference was reduced and by the 7th hour no difference among the control and other treatments were observed (Fig. 1). Epinephrine alone at concentrations of 50 μM did not have any effect on ETEC growth dynamics, and when combined with the conditioned medium the growth curve was similar to the
Discussion
Studies have shown that the use of catecholamines in culture media promotes the multiplication, motility and expression of genes involved in bacterial virulence (Toscano et al., 2007, Bearson and Bearson, 2008, Bearson et al., 2010). The results presented in this study suggest that ETEC E68 may utilize epinephrine, as well as its own molecules (conditioned medium, e.g., AI-3) as an environmental cue to modulate virulence factors essential for its pathogenesis. We have found that the combination
Conclusion
Given the presence of catecholamine hormones and autoinducers in the gastrointestinal tract, findings from this study suggest that their combination within the host may increase the expression of ETEC E68 virulence factors. We observed that the presence of epinephrine and conditioned medium increases the ETEC growth rate. Additionally, their association was able to modulate the expression of the major ETEC virulence factors: F4 fimbriae and LT. The results of this study help to better
Conflict of interest
The authors declare no conflict of interest.
Acknowledgements
The authors would like to thank the Federal University of Pelotas, the graduate program in Biotechnology, and CAPES for Sturbelle, R.T. scholarship and research grant.
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