Intrahost milieu modulates production of outer membrane vesicles, vesicle‐associated Shiga toxin 2a and cytotoxicity in Escherichia coli O157:H7 and O104:H4

Abstract

Outer membrane vesicles (OMVs) are important virulence tools of enterohaemorrhagic Escherichia coli (EHEC), but other biological functions of these nanostructures are unknown. We tested the hypothesis that modulation of OMV production enables EHEC to resist the intrahost environment during infection by investigating if simulated human gastrointestinal conditions affect OMV production in EHEC O157:H7 and O104:H4. All the conditions tested including a low pH, simulated ileal and colonic media, presence of mucin, intestinal epithelial cell lysate or antimicrobial peptides, as well as iron limitation, significantly increased OMV production by these pathogens. Accordingly, a maximum vesiculation in EHEC O104:H4 was observed immediately after its isolation from a patient's intestine, and rapidly decreased during passages in vitro. Most of the simulated intrahost conditions also upregulated the OMV-associated Shiga toxin 2a (Stx2a), the major EHEC virulence factor, and, as a result, OMV cytotoxicity. The data indicates that upregulation of OMV production by the human gastrointestinal milieu contributes to EHEC survival and adaptation within the host during infection. Moreover, the intrahost increase of vesiculation and OMV-associated Stx2a may augment EHEC virulence.

Bacterial outer membrane vesicles provide an alternative pathway for trafficking of type III secreted effectors into epithelial cells

“…OMVs are constantly produced, but the transition to the intra-host milieu and exposure to cues such as colonic medium and mucin significantly increases vesiculation. OMV production is highest in host-isolated EHEC and decreases during lab cultivation (40). Similarly, antibiotic treatment increases OMV production and the amount of OMV-associated Shiga-like toxins (40).…”

“…OMV production is highest in host-isolated EHEC and decreases during lab cultivation (40). Similarly, antibiotic treatment increases OMV production and the amount of OMV-associated Shiga-like toxins (40). These quantitative differences between OMV production in laboratory culture and in vivo are likely the reason why OMV mediated trafficking of T3SS effectors has thus far eluded detection in conventional in vitro culture assays.…”

Role of Shiga Toxins in Cytotoxicity and Immunomodulatory Effects of Escherichia coli O157:H7 during Host-Bacterial Interactions in vitro

“…The plasmid was constructed by standard cloning techniques, according to the NIH policy manual on Working Safely with Hazardous Biological Materials. The complete stx 2a sequence was amplified by PCR from total DNA from E. coli C600Φ933W, using the primers stx 2 Fw (5 -GAATTCATTATGCGTTGTTAG-3 ) and stx 2 R (5 -GAATTCTCAGTCATTATTAAACTG-3 ), both containing an EcoRI restriction site [18]. The resulting fragment (1413 bp) was cloned in a pGEM-T Easy vector (Promega Inc., Madison, WI, USA), generating the plasmid pStx2.…”

“…Transcription of stx 2 is highly dependent on induction of the phage lytic cycle, as it is mainly governed by the late phage promoter pR' [11]. In addition, it has been recently demonstrated that Stx2a and/or Stx2c from periplasmic space could be delivered by outer membrane vesicles (OMVs) [7,18]. A comprehensive understanding of early events during EHEC colonization that lead to HUS could aid in the development of new strategies to prevent and treat the disease.…”

Secretion and Delivery of Intestinal Pathogenic Escherichia coli Virulence Factors via Outer Membrane Vesicles

“…OMVs carry both bacterial toxins (Horstman and Kuehn, 2000;Wai et al, 2003;Balsalobre et al, 2006;Kouokam et al, 2006;Aldick et al, 2009;Ellis and Kuehn, 2010;Chatterjee and Chaudhuri, 2011;Rompikuntal et al, 2012;Guidi et al, 2013;Kunsmann et al, 2015;Bielaszewska et al, 2017) and other virulence factors such as adhesins, invasins, outer membrane proteins, lipopolysaccharide (LPS), flagellin, and proteases (Kadurugamuwa and Beveridge, 1995;Bomberger et al, 2009;Ellis and Kuehn, 2010;Rolhion et al, 2010;Kunsmann et al, 2015;Rompikuntal et al, 2015;Vanaja et al, 2016;Bielaszewska et al, 2017). Secretion of OMVs is generally considered to be an adaptive response to environmental stress and often occurs during infection when the bacteria are exposed to the host's defense mechanisms (MacDonald and Kuehn, 2012;Orench-Rivera and Kuehn, 2016;Bauwens et al, 2017b). In the presence of antimicrobial peptides or bacteriophages, increased production of membrane vesicles correlates with improved fitness and increased survival (Manning and Kuehn, 2011;Duperthuy et al, 2013).…”