The effects of vitamin K on enterohemorrhagic Escherichia coli

Abstract

Enterohemorrhagic Escherichia coli (EHEC) is the cause of foodborne outbreaks, hospitalizations, and deaths worldwide. Today, no optimal treatment for EHEC infections exist, as antibiotics may increase production of Shiga toxin (Stx), the most important virulence factor of EHEC. Ruminants are healthy reservoirs for EHEC, and transmission to humans are typically by the fecal-oral route. Vitamin K, a biomolecule found in the intestinal tract of both humans and ruminants, was previously identified by our research group as an inhibitor of Stx production. While vitamin K levels generally are high in the ruminant intestine, they vary in the human intestine due to factors such as age, diet, and intestinal microbiota. This thesis aims to address if and how exposure to the different vitamin K analogs; phylloquinone, menaquinone, menadione and menadione sodium bisulfite (MSB), influence the growth and virulence potential of EHEC under antibiotic- and H2O2-stress induced conditions. The working hypothesis was that vitamin K would reduce Stx production, and possibly other virulence factors as well. Our initial studies showed that exposure to all vitamin K analogs tested influenced the level of Stx produced by three different EHEC strains, including the reference strain EHEC O157:H7 strain EDL933. This strain was therefore used as a model strain throughout this study. The growth of EHEC was influenced to varying extents by the presence of vitamin K analogs in the growth medium. Phylloquinone showed a modest but positive effect on growth, menaquinone no effect, whereas menadione and MSB inhibited growth. The transcription of the stx gene and the levels of Stx produced were markedly reduced in EHEC cultures exposed to sub-inhibitory concentrations of vitamin K when induced by ciprofloxacin and H2O2. There was also a concomitant 2 to 40 times reduction in phage production in the vitamin K treated cultures. Both ciprofloxacin and mitomycin C induced cellular filamentation, a morphological alteration that promote surface adhesion and biofilm formation. This phenotypic change was inhibited when the antibiotic was combined with menadione or MSB. Menadione and MSB also increased survival of EHEC in the presence of H2O2 and ciprofloxacin. To get insight into the mechanisms of vitamin K's effects on EHEC, full transcriptomic (RNA sequencing) and proteomic (LC-MS) analyses were performed. Prior to investigating the impact of vitamin K analogs on EHEC, insights into the organism's response to the virulence inducing agents, H2O2 and ciprofloxacin, was important. Thus, cultures treated with H2O2 and ciprofloxacin were analyzed first. H2O2 and ciprofloxacin were found to alter expression of 42% and 25% of the genes in the EDL933 genome, respectively. Samples treated with H2O2 showed the largest changes in gene and protein expression, particularly in pathways associated with iron metabolism, ribosome biosynthesis, SOS response and oxidative stress response. For ciprofloxacin treated samples, the most strongly regulated genes and proteins were mainly phage-associated. Although the SOS-response was activated by ciprofloxacin, it was less pronounced than that seen in the H2O2 treated samples. Ciprofloxacin increased expression of numerous virulence factors suggesting that its virulence promoting effect is not only due to stimulation of Stx production. Phylloquinone and menaquinone had no significant effect on gene expression in either uninduced or H2O2-treated samples. However, in H2O2-induced samples with menadione or MSB, there were 1589 and 1462 differentially expressed genes (DEGs), respectively, compared to samples without vitamin K analogs. The expression of motility and chemotaxis-associated genes as well as genes located on the virulence plasmid pO157 were downregulated in the presence of MSB and menadione, as well as genes encoding proteins involved in iron acquisition and toxin-antitoxin systems. The genes in the Locus of Enterocyte Effacement (LEE)-encoded type three secretion system (T3SS) were, on the other hand, upregulated in the presence of either MSB or menadione. The two vitamin K3 analogs demonstrated opposite effects on expression of phage-encoded genes, while menadione increased their transcription, MSB suppressed it. In ciprofloxacin-treated samples, the most noticeable change in transcription caused by MSB was the downregulation of phage genes. Additionally, there was a 2.6-fold average downregulation of genes that has previously been shown to be overexpressed in hypervirulent EHEC strains, indicating that vitamin K could counteract the stress-induced expression of virulence traits. Together, the results suggest that vitamin K inhibits key virulence traits such as toxin and phage production, counteracts filamentation and increase survival in the presence of phage–inducing compounds. In the situation of a human infection, such inhibitory effects could improve disease outcome due to a lower level of Stx released and less spreading of infectious phages. At the same time, the presence of vitamin K leads to less lysis of EHEC cells, which can promote ruminant carriage and generate persister cells, but also less growth of EHEC in general. Although additional research is needed, the findings of the study suggest that vitamin K analogs or their derivatives could be promising for the treatment of EHEC infections, given their ability to reduce the expression of numerous genes associated with virulence.

Enterohemorrhagisk Escherichia coli (EHEC) forårsaker matbårne utbrudd, sykehusinnleggelser og dødsfall over hele verden. I dag finnes ingen optimal behandling for EHEC-infeksjoner, da antibiotika kan øke produksjonen av Shigatoksinet (Stx), den viktigste virulensfaktoren til EHEC. Drøvtyggere er friske reservoarer for EHEC, og fekalt kontaminert mat og drikke er hovedkilden for EHEC infeksjoner. Vitamin K, et biomolekyl som finnes i tarmkanalen til både mennesker og drøvtyggere, har tidligere blitt identifisert som en hemmer av Stx-produksjon av vår forskningsgruppe. Nivåene av vitamin K er generelt høye i drøvtyggertarmen, men varierer i mennesketarmen på grunn av faktorer som alder, kosthold og tarmmikrobiota. Denne avhandlingen har som mål å undersøke om, og hvordan, eksponering for de ulike vitamin K-analogene; fyllokinon, menakinon, menadion og menadion natriumbisulfitt (MSB), påvirker vekst og virulenspotensiale til EHEC under stress påført av behandling med antibiotika og hydrogenperoksid (H2O2). Arbeidshypotesen var at vitamin K ville redusere Stx-produksjonen, og muligens også andre virulensfaktorer.