GLOBAL EXPRESSION PROFILE OF HYPERVIRULENT ENTEROHEMORRAGIC ESCHERICHIA COLI O157:H7 IN PHAGOSOME OF MURINE MACROPHAGE

NAHUEL RIVIERE; LIBIA SMITH ; CAROLINA CASSABONE; WANDERSON MARQUES DA SILVA; ANGEL CATALDI

Foz de Iguazú

Congreso; 32° CONGRESO BRASILEIRO DE MICROBIOLOGÍA; 2023

Sociedade Brasileiera de Microbiología

Enterohemorrhagic Escherichia coli(EHEC) is a foodborne pathogen of worldwide importance associated with bloody diarrhea and Hemolytic Uremic Syndrome (HUS) outbreaks in humans. The main virulence factor of EHEC O157:H7 is the Shiga toxin (stx) encoded by lysogenic lambdoid bacteriophage. EHEC pass through gastrointestinal tract and survives the acidic condition of the stomach. The intestinal adhesion occurs in regions with follicle-associated epithelium, which overlies Peyer´s patches. These follicles present M cells specialized in the translocation of pathogens from the intestinal lumen to the basolateral side of the epithelium where the bacteria are then phagocytized by macrophages. One way to understand host-pathogen interaction is to elucidate the molecular mechanisms by focusing on gene expression. For this purpose RNA seq was performed, EHEC was incubated with murine macrophages. RAW 264.7 cells were seeded at 1x106 per well in a 12-well plate and co-incubated with a EHEC bacterial culture with OD 0.6 nm and MOI=100. After 2 h of incubation at 37°C and 5% CO2, total RNA extraction was performed with trizol. Subsequently, bacterial RNA enrichment was performed with the MicrobEnrich kit. The cDNA was synthesized and sequenced using the Illumina Nextseq 500 platform. Transcriptomic results for phagocytosed EHEC O157:H7 showed up-regulation of genes associated with response to oxidative stress such as oxyR, soxRS and rpoS expression. The up regulation of norV (flavorubredoxin), a NO detoxification-associated enzyme, indicate the exposure of EHEC O157:H7 to macrophage-generated nitric oxide. Furthermore, these nitric stress conditions could explain the activation of SoxR generating an elevated fold-change of the soxS transcript observed in EHEC O157:H7 under the phagosome. Tree susceptible enzymes as AroF (DAHP synthase), TyrA (chorismate mutase/prephenate dehydrogenase) and MetE (homocysteine transmethylase), implicated in synthesis of amino acids, were up-regulated in EHEC O157:H7 under the phagosome. The enzymes transcription prevents the inactivity produced by stress. LpxC enzyme implicated in the catalysis of lipid A biosynthesis was up-regulated in EHEC O157:H7 inside the phagosome. In turn, SoxR proved to be involved in the transcription of divalent ion transporter genes such zinT and znuABC for Zn2+, and mgtA for Mg2+ transportation in phagocytosed EHEC O157:H7. Several enzymes that are activated in stress conditions dependent on Zn2+ as TrxC, LpxC and MetE to act properly. In macrophage-phagocytosed EHEC O157:H7 samples, overexpression of genes was observed associated with SOS response. It was seen that the type 3 and 6 secretion system is downregulated. We observed that EHEC vs macrophages downregulated of flagellar genes was seen. These results show that when EHEC is phagocytosed, genes that favor the survival of the pathogen are turned on. In turn, many virulence factors downregulate their expression, which would prevent the immune response.