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Modulation of AggR levels reveals features of virulence regulation in enteroaggregative E. coli

ABSTRACT: Enteroaggregative Escherichia coli (EAEC) strains are one of the diarrheagenic pathotypes. EAEC strains harbor a virulence plasmid (pAA2) that encodes, among other virulence determinants, the aggR gene. The expression of the AggR protein leads to the expression of several virulence determinants in both plasmids and chromosomes. In this work, we describe a novel mechanism that influences AggR expression. Because of the absence of a Rho-independent terminator in the 3′UTR, aggR transcripts extend far beyond the aggR ORF. These transcripts are prone to PNPase-mediated degradation. Structural alterations in the 3′UTR result in increased aggR transcript stability, leading to increased AggR levels. We therefore investigated the effect of increased AggR levels on EAEC virulence. Upon finding the previously described AggR-dependent virulence factors, we detected novel AggR-regulated genes that may play relevant roles in EAEC virulence. Mutants exhibiting high AggR levels because of structural alterations in the aggR 3′UTR show increased mobility and increased pAA2 conjugation frequency. Furthermore, among the genes exhibiting increased fold change values, we could identify those of metabolic pathways that promote increased degradation of arginine, fatty acids and gamma-aminobutyric acid (GABA), respectively. In this paper, we discuss how the AggR-dependent increase in specific metabolic pathways activity may contribute to EAEC virulence. Studying Enteroaggregative E. coli (EAEC), Prieto et al have found that the 3′UTR of the gene that encodes the transcriptional activator AggR, regulates aggR transcript stability. They further demonstrate that enhanced levels of AggR leads to increased motility and virulence and identify novel AggR–regulated genes that may be important for EAEC virulence.

SUBMITTER: Prieto A

PROVIDER: S-EPMC8595720 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Enteroaggregative Escherichia coli (EAggEC) has been associated with persistent pediatric diarrhea in the developing world, yet the pathogenetic mechanisms of EAggEC infection are unknown. Our previous data have suggested that aggregative adherence of some EAggEC strains to HEp-2 cells is mediated by flexible, bundle-forming fimbriae, which we have termed aggregative adherence fimbriae I (AAF/I). Genes sufficient to confer expression of AAF/I are located on the 60-MDa plasmid of EAggEC 17-2; AAF/I genes are present as two unlinked plasmid regions (regions 1 and 2), separated by 9 kb of DNA. Here we report the complete DNA sequencing of region 2 and the identification of an open reading frame which is involved in the expression of AAF/I. One open reading frame of 794 bp encodes a protein (designated AggR) with a predicted molecular size of 29.4 kDa, which shows a high degree of amino acid sequence identity to CfaR and other members of the AraC class of gene regulators. The cloned aggR gene (or, alternatively, a cloned cfaR gene) was sufficient to complement a region 1 clone to confer AAF/I expression. To further substantiate the role of aggR in the regulation of AAF/I, we constructed a 289-bp in-frame aggR deletion and replaced the native gene in 17-2 by allelic exchange, using the temperature-sensitive vector pIB307. The resulting aggR deletions were negative for AAF/I expression, but expression was restored when the aggR gene (cloned into pBluescript II SK) was reintroduced into the aggR mutant. RNA slot blot experiments using a probe for the putative AAF/I pilin subunit (aggA) revealed that aggR operates as a transcriptional activator of aggA expression. aggA::phoA fusions were constructed in 17-2 and in 17-2 delta aggR. AggR was found to promote expression of the aggA gene under a variety of conditions of temperature, osmolarity, oxygen tension, and medium. At acid pH, aggA expression was maximal and was regulated by both AggR-dependent and AggR-independent mechanisms.

Project description:BackgroundEscherichia coli is a normal inhabitant of the gut which upon acquiring virulence factors becomes potentially able to cause diseases. Although E. coli population augments in Crohn's disease (CD), the reason of this proliferation is not yet clear. CD associated E. coli shows features of extraintestinal pathogenic categories (ExPEC), and eventually the ability to invade cultured epithelial cells, a property observed among diarrheagenic E. coli (DEC). In this work, data on the characterization of an E. coli isolate from a CD patient reveal that, besides invasiveness, CD associated E. coli may harbor other typical DEC markers, namely those defining enterohemorragic (EHEC) and enteroaggregative (EAEC) pathotypes.ResultsThe studied strain, detected both in an ileum biopsy and stools, belonged to the B2 E. coli reference collection (EcoR) phylogroup and harbored the intimin, Shiga cytotoxin 1, and AggR transcriptional activator encoding genes (eae, stx1, aggR, respectively); displayed aggregative adherence to Hep-2 cells and an ability to enter Caco-2 cells four times as high as that of EIEC reference strain and half of invasiveness of AIEC LF82. It was able to enter and replicate in J774 macrophages with invasiveness 85 times as high as that of LF82, but with only one sixth of the intracellular proliferation ability of the later. Extracellular products with cytotoxic activity on Vero cells were detected in strain's cultures. Preliminary analysis indicated similarity of this strain's genome with that of O104:H4/2011C-3493.MethodsFollowing its isolation from a resected CD patient, the strain was characterized by in vitro adhesion and invasion assays to Hep-2, invasion to Caco-2 cells and to J774 macrophages and tested for the ability to form biofilm and to produce Shiga cytotoxins. PCRs were carried out to identify virulence genetic markers and for EcoR phylogrouping. The strain's genome was sequenced by means of Ion torrent PGM platform.ConclusionThe detection, in a CD patient, of an E. coli combining virulence features of multiple DEC pathotypes seems not only to stress the relevance of E. coli to CD etiopathogenesis but also to indicate the existence of new and potentially more virulent strains putatively associated with this disease.

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Modulation of AggR levels reveals features of virulence regulation in enteroaggregative E. coli

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