Analysis of Global Transcriptional Profiles of Enterotoxigenic Escherichia coli Isolate E24377A

Abstract

Enterotoxigenic Escherichia coli (ETEC) is an important pathogenic variant (pathovar) of E. coli in developing countries from a human health perspective, causing significant morbidity and mortality. Previous studies have examined specific regulatory networks in ETEC, although little is known about the global effects of inter-and intrakingdom signaling on the expression of virulence and colonization factors in ETEC. In this study, an E. coli/Shigella pan-genome microarray, combined with quantitative reverse transcriptase PCR (qRT-PCR) and RNA sequencing (RNA-seq), was used to quantify the expression of ETEC virulence and colonization factors. Biologically relevant chemical signals were combined with ETEC isolate E24377A during growth in either Luria broth (LB) or Dulbecco's modified Eagle medium (DMEM), and transcription was examined during different phases of the growth cycle; chemical signals examined included glucose, bile salts, and preconditioned media from E. coli/Shigella isolates. The results demonstrate that the presence of bile salts, which are found in the intestine and thought to be bactericidal, upregulates the expression of many ETEC virulence factors, including heat-stable (estA) and heat-labile (eltA) enterotoxin genes. In contrast, the ETEC colonization factors CS1 and CS3 were downregulated in the presence of bile, consistent with findings in studies of other enteric pathogens. RNA-seq analysis demonstrated that one of the most differentially expressed genes in the presence of bile is a unique plasmid-encoded AraC-like transcriptional regulator (peaR); other previously unknown genetic elements were found as well. These results provide transcriptional targets and putative mechanisms that should help improve understanding of the global regulatory networks and virulence expression in this important human pathogen. Despite the high incidence of death resulting from enterotoxigenic Escherichia coli (ETEC) infection, especially in children in developing countries (55), the mechanisms and regulatory networks of ETEC virulence are still relatively unexplored on a genomic scale. ETEC bacteria are characterized by the production of the plasmid-encoded heat-stable (ST) and/or heat-labile (LT) enterotoxin (11). ETEC release ST and/or LT into the host epithelium, which elevates intracellular cyclic AMP (cAMP) that then activates the cystic fibrosis transmembrane regulator (CFTR) chloride channel; this activation leads to the secretion of electrolytes and, subsequently, water as diarrhea (43). In addition to ST and LT, the enteroaggregative E. coli (EAEC) heat-stable enterotoxin 1 (EAST1) protein, first identified in enteroaggregative E. coli (45), has been found in many ETEC isolates with defined colonization factor profiles (58); however, the contribution of EAST1 to ETEC diarrheal disease is currently unknown (11).ETEC bacteria adhere to intestinal epithelium by fimbrial appendages known as colonization factors (CFs) (12). CFs are structurally and genetically diverse; to date, more than 25 CFs have been id...

IscR Regulates Synthesis of Colonization Factor Antigen I Fimbriae in Response to Iron Starvation in Enterotoxigenic Escherichia coli

“…Iron starvation led to increased production of the CFA/I fimbriae but inhibited LT secretion, supporting the hypothesis that ETEC-specific fimbriae and toxins may be differentially regulated. Indeed, differential expression between CFs and the LT has been previously observed in vitro in the presence of glucose or bile salts (10,13,44).…”

“…To determine if cfaABCE (CFA/I) or eltAB (LT) operon expression was modified in response to iron starvation, RT-qPCR analysis was performed. Gene expression was evaluated in both exponential and early stationary growth phases, as several ETEC virulence genes have previously been shown to be growth phase regulated (44). As this is the first study to evaluate the impact of iron starvation on gene expression in ETEC, we first analyzed the expression levels of ironregulated genes (fur, ryhB, fepA, fyuA, and ompW) previously characterized in other E. coli strains (16,45,46).…”

Understanding the commonalities and differences in genomic organizations across closely related bacteria from an energy perspective

“…Five hundred and fifty-five microarray gene-expression datasets of four E. coli strains are retrieved from the GEO database [46] and the M3D database [47] for estimating the frequencies of transcription activation of individual pathways. Specifically, 40 datasets are found and retrieved for K_12_ W3110 [48], 26 sets for O157_H7_Sakai [49], 23 sets for E24377A [50] and 466 sets for K_12_MG1655 [47]. Biological pathway information used in this study was collected from the KEGG database [7].…”

Transcriptional Modulation of Enterotoxigenic Escherichia coli Virulence Genes in Response to Epithelial Cell Interactions

“…To compare the transcriptomes of adherent and planktonic ETEC E24377A following infection of Caco-2 intestinal epithelial cells, we used the FDA-E. coli-Shigella (FDA-ECSG) Affymetrix array (http://pfgrc.jcvi.org/index.php/microarray/affy_array _description/ecoli_shigella/version1.html), which represents genes from multiple E. coli pathogens, including those from ETEC strain E24377A (18). Array hybridizations were carried out as previously described (19). Briefly, cDNA was synthesized with random hexamers and partially digested with DNase to obtain 20-to 200-bp fragments, end labeled with biotin-11-ddATP, and hybridized for 16 h at 45°C.…”

“…Array analysis was carried out a previously described (19). Briefly, array scans were acquired in Affymetrix GCOS software, and expression was normalized using the simpleaffy Bioconductor R package (20).…”