RNA-Seq analysis of isolate- and growth phase-specific differences in the global transcriptomes of enteropathogenic Escherichia coli prototype isolates.

Tracy H Hazen,Owen White,Sean C Daugherty,Amol Shetty,James B Kaper,David A Rasko,Anup A Mahurkar

doi:10.3389/fmicb.2015.00569

Abstract

Enteropathogenic Escherichia coli (EPEC) are a leading cause of diarrheal illness among infants in developing countries. E. coli isolates classified as typical EPEC are identified by the presence of the locus of enterocyte effacement (LEE) and the bundle-forming pilus (BFP), and absence of the Shiga-toxin genes, while the atypical EPEC also encode LEE but do not encode BFP or Shiga-toxin. Comparative genomic analyses have demonstrated that EPEC isolates belong to diverse evolutionary lineages and possess lineage- and isolate-specific genomic content. To investigate whether this genomic diversity results in significant differences in global gene expression, we used an RNA sequencing (RNA-Seq) approach to characterize the global transcriptomes of the prototype typical EPEC isolates E2348/69, B171, C581-05, and the prototype atypical EPEC isolate E110019. The global transcriptomes were characterized during laboratory growth in two different media and three different growth phases, as well as during adherence of the EPEC isolates to human cells using in vitro tissue culture assays. Comparison of the global transcriptomes during these conditions was used to identify isolate- and growth phase-specific differences in EPEC gene expression. These analyses resulted in the identification of genes that encode proteins involved in survival and metabolism that were coordinately expressed with virulence factors. These findings demonstrate there are isolate- and growth phase-specific differences in the global transcriptomes of EPEC prototype isolates, and highlight the utility of comparative transcriptomics for identifying additional factors that are directly or indirectly involved in EPEC pathogenesis.

Highlights

Enteropathogenic Escherichia coli (EPEC) have been associated with moderate to severe cases of diarrhea and are a leading cause of lethal diarrheal illness among young children in developing countries (Ochoa and Contreras, 2011; Kotloff et al, 2013)
Comparison of the genomic content of the four EPEC prototype isolates (E2348/69, B171, C581-05, E110019) using large-scale BLAST score ratio (LS-BSR) demonstrated there are 3836 genes that are present in all four isolates with significant similarity (LS-BSR ≥ 0.8) (Figure 1)
Comparison of the Global Virulence Regulons of EPEC Prototype Isolates During Growth in Laboratory Conditions To investigate isolate-specific differences in the expression of known virulence factors, and to identify additional genes that are coordinately-expressed with the known virulence factors, we identified the differences in expression of all protein-encoding genes of the four EPEC prototype isolates during exponential growth in Dulbecco’s Modified Eagle’s Medium (DMEM) compared to LB during exponential growth (OD600 = 0.5) (Table 1, Supplemental Data Sets 3–6)

Summary

Introduction

Enteropathogenic Escherichia coli (EPEC) have been associated with moderate to severe cases of diarrhea and are a leading cause of lethal diarrheal illness among young children in developing countries (Ochoa and Contreras, 2011; Kotloff et al, 2013). EPEC are identified by the presence of the locus of enterocyte effacement (LEE), which encodes a type III secretion system (T3SS) and the intimin adherence factor, which are involved in translocation of bacterial factors into host cells, and adherence to the surface of host cells. EPEC are characterized by the absence of the Shiga-toxin genes, which is typically present in the enterohemorrhagic E. coli (EHEC) (Nataro and Kaper, 1998; Kaper et al, 2004). EPEC are further identified as typical EPEC by the presence of genes encoding the bundle-forming pilus (BFP), which is a type IV pilus typically carried by the EPEC adherence factor (EAF) plasmid. E. coli isolates that contain the LEE and do not carry the Shiga-toxin phage or the BFP genes are considered atypical EPEC (Nataro and Kaper, 1998). We have previously demonstrated that the atypical EPEC includes isolates with genomic similarity to typical EPEC and EHEC (Hazen et al, 2013)

Methods

Results

Conclusion