Differences in virulence gene expression between atypical enteropathogenic Escherichia coli strains isolated from diarrheic and healthy ruminants

Abstract

Differences in the pathogenicity of atypical enteropathogenic Escherichia coli (EPEC) strains may be due, at least partially, to different expression patterns of some virulence genes. To investigate this hypothesis, the virulence gene expression patterns of 6 atypical EPEC strains isolated from healthy and diarrheic ruminants were compared using quantitative real-time reverse transcription polymerase chain reaction after growing the bacteria in culture medium alone or after binding it to HeLa epithelial cells. Some virulence genes in strains from diarrheic animals were upregulated relative to their expression in strains from healthy animals. When bacteria were cultured in the presence of HeLa cells, the ehxA and efa1/lifA genes, previously associated with the production of diarrhea, were expressed at higher levels in strains from diarrheic animals than in strains from healthy animals. Thus, the expression levels of some virulence genes may help determine which atypical EPEC strains cause diarrhea in ruminants.

Atypical enteropathogenic Escherichia coli (EPEC) are a cause of diarrhea in ruminants and humans, although these bacteria can also be isolated from healthy ruminants and humans (1,2). These bacteria cause a characteristic attaching and effacing (AE) lesion in the gut mucosa because of the intimate bacterial adhesion to the enterocytes and effacement of the brush border microvilli (2). Formation of AE lesions is governed by the locus of enterocyte effacement (LEE). This locus contains the eae gene, which encodes an outer membrane protein called intimin, necessary for intimate attachment to epithelial cells (2). The LEE also encodes a type III secretion system; EPEC-secreted proteins (Esp), such as EspA, which is essential for the delivery of proteins into the host cell; and LEE-encoded regulator (Ler), which positively regulates virulence factors (3,4). Additional non-LEE-encoded virulence factors have been described, such as Efa1/LifA, which is encoded by the pathogenicity island PAI OI-122, and enterohemolysin, which is encoded by large plasmids, such as pO157 (5,6).

Molecular characterization of atypical EPEC strains isolated from humans and ruminants has revealed a tremendous diversity of virulence genes among different strains (1,6,7). Therefore, it is possible that different atypical EPEC strains have different pathogenic potential and that only some of them can cause disease in humans and animals. It seems likely that only the atypical EPEC strains possessing certain virulence genes, such as efa1/lifA and ehxA, can cause disease (1,5,6). However, it is also possible that differences in pathogenicity among strains may be due, at least partially, to different expression patterns of some virulence genes. In support of this hypothesis, a previous study has found that expression of virulence genes differs among enterohemorrhagic E. coli O157:H7 strains depending on whether their genotype is predominant among human clinical cases or among bovines (8). Despite this evidence suggesting a link between expression profiles of virulence genes and pathogenicity, the authors are unaware of studies that compare levels of virulence gene expression among atypical EPEC strains isolated from healthy and diarrheic ruminants.

The aim of this study was to establish whether differences in virulence gene expression can be detected between atypical EPEC strains that share the same serotype and virulence gene profile, but differ in whether they were isolated from healthy or diarrheic ruminants.

Six strains were tested in this study in pairs, with one strain coming from a diarrheic ruminant and the paired strain coming from a normal ruminant. The strain pairs were as follows: O153:HNM strains (NM indicates non-motile) isolated from a diarrheic lamb and a healthy sheep, O26:HNM strains isolated from a diarrheic calf and a healthy cow, and O26:H11 strains isolated from a diarrheic lamb and a healthy goat. The genes analyzed were eae, espA, ler, efa1/lifA, and ehxA, which encode enterohemolysin. The strains used in this study have previously been characterized (1) and possess all 5 genes analyzed, except for the O26:HNM strains, which lack ehxA.

Virulence gene expression in enteropathogenic bacteria is regulated by environmental conditions (9), so expression was analyzed in the present study under 2 sets of conditions: after culturing the bacteria in Dulbecco’s modified Eagle medium (DMEM; Lonza, Verviers, Belgium) and after co-culturing them in the same medium together with HeLa cells. Dulbecco’s modified Eagle medium was chosen because it provides a controlled environment with minimal variation in growth conditions, and it has also been shown to induce expression of LEE genes (8,10). The HeLa epithelial cells were used because growth in the presence of epithelial cells may better simulate the gastrointestinal tract environment than growth in DMEM (8). In the first set of conditions, strains were grown to exponential phase in DMEM, the bacteria were sedimented by centrifugation (5000 × g for 5 min) and the pellet was stored in RNA stabilization solution (RNA later; Invitrogen, Prat de Llobregat, Barcelona, Spain) for subsequent RNA purification. In parallel, under the second set of conditions, HeLa cells were infected with “preactivated” bacteria, which had been grown to early- or mid-log phase at 37°C in DMEM (9), at a multiplicity of infection of 100:1. After 5 h of incubation, the HeLa cells were washed vigorously to remove nonadhering bacteria, and cells with adhered bacteria were collected in RNA stabilization solution until RNA isolation. Culturing of the atypical EPEC strains alone in DMEM or in the presence of HeLa cells was carried out at 37°C in an atmosphere of 5% CO₂.

Total bacterial RNA was extracted using a commercial RNA isolation kit (High Pure RNA Isolation kit; Roche Applied Science, Sant Cugat del Vallés, Barcelona, Spain) and cDNA was synthesized using a commercial kit (qScript cDNA SuperMix kit; Quanta Biosciences, Gaithersburg, Maryland, USA) according to the manufacturer’s instructions. Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) was done using a RT-PCR thermocycler (7300 Real-Time PCR System; Applied Biosystems, Tres Cantos, Madrid, Spain) and a commercial dye (FastStart Universal SYBR Green Master Mix; Roche Applied Science). Samples were tested in duplicate. Previously published primers were used to amplify the eae, ler, and ehxA genes (11–13). In addition, primers were designed in the present study to amplify the espA gene (espAq-F: 5′-GATGCCTCATTCATATCAGC-3′ and espAq-R: 5′-TCGGTGTTTTTCAGGCT GC-3′) and efa1/lifA gene (efaF-RT: 5′-TGGTAGTCAGGTATACATCCGTATTTC-3′ and efaR-RT: 5′-GCTGAAAACCGGCACAAT-3′). Data were analyzed using Gene Expression’s CT Difference method and individual PCR efficiencies were determined using LinRegPCR, as previously described (14). The 16S rRNA housekeeping gene was used as the internal control (15). Genes whose expression in strains from diarrheic animals relative to their expression in strains from healthy animals that changed 0.5- to 1.5-fold, were classified as unchanged.

When cultured in DMEM, there was no difference in gene expression except for eae, espA, and ler in the pair of O26:H11 strains (Figure 1A). These 3 genes were expressed at levels 3- to 6-fold higher in bacteria isolated from a diarrheic lamb than from a healthy goat.

Figure 1.

Expression of virulence genes in strains of atypical enteropathogenic Escherichia coli isolated from diarrheic ruminants relative to their expression in strains isolated from healthy ruminants. A — Strains grown in Dulbecco’s modified Eagle medium. B — Strains grown in contact with HeLa cells. NM — nonmotile.

When virulence gene expression was compared between strains from diarrheic and healthy hosts after being cultured in contact with HeLa cells, the eae and espA genes were expressed at similar levels between strains, whereas ler, ehxA, and efa1/lifA were expressed at levels 2- to 7-fold higher in O153:HNM and O26:H11 strains isolated from diarrheic animals (Figure 1B). The presence of the ehxA and efa1/lifA genes has been previously associated with diarrhea caused by atypical EPEC in children and neonatal ruminants (1,5,6). In addition, the results of this study suggest that overexpression of these genes may also determine whether atypical EPEC strains can cause diarrhea in neonatal ruminants. Our results on the expression of the eae and espA genes in the pair of O26:H11 strains grown in contact with epithelial cells are not in agreement with those results obtained using the same strains grown in DMEM or with the results of Vanaja et al (8), who found an association between overexpression of LEE genes in E. coli O157:H7 strains grown in DMEM and virulence. It is possible that these differences may be due to the influence of environmental conditions on bacterial gene expression. Thus explaining how Jandu et al (16) were able to find differences in gene expression when E. coli O157:H7 strains were cultured in the presence and absence of epithelial cells.

In summary, the results of this study show that some virulence genes are upregulated in strains isolated from diarrheic animals relative to their expression in strains isolated from healthy animals. Thus, the expression levels of some virulence genes may help determine which atypical EPEC strains cause diarrhea in ruminants. Since this is a preliminary study involving a small number of strains, further studies are required to confirm the role of virulence gene expression in the production of diarrhea by atypical EPEC strains in neonatal ruminants.