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Distribution and expression of the astA gene (EAST1 toxin) in Escherichia coli and Salmonella
IJMM IJ
Int. J. Med. Microbiol. 291, 15-20 (2001) © Urban & Fischer Verlag http://www.urbanfischer.de/journals/ijmm
Distribution and expression of the astA gene (EAST1 toxin) in Escherichia coli and Salmonella Cristina Paiva de Sousa1, J. Daniel Dubreuil2 1 2
Departamento de Nutrição, Centro de Ciências da Saúde, Universidade Federal da Paraíba, Campus Universitário I S/N, João Pessoa, PB, 58.970-900, Brasil Department of Pathology and Microbiology, Faculty of Veterinary Medicine, Montréal University, 3200 rue Sicotte, St-Hyacinthe, Québec, Canada J2S 7C6
Received August 28, 2000 · Revision received December 6, 2000 · Accepted December 6, 2000
Abstract The distribution and expression of the astA gene (EAST1 toxin) among 358 strains of Enterobacteriaceae were studied. The gene was found in 32.6 % and 11.9 % of Escherichia coli and Salmonella strains, respectively. The majority of E. coli EAST1-positive strains were found among EHEC (88.0 %), EAggEC (86.6 %), and A-EPEC (58.3 %). The gene was present in 16.6 % of E. coli strains without known virulence genes. There was no significant variation among the different serotypes of E. coli tested regarding the presence of the gene. For EPEC, 13.7 % of the tested strains were astA-positive. Among atypical EPEC (eae+, bfp–, EAF–) and (eae+, bfp+, EAF–) 46.2 and 72.7 %, respectively, were positive. The majority of the A-EPEC (87 %) and EaggEC (83 %) strains expressed the EAST-1 toxin as judged from Ussing chamber experiments. Of 32 EIEC strains studied, 2 possessed and expressed the gene as determined in Ussing chamber experiments. Among the Salmonella strains studied, five strains isolated from food were positive for astA and one strain of S. agona showed biological activity in Ussing chamber experiments. Key words: Escherichia coli – Salmonella – EAST 1 toxin – toxin gene
Introduction The Escherichia coli population is composed of commensal organisms that live harmlessly in the gut, opportunistic pathogens that occasionally infect normally sterile tissues, and specialized pathogens that possess specific virulence attributes promoting the ability of the organisms to cause disease. Enteroaggregative E. coli (EaggEC) are defined by their distinctive adherence pattern on HEp-2 cells in culture. The essential element of the aggregative phenotype is the stacked brick adherence pattern, in which
bacterial cells are seen to be lying side-by-side with an appreciable distinction of where one bacterium begins and another ends. EAggEC have epidemiologic properties that distinguish them from other categories of HEp-2 adherent E. coli. In several studies, EAggEC have been specifically associated with persistent diarrhea with variable intensity among children in the developing world (Bhan et al., 1989a, b; Cravioto et al., 1991; Wanke et al., 1991). However, recent outbreaks and volunteer studies suggest that EAggEC strains are virulent in adults (Smith et al., 1990, 1997) and have a global distribution (Itoh et al., 1997).
Corresponding author: Prof. Dr. J. Daniel Dubreuil, Department of Pathology and Microbiology, Faculty of Veterinary Medicine, Montréal University, 3200 rue Sicotte, St-Hyacinthe, Québec, Canada J2S 7C6, E-mail: [email protected] 1438-4221/01/291/1-015 $ 15.00/0
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C. Paiva de Sousa, J. Daniel Dubreuil
The importance of colonization and its features have now been studied and it underscores the importance of the aggregative phenotype. Three possible mechanisms of enteric bacterial pathogenesis are generally accepted: toxin production, invasion, and the signal transduction phenotype seen in enteropathogenic E. coli (EPEC). There is, as yet, no evidence for the presence of a signal trasduction mechanism among EAggEC. Invasion has been described in HEp-2 cells by Benjamin et al. (1995). There is substantial evidence for the presence of enterotoxins elaborated by EAggEC. Savarino et al. (1993) described a homolog of the enterotoxigenic ETEC heat-stable (ST) toxin (designated EAST1 for enteroaggregative thermostable enterotoxin), which is produced by about 40 % of EAggEC strains associated with persistent diarrhea in children in Chile. They also reported that the prototype strain 17-2 (O3 : H2) produced an enterotoxin (4.1 kDa) comprising 38 amino acids (Savarino et al., 1991). The astA gene encodes the EAST1 enterotoxin, and cGMP is the intracellular mediator of the enterotoxic efect of EAST1 (Savarino et al., 1991). Although STa and EAST1 activate guanylate cyclase, they differ at the nucleotide level, suggesting that EAST1 evolved in a genetically different way of STa (Fasano, 1997). No co-hybridisation were noted for those toxins. More recently, Nataro and Kaper (1998), proposed a three stages model for the pathogenesis of EAggEc: i) initial adhesion to the intestinal mucosa; aggregative adherence fimbria type I (AAF/I) and type II (AAF/II) could facilitate the initial colonization; ii) increase in mucus production and biofilm formation; iii) cytotoxin production with damage to the intestinal cells. It was shown that most EAggEC strains harbor a 60 to 65 MDa plasmid which may encode the AA fimbria AAF/I (Nataro et al., 1993) or AAF/II (Czeczulin et al., 1997), and in some cases, EAST1 (Savarino et al., 1991). According to Czeczulin et al. (1999), EAggEC strains are a heterogeneous group of organisms with respect to chromosomal and plasmid-borne genes but the majority harbor a member of a conserved family of virulence plasmids. Comparison of plasmid and chromosomal relatedness of strains suggests clonality of chromosomal markers and a limited transfer model of plasmid distribution. The infection site of EAggEC is the human intestine. EAggEC strains are capable to adhere to the mucosa of the small bowel as well as to that of the large bowel (Nataro and Kaper, 1998). Classical EPEC share the eaeA and bfp genes and the EAF plasmid. They are responsible for diarrhea in children in developing countries. Atypical EPEC are EPEC that have lost the EAF plasmid. ETEC produce heatlabile (LT) or heat-stable toxins (STa and STb) that are also causing diarrhea. EHEC strains are implicated in food-borne diseases principally due to ingestion of un-
cooked minced meat and raw milk. Those strains produce shiga-like toxin 1 (stx1), shiga-like toxin 2 (stx2) and variants thereof. They are involved in episodes of diarrhea with complications. Serotype O157 : H7 is the prototype of increasing importance and is associated with hemorrhagic colitis, bloody diarrhea and the hemolytic uremic syndrome (HUS). EIEC cause a broad spectrum of human diseases. They are closely related to Shigella spp. Their invasive capacity is conferred by the inv plasmid. DAEC strains are diffusely adhering E. coli that are also implicated with episodes of diarrhea. This study was undertaken to determine the distribution and expression of the astA gene among different classes of E. coli and in Salmonella.
Materials and methods Bacteria and culture media Three hundred and fifty eight bacterial isolates belonging to the Enterobacteriaceae were studied, including Escherichia coli [218], Salmonella [42], Shigella [13], Edwardsiella [10], Klebsiella [11], Proteus [21], Morganella [13], Citrobacter [2], and Enterobacter [28]. The E. coli strains studied were previously classified as EPEC [51], atypical EPEC (A-EPEC) [24], enterohaemorrhagic E. coli (EHEC) [25], EAggEC [15], enteroinvasive E. coli (EIEC) [32], ETEC [29] and strains without known virulence genes (non-DEC) [42]. All E. coli strains were from Dr. Trabulsi’s laboratory (University of São Paulo) and other strains were kindly provided by Dr. Iaria’s lab (University of São Paulo). The majority of strains were from diseased humans. All bacterial strains were kept on agar slants at room temperature until the moment of use. DNA probe and colony blot The DNA probe was constructed using the prototype strain EAggEC 17-2. We amplified the astA gene using the following primers: EAST11a (5-CCATCAACACAGTATAT-3) and EAST11b (5-GGTCGCGAGTGACGGCTTTGT-3) (Itoh et al., 1997)). These primers gave a product of 111 bp, that was visualized after electrophoresis on 2 % agarose gels stained with ethidium bromide. The DNA fragment was excized from the gel, purified with the PCR Wizard Kit (Promega), and used as a DNA probe. The probe was labeled with [α-32P]dATP by random priming using a Stratagene kit, according to the manufacturer’s instructions. Colony blots onto Whatman filter n° 541 were prepared according to Maas (1983). Hybridizations and washes were done under stringent conditions (Grunstein and Hogness, 1975). PCR The primers used are described in the preceding section. Cycle conditions used were: denaturation 30 s 95 °C, annealing 120 s 55 °C and extension 120 s 72 °C for 30 cycles. The reaction conditions were: buffer 1, 100 pmol of each primer, 200 µM of each dNTPs, 1.0 mM MgCl2 and 1 U of Taq poly-
Distribution and expression of the astA gene (EAST1 toxin) in Escherichia coli and Salmonella
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merase. The amplified product was analyzed by electrophoresis on a 2 % agarose gel, stained with ethidium bromide and visualized with UV. We used a 1-kb DNA ladder as molecular marker (Life Technologies, Gaithersburg, MD). To detect the astA sequence in Salmonella, we modified the annealing temperature from 55 to 50 °C (see Fig. 2). All other conditions were the same as described before. Ussing chamber experiments Experiments on the biological activity of culture filtrates were conducted in Dr. Fasano’s Laboratory in the Center for Vaccine Development (CVD) at the University of Maryland (Baltimore). Bacterial strains were grown in Luria Bertani Broth at 37 °C for 18 hours with shaking (500 rpm). Culture filtrates were prepared by centrifugation of the cell suspension (Sorvall RT 6000 D) and passage of the supernatant through a Mr 20,000 cut-off Centristar I filter (Sartorius), under conditions suggested by the manufacturer. Ussing chamber experiments were done as previously described, using stripped rabbit ileal tissue (Trucksis et al., 1993). Potential difference and short-circuit current (Isc) were measured, and tissue conductance was calculated (data not shown). Once the tissue reached the equilibrium, 300 µl of the culture filtrate previously described was added on both sides of the tissue to preserve the osmotic balance. Only tissues that showed an increase in Isc in response to glucose (indicating tissue viability) were included in the analysis (Fasano et al., 1991; Trucksis et al., 1993). The positive control was the EAST1 prototype strain 17-2.
Fig. 1. Frequence of the astA gene among different categories of diarrheagenic Escherichia coli.
1 2 3 4 5 6 7 8 9 10
Plasmid extraction Plasmid extraction was done using the QIAprep Miniprep Kit protocol.
Results The 358 bacterial strains studied using genetic probes and PCR showed that the astA gene was present in E. coli and Salmonella. We found the astA gene in 71 E. coli (32.6 %) and in 5 (11.9 %) Salmonella spp. strains. The astA gene was not found in the Shigella, Edwardsiella, Klebsiella, Proteus, Morganella, Enterobacter, and Citrobacter strains studied. Figure 1 shows the distribution of the astA gene among different categories of diarrheagenic E. coli. Most of the strains belonging to EHEC, EAggEC, and A-EPEC were positive for the astA gene. However, the astA gene was also found in a small proportion of EPEC, ETEC and non diarrheagenic E. coli (nonDEC). The gene was present in 2 of the 32 strains of EIEC. Table 1 shows the distribution of the astA gene according to O : H serotypes. There was no significant correlation among serotypes belonging to each E. coli category regarding the presence of the astA gene. The only exception was found in EPEC, where isolates be-
Fig. 2. PCR amplification results for the Salmonella agona strain. Lane 1 = Molecular size standard (1-kb DNA ladder); Subsequent lanes represent 50°C, 45°C and 40°C as annealing temperature, respectively. Lanes 2, 3 and 4 = negative controls; 5, 6 and 7 = positive controls; 8, 9 and 10 = Salmonella agona strain.
longing to serotypes O86 : H34, O111 : H32 and O127 : H40 did not possess the gene. The gene was found in one strain each of O55 : H6, O119 : H6, O142 : H6 and O142 : H34 serotypes. Among 5 strains of O127 : H6 serotype 3 isolates were astA-positive. To optimize the conditions for Salmonella, we modified the PCR annealing temperature to 50 °C. At this temperature, a band corresponding to the positive control astA gene was observed for Salmonella (S. agona) (Fig. 2, lane 8). In order to verify if the strains possessed biological activity, we performed Ussing chamber experiments with some serotypes of A-EPEC, EHEC, EAggEC,
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C. Paiva de Sousa, J. Daniel Dubreuil
Table 1. Distribuction of the astA sequence in EPEC, atypical-EPEC, EHEC, EAggEC, EIEC, ETEC and E. coli without virulence genes. Category
Serotype
N° of astA/total strains
EPEC eae, bfp, EAF
O55:H6 O86:H34 O111:H32 O119:H6 O127:H6 O127:H40 O142:H6 O142:H34
1/3 0/10 0/8 1/6 3/5 0/10 1/5 1/4 Total 7/51 (13.7%)
Atypical EPEC eae
bfp–, EAF–
O55:[H7] O111:[H9]
6/10 0/3 Total 6/13 (46.2%)
bfp, EAF–
O119:H2 O128:H2
5/5 3/6 Total 8/11 (72.7%)
EHEC eae, sxt
O26:H11 O111ac:[H8] O157:H7
4/6 5/6 13/13 Total 22/25 (88.0%)
EAggEC
AA, aaa– AA, aaa
O111:H12 O111:H21 O125:H21
6/6 2/2 5/7 Total 13/15 (86.6%) Total 2*/321 (6.3%)
EIEC ETEC
O78:H12 O6:H16 O159:H4
Others2
4/4 1/7 1/1 0/17 Total 6/29 (20.7%)
E. coli without known virulence genes
Total 7/42 (16.6%)
* Serotypes () = O144:H- (1) and O164:H- (1) 1 Serotypes (–) = O28ac:H- (3), O29:H- (3), O124ac:H- (3), O136ac:H- (3), O143ac:H- (3), O152ac:H- (3), O112ac:H- (4), O167ac:H- (4), O164:H- (4). 2 Serotypes (–) = O6:H? (2), O6:H6 (1), O153:H45 (4), O27:H7 (1), O78:H10 (1), O128ac:H7 (1), O128ac:H12 (1), O29:H21 (1), O27:H- (1) others (4).
EIEC, ETEC, and also with Salmonella strains carrying the astA gene. The majority of the strains of A-EPEC (87 %) and EaggEC (83 %) expressed the EAST1 toxin as judged from the positive response in Ussing chamber experiments. Two EIEC strains tested expressed the toxin, and one isolate of Salmonella agona (Isc 93.0) showed even a higher biological activity than the positive control (E. coli 17-2) (Isc 78.2).
Discussion Till now, it was accepted that only E. coli carry the astA gene. Our results, however, show that Salmonella also possess the gene, and at least one strain of S. agona was biologically active when tested in Ussing chamber experiments. Among the different E. coli categories, A-EPEC had not yet been investigated for the presence of the astA gene. We observed for the first time that A-EPEC possess the astA gene at a very high frequency, comparable to that found in EHEC and EAggEC. The percentage of EAST1 in EaggEC strains observed in this study (86.6 %) could be explained, in part, by the serotypes studied. Using a DNA probe it was shown that the astA gene was found in certain diarrheagenic E. coli categories and also in non-diarrheagenic E. coli (nonDEC) (Savarino et al., 1996). Our results confirm these observations as we found the astA gene in 16.6 % of non-DEC. The present study demonstrates that some EIEC strains possess the astA gene. Our results do not agree with those of other authors (Savarino et al., 1996), who did not find the gene in 55 EIEC strains studied. Some EPEC strains had the capacity to produce EAST1. It was reported that 14 of 65 strains of EPEC hybridized with the astA probe (Savarino et al., 1996). The significance of this toxin in EPEC pathogenesis is not known, but enteroadhesive factor-negative (EAFnegative) A-EPEC implicated in two diarrhoea cases in adults in Minnesota (USA) and in Finland possessed the astA gene (Hedberg et al., 1997). Our results show a low frequency of the gene in EPEC (13.7 %). There is some evidence that serotype O157 : H7 is presumably derived from O55 : H7. The O157 : H7 serotype is incriminated to cause food-related outbreaks, particularly related to undercooked minced meat and raw milk (Whittam et al., 1993). According to these studies, the same intimin is found in both serotypes (Pelayo, 1997). The results of our work show that all 13 EHEC strains belonging to O157 : H7 serotype had the astA gene. The implication of EAST1 in EHEC illness is not yet known but could probably explain the non-bloody diarrhoea seen in infected individuals. For the O157 : H7 serotype it was shown that the astA gene was on the chromosome, and in other E. coli categories, the gene was plasmid encoded. These results suggest that EAST1 could have been acquired by the O157 : H7 serotype during clonal evolution (Fasano, 1997). Some studies (Gomes et al., 1989; Pelayo, 1997; Scotland et al., 1991; Smith et al., 1990) had shown that probably A-EPEC is another EPEC category associated with diarrhea of clinical importance. It is probably rather early to try to classify A-EPEC (without the
Distribution and expression of the astA gene (EAST1 toxin) in Escherichia coli and Salmonella
EAF plasmid or with an incomplete plasmid, that is, without the EAF locus). A-EPEC strains could have evolved from an EPEC strain (that lost the EAF plasmid) or from an EHEC strain (that lost the phage that encodes Shiga toxin, -Stx). We reported (Sousa et al., 1997) that certain A-EPEC isolates possess the astA gene at a high frequency, similar to that found in EAggEC and in EHEC. Other results suggest similarities between A-EPEC and EHEC (Campos et al., 1994; Gonçalves et al., 1997; Rodrigues et al., 1996). It was shown that some strains of ETEC could express the EAST1 toxin (Savarino et al., 1996; Sousa et al., 1997) and other heat-stable and heat-labile toxins (Savarino et al., 1996; Savkovic et al., 1996). Among ETEC the astA prevalence varied according to the toxin phenotype. These data reported by other authors (Yamamoto and Echeverria, 1996; Yamamoto and Nakazawa, 1997) suggest that EAST1 is of pathophysiological significance. Our data shows that 20.7 % of ETEC strains possessed the astA gene. Statistical analysis (Chi-square tests) of the frequency of the astA gene revealed no significant differences between EHEC and A-EPEC (p 0.001) and EHEC and EAggEC (p 0.0001). In other categories of diarrheagenic E. coli the differences compared to EHEC, A-EPEC and EAggEC were significant. The same holds true for the differences between the E. coli strains studied (71/218) and the Salmonella strains (5/42) (p 0.0428). The virulence genes in Salmonella spp. are regulated by many environmental factors. For example, the expression of the inv gene is downregulated by anaerobic conditions. The expression of spvR, a regulator gene that controls the spv operon is stimulated by Rpos, a sigma factor that is believed to be produced in response to low concentration of nutrients (Gulig et al., 1993). The PhoP/PhoQ system that regulates the prg and pag genes seems to sense the pH, anaerobic conditions and carbon concentration. The Fur repressor, which is responsible for the regulation of the genes that are iron regulated, seems to regulate the acid-tolerance response (Francis et al., 1992). These environmentcontrolled mechanisms may explain the production of the EAST-1 toxin in Salmonella and the consequent importance of the astA gene for this bacterium. The astA gene has probably some importance in the pathogenesis of Salmonella-induced diarrhoea. Further experiments are needed to determine this more precisely. The virulence potential of the EAST1 toxin is not completely known. Because this toxin was found in both, ill and healthy individuals, we cannot discard the possibility that this toxin is capable to cause illness. In a recent study, Stephan and Untermann (1999) characterized fourteen verotoxin-producing E. coli strains isolated from stool samples of 14 different asymptomatic
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human carriers. Interestingly, these authors found the astA gene in the O113 : H4 serotype, that harbored three of the four additional virulence factors tested (60 MDa plasmid, E-hlyA and eae). These authors suggested that the astA gene could represent an additional pathogenesis determinant in E. coli diarrhea. As mentioned before, little is known about the role of the EAST1 toxin in the virulence of E. coli. Its high incidence in A-EPEC, EAggEC and EHEC strains may provide us with hints for a possible role played by this toxin in pathogenesis. Acknowledgements. This work was supported in part by grants n° 96/4185-5 from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP). We thank Dr. L. R. Trabulsi (Universidade de São Paulo) for providing lab facilities, Dr. T. A. T. Gomes (Universidade Federal de São Paulo) for colony hybridizations, and Dr. Alessio Fasano and his technician Klara Margaretten (Center for Vaccine Development, University of Maryland at Baltimore) for Ussing chamber experiments.
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Int. J. Med. Microbiol. 291, 15-20 (2001) © Urban & Fischer Verlag http://www.urbanfischer.de/journals/ijmm
Distribution and expression of the astA gene (EAST1 toxin) in Escherichia coli and Salmonella Cristina Paiva de Sousa1, J. Daniel Dubreuil2 1 2
Departamento de Nutrição, Centro de Ciências da Saúde, Universidade Federal da Paraíba, Campus Universitário I S/N, João Pessoa, PB, 58.970-900, Brasil Department of Pathology and Microbiology, Faculty of Veterinary Medicine, Montréal University, 3200 rue Sicotte, St-Hyacinthe, Québec, Canada J2S 7C6
Received August 28, 2000 · Revision received December 6, 2000 · Accepted December 6, 2000
Abstract The distribution and expression of the astA gene (EAST1 toxin) among 358 strains of Enterobacteriaceae were studied. The gene was found in 32.6 % and 11.9 % of Escherichia coli and Salmonella strains, respectively. The majority of E. coli EAST1-positive strains were found among EHEC (88.0 %), EAggEC (86.6 %), and A-EPEC (58.3 %). The gene was present in 16.6 % of E. coli strains without known virulence genes. There was no significant variation among the different serotypes of E. coli tested regarding the presence of the gene. For EPEC, 13.7 % of the tested strains were astA-positive. Among atypical EPEC (eae+, bfp–, EAF–) and (eae+, bfp+, EAF–) 46.2 and 72.7 %, respectively, were positive. The majority of the A-EPEC (87 %) and EaggEC (83 %) strains expressed the EAST-1 toxin as judged from Ussing chamber experiments. Of 32 EIEC strains studied, 2 possessed and expressed the gene as determined in Ussing chamber experiments. Among the Salmonella strains studied, five strains isolated from food were positive for astA and one strain of S. agona showed biological activity in Ussing chamber experiments. Key words: Escherichia coli – Salmonella – EAST 1 toxin – toxin gene
Introduction The Escherichia coli population is composed of commensal organisms that live harmlessly in the gut, opportunistic pathogens that occasionally infect normally sterile tissues, and specialized pathogens that possess specific virulence attributes promoting the ability of the organisms to cause disease. Enteroaggregative E. coli (EaggEC) are defined by their distinctive adherence pattern on HEp-2 cells in culture. The essential element of the aggregative phenotype is the stacked brick adherence pattern, in which
bacterial cells are seen to be lying side-by-side with an appreciable distinction of where one bacterium begins and another ends. EAggEC have epidemiologic properties that distinguish them from other categories of HEp-2 adherent E. coli. In several studies, EAggEC have been specifically associated with persistent diarrhea with variable intensity among children in the developing world (Bhan et al., 1989a, b; Cravioto et al., 1991; Wanke et al., 1991). However, recent outbreaks and volunteer studies suggest that EAggEC strains are virulent in adults (Smith et al., 1990, 1997) and have a global distribution (Itoh et al., 1997).
Corresponding author: Prof. Dr. J. Daniel Dubreuil, Department of Pathology and Microbiology, Faculty of Veterinary Medicine, Montréal University, 3200 rue Sicotte, St-Hyacinthe, Québec, Canada J2S 7C6, E-mail: [email protected] 1438-4221/01/291/1-015 $ 15.00/0
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The importance of colonization and its features have now been studied and it underscores the importance of the aggregative phenotype. Three possible mechanisms of enteric bacterial pathogenesis are generally accepted: toxin production, invasion, and the signal transduction phenotype seen in enteropathogenic E. coli (EPEC). There is, as yet, no evidence for the presence of a signal trasduction mechanism among EAggEC. Invasion has been described in HEp-2 cells by Benjamin et al. (1995). There is substantial evidence for the presence of enterotoxins elaborated by EAggEC. Savarino et al. (1993) described a homolog of the enterotoxigenic ETEC heat-stable (ST) toxin (designated EAST1 for enteroaggregative thermostable enterotoxin), which is produced by about 40 % of EAggEC strains associated with persistent diarrhea in children in Chile. They also reported that the prototype strain 17-2 (O3 : H2) produced an enterotoxin (4.1 kDa) comprising 38 amino acids (Savarino et al., 1991). The astA gene encodes the EAST1 enterotoxin, and cGMP is the intracellular mediator of the enterotoxic efect of EAST1 (Savarino et al., 1991). Although STa and EAST1 activate guanylate cyclase, they differ at the nucleotide level, suggesting that EAST1 evolved in a genetically different way of STa (Fasano, 1997). No co-hybridisation were noted for those toxins. More recently, Nataro and Kaper (1998), proposed a three stages model for the pathogenesis of EAggEc: i) initial adhesion to the intestinal mucosa; aggregative adherence fimbria type I (AAF/I) and type II (AAF/II) could facilitate the initial colonization; ii) increase in mucus production and biofilm formation; iii) cytotoxin production with damage to the intestinal cells. It was shown that most EAggEC strains harbor a 60 to 65 MDa plasmid which may encode the AA fimbria AAF/I (Nataro et al., 1993) or AAF/II (Czeczulin et al., 1997), and in some cases, EAST1 (Savarino et al., 1991). According to Czeczulin et al. (1999), EAggEC strains are a heterogeneous group of organisms with respect to chromosomal and plasmid-borne genes but the majority harbor a member of a conserved family of virulence plasmids. Comparison of plasmid and chromosomal relatedness of strains suggests clonality of chromosomal markers and a limited transfer model of plasmid distribution. The infection site of EAggEC is the human intestine. EAggEC strains are capable to adhere to the mucosa of the small bowel as well as to that of the large bowel (Nataro and Kaper, 1998). Classical EPEC share the eaeA and bfp genes and the EAF plasmid. They are responsible for diarrhea in children in developing countries. Atypical EPEC are EPEC that have lost the EAF plasmid. ETEC produce heatlabile (LT) or heat-stable toxins (STa and STb) that are also causing diarrhea. EHEC strains are implicated in food-borne diseases principally due to ingestion of un-
cooked minced meat and raw milk. Those strains produce shiga-like toxin 1 (stx1), shiga-like toxin 2 (stx2) and variants thereof. They are involved in episodes of diarrhea with complications. Serotype O157 : H7 is the prototype of increasing importance and is associated with hemorrhagic colitis, bloody diarrhea and the hemolytic uremic syndrome (HUS). EIEC cause a broad spectrum of human diseases. They are closely related to Shigella spp. Their invasive capacity is conferred by the inv plasmid. DAEC strains are diffusely adhering E. coli that are also implicated with episodes of diarrhea. This study was undertaken to determine the distribution and expression of the astA gene among different classes of E. coli and in Salmonella.
Materials and methods Bacteria and culture media Three hundred and fifty eight bacterial isolates belonging to the Enterobacteriaceae were studied, including Escherichia coli [218], Salmonella [42], Shigella [13], Edwardsiella [10], Klebsiella [11], Proteus [21], Morganella [13], Citrobacter [2], and Enterobacter [28]. The E. coli strains studied were previously classified as EPEC [51], atypical EPEC (A-EPEC) [24], enterohaemorrhagic E. coli (EHEC) [25], EAggEC [15], enteroinvasive E. coli (EIEC) [32], ETEC [29] and strains without known virulence genes (non-DEC) [42]. All E. coli strains were from Dr. Trabulsi’s laboratory (University of São Paulo) and other strains were kindly provided by Dr. Iaria’s lab (University of São Paulo). The majority of strains were from diseased humans. All bacterial strains were kept on agar slants at room temperature until the moment of use. DNA probe and colony blot The DNA probe was constructed using the prototype strain EAggEC 17-2. We amplified the astA gene using the following primers: EAST11a (5-CCATCAACACAGTATAT-3) and EAST11b (5-GGTCGCGAGTGACGGCTTTGT-3) (Itoh et al., 1997)). These primers gave a product of 111 bp, that was visualized after electrophoresis on 2 % agarose gels stained with ethidium bromide. The DNA fragment was excized from the gel, purified with the PCR Wizard Kit (Promega), and used as a DNA probe. The probe was labeled with [α-32P]dATP by random priming using a Stratagene kit, according to the manufacturer’s instructions. Colony blots onto Whatman filter n° 541 were prepared according to Maas (1983). Hybridizations and washes were done under stringent conditions (Grunstein and Hogness, 1975). PCR The primers used are described in the preceding section. Cycle conditions used were: denaturation 30 s 95 °C, annealing 120 s 55 °C and extension 120 s 72 °C for 30 cycles. The reaction conditions were: buffer 1, 100 pmol of each primer, 200 µM of each dNTPs, 1.0 mM MgCl2 and 1 U of Taq poly-
Distribution and expression of the astA gene (EAST1 toxin) in Escherichia coli and Salmonella
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merase. The amplified product was analyzed by electrophoresis on a 2 % agarose gel, stained with ethidium bromide and visualized with UV. We used a 1-kb DNA ladder as molecular marker (Life Technologies, Gaithersburg, MD). To detect the astA sequence in Salmonella, we modified the annealing temperature from 55 to 50 °C (see Fig. 2). All other conditions were the same as described before. Ussing chamber experiments Experiments on the biological activity of culture filtrates were conducted in Dr. Fasano’s Laboratory in the Center for Vaccine Development (CVD) at the University of Maryland (Baltimore). Bacterial strains were grown in Luria Bertani Broth at 37 °C for 18 hours with shaking (500 rpm). Culture filtrates were prepared by centrifugation of the cell suspension (Sorvall RT 6000 D) and passage of the supernatant through a Mr 20,000 cut-off Centristar I filter (Sartorius), under conditions suggested by the manufacturer. Ussing chamber experiments were done as previously described, using stripped rabbit ileal tissue (Trucksis et al., 1993). Potential difference and short-circuit current (Isc) were measured, and tissue conductance was calculated (data not shown). Once the tissue reached the equilibrium, 300 µl of the culture filtrate previously described was added on both sides of the tissue to preserve the osmotic balance. Only tissues that showed an increase in Isc in response to glucose (indicating tissue viability) were included in the analysis (Fasano et al., 1991; Trucksis et al., 1993). The positive control was the EAST1 prototype strain 17-2.
Fig. 1. Frequence of the astA gene among different categories of diarrheagenic Escherichia coli.
1 2 3 4 5 6 7 8 9 10
Plasmid extraction Plasmid extraction was done using the QIAprep Miniprep Kit protocol.
Results The 358 bacterial strains studied using genetic probes and PCR showed that the astA gene was present in E. coli and Salmonella. We found the astA gene in 71 E. coli (32.6 %) and in 5 (11.9 %) Salmonella spp. strains. The astA gene was not found in the Shigella, Edwardsiella, Klebsiella, Proteus, Morganella, Enterobacter, and Citrobacter strains studied. Figure 1 shows the distribution of the astA gene among different categories of diarrheagenic E. coli. Most of the strains belonging to EHEC, EAggEC, and A-EPEC were positive for the astA gene. However, the astA gene was also found in a small proportion of EPEC, ETEC and non diarrheagenic E. coli (nonDEC). The gene was present in 2 of the 32 strains of EIEC. Table 1 shows the distribution of the astA gene according to O : H serotypes. There was no significant correlation among serotypes belonging to each E. coli category regarding the presence of the astA gene. The only exception was found in EPEC, where isolates be-
Fig. 2. PCR amplification results for the Salmonella agona strain. Lane 1 = Molecular size standard (1-kb DNA ladder); Subsequent lanes represent 50°C, 45°C and 40°C as annealing temperature, respectively. Lanes 2, 3 and 4 = negative controls; 5, 6 and 7 = positive controls; 8, 9 and 10 = Salmonella agona strain.
longing to serotypes O86 : H34, O111 : H32 and O127 : H40 did not possess the gene. The gene was found in one strain each of O55 : H6, O119 : H6, O142 : H6 and O142 : H34 serotypes. Among 5 strains of O127 : H6 serotype 3 isolates were astA-positive. To optimize the conditions for Salmonella, we modified the PCR annealing temperature to 50 °C. At this temperature, a band corresponding to the positive control astA gene was observed for Salmonella (S. agona) (Fig. 2, lane 8). In order to verify if the strains possessed biological activity, we performed Ussing chamber experiments with some serotypes of A-EPEC, EHEC, EAggEC,
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C. Paiva de Sousa, J. Daniel Dubreuil
Table 1. Distribuction of the astA sequence in EPEC, atypical-EPEC, EHEC, EAggEC, EIEC, ETEC and E. coli without virulence genes. Category
Serotype
N° of astA/total strains
EPEC eae, bfp, EAF
O55:H6 O86:H34 O111:H32 O119:H6 O127:H6 O127:H40 O142:H6 O142:H34
1/3 0/10 0/8 1/6 3/5 0/10 1/5 1/4 Total 7/51 (13.7%)
Atypical EPEC eae
bfp–, EAF–
O55:[H7] O111:[H9]
6/10 0/3 Total 6/13 (46.2%)
bfp, EAF–
O119:H2 O128:H2
5/5 3/6 Total 8/11 (72.7%)
EHEC eae, sxt
O26:H11 O111ac:[H8] O157:H7
4/6 5/6 13/13 Total 22/25 (88.0%)
EAggEC
AA, aaa– AA, aaa
O111:H12 O111:H21 O125:H21
6/6 2/2 5/7 Total 13/15 (86.6%) Total 2*/321 (6.3%)
EIEC ETEC
O78:H12 O6:H16 O159:H4
Others2
4/4 1/7 1/1 0/17 Total 6/29 (20.7%)
E. coli without known virulence genes
Total 7/42 (16.6%)
* Serotypes () = O144:H- (1) and O164:H- (1) 1 Serotypes (–) = O28ac:H- (3), O29:H- (3), O124ac:H- (3), O136ac:H- (3), O143ac:H- (3), O152ac:H- (3), O112ac:H- (4), O167ac:H- (4), O164:H- (4). 2 Serotypes (–) = O6:H? (2), O6:H6 (1), O153:H45 (4), O27:H7 (1), O78:H10 (1), O128ac:H7 (1), O128ac:H12 (1), O29:H21 (1), O27:H- (1) others (4).
EIEC, ETEC, and also with Salmonella strains carrying the astA gene. The majority of the strains of A-EPEC (87 %) and EaggEC (83 %) expressed the EAST1 toxin as judged from the positive response in Ussing chamber experiments. Two EIEC strains tested expressed the toxin, and one isolate of Salmonella agona (Isc 93.0) showed even a higher biological activity than the positive control (E. coli 17-2) (Isc 78.2).
Discussion Till now, it was accepted that only E. coli carry the astA gene. Our results, however, show that Salmonella also possess the gene, and at least one strain of S. agona was biologically active when tested in Ussing chamber experiments. Among the different E. coli categories, A-EPEC had not yet been investigated for the presence of the astA gene. We observed for the first time that A-EPEC possess the astA gene at a very high frequency, comparable to that found in EHEC and EAggEC. The percentage of EAST1 in EaggEC strains observed in this study (86.6 %) could be explained, in part, by the serotypes studied. Using a DNA probe it was shown that the astA gene was found in certain diarrheagenic E. coli categories and also in non-diarrheagenic E. coli (nonDEC) (Savarino et al., 1996). Our results confirm these observations as we found the astA gene in 16.6 % of non-DEC. The present study demonstrates that some EIEC strains possess the astA gene. Our results do not agree with those of other authors (Savarino et al., 1996), who did not find the gene in 55 EIEC strains studied. Some EPEC strains had the capacity to produce EAST1. It was reported that 14 of 65 strains of EPEC hybridized with the astA probe (Savarino et al., 1996). The significance of this toxin in EPEC pathogenesis is not known, but enteroadhesive factor-negative (EAFnegative) A-EPEC implicated in two diarrhoea cases in adults in Minnesota (USA) and in Finland possessed the astA gene (Hedberg et al., 1997). Our results show a low frequency of the gene in EPEC (13.7 %). There is some evidence that serotype O157 : H7 is presumably derived from O55 : H7. The O157 : H7 serotype is incriminated to cause food-related outbreaks, particularly related to undercooked minced meat and raw milk (Whittam et al., 1993). According to these studies, the same intimin is found in both serotypes (Pelayo, 1997). The results of our work show that all 13 EHEC strains belonging to O157 : H7 serotype had the astA gene. The implication of EAST1 in EHEC illness is not yet known but could probably explain the non-bloody diarrhoea seen in infected individuals. For the O157 : H7 serotype it was shown that the astA gene was on the chromosome, and in other E. coli categories, the gene was plasmid encoded. These results suggest that EAST1 could have been acquired by the O157 : H7 serotype during clonal evolution (Fasano, 1997). Some studies (Gomes et al., 1989; Pelayo, 1997; Scotland et al., 1991; Smith et al., 1990) had shown that probably A-EPEC is another EPEC category associated with diarrhea of clinical importance. It is probably rather early to try to classify A-EPEC (without the
Distribution and expression of the astA gene (EAST1 toxin) in Escherichia coli and Salmonella
EAF plasmid or with an incomplete plasmid, that is, without the EAF locus). A-EPEC strains could have evolved from an EPEC strain (that lost the EAF plasmid) or from an EHEC strain (that lost the phage that encodes Shiga toxin, -Stx). We reported (Sousa et al., 1997) that certain A-EPEC isolates possess the astA gene at a high frequency, similar to that found in EAggEC and in EHEC. Other results suggest similarities between A-EPEC and EHEC (Campos et al., 1994; Gonçalves et al., 1997; Rodrigues et al., 1996). It was shown that some strains of ETEC could express the EAST1 toxin (Savarino et al., 1996; Sousa et al., 1997) and other heat-stable and heat-labile toxins (Savarino et al., 1996; Savkovic et al., 1996). Among ETEC the astA prevalence varied according to the toxin phenotype. These data reported by other authors (Yamamoto and Echeverria, 1996; Yamamoto and Nakazawa, 1997) suggest that EAST1 is of pathophysiological significance. Our data shows that 20.7 % of ETEC strains possessed the astA gene. Statistical analysis (Chi-square tests) of the frequency of the astA gene revealed no significant differences between EHEC and A-EPEC (p 0.001) and EHEC and EAggEC (p 0.0001). In other categories of diarrheagenic E. coli the differences compared to EHEC, A-EPEC and EAggEC were significant. The same holds true for the differences between the E. coli strains studied (71/218) and the Salmonella strains (5/42) (p 0.0428). The virulence genes in Salmonella spp. are regulated by many environmental factors. For example, the expression of the inv gene is downregulated by anaerobic conditions. The expression of spvR, a regulator gene that controls the spv operon is stimulated by Rpos, a sigma factor that is believed to be produced in response to low concentration of nutrients (Gulig et al., 1993). The PhoP/PhoQ system that regulates the prg and pag genes seems to sense the pH, anaerobic conditions and carbon concentration. The Fur repressor, which is responsible for the regulation of the genes that are iron regulated, seems to regulate the acid-tolerance response (Francis et al., 1992). These environmentcontrolled mechanisms may explain the production of the EAST-1 toxin in Salmonella and the consequent importance of the astA gene for this bacterium. The astA gene has probably some importance in the pathogenesis of Salmonella-induced diarrhoea. Further experiments are needed to determine this more precisely. The virulence potential of the EAST1 toxin is not completely known. Because this toxin was found in both, ill and healthy individuals, we cannot discard the possibility that this toxin is capable to cause illness. In a recent study, Stephan and Untermann (1999) characterized fourteen verotoxin-producing E. coli strains isolated from stool samples of 14 different asymptomatic
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human carriers. Interestingly, these authors found the astA gene in the O113 : H4 serotype, that harbored three of the four additional virulence factors tested (60 MDa plasmid, E-hlyA and eae). These authors suggested that the astA gene could represent an additional pathogenesis determinant in E. coli diarrhea. As mentioned before, little is known about the role of the EAST1 toxin in the virulence of E. coli. Its high incidence in A-EPEC, EAggEC and EHEC strains may provide us with hints for a possible role played by this toxin in pathogenesis. Acknowledgements. This work was supported in part by grants n° 96/4185-5 from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP). We thank Dr. L. R. Trabulsi (Universidade de São Paulo) for providing lab facilities, Dr. T. A. T. Gomes (Universidade Federal de São Paulo) for colony hybridizations, and Dr. Alessio Fasano and his technician Klara Margaretten (Center for Vaccine Development, University of Maryland at Baltimore) for Ussing chamber experiments.
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