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Identification and characterization of “pathoadaptive mutations” of the cadBA operon in several intestinal Escherichia coli
ARTICLE IN PRESS
International Journal of Medical Microbiology 296 (2006) 547–552 www.elsevier.de/ijmm
SHORT COMMUNICATION
Identification and characterization of ‘‘pathoadaptive mutations’’ of the cadBA operon in several intestinal Escherichia coli Joerg Joresa,, Alfredo G. Torresb, Sylke Wagnera, Christopher B. Tuttb, James B. Kaperc, Lothar H. Wielera a
Institut fu¨r Mikrobiologie und Tierseuchen, Freie Universita¨t Berlin, Philippstraße 13, D-10115 Berlin, Germany Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX 77555-1070, USA c Center for Vaccine Development, School of Medicine, University of Maryland, 685 W. Baltimore Street, Baltimore, MD 21201, USA b
Received 27 September 2005; received in revised form 27 June 2006; accepted 13 July 2006
Abstract The dysenteric Shigella spp. and enteroinvasive Escherichia coli (EIEC) have evolved from commensal E. coli by the acquisition of a virulence plasmid and inactivation of genes of the cad locus encoding lysine decarboxylase (LDC) by so-called pathoadaptive mutation. As horizontal gene transfer and recombination occurs frequently in E. coli we were interested to see if similar pathoadaptive mutations are commonly present in other intestinal pathotypes. Therefore, we examined 140 intestinal E. coli strains of various pathotypes and the ECOR collection for their ability to decarboxylate lysine, and identified 25 strains that were unable to do so. Complementation of a Shiga toxin-producing E. coli and two enteropathogenic E. coli strains, both LDC-negative, with the intact cad locus restored LDC activity and resulted in a reduction in adherence to tissue culture cells. We investigated the cad locus for possible alterations by using hybridization and PCR techniques and compared the results with the alterations reported for Shigella spp. and EIEC strains. Interestingly, the alterations of the cad genes were similar to those previously reported, pointing towards a parallel evolution of LDC silencing in different intestinal E. coli pathotypes. r 2006 Elsevier GmbH. All rights reserved. Keywords: cadBA; Lysine decarboxylase; E. coli; Pathoadaptive mutation
Introduction Escherichia coli is one of the most fascinating members of the prokaryotes, since this species is an important representative of the autochthonous mammalian gut flora, and in addition it harbours a broad spectrum of diverse pathotypes, causing intestinal as Corresponding author. Present address: International Livestock Research Institute, Box 30709, Nairobi 00100, Kenya. Tel.: +254 20 422 3000; fax: +254 2 422 3001. E-mail address: [email protected] (J. Jores).
1438-4221/$ - see front matter r 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.ijmm.2006.07.002
well as extra-intestinal disease. The different pathotypes are characterized by a specific pattern of virulence traits, which are a result of lateral gene transfer (Kaper et al., 2004). Another source of specification reported for members of the enteroinvasive E. coli (EIEC) and several Shigella spp. are alterations encompassing the cadBA operon encoding lysine decarboxylase (LDC) (Maurelli et al., 1998). This enzyme synthesizes the polyamine cadaverine, which was shown to act in an antagonistic fashion to the enterotoxins of Shigella flexneri (Maurelli et al., 1998) and to inhibit the induction of transepithelial migration of
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polymorphonuclear neutrophils (McCormick et al., 1999). Transepithelial neutrophil migration has also been reported for enteropathogenic E. coli (EPEC) (Savkovic et al., 1996; Michail et al., 2003), raising the question of the evolution of similar pathoadaptive mutations in these pathogens. Further, it has been shown that the inactivation of the cadA gene in enterohemorrhagic E. coli (EHEC) O157:H7 had an effect on the ability of this strain to adhere to HeLa cells (Torres and Kaper, 2003) and that pathoadaptive mutations of the cadBA operon mediate increased adherence to HeLa cells in three out of four STEC strains of serogroups O26 and O111 (Torres et al., 2005). In Shigella spp. mostly deletions and rearrange ments of the cadBA operon are responsible for a silencing of LDC (Day et al., 2001), while in EIEC insertions of IS within the cadC gene as well as point mutations of its promotor account for this phenotype (Casalino et al., 2003). The Shigella/EIEC pathotype has originated several times by convergent evolution (Lan and Reeves, 2002; Lan et al., 2004; Wirth et al., 2006), giving an explanation for the different cadBA silencing strategies within the Shigella/EIEC pathotype. Since different E. coli pathotypes cause diarrhoea/enteritis and horizontal gene transfer and recombinations occur frequently in E. coli we were interested in ascertaining the distribution of ‘‘pathoadaptive mutations’’ in other intestinal E. coli. Therefore, 212 E. coli strains, representing many currently known intestinal pathotypes and the ECOR collection (Ochman and Selander, 1984), were screened for their LDC activity. Using PCR and hybridization experiments, we determined the alterations of the cad locus in those strains showing an LDC-negative phenotype.
Material and methods Bacterial strains We investigated 212 E. coli strains including 140 intestinal E. coli displaying 81 serotypes and the ECOR collection (Ochman and Selander, 1984). The strains tested are listed in Table S1 (supplementary online material). Bacterial strains were routinely grown in Luria–Bertani (LB) broth or on LB agar at 37 1C. When needed, ampicillin was added to media at 100 mg/ml. The plasmids pBR322 and pCadABC (Torres et al., 2005) were introduced into the LDC-negative EPEC and STEC strains by electroporation as described by Dower et al. (1988). Bacterial growth was monitored for all strains to confirm that the presence of the plasmids did not have an effect on the ability of the strains to grow in different media.
Bacterial adhesion to epithelial cells For quantitative adhesion assays, E. coli strains were evaluated for their ability to adhere to HeLa cell monolayers as previously described (Torres et al., 2005). Briefly, the strains were grown in LB broth overnight at 37 1C and added to tissue culture cells replenished with fresh DMEM at a concentration of 1 107 bacteria per well for 3 h at 37 1C. The monolayers were washed, fixed and stained with Giemsa solution for microscopic evaluation, or bacteria were recovered with 0.1% Triton X-100 in phosphatebuffered saline (PBS, pH 7.4) and plated on LB agar plates for quantification.
Lysine decarboxylase assay To determine the LDC activity, strains were grown in Moeller decarboxylase broth with lysine (BD BBL, Fisher Scientific). Results were read after incubation for 24 or 48 h at 37 1C. S. flexneri 2457T served as negative, and E. coli MG1655 as positive control.
Polymerase chain reaction (PCR) and DNA–DNA hybridization HerkulaseTM Enhanced Polymerase Blend (Stratagene, Amsterdam, The Netherlands) was used for PCR experiments as recommended by the manufacturer. Hybridization was performed with digoxigenin-labelled probes by using the Roche Labeling and Detection Kit (Roche, Mannheim, Germany) according to the vendor’s protocols and 500 ng of total bacterial DNA per dot. The cadA-specific probe was synthesized using PCR employing primers A1–A2, while probe cadB was generated by using primers B1–B3 (Casalino et al., 2003). We used E. coli K-12 strain MG1655 as template for generating both probes. The region between the pheU and cadB gene encompassing the cadC gene was amplified using primers B2 and C2 (Casalino et al., 2003), and presence of pINV was examined by PCR employing primers specific for ipaC (Boudeau et al., 1999).
Multilocus sequence typing Multilocus sequence typing (MLST) has been carried out as recently described (Wirth et al., 2006).
Results and discussion Out of 212 E. coli strains tested for LDC activity, 25 strains belonging to the EHEC, EPEC, enterotoxigenic E. coli (ETEC) pathotypes and non-pathogenic
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Table 1.
Characteristics of LCD-negative E. coli
Strain designation
Serotype
Country
Pathotype
cadA/cadB
0202-1 0421-1 0471-1 055/89 1112-6 1582-4 3632-2 CL-37 DEC8A DEC8B DEC8E DEC9B ECOR17 GS1128-1 GS1187-1 IHIT0554 IHIT0597 IHIT1190 IHIT1703 IHIT1968 IHIT2115 IHIT2430 PS37 WH 02/24/007-2 WUS-02/09/010-1
R:H11,21,41 O162:H33 NT:H19 O2:H5 R:H11,21 O162:NM NT:H19 O111:H8 O111:NM O111:H8 O111:H8 O26:NM O106:NM On.t.:NM On.t.:NM On.t.:NM O157:NM O92:NM O111:H2 O119:H25 O4:NM O80:NM O9:K35:K99 O165:H25 O165:H25
Brazil Brazil Brazil Germany Brazil Brazil Brazil Canada USA USA Denmark USA Indonesia Germany Germany Germany Germany Germany Germany Germany Germany Germany Germany Germany Germany
EPEC EPEC EPEC STEC EPEC EPEC EPEC EHEC EHEC EHEC EHEC EHEC
No No No Yes No No Yes No No No No No No Yes Yes No No No No No No No No No No
a
549
EHEC EHEC EHEC EHEC EHEC EHEC EHEC EHEC EHEC ETEC EHEC EHEC
ST-Clpx/STa
None/1 10/378 29/16 29/16 29/294 29/29 None/47
10/304 10/165 306/300 29/16 306/300 10/301 10/301 None/120 None/119 None/119
ST-Clpx-sequence type complex/ST-sequence type according to http://web.mpib-berlin.mpg.de/mlst/dbs/Ecoli.
ECOR17 (see Table 1) were LDC-negative. Out of these, four common diarrhoeagenic E. coli (DEC) strains (Whittam et al., 1993), namely DEC8A, DEC8B, DEC8E and DEC9B belonging to the important serogroups O111 and O26 were LDCnegative, which is in good accordance to results reported recently (Torres et al., 2005). An LDC-negative phenotype has also been previously reported in three EHEC isolated from children in Brazil, but the cadBA operon has not been investigated in these strains (Guth et al., 2002). To gain insight into the nature of the LDC-negative phenotype, we investigated the cadBA operon more thoroughly by using dot blot hybridization of total genomic DNA and probes specific for the cadA and cadB gene (Fig. 1A,B). Twenty-one strains did not react with either the cadA or cadB probe indicating a deletion similar to the one reported for Shigella boydii 18 (Day et al., 2001). In contrast, four out of all 25 LDC-negative strains reacted with both probes. We therefore investigated the cadC gene encoding the CadBA regulator of these strains by PCR employing primers B2 and C2 (Casalino et al., 2003). Interestingly, STEC 055/89 (O2:H5) showed a PCR amplicon of 3.8 kb in size instead of
the expected 2.5 kb fragment (data not shown), suggesting that an insertion in the cadC gene has occurred, probably an IS element as previously described for EIEC (Casalino et al., 2003). Sequence analysis of the 3.8-kb PCR amplicon confirmed the insertion of an IS element (showing compelling similarity to IS2), 303 bp downstream of the translational start site of cadC. It was recently proposed that inactivation of the cadC gene is the first step in silencing of the cadBA operon followed by a spread of IS elements into cadA and cadB (Casalino et al., 2005). The remaining three strains (3632-2, GS11281 and GS1187-1) showed neither deletions nor an inactivated regulator, although they clearly reacted negatively in their ability to decarboxylate lysine. After introduction of the plasmid pCadABC into EPEC 36322 (see details below) LDC activity was restored, indicating that a mutation(s) in the cadBA operon or its regulator gene account for the LDC silencing as speculated already for some EIEC strains (Casalino et al., 2003). In order to determine whether the observed LDCnegative phenotype represents a pathoadaptive mutation, we investigated the adhesion ability of three selected strains, namely EPEC 3632-2, EPEC 1112-6,
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A
B
C
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
27
160
CFU / ml ( x106)
140 *
120
**
100 80
***
60 40 20
055 / 89 (pCadABC)
055 / 89 (pBR322)
3632 - 2 (pCadABC)
3632 - 2 (pBR322)
1112 - 6 (pCadABC)
1112-6 (pBR322)
0
* P < 0.05; ** P > 0.05; *** P < 0.01
Fig. 1. Dot blot hybridization, using probes specific for detection of cadA (A) and cadB (B). DNA from lysine decarboxylase negative E. coli was tested: (1) IHIT0597; (2) IHIT0554; (3) IHIT1190; (4) IHIT1703; (5) IHIT1968; (6) IHIT2115; (7) IHIT2430; (8) WH 02/24/007-2; (9) WUS-02/09/010-1; (10) PS37; (11) DEC8E; (12) DEC8B; (13) GS1128-1; (14) GS1187-1; (15) DEC8A; (16) DEC9B; (17) 0202-1; (18) 0421-1; (19) 0471-1; (20) 1112-6; (21) 1582-4; (22) 3632-2; (23) CL-37; (24) ECOR17; (25) 055/89; (26) MG1655; and (27) Lambda. (C) Chart displaying the adherence to HeLa cells of STEC 055/89 (white columns), EPEC 1112-6 (grey columns) and EPEC 3632-2 (black columns) carrying plasmids pBR322 or pCadABC. The statistical difference between each strain containing pCadABC vs. pBR322 was determined by a Student’s t-test analysis.
and STEC 55/89. We selected these strains, because they belong to pathotypes so far not reported to be LDCnegative and showed different alterations of the cadBA operon. After introduction of plasmid pCadABC carrying the cadBA operon the LDC-positive phenotype was restored. Further, we observed a reduction in adhesion properties in such strains, suggesting the existence of pathoadaptive mutations affecting the adherence properties of these strains (Fig. 1C). Because it was previously shown for STEC strains of the serogroup O111 that a reduction in intimin expression due to LDC activity has an effect in adhesion (Torres et al., 2005), we investigated whether changes in intimin expression were responsible for the reduction in adhesion observed in our EPEC strains (3632-2 and 1112-6). No reduction in
intimin expression was observed in the EPEC strains harbouring the plasmid pCadABC as compared to the pBR322-containing control strains (data not shown). We speculate that this result is due to differences in the in vitro regulatory mechanisms controlling intimin expression in EPEC strains or because the cad locus is controlling the expression of other adhesins in EPEC. So far, our data showed that the so-called pathoadaptive alterations of the cad loci are not restricted to EIEC, EHEC and Shigella spp., but can also be found in at least three other pathotypes, namely EPEC, STEC and ETEC. In S. flexneri the absence of cadaverine has been demonstrated to enhance the impact of its invasion process (Maurelli et al., 1998; McCormick et al., 1999).
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Since horizontal gene transfer is frequently observed in E. coli, we investigated the possible presence of the virulence plasmid pINV in the 25 LDC-negative E. coli. None of the 25 strains possessed the virulence plasmid, pointing towards another evolutionary force leading to the inactivation of the cadBA operon. Additionally, we MLST-typed 17 out of the 25 LDC-negative strains (see Table 1) according to the MLST scheme published recently (Wirth et al., 2006). By doing so, we could prove these 17 strains belong to three different sequence type complexes as well as 12 different sequence types. Therefore we propose a parallel evolution of LDC silencing within the E. coli population. Interestingly, different strains with the same sequence type (ST29 and ST300, see Table S1, supplementary online material) can be either LDCnegative or LDC-positive, in contrast to Shigella spp. and EIEC. Obviously, the cadBA operon alterations within Shigella spp. and EIEC are rather fixed in contrast to other E. coli pathotypes. Moreover it is likely that recombination events as a result of horizontal gene transfer could be attributed to the cadBA alterations reported here. This could be a hitchhiking effect due to the insertion of foreign DNA into the pheU tRNA gene, which often serves as target for pathogenicity islands in E. coli and in Shigella spp. (Rumer et al., 2003; Schmidt and Hensel, 2004). As we discovered similar mutations in several independently evolved lines (different serotypes, pathotypes and sequence type complexes), we propose that these mutations are beneficial for the bacteria in ways either involving pathogenicity or in terms of competition with other bacteria occupying the same ecological niche. It is also likely that such mutations represent a pivotal evolutionary step for the pathogens competing with other bacteria for the same ecological niche, because several gastrointestinal bacteria produce cadaverine, which theoretically could hamper the virulence of pathogens such as Shigella and EIEC. Future work is needed to elucidate the exact role of these ‘‘pathoadaptive mutations’’ in non-Shigella/EIEC members of the E. coli population.
Acknowledgment This work was supported by Grant WI 1436/3-2 from the Deutsche Forschungsgemeinschaft and by the Welcome Trust financed IPRAVE Consortium. Sylke Wagner was financed from NaFo¨G. Alfredo Torres’ laboratory is supported in part by institutional funds from the UTMB John Sealy Memorial Endowment Fund for Biomedical Research. We thank Dr. Fasano, Dr. Whittam, Dr. Gomes, Dr. Gene and Dr. Karch for providing strains and Ju¨rgen Eichberg for excellent technical support. James Kaper’s laboratory is sup-
551
ported in part by grants NIH Grants AI21657 and DK58957.
Appendix A. Supplementary material The online version of this article contains additional supplementary data. Please visit doi:10.1016/ j.ijmm.2006.07.002.
References Boudeau, J., Glasser, A.L., Masseret, E., Joly, B., DarfeuilleMichaud, A., 1999. Invasive ability of an Escherichia coli strain isolated from the ileal mucosa of a patient with Crohn’s disease. Infect. Immun. 67, 4499–4509. Casalino, M., Latella, M.C., Prosseda, G., Colonna, B., 2003. CadC is the preferential target of a convergent evolution driving enteroinvasive Escherichia coli toward a lysine decarboxylase-defective phenotype. Infect. Immun. 71, 5472–5479. Casalino, M., Latella, M.C., Prosseda, G., Ceccarini, P., Grimont, F., Colonna, B., 2005. Molecular evolution of the lysine decarboxylase-defective phenotype in Shigella sonnei. Int. J. Med. Microbiol. 294, 503–512. Day Jr., W.A., Fernandez, R.E., Maurelli, A.T., 2001. Pathoadaptive mutations that enhance virulence: genetic organization of the cadA regions of Shigella spp. Infect. Immun. 69, 7471–7480. Dower, W.J., Miller, J.F., Ragsdale, C.W., 1988. High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res. 16, 6127–6145. Guth, B.E., Ramos, S.R., Cerqueira, A.M., Andrade, J.R., Gomes, T.A., 2002. Phenotypic and genotypic characteristics of shiga toxin-producing Escherichia coli strains isolated from children in Sao Paulo, Brazil. Mem. Inst. Oswaldo Cruz 97, 1085–1089. Kaper, J.B., Nataro, J.P., Mobley, H.L., 2004. Pathogenic Escherichia coli. Nat. Rev. Microbiol. 2, 123–140. Lan, R., Reeves, P.R., 2002. Escherichia coli in disguise: molecular origins of Shigella. Microbes Infect. 4, 1125–1132. Lan, R., Alles, M.C., Donohoe, K., Martinez, M.B., Reeves, P.R., 2004. Molecular evolutionary relationships of enteroinvasive Escherichia coli and Shigella spp. Infect. Immun. 72, 5080–5088. Maurelli, A.T., Fernandez, R.E., Bloch, C.A., Rode, C.K., Fasano, A., 1998. ‘‘Black holes’’ and bacterial pathogenicity: a large genomic deletion that enhances the virulence of Shigella spp. and enteroinvasive Escherichia coli. Proc. Natl. Acad. Sci. USA 95, 3943–3948. McCormick, B.A., Fernandez, M.I., Siber, A.M., Maurelli, A.T., 1999. Inhibition of Shigella flexneri-induced transepithelial migration of polymorphonuclear leucocytes by cadaverine. Cell. Microbiol. 1, 143–155. Michail, S.K., Halm, D.R., Abernathy, F., 2003. Enteropathogenic Escherichia coli: stimulating neutrophil migration across a cultured intestinal epithelium without altering transepithelial conductance. J. Pediatr. Gastroenterol. Nutr. 36, 253–260.
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Ochman, H., Selander, R.K., 1984. Standard reference strains of Escherichia coli from natural populations. J. Bacteriol. 157, 690–693. Rumer, L., Jores, J., Kirsch, P., Cavignac, Y., Zehmke, K., Wieler, L.H., 2003. Dissemination of pheU- and pheVlocated genomic islands among enteropathogenic (EPEC) and enterohemorrhagic (EHEC) E. coli and their possible role in the horizontal transfer of the locus of enterocyte effacement (LEE). Int. J. Med. Microbiol. 292, 463–475. Savkovic, S.D., Koutsouris, A., Hecht, G., 1996. Attachment of a noninvasive enteric pathogen, enteropathogenic Escherichia coli, to cultured human intestinal epithelial monolayers induces transmigration of neutrophils. Infect. Immun. 64, 4480–4487. Schmidt, H., Hensel, M., 2004. Pathogenicity islands in bacterial pathogenesis. Clin. Microbiol. Rev. 17, 14–56.
Torres, A.G., Kaper, J.B., 2003. Multiple elements controlling adherence of enterohemorrhagic Escherichia coli O157:H7 to HeLa cells. Infect. Immun. 71, 4985–4995. Torres, A.G., Vazquez-Juarez, R.C., Tutt, C.B., GarciaGallegos, J.G., 2005. Pathoadaptive mutation that mediates adherence of shiga toxin-producing Escherichia coli O111. Infect. Immun. 73, 4766–4776. Whittam, T.S., Wolfe, M.L., Wachsmuth, I.K., Orskov, F., Orskov, I., Wilson, R.A., 1993. Clonal relationships among Escherichia coli strains that cause hemorrhagic colitis and infantile diarrhea. Infect. Immun. 61, 1619–1629. Wirth, T., Falush, D., Lan, R., Mensa, P., Wieler, L.H., Karch, H., Reeves, P.R., Maiden, M.C.J., Ochman, H., Achtmann, M., 2006. Sex and virulence in Escherichia coli: an evolutionary perspective. Mol. Microbiol. 60, 1136–1151.
International Journal of Medical Microbiology 296 (2006) 547–552 www.elsevier.de/ijmm
SHORT COMMUNICATION
Identification and characterization of ‘‘pathoadaptive mutations’’ of the cadBA operon in several intestinal Escherichia coli Joerg Joresa,, Alfredo G. Torresb, Sylke Wagnera, Christopher B. Tuttb, James B. Kaperc, Lothar H. Wielera a
Institut fu¨r Mikrobiologie und Tierseuchen, Freie Universita¨t Berlin, Philippstraße 13, D-10115 Berlin, Germany Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX 77555-1070, USA c Center for Vaccine Development, School of Medicine, University of Maryland, 685 W. Baltimore Street, Baltimore, MD 21201, USA b
Received 27 September 2005; received in revised form 27 June 2006; accepted 13 July 2006
Abstract The dysenteric Shigella spp. and enteroinvasive Escherichia coli (EIEC) have evolved from commensal E. coli by the acquisition of a virulence plasmid and inactivation of genes of the cad locus encoding lysine decarboxylase (LDC) by so-called pathoadaptive mutation. As horizontal gene transfer and recombination occurs frequently in E. coli we were interested to see if similar pathoadaptive mutations are commonly present in other intestinal pathotypes. Therefore, we examined 140 intestinal E. coli strains of various pathotypes and the ECOR collection for their ability to decarboxylate lysine, and identified 25 strains that were unable to do so. Complementation of a Shiga toxin-producing E. coli and two enteropathogenic E. coli strains, both LDC-negative, with the intact cad locus restored LDC activity and resulted in a reduction in adherence to tissue culture cells. We investigated the cad locus for possible alterations by using hybridization and PCR techniques and compared the results with the alterations reported for Shigella spp. and EIEC strains. Interestingly, the alterations of the cad genes were similar to those previously reported, pointing towards a parallel evolution of LDC silencing in different intestinal E. coli pathotypes. r 2006 Elsevier GmbH. All rights reserved. Keywords: cadBA; Lysine decarboxylase; E. coli; Pathoadaptive mutation
Introduction Escherichia coli is one of the most fascinating members of the prokaryotes, since this species is an important representative of the autochthonous mammalian gut flora, and in addition it harbours a broad spectrum of diverse pathotypes, causing intestinal as Corresponding author. Present address: International Livestock Research Institute, Box 30709, Nairobi 00100, Kenya. Tel.: +254 20 422 3000; fax: +254 2 422 3001. E-mail address: [email protected] (J. Jores).
1438-4221/$ - see front matter r 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.ijmm.2006.07.002
well as extra-intestinal disease. The different pathotypes are characterized by a specific pattern of virulence traits, which are a result of lateral gene transfer (Kaper et al., 2004). Another source of specification reported for members of the enteroinvasive E. coli (EIEC) and several Shigella spp. are alterations encompassing the cadBA operon encoding lysine decarboxylase (LDC) (Maurelli et al., 1998). This enzyme synthesizes the polyamine cadaverine, which was shown to act in an antagonistic fashion to the enterotoxins of Shigella flexneri (Maurelli et al., 1998) and to inhibit the induction of transepithelial migration of
ARTICLE IN PRESS 548
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polymorphonuclear neutrophils (McCormick et al., 1999). Transepithelial neutrophil migration has also been reported for enteropathogenic E. coli (EPEC) (Savkovic et al., 1996; Michail et al., 2003), raising the question of the evolution of similar pathoadaptive mutations in these pathogens. Further, it has been shown that the inactivation of the cadA gene in enterohemorrhagic E. coli (EHEC) O157:H7 had an effect on the ability of this strain to adhere to HeLa cells (Torres and Kaper, 2003) and that pathoadaptive mutations of the cadBA operon mediate increased adherence to HeLa cells in three out of four STEC strains of serogroups O26 and O111 (Torres et al., 2005). In Shigella spp. mostly deletions and rearrange ments of the cadBA operon are responsible for a silencing of LDC (Day et al., 2001), while in EIEC insertions of IS within the cadC gene as well as point mutations of its promotor account for this phenotype (Casalino et al., 2003). The Shigella/EIEC pathotype has originated several times by convergent evolution (Lan and Reeves, 2002; Lan et al., 2004; Wirth et al., 2006), giving an explanation for the different cadBA silencing strategies within the Shigella/EIEC pathotype. Since different E. coli pathotypes cause diarrhoea/enteritis and horizontal gene transfer and recombinations occur frequently in E. coli we were interested in ascertaining the distribution of ‘‘pathoadaptive mutations’’ in other intestinal E. coli. Therefore, 212 E. coli strains, representing many currently known intestinal pathotypes and the ECOR collection (Ochman and Selander, 1984), were screened for their LDC activity. Using PCR and hybridization experiments, we determined the alterations of the cad locus in those strains showing an LDC-negative phenotype.
Material and methods Bacterial strains We investigated 212 E. coli strains including 140 intestinal E. coli displaying 81 serotypes and the ECOR collection (Ochman and Selander, 1984). The strains tested are listed in Table S1 (supplementary online material). Bacterial strains were routinely grown in Luria–Bertani (LB) broth or on LB agar at 37 1C. When needed, ampicillin was added to media at 100 mg/ml. The plasmids pBR322 and pCadABC (Torres et al., 2005) were introduced into the LDC-negative EPEC and STEC strains by electroporation as described by Dower et al. (1988). Bacterial growth was monitored for all strains to confirm that the presence of the plasmids did not have an effect on the ability of the strains to grow in different media.
Bacterial adhesion to epithelial cells For quantitative adhesion assays, E. coli strains were evaluated for their ability to adhere to HeLa cell monolayers as previously described (Torres et al., 2005). Briefly, the strains were grown in LB broth overnight at 37 1C and added to tissue culture cells replenished with fresh DMEM at a concentration of 1 107 bacteria per well for 3 h at 37 1C. The monolayers were washed, fixed and stained with Giemsa solution for microscopic evaluation, or bacteria were recovered with 0.1% Triton X-100 in phosphatebuffered saline (PBS, pH 7.4) and plated on LB agar plates for quantification.
Lysine decarboxylase assay To determine the LDC activity, strains were grown in Moeller decarboxylase broth with lysine (BD BBL, Fisher Scientific). Results were read after incubation for 24 or 48 h at 37 1C. S. flexneri 2457T served as negative, and E. coli MG1655 as positive control.
Polymerase chain reaction (PCR) and DNA–DNA hybridization HerkulaseTM Enhanced Polymerase Blend (Stratagene, Amsterdam, The Netherlands) was used for PCR experiments as recommended by the manufacturer. Hybridization was performed with digoxigenin-labelled probes by using the Roche Labeling and Detection Kit (Roche, Mannheim, Germany) according to the vendor’s protocols and 500 ng of total bacterial DNA per dot. The cadA-specific probe was synthesized using PCR employing primers A1–A2, while probe cadB was generated by using primers B1–B3 (Casalino et al., 2003). We used E. coli K-12 strain MG1655 as template for generating both probes. The region between the pheU and cadB gene encompassing the cadC gene was amplified using primers B2 and C2 (Casalino et al., 2003), and presence of pINV was examined by PCR employing primers specific for ipaC (Boudeau et al., 1999).
Multilocus sequence typing Multilocus sequence typing (MLST) has been carried out as recently described (Wirth et al., 2006).
Results and discussion Out of 212 E. coli strains tested for LDC activity, 25 strains belonging to the EHEC, EPEC, enterotoxigenic E. coli (ETEC) pathotypes and non-pathogenic
ARTICLE IN PRESS J. Jores et al. / International Journal of Medical Microbiology 296 (2006) 547–552
Table 1.
Characteristics of LCD-negative E. coli
Strain designation
Serotype
Country
Pathotype
cadA/cadB
0202-1 0421-1 0471-1 055/89 1112-6 1582-4 3632-2 CL-37 DEC8A DEC8B DEC8E DEC9B ECOR17 GS1128-1 GS1187-1 IHIT0554 IHIT0597 IHIT1190 IHIT1703 IHIT1968 IHIT2115 IHIT2430 PS37 WH 02/24/007-2 WUS-02/09/010-1
R:H11,21,41 O162:H33 NT:H19 O2:H5 R:H11,21 O162:NM NT:H19 O111:H8 O111:NM O111:H8 O111:H8 O26:NM O106:NM On.t.:NM On.t.:NM On.t.:NM O157:NM O92:NM O111:H2 O119:H25 O4:NM O80:NM O9:K35:K99 O165:H25 O165:H25
Brazil Brazil Brazil Germany Brazil Brazil Brazil Canada USA USA Denmark USA Indonesia Germany Germany Germany Germany Germany Germany Germany Germany Germany Germany Germany Germany
EPEC EPEC EPEC STEC EPEC EPEC EPEC EHEC EHEC EHEC EHEC EHEC
No No No Yes No No Yes No No No No No No Yes Yes No No No No No No No No No No
a
549
EHEC EHEC EHEC EHEC EHEC EHEC EHEC EHEC EHEC ETEC EHEC EHEC
ST-Clpx/STa
None/1 10/378 29/16 29/16 29/294 29/29 None/47
10/304 10/165 306/300 29/16 306/300 10/301 10/301 None/120 None/119 None/119
ST-Clpx-sequence type complex/ST-sequence type according to http://web.mpib-berlin.mpg.de/mlst/dbs/Ecoli.
ECOR17 (see Table 1) were LDC-negative. Out of these, four common diarrhoeagenic E. coli (DEC) strains (Whittam et al., 1993), namely DEC8A, DEC8B, DEC8E and DEC9B belonging to the important serogroups O111 and O26 were LDCnegative, which is in good accordance to results reported recently (Torres et al., 2005). An LDC-negative phenotype has also been previously reported in three EHEC isolated from children in Brazil, but the cadBA operon has not been investigated in these strains (Guth et al., 2002). To gain insight into the nature of the LDC-negative phenotype, we investigated the cadBA operon more thoroughly by using dot blot hybridization of total genomic DNA and probes specific for the cadA and cadB gene (Fig. 1A,B). Twenty-one strains did not react with either the cadA or cadB probe indicating a deletion similar to the one reported for Shigella boydii 18 (Day et al., 2001). In contrast, four out of all 25 LDC-negative strains reacted with both probes. We therefore investigated the cadC gene encoding the CadBA regulator of these strains by PCR employing primers B2 and C2 (Casalino et al., 2003). Interestingly, STEC 055/89 (O2:H5) showed a PCR amplicon of 3.8 kb in size instead of
the expected 2.5 kb fragment (data not shown), suggesting that an insertion in the cadC gene has occurred, probably an IS element as previously described for EIEC (Casalino et al., 2003). Sequence analysis of the 3.8-kb PCR amplicon confirmed the insertion of an IS element (showing compelling similarity to IS2), 303 bp downstream of the translational start site of cadC. It was recently proposed that inactivation of the cadC gene is the first step in silencing of the cadBA operon followed by a spread of IS elements into cadA and cadB (Casalino et al., 2005). The remaining three strains (3632-2, GS11281 and GS1187-1) showed neither deletions nor an inactivated regulator, although they clearly reacted negatively in their ability to decarboxylate lysine. After introduction of the plasmid pCadABC into EPEC 36322 (see details below) LDC activity was restored, indicating that a mutation(s) in the cadBA operon or its regulator gene account for the LDC silencing as speculated already for some EIEC strains (Casalino et al., 2003). In order to determine whether the observed LDCnegative phenotype represents a pathoadaptive mutation, we investigated the adhesion ability of three selected strains, namely EPEC 3632-2, EPEC 1112-6,
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A
B
C
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
27
160
CFU / ml ( x106)
140 *
120
**
100 80
***
60 40 20
055 / 89 (pCadABC)
055 / 89 (pBR322)
3632 - 2 (pCadABC)
3632 - 2 (pBR322)
1112 - 6 (pCadABC)
1112-6 (pBR322)
0
* P < 0.05; ** P > 0.05; *** P < 0.01
Fig. 1. Dot blot hybridization, using probes specific for detection of cadA (A) and cadB (B). DNA from lysine decarboxylase negative E. coli was tested: (1) IHIT0597; (2) IHIT0554; (3) IHIT1190; (4) IHIT1703; (5) IHIT1968; (6) IHIT2115; (7) IHIT2430; (8) WH 02/24/007-2; (9) WUS-02/09/010-1; (10) PS37; (11) DEC8E; (12) DEC8B; (13) GS1128-1; (14) GS1187-1; (15) DEC8A; (16) DEC9B; (17) 0202-1; (18) 0421-1; (19) 0471-1; (20) 1112-6; (21) 1582-4; (22) 3632-2; (23) CL-37; (24) ECOR17; (25) 055/89; (26) MG1655; and (27) Lambda. (C) Chart displaying the adherence to HeLa cells of STEC 055/89 (white columns), EPEC 1112-6 (grey columns) and EPEC 3632-2 (black columns) carrying plasmids pBR322 or pCadABC. The statistical difference between each strain containing pCadABC vs. pBR322 was determined by a Student’s t-test analysis.
and STEC 55/89. We selected these strains, because they belong to pathotypes so far not reported to be LDCnegative and showed different alterations of the cadBA operon. After introduction of plasmid pCadABC carrying the cadBA operon the LDC-positive phenotype was restored. Further, we observed a reduction in adhesion properties in such strains, suggesting the existence of pathoadaptive mutations affecting the adherence properties of these strains (Fig. 1C). Because it was previously shown for STEC strains of the serogroup O111 that a reduction in intimin expression due to LDC activity has an effect in adhesion (Torres et al., 2005), we investigated whether changes in intimin expression were responsible for the reduction in adhesion observed in our EPEC strains (3632-2 and 1112-6). No reduction in
intimin expression was observed in the EPEC strains harbouring the plasmid pCadABC as compared to the pBR322-containing control strains (data not shown). We speculate that this result is due to differences in the in vitro regulatory mechanisms controlling intimin expression in EPEC strains or because the cad locus is controlling the expression of other adhesins in EPEC. So far, our data showed that the so-called pathoadaptive alterations of the cad loci are not restricted to EIEC, EHEC and Shigella spp., but can also be found in at least three other pathotypes, namely EPEC, STEC and ETEC. In S. flexneri the absence of cadaverine has been demonstrated to enhance the impact of its invasion process (Maurelli et al., 1998; McCormick et al., 1999).
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Since horizontal gene transfer is frequently observed in E. coli, we investigated the possible presence of the virulence plasmid pINV in the 25 LDC-negative E. coli. None of the 25 strains possessed the virulence plasmid, pointing towards another evolutionary force leading to the inactivation of the cadBA operon. Additionally, we MLST-typed 17 out of the 25 LDC-negative strains (see Table 1) according to the MLST scheme published recently (Wirth et al., 2006). By doing so, we could prove these 17 strains belong to three different sequence type complexes as well as 12 different sequence types. Therefore we propose a parallel evolution of LDC silencing within the E. coli population. Interestingly, different strains with the same sequence type (ST29 and ST300, see Table S1, supplementary online material) can be either LDCnegative or LDC-positive, in contrast to Shigella spp. and EIEC. Obviously, the cadBA operon alterations within Shigella spp. and EIEC are rather fixed in contrast to other E. coli pathotypes. Moreover it is likely that recombination events as a result of horizontal gene transfer could be attributed to the cadBA alterations reported here. This could be a hitchhiking effect due to the insertion of foreign DNA into the pheU tRNA gene, which often serves as target for pathogenicity islands in E. coli and in Shigella spp. (Rumer et al., 2003; Schmidt and Hensel, 2004). As we discovered similar mutations in several independently evolved lines (different serotypes, pathotypes and sequence type complexes), we propose that these mutations are beneficial for the bacteria in ways either involving pathogenicity or in terms of competition with other bacteria occupying the same ecological niche. It is also likely that such mutations represent a pivotal evolutionary step for the pathogens competing with other bacteria for the same ecological niche, because several gastrointestinal bacteria produce cadaverine, which theoretically could hamper the virulence of pathogens such as Shigella and EIEC. Future work is needed to elucidate the exact role of these ‘‘pathoadaptive mutations’’ in non-Shigella/EIEC members of the E. coli population.
Acknowledgment This work was supported by Grant WI 1436/3-2 from the Deutsche Forschungsgemeinschaft and by the Welcome Trust financed IPRAVE Consortium. Sylke Wagner was financed from NaFo¨G. Alfredo Torres’ laboratory is supported in part by institutional funds from the UTMB John Sealy Memorial Endowment Fund for Biomedical Research. We thank Dr. Fasano, Dr. Whittam, Dr. Gomes, Dr. Gene and Dr. Karch for providing strains and Ju¨rgen Eichberg for excellent technical support. James Kaper’s laboratory is sup-
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ported in part by grants NIH Grants AI21657 and DK58957.
Appendix A. Supplementary material The online version of this article contains additional supplementary data. Please visit doi:10.1016/ j.ijmm.2006.07.002.
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