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Occurrence and characteristics of the cytolysin A gene in Shigella strains and other members of the family Enterobacteriaceae

Christine Von Rhein, Susanne Bauer, Valeska Simon, Albrecht Ludwig
DOI: http://dx.doi.org/10.1111/j.1574-6968.2008.01290.x 143-148 First published online: 1 October 2008

Abstract

Cytolysin A (ClyA, HlyE, SheA) is a hemolytic pore-forming toxin found in Escherichia coli and Salmonella enterica serovars Typhi and Paratyphi A. In the present study, analysis of several Shigella strains revealed that they harbor only nonfunctional clyA gene copies that have been inactivated either by the integration of insertion sequence (IS) elements (Shigella dysenteriae, Shigella boydii, and Shigella sonnei strains) or by a frameshift mutation (Shigella flexneri). Shigella dysenteriae and S. boydii strains also exhibited IS-associated deletions at the clyA locus. PCR and Southern blot analyses as well as database searches indicated that clyA-related DNA sequences are completely absent in strains belonging to various other genera of the family Enterobacteriaceae. According to these data, ClyA may play a role only for a rather small subset of the enteric bacteria.

Keywords
  • cytolysin
  • pore-forming toxin
  • ClyA
  • Escherichia coli
  • Shigella
  • Enterobacteriaceae

Introduction

Cytolytic toxins are major virulence factors of many bacterial pathogens. Recently, a novel pore-forming cytolytic toxin, cytolysin A (ClyA, also designated HlyE or SheA), has been detected in Escherichia coli and Salmonella enterica serovars Typhi (S. Typhi) and Paratyphi A (S. Paratyphi A). ClyA is an exported 34-kDa protein that shows hemolytic, cytotoxic, and apoptogenic activity based on its ability to assemble into ring-shaped oligomers that form pores in target cell membranes (del Castillo et al., 1997; Ludwig et al., 1999; Oscarsson et al., 1999, 2002; Lai et al., 2000; Wallace et al., 2000; Wai et al., 2003; Eifler et al., 2006; Tzokov et al., 2006; von Rhein et al., 2008). The prototypical E. coli clyA gene is found in E. coli K-12 and in different types of intestinal pathogenic E. coli strains such as enterohemorrhagic (Shiga toxin-producing), enteroinvasive, enterotoxigenic, and enteroaggregative strains, while various uropathogenic and newborn meningitis-causing E. coli strains harbor only clyA fragments due to deletions at the clyA locus (del Castillo et al., 1997, 2000; Ludwig et al., 2004). A functional clyA homolog has been detected in avian pathogenic E. coli (Reingold et al., 1999; Wyborn et al., 2004a). Furthermore, intact clyA homologs are present in S. Typhi, the etiological agent of typhoid fever, and in S. Paratyphi A, which causes paratyphoid fever, while in various other Salmonella serovars, clyA appears to be completely absent (Oscarsson et al., 2002; von Rhein et al., 2006, 2008).

Under standard in vitro growth conditions, ClyA production is usually strongly downregulated in E. coli and Salmonella strains, and clyA transcription in E. coli K-12 has particularly been shown to be repressed under these conditions by the global regulator H-NS (histone-like nucleoid-structuring protein) (Westermark et al., 2000; Oscarsson et al., 2002; von Rhein et al., 2008). Nevertheless, E. coli clyA expression has also been shown to be positively controlled by several proteins, namely SlyA, which directly antagonizes the H-NS-mediated repression by competing with H-NS for binding sites at the clyA promoter, and the transcription factors CRP (cyclic AMP receptor protein) and FNR (fumarate and nitrate reduction regulator), which activate clyA expression in response to glucose and oxygen starvation, respectively (Ludwig et al., 1995, 1999; Oscarsson et al., 1996; Green & Baldwin, 1997; Westermark et al., 2000; Wyborn et al., 2004b; Lithgow et al., 2007). Expression of the S. Typhi and S. Paratyphi A clyA homologs has also been shown to be activated by SlyA (von Rhein et al., 2008). Furthermore, serologic data and results of proteomics analyses have indicated that these two Salmonella serovars produce substantial amounts of ClyA during infection, suggesting that ClyA might play a role in typhoid Salmonella pathogenesis (von Rhein et al., 2006; Ansong et al., 2008).

It is presently unknown whether organisms other than members of E. coli and S. enterica are able to produce functional ClyA or related toxins. The bacteria of the genus Shigella, which cause shigellosis, or bacillary dysentery, are of particular interest in this context as they are very closely related to E. coli. Molecular evidence actually indicates that E. coli and all shigellae belong to the same species (Ochman et al., 1983; Pupo et al., 1997). Some Shigella flexneri strains have been reported to harbor a mutant clyA variant exhibiting an 11-bp deletion (del Castillo et al., 2000; Wallace et al., 2000), but the occurrence and characteristics of clyA in other shigellae have not been well studied. Here, we characterized the clyA homologs of strains from all four Shigella species, i.e. Shigella dysenteriae, S. flexneri, Shigella boydii, and Shigella sonnei (also known as Shigella subgroups A, B, C, and D, respectively) and tested a number of strains belonging to other genera of the family Enterobacteriaceae for the presence of clyA-related DNA sequences.

Materials and methods

Bacterial strains and culture conditions

The bacterial strains analyzed in this study are listed in Table 1. Escherichia coli K-12 strain DH5α [Fφ80lacZΔM15 Δ (lacZYAargF) U169 deoR recA1 endA1 hsdR17 (rk, mk+) phoA supE44λthi-1 gyrA96 relA1] (Invitrogen) was used as the cloning host and for the propagation of all plasmids. All bacteria were grown aerobically at 37 °C in double-concentrated yeast extract–tryptone (2 × YT) medium, on YT agar, or on YT-horse blood agar (Ludwig et al., 2004). Antibiotic selection was carried out using ampicillin at a final concentration of 100 μg mL−1.

View this table:
Table 1

Bacterial strains analyzed in this study

SpeciesStrainsclyAReferences or sources
Shigella dysenteriae60R(+)Greco (2004)
Shigella flexneriM90T (ipaB mutant)(+)Menard (1993)
Shigella boydiiST1139(+)IMMF
Shigella sonneiST2757, ST3112, ST3135A(+)IMMF
Citrobacter amalonaticusDSM 4593 (ATCC 25405)TDSMZ
Citrobacter braakiiPEG-38893IMMF
Citrobacter freundiiDSM 30039 (ATCC 8090)TDSMZ
PEG-40690, PEG-41830, PEG-41867, MSD-41, MSD-44IMMF
Citrobacter gilleniiDSM 13694 (ATCC 51117)TDSMZ
Citrobacter koseriDSM 4570 (ATCC 27156), DSM 4595 (ATCC 27028)T, DSM 4596 (ATCC 25408)DSMZ
PEG-40047IMMF
Citrobacter murliniaeDSM 13695 (ATCC 51118)TDSMZ
Klebsiella oxytocaPEG-39836, PEG-41381IMMF
Klebsiella pneumoniaePEG-40301, PEG-40400, PEG-41480, PEG-41587IMMF
Enterobacter cloacaeMSD-8IMMF
Serratia marcescensMSD-6IMMF
Hafnia alveiRV3/97IMMF
Proteus mirabilisMSD-1IMMF
Morganella morganiiFR301IMMF
Yersinia enterocoliticaRV2/00IMMF
  • * Type strains are denoted by superscript T. The Citrobacter koseri strains DSM 4570 and DSM 4596 are type strains of Citrobacter diversus and Levinea malonatica, respectively. American Type Culture Collection (ATCC) strain numbers are indicated where applicable. The Shigella boydii and Shigella sonnei strains were isolated in 2001 at the University Hospital of Frankfurt am Main from stool samples of patients who had contracted the Shigella infections abroad at different times and at different places.

  • (+), clyA present but defective; −, clyA absent. Results obtained by DNA sequencing (Shigella strains) and Southern blot analysis (all others).

  • IMMF, Institute of Medical Microbiology, University Hospital of Frankfurt am Main, Frankfurt am Main, Germany; DSMZ, Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany.

Molecular genetic techniques

DNA manipulations were performed using standard protocols (Sambrook & Russell, 2001). PCR was conducted with high-fidelity thermophilic DNA polymerases. Nucleotide sequences were determined by cycle sequencing as described previously (Ludwig et al., 2004). Southern blot hybridization was carried out under conditions of low stringency, using the ECL direct nucleic acid labeling and detection system (Amersham Biosciences) according to the manufacturer's instructions.

Construction of plasmids

clyA of S. dysenteriae (clyASd) was amplified by PCR from strain 60R (Greco et al., 2004), using primers binding 262–244 bp upstream and 55–24 bp downstream of E. coli K-12 clyA (clyAK-12). clyA of S. flexneri (clyASf) was amplified from an ipaB mutant of strain M90T (Menard et al., 1993) with primers binding 563–542 bp upstream and 29–13 bp downstream of clyAK-12. The PCR products (1.36 and 1.49 kb, respectively) were cloned into the SmaI site of pUC18 (ampicillinr, New England Biolabs), resulting in the plasmids pAL209 and pAL210, which carry clyASd and clyASf, respectively, downstream from the lacZ promoter of the vector.

Isolation and analysis of proteins

Protein isolation, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and Western blotting were conducted as described previously (Ludwig et al., 2004). Immunoblot analysis of ClyA was performed using a rabbit anti-ClyAK-12 antiserum (Ludwig et al., 2004) and horseradish peroxidase-conjugated secondary antibodies, both at a final dilution of 1 : 2000. Immunoreactive proteins were visualized by conventional chromogenic detection (Ludwig et al., 2004).

Determination of hemolytic activity

Phenotypes of recombinant E. coli strains were determined using individual colonies grown overnight on YT-horse blood agar. Quantitative hemolytic activity assays were performed as described previously with E. coli cell lysates that were obtained by sonication of bacteria suspended in phosphate-buffered saline (Ludwig et al., 2004).

Computer-assisted analyses

Database searches for clyA-related DNA sequences were conducted with the NCBI blast sequence analysis tool (http://blast.ncbi.nlm.nih.gov/Blast.cgi), using the blastn program and the Nucleotide collection (nr/nt) database (all GenBank+EMBL+DDBJ+PDB sequences). Theoretical molecular masses of proteins were determined using the ExPASy proteomics server (http://www.expasy.ch).

Nucleotide sequence accession numbers

The nucleotide sequences of clyA from S. dysenteriae 60R, S. flexneri M90T, S. boydii strain ST1139, and S. sonnei strains ST2757 and ST3112 have been submitted to the GenBank database (http://www.ncbi.nlm.nih.gov) (accession nos. EU236934–EU236938).

Results

Characterization of clyA homologs of Shigella strains

The clyA homologs of several Shigella strains were amplified by PCR using primers based on the E. coli K-12 genome, and analyzed by DNA sequencing. In clyA of S. dysenteriae serotype 1 strain 60R, we found a 632-bp deletion running from the second nucleotide of codon 78 to the end of codon 288. In addition, a copy of the 766-bp insertion sequence (IS) element iso-IS1 of S. dysenteriae was detected at the deletion site, suggesting that the insertion of iso-IS1 caused the deletion (Fig. 1). As a result of these sequence changes, the ORF of clyA from S. dysenteriae 60R is terminated immediately after codon 78 by a random stop codon, while clyA normally encodes a 303-residue protein. clyA of S. flexneri serotype 5a strain M90T exhibited an 11-bp deletion running from the second nucleotide of codon 112 to the end of codon 115, as also observed previously for clyA of S. flexneri serotype 2a and 2b strains (del Castillo et al., 2000; Wallace et al., 2000). The frameshift caused by this deletion generated a stop codon just after codon 113. In the case of S. boydii strain ST1139, clyA proved to be destructed by the insertion of IS1 of S. boydii (a 768-bp element), which apparently caused the deletion of a 3089-bp fragment comprising the 604 3′-terminal base pairs of clyA (i.e. the clyA sequence following the second nucleotide of codon 103) and the first 2485 bp downstream of clyA, as suggested by sequence comparison with the genome of E. coli strain K-12 substrain MG1655. The IS1 element present in place of this missing DNA fragment restored codon 103 of clyA and caused premature termination of the clyA ORF after six additional random codons.

Figure 1

Characteristics of clyA in Shigella spp. Chromosomal DNA fragments carrying clyA of Shigella dysenteriae (clyASd), Shigella flexneri (clyASf), Shigella boydii (clyASb), and Shigella sonnei (clyASs) are schematically aligned with the clyA-carrying genome region of Escherichia coli K-12 MG1655. clyA is depicted as a shaded arrow. Deletions are indicated by dotted lines. The position and orientation of IS elements is shown, but these elements are not drawn true to scale. The presented sequence characteristics particularly have been observed for the S. dysenteriae strains 60R and Sd197, the S. flexneri strains M90T, 301, 2457T, and 8401, the S. boydii strains ST1139 and Sb227, and the S. sonnei strains ST2757, ST3135A, and Ss046. Another S. sonnei strain, ST3112, was found to contain in its clyA gene only one of the two IS1 copies shown in the figure, namely that located closer to the 3′-end of clyA. In S. boydii strain CDC 3083-94 (BS512), clyA proved to be completely absent due to an IS1-associated 1153-bp deletion beginning 163 bp upstream and ending 78 bp downstream of clyA. The S. boydii IS1 element present at this deletion site is oppositely oriented relative to the absent clyA gene.

The clyA homologs of the analyzed S. sonnei strains showed IS insertions that were not associated with deletions. In the strains ST2757 and ST3135A, two copies of IS1 of S. sonnei (768-bp element) were found in clyA: one after the second nucleotide of codon 27 and the other directly after codon 118. These are flanked on either side by direct repeats of 8 and 9 bp, respectively, consistent with the fact that during the transposition of IS1 an 8- or a 9-bp sequence at the target site is duplicated. The upper IS1 insertion converted codon 27 into a stop codon. clyA of strain ST3112 showed only the IS1 insertion directly after codon 118, which fused the clyA codons 1–118 with eight random codons that precede a stop codon.

Apart from these mutations, the clyA sequences and flanking DNA regions of the Shigella strains proved to be very similar to those of E. coli strains. Without the IS1 insertions, clyA of the analyzed S. sonnei strains would be about 99% identical to clyAK-12. clyA of S. flexneri M90T shares 97.9% sequence identity with clyAK-12. The clyA fragments and flanking DNA sequences of S. dysenteriae 60R particularly resemble the sequences present in Shiga toxin-producing E. coli (STEC) strains of serogroup O157, even though clyA is intact in most O157 STEC strains tested so far (del Castillo et al., 2000; Ludwig et al., 2004). The putative −10 and −35 promoter elements and the binding sites for H-NS, SlyA, and CRP/FNR identified in the clyAK-12 promoter region (Ludwig et al., 1999; Westermark et al., 2000; Wyborn et al., 2004b; Lithgow et al., 2007) are highly conserved in all Shigella strains analyzed, suggesting that the mutant Shigella clyA genes may be expressed under certain environmental conditions.

Comparison of the Shigella DNA sequences determined in the present study with complete Shigella genome sequences available from databases revealed that S. dysenteriae serotype 1 strain Sd197 and S. boydii serotype 4 strain Sb227 (Yang et al., 2005) have the same IS insertions and associated deletions at their clyA loci as S. dysenteriae 60R and S. boydii ST1139, respectively. The S. flexneri 2a strains 301 and 2457 T (Jin et al., 2002; Wei et al., 2003) and S. flexneri 5b strain 8401 (Nie et al., 2006) turned out to have the same 11-bp deletion in clyA as S. flexneri 5a strain M90T, and S. sonnei strain Ss046 (Yang et al., 2005) proved to have the same two IS1 insertions in clyA that we found in the strains ST2757 and ST3135A. A clyA homolog corresponding to that of S. sonnei strain ST3112 (i.e. with a single IS1 insertion) was, however, not detectable in published DNA sequences. Inspection of the recently published complete genome of S. boydii serotype 18 strain CDC 3083-94 (GenBank CP001063) revealed that in this strain clyA is completely absent due to an IS1-associated 1153-bp deletion that begins 163 bp upstream of the clyA start codon and that ends 78 bp downstream of the clyA stop codon.

Expression of Shigella clyA in E. coli

The characteristics of the clyA variants found in Shigella strains suggested that their protein products are nonfunctional. To confirm this, we cloned the mutant clyA genes of S. dysenteriae and S. flexneri, clyASd and clyASf, and expressed them in E. coli DH5α. As shown previously (Ludwig et al., 1999, 2004), E. coli DH5α carrying clyAK-12 under the control of the lacZ promoter on plasmid pAL201 (a pUC18 derivative) produces large amounts of ClyAK-12 when grown in or on standard medium, giving rise to a strongly hemolytic phenotype on blood agar. Escherichia coli DH5α transformed with the isogenic plasmids pAL209, carrying clyASd, or pAL210, carrying clyASf, remained nonhemolytic on blood agar. In contrast to DH5α/pAL201, the clones harboring pAL209 and pAL210 also did not show significant intracellular hemolytic activity. Furthermore, the predicted products of clyASd (8.9 kDa polypeptide) and clyASf (12.9 kDa polypeptide) were not detectable using SDS-PAGE and Western blot analysis in culture supernatants or cell lysates of these E. coli clones (data not shown), suggesting that the truncated ClyA proteins are unstable and rapidly degraded.

Analysis of the presence of clyA in other members of the family Enterobacteriaceae

Twenty-six bacterial strains from the genera Citrobacter, Klebsiella, Enterobacter, Serratia, Hafnia, Proteus, Morganella, and Yersinia (see Table 1) were tested for the presence of clyA-related DNA sequences. Attempts to amplify such sequences from these strains by PCR, using various primers deduced from clyAK-12 or from clyA of S. Typhi (clyASTy) or S. Paratyphi A (clyASPaA), were unsuccessful. Therefore, we searched for clyA-related sequences by Southern blot analysis of EcoRI/HindIII-digested genomic DNA, using as probes a 712-bp fragment of clyAK-12 (nucleotides 112–823) amplified from E. coli K-12 strain CC118 (Ludwig et al., 1999) and a 909-bp fragment amplified from S. Paratyphi A strain ATCC 9150 (McClelland et al., 2004), which comprised almost the complete clyASPaA gene (nucleotides 3–911). While EcoRI/HindIII-digested genomic DNA of E. coli CC118 and of strain ATCC 9150 showed the expected hybridizations with both probes, no hybridization signals were detectable in the case of the other 26 strains (data not shown), suggesting that they do not possess clyA-related sequences. Furthermore, DNA database searches (NCBI blast searches, 06/2008) using clyAK-12 and consecutive 100-bp fragments of it as query sequences did not yield any significant clue for the presence of clyA-related sequences in organisms other than E. coli, Shigella spp., S. Typhi, and S. Paratyphi A. Thus, strains from other genera of the family Enterobacteriaceae, whose genomes have been completely sequenced (strains of Citrobacter koseri, Klebsiella pneumoniae ssp. pneumoniae, Enterobacter sakazakii, Erwinia tasmaniensis, Serratia proteamaculans, Yersinia enterocolitica ssp. enterocolitica, Yersinia pestis, and Yersinia pseudotuberculosis), obviously do not harbor clyA.

Discussion

ClyA has been recognized recently as a novel pore-forming toxin that is found in several types of E. coli as well as in S. Typhi and S. Paratyphi A, where it might play a role as a virulence factor. Here we observed that randomly tested strains of all four Shigella species harbor only mutant clyA gene copies, which extends findings about Shigella clyA homologs obtained previously by specific analysis of S. flexneri strains (del Castillo et al., 2000; Wallace et al., 2000) or deducible from complete Shigella genome sequences (Jin et al., 2002; Wei et al., 2003; Yang et al., 2005; Nie et al., 2006). Altogether, it is striking that clyA is defective or absent in all Shigella strains tested so far. The collection of strains analyzed to date is certainly not large enough to draw the conclusion that clyA is generally inactivated or absent in Shigella, but the available data suggest that ClyA is dispensable, and hence not of major importance, for Shigella infections.

Interestingly, the mutations found in clyA are quite different in S. dysenteriae, S. flexneri, S. boydii, and S. sonnei strains, indicating that clyA has been independently inactivated during the evolution of these bacteria. This is consistent with the assumption that the shigellae emerged from different strains of E. coli and became highly specific human pathogens through convergent evolution (Pupo et al., 2000). It is tempting to speculate that clyA has been inactivated in the course of the adaptation of the shigellae to their particular, intracellular lifestyle in the human host. On the other hand, a specific relationship between this lifestyle and the presence or absence of functional clyA is not directly evident as enteroinvasive E. coli (EIEC) strains, which show a similar lifestyle and mode of pathogenesis as Shigella spp., typically or at least frequently possess a functional clyA gene (Ludwig et al., 2004). Whether ClyA really plays a significant role in EIEC infections is, however, unclear at present.

The G+C content of clyA of E. coli strains, Shigella spp., S. Typhi, and S. Paratyphi A (39–41%) is about 10–14% lower than the average for the corresponding bacterial chromosomes, which suggests that clyA has been acquired from another organism. In the present study, we did not detect clyA-related DNA sequences using PCR and Southern blot analysis in 26 strains from eight other genera of the family Enterobacteriaceae, and DNA database searches did not yield significant hints for the existence of such sequences in organisms other than E. coli, Shigella, S. Typhi, and S. Paratyphi A, so that the origin of clyA remains unclear. Obviously, larger collections of strains from different bacterial taxa need to be analyzed in order to further elucidate the distribution of the clyA gene, but the present data nevertheless suggest that this gene is not widespread among the members of the Enterobacteriaceae and that ClyA may play a role only for a rather small subset of the enteric bacteria, in particular certain E. coli and Salmonella strains. Studies using defined clyA knock-out mutants of E. coli, S. Typhi, and S. Paratyphi A strains should provide further clues about the importance of ClyA in infections caused by these pathogens.

Acknowledgements

We thank Jörg Hacker for providing S. dysenteriae 60R, and Philippe J. Sansonetti and Werner Goebel for providing the ipaB mutant of S. flexneri M90T. This work was supported by grants from the Deutsche Forschungsgemeinschaft (LU 842/1-1).

Footnotes

  • Editor: Klaus Hantke

References

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