Department of Microbiology, National University of Ireland, University College Cork, Cork, Ireland1
Howard Hughes Medical Institute and Division of Geographic Medicine and Infectious Diseases, Tufts-New England Medical Center and Tufts University School of Medicine, 750 Washington Street, Boston, MA 02111, USA2
Author for correspondence: E. Fidelma Boyd. Tel: +353 21 4903624. Fax: +353 21 4903624. e-mail: f.boyd{at}ucc.ie
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ABSTRACT |
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Keywords: pathogenesis, intestinal colonization, CTXphi receptor
Abbreviations: CT, cholera toxin; TCP, toxin-coregulated pilus; VPI, Vibrio pathogenicity island
The GenBank accession numbers for the sequences reported in this paper are AY078355AY078358.
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INTRODUCTION |
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The biogenesis and regulation of TCP production involves at least 15 genes encoded in the tcp gene cluster as well as several unlinked genes (Kaufman et al., 1993 ; Manning, 1997
; Peek & Taylor, 1992
). The tcp gene cluster is part of a large pathogenicity island termed the TCP-ACF element, or the Vibrio pathogenicity island (VPI) (Brown & Taylor, 1995
; Karaolis et al., 1998
; Kovach et al., 1996
). The V. cholerae O1 serogroup is divided into two biotypes, classical and El Tor, on the basis of a number of phenotypic differences. At most loci, the two V. cholerae O1 biotypes are thought to be nearly identical (Beltran et al., 1999
; Byun et al., 1999
); however, the amino acid sequences of the major pilin protein TcpA are known to differ considerably between the El Tor and classical biotypes (75% similarity at the nucleotide level) (Iredell & Manning, 1997
; Rhine & Taylor, 1994
). Indeed, reports have identified several variant TcpA sequences among non-O1/non-O139 serogroup isolates (Ghosh et al., 1997
; Nandi et al., 2000
; Chakraborty et al., 2000
; Mukhopadhyay et al., 2001
). Furthermore, it is known that there are marked differences in in vitro growth conditions under which optimal expression of TCP occurs in each biotype (Manning, 1997
). For example, using the most common in vitro growth conditions, El Tor strains do not produce TCP or CT. Expression of TCP and CT are co-ordinately regulated by ToxR, a transmembrane DNA-binding protein (Skorupski & Taylor, 1997
). Direct activation of transcription of the genes encoding these virulence factors requires ToxT, a ToxR-regulated cytoplasmic transcription factor that is encoded within the VPI (Skorupski & Taylor, 1997
).
Interestingly, TCP is also a receptor for CTX, the temperate filamentous phage that carries the CT genes (ctxAB) (Waldor & Mekalanos, 1996
). Lysogenic conversion of non-toxigenic strains to toxigenicity by CTX
infection appears to be a crucial step in the evolution of fully pathogenic V. cholerae. After infection, CTX
can replicate as a plasmid or integrate at a specific attachment site, attRS, within the V. cholerae genome (Pearson et al., 1993
; Waldor & Mekalanos, 1996
). The O1 classical strains of V. cholerae do not produce CTX
, although they produce CT and they contain CTX prophages integrated at two sites (Davis et al., 2000
). It is believed that there are several similar steps in the pathways by which CTX
infects V. cholerae and the F-pilus-specific filamentous phages, Ff phages, infect E. coli. Both phages are thought to first bind to a pilus receptor, the F pilus for Ff phages and TCP for CTX
(Jacobson, 1972
; Waldor & Mekalanos, 1996
). Then, both types of phages require a second receptor, a complex of the periplasmic and inner-membrane proteins TolQ, TolR and TolA for infection to occur (Heilpern & Waldor, 2000
; Sun & Webster, 1986
, 1987
). For the Ff phages, a minor coat protein, pIII, located at one end of the phage particle, binds to both the F pilus and TolA (Riechmann & Holliger, 1997
). The OrfU protein of CTX
is thought to be functionally equivalent to pIII of Ff (Boyd et al., 2000
; Heilpern & Waldor, 2000
; Holliger & Riechmann, 1997
; Waldor & Mekalanos, 1996
). Therefore, CTX
infection of V. cholerae is believed to involve OrfU binding to TcpA and to the V. cholerae orthologue of TolA (Heilpern & Waldor, 2000
).
The discovery of variable CTX orfU sequences between the two O1 biotypes and among a number of toxigenic non-O1/non-O139 V. cholerae isolates (Boyd et al., 2000
) prompted us to examine the tcpA gene in these isolates. Here we show the presence of highly variable tcpA sequences among a number of non-O1/non-O139 serogroup isolates. Functional studies with these strains revealed that the expression of the variant tcpA sequences was ToxT-dependent and that the variant TcpA proteins can act as receptors for CTX
. Interestingly, all the non-O1/non-O139 serogroup isolates were capable of colonizing the suckling mouse intestine.
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METHODS |
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Sequence analysis.
Comparisons and phylogenetic analysis of the V. cholerae aldA and tcpA sequences were carried out using the MEGA program (S. Kumar, K. Tamura & M. Nei; http://wwwmegasoftware.net/). Multiple sequence alignment was performed on the tcpA gene sequences and the deduced amino acid sequences using CLUSTAL W (Higgins et al., 1996 ). Gene trees were constructed from pairwise comparisons by the neighbour-joining algorithm (Saitou & Nei, 1987
). The predicted secondary structure of the TcpA protein was obtained from http://www.biochem.ucl.ac.uk/bsm/pdbsum/1qqz/main.html and used to map the position of polymorphic sites along the protein. The theoretical three-dimensional structures of TcpA and the TCP fibre (accession nos PDB ID 1QQZ and RCSB 001169, respectively) were analysed and coloured using RASMOL (Sayle & Milner-White, 1995
; http://www.umass.edu/microbio/rasmol/).
CTX transduction assay V. cholerae strains with variant tcpA sequence.
Filtered cell-free supernatants from a strain [O395(pCTX-Kn)] producing a Kn-marked El Tor CTX (denoted here as CTXET-Kn
) and O139 Calcutta CTX
(denoted here as CTXCalc-Kn
) were used to transduce various potential recipients to KnR using a previously described assay (Waldor & Mekalanos, 1996
; Davis et al., 1999
; Heilpern & Waldor, 2000
). Briefly, 75 µl sterile supernatant from overnight cultures of O395(pCTXET-Kn) was mixed with 75 µl recipient cells, each of which harboured pMT5 and had been grown overnight at 30 °C in Ap and IPTG. This plasmid (pMT5) contains an IPTG-inducible toxT (DiRita et al., 1996
). The phage and recipient cells were gently mixed for 30 min at room temperature. Appropriate dilutions of the mixture were then plated on LB agar containing Kn to enumerate the transductants. Also, dilutions of the recipient strains were plated on LB agar containing Sm to determine the total number of potential recipient bacteria. The frequency of infection was determined by dividing the number of transductants ml-1 (Knr c.f.u.) by the number of recipients ml-1 (Smr c.f.u.). All strains were tested using the same phage lysate.
Mouse colonization assay.
Five-day-old suckling CD1 mice were used to study the intestinal colonization properties of spontaneous Smr derivatives of strains encoding TcpA variants. In this single-strain inoculation assay, 1/100 dilutions of overnight cultures of individual test strains grown at 30 °C in LB broth were used to intragastrically inoculate suckling mice. After 24 h, the small intestines were removed and mechanically homogenized in 7 ml LB with a Tissue Tearor (Biospec Products). To enumerate the V. cholerae in the homogenates serial dilutions were plated on LB agar containing Sm. Four animals were used per group.
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RESULTS AND DISCUSSION |
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Distribution of polymorphic sites along the TcpA protein
Some regions of the TcpA variants were conserved. In fact, there were no polymorphisms in the N-terminal 38 aa of these TcpA sequences. This part of the protein is thought to form an -helix (H1in Fig. 2A
) (http://www.biochem.ucl.ac.uk/bsm/pdbsum/1qqz/main.html). The last four predicted ß-sheets in TcpA also lacked polymorphisms (Fig. 2A
). The latter four regions are all likely to be buried within the TcpA molecule (Fig. 2B
). Certain amino acids were conserved in all of the TcpA sequences. Several of these were shown to be required for pilin stability by mutational analysis by Taylor and colleagues (Kirn et al., 2000
). For example, the cysteine residues at positions 120 and 186 are present in all the TcpA sequences. These two cysteine residues are thought to form a disulphide bond that is essential for pilin stability (Kirn et al., 2000
). Similarly, the K at position 165, which is required for pilin stability (Kirn et al., 2000
), is conserved in all the TcpA sequences. All the TcpA sequences also contained conserved proline residues at positions 58, 87, 99 (except strain V54) and 132 (Fig. 2A
).
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Next, we carried out comparative sequence analysis on all tcpA sequences presently available in the database. An additional 10 strains were examined to determine whether they clustered in similar phylogenetic groups based on tcpA sequences. In all cases the epidemic V. cholerae isolates clustered within the two previously identified lineages I and IV, whereas most of the non-O1/non-O139 serogroup isolates clustered within the previously identified lineages II, III and V (Fig. 6). Furthermore, the non-O1/non-O139 serogroup isolates did not form distinct lineages based on their source of isolation (environmental versus clinical) or serogroup designation.
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Analysis of CTX infection of V. cholerae strains that contain variant TcpA sequence
A transduction assay was used to test whether TcpA variant strains 151, 208, V46, V52 and V54 could be infected by CTX. The test strains were examined relative to two control strains: LAC1, a lacZ mutant derivative of O395, and TCP2, a TCP-deficient mutant of O395 (Herrington et al., 1988
; Waldor et al., 1994
). Cell-free supernatants containing two different Kn-marked CTX
s, CTXET-Kn
and CTXCalc-Kn
(Waldor & Mekalanos, 1996
; Davis et al., 1999
), were used to transduce recipient strains to Knr. CTXclass
was not used in this assay since the CTX prophage arrangement in classical V. cholerae isolates does not yield extrachromosomal CTX DNA and thus does not yield virions. To help ensure production of TCP for these in vitro transduction assays, pMT5, a plasmid containing toxT under the control of an IPTG-inducible promoter, was introduced into all the recipient strains. When strains were grown in the absence of IPTG, none of the five test strains yielded Knr transductants with either phage (data not shown). However, after growth in the presence of IPTG, Knr transductants were found for all recipients with the exception of TCP2, the TCP-deficient mutant (Table 3
). CTXET-Kn
infected strains 151 and V46 with 410-fold lower frequencies than LAC1, the positive control, whereas strains V52, 208 and V54 were infected three to five orders of magnitude less efficiently than LAC1. The frequency of CTXCalc-Kn
infection of 151, V46, V52 and V54 was similar to that of CTXET-Kn
with the notable exception of strain LAC1, which showed a threefold decrease in CTXCalc-Kn
uptake. Furthermore, 208 did not produce any Knr colonies upon repeated assays with CTXCalc-Kn
(Table 3
). The mechanisms accounting for the difference in infection frequency between the different strains are unknown. Some of the differences may be attributable to differing capacities of the variant TCPs to act as CTX
receptors. Also, phage immunity and heteroimmunity may account for some of the differences (Davis et al., 1999
; Kimsey & Waldor, 1998
). Overall, these results indicate that in these non-O1 strains, ToxT can augment TCP production and that to varying degrees all of the variant TCPs can serve as receptors for CTX
.
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To date only two V. cholerae serogroups O1 and O139 are known to cause epidemic cholera, whereas all other toxigenic V. cholerae isolates are only associated with sporadic cholera outbreaks. The lack of epidemic spread among the toxigenic non-O1/non-O139 serogroup isolates may result from a superior intestinal colonization ability of O1 and O139 serogroup strains, which needs to be examined. Furthermore, the pathogenesis of V. cholerae is not completely understood and there are undoubtedly factor(s) involved in V. cholerae persistence during inter-epidemic periods, virulence and spread that are not yet characterized and that are absent in non-epidemic isolates.
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ACKNOWLEDGEMENTS |
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Received 16 November 2001;
revised 18 February 2002;
accepted 25 February 2002.