Department of Microbiology, University College Cork, National University of Ireland, Cork, Ireland1
Author for correspondence: E. Fidelma Boyd. Tel: +353 21 4903624. Fax: +353 21 4903101. e-mail: f.boyd{at}ucc.ie
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ABSTRACT |
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Keywords: virulence factors, bacteriophage, restriction modification
Abbreviations: CT, cholera toxin; IS, insertion sequence; TCP, toxin coregulated pilus; Vibrio pathogenicity island
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INTRODUCTION |
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Interestingly, the main virulence factors of V. cholerae are also encoded on mobile genetic elements and are acquired via horizontal gene transfer. For example, the major symptoms of cholera result from the production of cholera toxin (CT) in the small intestine (Sears & Kaper, 1996 ) and the ctxAB genes encoding CT reside on a lysogenic filamentous phage, CTX
(Waldor & Mekalanos, 1996
). The receptor for CTX
on the V. cholerae cell is a type IV pilus, the toxin-coregulated pilus (TCP), that also functions as an essential intestinal colonization factor (Taylor et al., 1987
). The genes encoding the biosynthesis of TCP were initially shown to reside on a pathogenicity island, designated the Vibrio pathogenicity island (VPI) (Kovach et al., 1996
; Karaolis et al., 1998
). As defined by Hacker et al. (1997)
a pathogenicity island is a large unstable chromosomal region that encodes several virulence genes; is present in pathogenic isolates and absent from non-pathogenic isolates; has a G+C content that differs from the rest of the genome; is associated with a tRNA gene; has IS and/or repeat sequences near the site of integration; and contains a bacteriophage-like integrase. The VPI fulfils all the criteria of a pathogenicity island and the identification of integrase and transposase genes at each end of the VPI suggests that they could be involved in the transfer and integration of this region (Karaolis et al., 1998
). More recently, Karaolis and colleagues have suggested that the VPI is actually the genome of a novel filamentous bacteriophage VPI
; however, transfer of VPI
between V. cholerae O1 and O139 serogroup isolates, the predominant cause of epidemic cholera, was not shown (Karaolis et al., 1999
). Instead, a recent report by OShea & Boyd (2002)
demonstrated efficient transfer of the VPI region between V. cholerae O1 serogroup strains via CP-T1 generalized transduction.
In Vibrio cholerae neuraminidase, encoded by nanH, is thought to increase the sensitivity of host cells to CT (Galen et al., 1992 ). It has been suggested that nanH from a number of bacterial pathogens has been acquired by horizontal gene transfer (Roggentin et al., 1993
). In this study, we examined the nanH gene and its flanking sequences among V. cholerae toxigenic (CTX
-positive) and non-toxigenic (CTX
-negative) isolates and found that the nanH gene is encoded within a 57·3 kb region that showed all the characteristics of a pathogenicity island which we named VPI-2 (Fig. 1
).
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METHODS |
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Sequence analysis.
A region spanning 57·3 kb from position 1896092 to 1953461 of the V. cholerae genome from strain N16961 (Heidelberg et al., 2000 ) was analysed for sequence similarities using the BLAST algorithm (Altschul et al., 1997
). DNA sequence analysis was carried out on both DNA strands of the fk3/fk4 purified PCR product by MWG-Biotech and the resulting sequence was examined using BLAST (Altschul et al., 1997
).
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RESULTS |
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We used PCR analysis to investigate whether the DNA sequence between the int and the nanH genes is present among V. cholerae isolates. Eight primer pairs (310) were designed to encompass the 35·9 kb region of interest (Fig. 1 and Table 2
). Positive PCR bands were obtained with all eight primer pairs for the 45 nanH- and int-positive V. cholerae strains. No PCR products were obtained for nanH- and int-negative V. cholerae strains. Similarly, no PCR products were obtained for the 13 nanH-negative O139 serogroup strains that tested positive for int (Table 3
). The absence of 35·9 kb between int and nanH among non-toxigenic V. cholerae was verified by Southern hybridization using six DNA probes (nint, hp3, hsd3, hsd1, 148F and nanH) derived from PCR fragments generated using primer pairs from Table 2
, which span the regions of interest (Fig. 1
). No hybridization fragments were obtained with the DNA probes (hp3, hsd1, hsd3 and 148F) for the nine nanH-negative strains tested (1528-79, SG3, SG6, SG7, SG10, AS207, AS209, MO10 and MO45) and, as expected, positive hybridization bands were obtained for all nanH-positive V. cholerae strains (Fig. 2b
and data not shown).
PCR analysis was also used to investigate whether the region immediately upstream of serine tRNA and int is present among V. cholerae isolates. The primer pair acrB1 and acrB2 was designed to PCR-amplify the gene VC1757 immediately 5' of the serine tRNA gene (Table 3). An identical 2·9 kb PCR band was obtained for all V. cholerae isolates examined (Fig. 3a
). Therefore, it appears that the serine tRNA gene marks the point of insertion of the 39·5 kb region (int-nanH) that is only associated with toxigenic V. cholerae isolates.
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Among the 13 V. cholerae O139 serogroup isolates that contained a deletion within VPI-2, a primer pair (fk3 and fk4) was used to determine whether the region between the int gene (VC1758) and the IS911-like element (VC1789) was empty in these isolates. Primer fk3 was designed 300 bp from the end of int (VC1758) and primer fk4 was designed within 100 bp from the start of IS911 (VC1789) (Fig. 1). A 3·8 kb PCR product was amplified from all 13 O139 serogroup isolates, indicating the absence of additional DNA at this site. The 3·8 kb PCR product was sequenced and analysed using the BLAST program (Altschul et al., 1997
) to reveal the presence of the VC1759 gene and a truncated VC1760 gene (Fig. 4
). Sequencing showed that the VC1760 gene has a 665 bp deletion in the 3' region of the gene and this partial VC1760 sequence is adjacent to the IS911-like sequence in the 13 O139 isolates (data not shown).
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VPI-2 contains a total of 52 ORFs and is 57·3 kb in length. The overall G+C content of VPI-2 (42 mol%) is lower than that of the entire V. cholerae genome (4749 mol%). Of the 52 ORFs (VC1758VC1809), 13 ORFs showed similarity to bacteriophage genes, 29 ORFs were homologous to genes of known function and 10 ORFs showed no significant matches in the database (Table 4). Among the 29 ORFs of known function, a number of gene clusters were observed. For example, downstream of the int gene (VC1758), a restriction modification system (VC1764VC1769), containing a type 1 restriction modification gene (hsdR) and its associated DNA methylase gene (hsdM) was identified, which showed sequence similarity to a restriction modification system from Xylella fastidiosa (Simpson et al., 2000
). Adjacent to this region is a cluster of 11 genes (VC1773VC1783) that showed considerable homology (2752% amino acid identity) to an equivalent gene cluster in the Haemophilus influenzae genome, encoding enzymes involved in the utilization of amino sugars (nan-nag region) (Fleischmann et al., 1995
). The gene order in H. influenzae (nanE-nanK-HI0143-nanA-nagB-nagA) is somewhat different to that in V. cholerae (nanA-VC1777 - VC1778 - VC1779 - VC1780 - nanE - nanK - nagC), but it suggests a remarkable grouping of ORFs of related function. The nan-nag gene cluster is located immediately upstream of nanH and may potentially be involved in the utilization of sialic acid released by the enzymic action of this neuraminidase.
An IS-like element (VC1789VC1790), located downstream of nanH, showed significant identity (66 and 72%) to the Shigella flexneri IS911 element (Prere et al., 1991 ). Adjacent to this IS911-like element within a region that encompasses 19 ORFs (VC1791VC1809), lie 10 ORFs that exhibit striking amino acid similarities (identities ranging from 21 to 50%) to ORFs of bacteriophage origin (Table 4
). These 10 ORFs include genes encoding phage regulatory and tail functions. For example, the amino acid sequence of the ORFs VC1791 and VC1792 share similarity (identity 35%, E value 4e-48; identity 33%, E value, 1e-05) with the gp42 protein and gp41 protein of Mu phage (Table 4
). Furthermore, the ORFs VC1793, VC1794, VC1795, VC1796 and VC1799 all exhibit sequence similarity to bacteriophage proteins and two of these (VC1793 and VC1799) show similarity to a transposase and integrase protein, respectively, suggesting that these genes may be involved in the mobilization and integration of this region (Table 4
). Additionally, the ORF VC1803 exhibits 33% similarity with a repressor protein from phage CP-933O and the amino acid sequence of ORF VC1809 has a 50% identity (E value 3e-11) with the Vis protein of P4. The IS911-like element is the first ORF that is present following the deletion of VC1761VC1788 in the nanH-negative O139 serogroup strains.
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DISCUSSION |
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In V. cholerae, VPI-2 is located within the 3' end of a serine tRNA gene. The tRNA loci serve as conserved genomic landmarks for the insertion of mobile genetic elements (bacteriophages and pathogenicity islands) in a range of bacterial pathogens. For example, among pathogenic E. coli isolates five pathogenicity islands, PAI-1 and LEE, PAI-2, PAI-4 and PAI-5 have insertion sites in selC, leuX, pheR and pheV, respectively (Blum et al., 1994 ; Hacker et al., 1997
; McDaniel & Kaper, 1997
). These same tRNA genes also serve as integration sites for different bacteriophages in E. coli K-12 and O157 strain EDL933 (Blattner et al., 1997
; Perna et al., 2001
). Furthermore, the serine tRNA locus in particular serves as the insertion site for the vap region from Dichelobacter nodosus (Cheetham et al., 1995
), SPI-5 in Salmonella enterica serovar Typhimurium (Wood et al., 1998
), an 84 kb pathogenicity island in E. coli O157 strain EDL933 (Perna et al., 2001
) and bacteriophage T12 of Streptococcus pyogenes (McShan et al., 1997
).
An additional characteristic of some pathogenicity islands is their instability and the VPI-2 region between VC1760 (helicase) and VC1789 (IS911-like element), which includes the restriction modification system and the nan-nag regions, is deleted from 13 of the 14 V. cholerae O139 serogroup strains examined. Of the 13 O139 strains with the truncated VPI-2 most were isolated after 1992. Only one O139 strain MO2, which was isolated in 1992, contains the entire 57·3 kb VPI-2. A recent study by Dziejman et al. (2002) analysing differences in gene content between endemic and pandemic cholera isolates identified the same deletion of the region spanning VC1761VC1786 in O139 serogroup strain MO10, consistent with our findings. In 1992 CT-producing V. cholerae O139 serogroup isolates emerged as the first non-O1 serogroup isolates to cause epidemic cholera in the Indian subcontinent, replacing the seventh pandemic V. cholerae O1 El Tor biotype strains (Albert et al., 1993
; Cholera Working Group, 1993
; Ramamurthy et al., 1993
). However, the El Tor biotype, which was the progenitor for the O139 epidemic clone, soon re-emerged as the dominant cause of cholera in these areas (Faruque et al., 1997
). A possible explanation for the re-emergence of El Tor cholera could be the presence of VPI-2 in the El Tor biotype and the deletion of most of VPI-2 from O139 serogroup strains.
The presence of nanH on a pathogenicity island suggests that it was acquired by horizontal transfer. Interestingly, it has been suggested that nanH from a number of bacterial pathogens was acquired by horizontal gene transfer (Hoyer et al., 1992 ; Roggentin et al., 1993
). On the S. enterica serovar Typhimurium LT2 genome the nanH gene is encoded within a prophage Fels-1 and shows a G+C content (47 mol%) that differs from the host genome (50 mol%) (McClelland et al., 2001
; Figueroa-Bossi, 2001
). Similarly, in Clostridium perfringens, the nanH gene is located near a phage attachment site on the chromosome and has a G+C content of 31·9 mol% compared to the chromosomal DNA G+C content of 2427 mol% (Canard & Cole, 1989
). In M. viridifaciens it has been observed that a region flanking the nanH gene shows significant homology to a transposase (Sakurada et al., 1992
).
In V. cholerae, it is hypothesized that the function of neuraminidase, encoded by nanH, is to act synergistically with CT by increasing the binding and penetration of the toxin to host enterocytes (Galen et al., 1992 ). However, these authors demonstrated only a modest effect of nanH on CT function in vitro and no significant effect in vivo in the suckling mouse model. Therefore, the true extent of the role of nanH in virulence remains unclear. We speculate that VPI-2 is likely to be important in pathogenesis, either directly in cholera virulence or indirectly in the transfer and integration of the island. The VPI-2 region may contribute to the survival of the bacterium in different ecological niches. For example, the product of nanH, neuraminidase, acts on higher order gangliosides converting them to GM1 gangliosides, with the subsequent release of sialic acid. The nan-nag region encodes proteins involved in the utilization of both N-acetylglucosamine and sialic acid. The ability to utilize the sialic acid by-product as an alternative nutrient source could convey a significant competitive advantage to pathogenic V. cholerae strains. VPI-2 also encodes a number of ORFs of unknown function and we are now in the process of examining these regions for a possible role in V. cholerae pathogenesis.
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ACKNOWLEDGEMENTS |
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Received 22 April 2002;
revised 2 July 2002;
accepted 30 July 2002.