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Infection and Immunity, November 2002, p. 6242-6250, Vol. 70, No. 11
0019-9567/02/$04.00+0 DOI: 10.1128/IAI.70.11.6242-6250.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Naval Medical Research Center, Silver Spring, Maryland 20910,1 AstraZeneca R & D Boston, Waltham, Massachusetts 024512
Received 25 February 2002/ Returned for modification 23 April 2002/ Accepted 29 July 2002
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ABSTRACT |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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was the host for cloning experiments. E. coli strains were grown on Luria broth supplemented with 50 µg of ampicillin per ml or 20 µg of chloramphenicol per ml when appropriate. Cloning and sequence analyses. Plasmid DNAs from C. jejuni were isolated using mini-Qiagen columns (Qiagen, Chatsworth, Calif.) as previously described (3). Restriction enzymes were purchased from New England Biolabs (Beverly, Mass.) and used as recommended by the supplier. Plasmid DNAs for sequence analyses were isolated using QIAprep spin miniprep columns (Qiagen). DNA sequencing was done using Big Dye sequencing kits (Perkin Elmer-Applied Biosystems, Foster City, Calif.) and an Applied Biosystems 373A or 3100 DNA sequencer. Sequencing of pVir inserts was performed by primer walking and by sequencing in both directions from within a chloramphenicol resistance cassette (41) following in vitro transposition, as described previously (15, 16). Custom DNA-sequencing primers were synthesized on an Applied Biosystems model 392 DNA synthesizer. The sequences of individual plasmid clones were assembled by either sequencing of overlapping cloned restriction fragments or PCR. The assembled sequence was analyzed and annotated manually. Open reading frames (ORFs) of greater than 30 residues were evaluated based on the presence of a suitable initiation codon with appropriate spacing to a ribosome-binding site as well as physical location to other ORFs. Similarity searches to the predicted proteins were performed using the BLAST-WU algorithm against all available public databases. Where appropriate, full-length proteins were aligned and analyzed using CLUSTALX.
Mutagenesis of pVir. Site-specific mutagenesis of pVir was performed using selected clones generated from the in vitro transposition outlined above. Plasmid DNAs containing transposon insertions were electroporated into C. jejuni 81-176 or NCTC 11168 as previously described with selection on MH agar containing 20 µg of chloramphenicol per ml (15, 16). Transformants were analyzed by PCR using primers flanking the insertion point to confirm that a double crossover event had occurred. To determine if the Cmr cassette exerted a polar effect on downstream genes, reverse transcriptase PCR (RT-PCR) was performed on the genes that are downstream of each of the insertionally inactivated genes in the five mutants that showed an invasion defect (see Table 2). RNA was isolated using RNAeasy kits (Qiagen). RT-PCR was done with the Prostar kit (Stratagene, La Jolla, Calif.) using Moloney murine leukemia virus RT as specified by the manufacturer. Denaturation was performed at 94°C and extension was performed at 72°C for 30s for all primers sets. The specific primers, annealing temperatures, and products were as follows. Expression of Cjp3 in the Cjp2 mutant was examined using primers 5'-CGACAACAAAATAGGACAAAATCGC-3' and 5'-CGTAAGGCATTCCGTATCTTTCAAG-3' at an annealing temperature of 51°C; the expected product size was 170 bp. Expression of Cjp16 in the Cjp15 mutant was examined using primers 5'-GAGCAATTTAATGATGGAATTTGC-3' and 5'-CGTAAGGCATTCCGTATCTTTCAAG-3' at an annealing temperature of 48°C; the expected product size was 453 bp. Expression of Cjp30 in a Cjp29 mutant was examined using primers 5'-CAGCAGGTATTCCTGTAATCGAC-3' and 5'-GACTTTATCTGTGTATTTCGTGCC-3' at an annealing temperature of 50°C; the expected product size was 341 bp. Expression of Cjp33 in a Cjp32 mutant was done with primers 5'-GGATAAGTGCAGTCGTAATTTTATTACTATGAG-3' and 5'-GCTTCAACCGTAGCCTTATTCATAG-3' at an annealing temperature of 49°C; the expected product size was 246 bp. Expression of Cjp48 in a Cjp49 mutant was done with primers 5'-GTGCTTTGGCTTTAGGAGCTATGAC-3' and 5'-GTCTTTATCGCAATAAACACTCACAAC-3' at an annealing temperature of 50°C; the expected product size was 317 bp. RT-PCR products were analyzed on 1.2% agarose. Postive controls were done using DNA as template and RNA from wild-type 81-176. The negative controls were the corresponding assays done without RT.
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Motility. The motility of all mutants defective in invasion was determined in motility agar (MH broth + 0.4% agar) as described previously (13, 14).
Statistical analyses. Percent invasion values of bacterial strains were compared using two-tailed t tests. Sample variance was assumed to be equal as determined by F-test analysis.
Nucleotide sequence accession number. The sequence of the pVir plasmid has been deposited in GenBank under accession no. AF472533.
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RESULTS AND DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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There are two large noncoding regions present on the pVir plasmid, the 1,147-bp region between Cjp28 and Cjp29 and the 1,049-bp intergenic region between Cjp49 and Cjp50. The region between Cjp28 and Cjp29, which has a G+C content of 19.8%, shows no homology to known DNA molecules. This stretch of DNA includes a complex region of direct repeats that is somewhat similar to the repeats that have been suggested as origins of replication of small cryptic campylobacter plasmids (25). The intergenic repetitive region spans 260 bp between Cjp28 and Cjp29 and is bracketed by perfect 52-bp direct repeats (5'-CCTATTTTACCATTACTTTAAGCTTTTTATAAGTCGCAAAATGTCGGTATAA-3'). The terminal 36 bases of the 52-mer are repeated once more within the 260-bp region, and the terminal 24 bases are repeated one more time within the same region. Interestingly, the 52-bp repeat is also found in the noncoding region between Cjp48 and Cjp49 (bp 31845 to 31896). The 36-bp part of the repeat is found again in the long noncoding intergenic region between Cjp49 and Cjp50 (bp 34216 to 34251), which has a similarly low G+C content of 18.3%. Thus, Cjp49 is bracketed by 36-bp direct repeats, an organization reminiscent of a transposable element (Fig. 1).
All of the predicted ORFs on pVir had suitable ribosome-binding sites (RBS) upstream of their predicted initiation codons. Although the spacing varied slightly (between 6 and 11 bases), the consensus RBS was AAGGA-N7-initiation codon. Only two of the predicted ORFs possessed an alternative start codon, Cjp31 with GUG and Cjp28 with UUG, although Cjp28 contains an authentic mutation causing an in-frame termination codon early in the protein (Table 1). This frequency of AUG initiation codons (96.3%) is higher than that seen for the NCTC 11168 chromosome (86.2%). There are 16 putative proteins encoded by pVir that are smaller than 100 residues and a further 19 that are between 100 and 200 residues. This represents an above-average proportion of low-molecular-weight proteins. However, the sequence quality of pVir was very high, and suitable RBSs preceded all these small proteins. The biological significance of the coding capacity of pVir being biased toward low-molecular-weight proteins is unknown.
The pVir proteins contain an amino acid bias that is driven by their low-G+C coding DNA content. There are high levels of Lys (11.8%) and Phe (6.6%) residues in the proteins, with 85% and 91.2% of the Lys and Phe residues being encoded by the AAA and UUU triplets, respectively. As expected, there is a strong bias in the third wobble position of the coding triplet, with this position being filled by an A or U base 81.5% of the time.
Genes encoding orthologs of NCTC 11168 genes. There were two genes that encoded proteins with homology to those encoded by the chromosome of C. jejuni NCTC 11168 (30). Cjp32 encodes a predicted protein of 239 amino acids which showed 21% identity and 40% similarity to the Cj0041 product (598 amino acids) over 98.7% of the total length of the Cjp32 protein, as well as 22% identity and 43% similarity over 209 amino acids to the Cj0320/FliH protein (276 amino acids), a protein involved in flagellin export. Cjp48 encodes a predicted protein of 130 amino acids which showed 90% identity and 94% similarity to the Cj1456c protein (104 amino acids) over 80% of the total length of the Cjp48 protein. Neither the Cj0041 nor Cj1456c protein show any significant homology to any other known protein. There were two possible initiation codons for Cjp48, one that matched Cj1456c and one that would have extended the NH2-terminal region of the protein by 14 residues. The internal Met residue was selected for Cjp48 due to the presence of a preferred RBS, as well as the fact that the homology to the NCTC 11168 chromosome deteriorated in the upstream residues including lacking the alternative Met residue. However, alignment of Cjp48 with the region in NCTC11168 demonstrated that the full-length homolog is present in the sequenced chromosome but is truncated due to a frameshift in a string of A residues (six in Cjp48 but only five in Cj1456c), suggesting that this gene is likely to be subject to slipped-strand regulation (30). Taking this frameshift into account, the identity of Cjp48 and the chromosomal ortholog increases to 92.3%. Further, the DNA homology to the NCTC chromosome extends for 66 bp downstream of the Cjp48 coding region.
Genes encoding type IV secretion system homologs. There are seven genes on pVir that encode homologs of type IV secretion proteins and are clustered in a region spanning 8.9 kb with an overall G+C content of 29.1%. The type IV genes in this region include the four genes described above (virB8/comB1, virB9/comB2, virB10/comB3, and virB11, ORFs Cjp1, Cjp2, Cjp3, and Cjp5, respectively). Orthologs of VirB8, VirB9, and VirB10 have been shown in other bacterial systems to encode pore-forming proteins, and VirB11 orthologs function as ATPases (6, 7). Just downstream of virB11, Cjp6 encodes an ortholog of VirD4 of H. pylori ATCC 55679 (36% identity and 55% similarity over 557 amino acids), another putative ATPase (6, 7). Cjp53 encodes an ortholog of VirB4 of H. pylori (33% identical and 54% similar over 842 amino acids), also a putative ATPase.
Located just upstream of virB8/comB1 on the circular map, Cjp54 encodes a small protein (Mr 4789) containing a lipobox (40). Although this protein showed no significant matches by BLASTP analyses, probably owing to its small size (42 amino acids), it showed 12% identity and 32% similarity to the lipoprotein VirB7 of Agrobacterium tumefaciens (11) by CLUSTAL analysis. In A. tumefaciens, a cysteine residue of VirB7 interacts with a cysteine of VirB9 to stabilize the complex of proteins that form the type IV transmembrane channel (2, 8, 35). Interestingly, Cjp54 contains three Cys residues across its 42-amino-acid length.
Additionally, there is one other gene encoding an ortholog of a type IV secretion system that is not linked to the others. This is Cjp31, which encodes a predicted protein with 32% identity and 49% similarity over 167 amino acids to trbM of plasmid RP4 (199 amino acids), a protein involved in conjugal transfer of this broad-host-range plasmid. The homology includes a stretch of 63 residues with 58.7% identity and 77.8% similarity to TrbM.
Genes encoding orthologs of H. pylori genes.
The type IV secretion system encoded by pVir shows its greatest similarities to type IV secretion proteins found in H. pylori. The pVir plasmid also encodes seven additional proteins whose best (and often only) homology is to other proteins identified in H. pylori (Table 1; Fig. 1). The Cjp11 protein displays a high level of homology to DNA topoisomerase I proteins, involved in DNA replication. However, it is significant that the topoisomerase I proteins with the highest level of similarity (
60%) are the "strain-specific" paralogs that have been identified in the plasticity zones of both sequenced H. pylori isolates, which are present in addition to the orthologous genes located in the conserved backbone of the H. pylori chromosome (1, 36).
The remaining six pVir proteins with orthologs in H. pylori are the Cjp8, Cjp10, Cjp26, Cjp33, Cjp49, and Cjp52 proteins (Table 1). Importantly, all of these H. pylori orthologs are found in the strain-specific plasticity zones of either H. pylori J99 (Cjp8 and Cjp10), H. pylori 26695 (Cjp33 and Cjp52), or both strains (Cjp26 and Cjp49). The Cjp26 protein shows similarity to HP1000 in strain 26695 and to JHP0935 in strain J99, which are homologs of ParA (partition) proteins that affect the proper distribution of newly replicated plasmids to daughter cells. The proteins with homology to the Cjp49 protein in strains 26695 and J99 are HP0996 and JHP0942, respectively, which are proteins of unknown function. However, in strain 26695 there is a second paralog (HP1004), which is also located in the plasticity zone. Analysis of 43 H. pylori isolates from Costa Rica demonstrated that at least one ortholog of this gene family was present in 19 of the strains (28). The two H. pylori J99 orthologs that were also present on pVir (Cjp8/JHP0926 and Cjp10/JHP0937) were looked at for their presence in the panel of 43 H. pylori isolated from Costa Rica (17 strains from cancer patients and 26 strains from gastritis patients) (28). Orthologs of JHP0926 (Cjp8) and JHP0937 (Cjp10) were found in 9 and 25 of the 43 strains, respectively (28).
It has been hypothesized that the variability of the H. pylori plasticity zone may be due, in part, to acquisition of sequences from self-replicating plasmids, since some plasticity zone genes have been identified on plasmids found in other H. pylori strains (1). Recently, proteins with high similarity to both chromosomal and plasticity zone-encoded proteins have also been found to be encoded by plasmids in certain H. pylori isolates (19), supporting the original theory that plasmid acquisition and genetic exchange plays a major role in the macrodiversity observed within the H. pylori population.
The presence of orthologs, from both plasticity zones, on a C. jejuni plasmid further strengthens this hypothesis and suggests the possibility that sequences may be exchanged between these two highly related species. Indeed, the presence of plasticity zone genes on a plasmid involved in pathogenesis of C. jejuni suggests that these regions in H. pylori may play a role in the varying level of disease caused by infection with different strains of this organism.
The plasticity zone is one of several regions in the H. pylori genome that is characterized by having a G+C content (35%) significantly lower than the remainder of the genome (39%), which is consistent with this region being introduced by horizontal transfer from another species. The plasticity zone orthologs on the pVir plasmid range in their G+C content from 25.3% (Cjp26) to 33.9% (Cjp10). Interestingly, the orthologs of the H. pylori J99 plasticity zone have significantly higher G+C content (Cjp8, 32.2%; Cjp10, 33.9%) than do the orthologs of the H. pylori 26695 plasticity zone (Cjp26, 25.3%; Cjp33, 26.6%; Cjp52, 28.8%). Whether the ancestral lineage of the pVir plasmid is married to the lineage of the C. jejuni species is unknown, but the low G+C content makes it unlikely and the evidence reported here strongly suggests that the pVir plasmid of C. jejuni and the plasticity zone of H. pylori have their origins from an as yet undiscovered source. There is considerable evidence suggesting that the diversity of the plasticity zone(s) of H. pylori strains is influenced by plasmid sequences (1, 19) and that each strain contains its own unique complement of genes, even though some of them are in common (28). Furthermore, recent evidence has demonstrated that H. pylori isolates that were taken from the J99 patient 6 years after the initial isolation of J99 and shown by sequence analysis of several regions totaling over 4 kb to be 100% identical to J99 have undergone several significant changes in the content of their plasticity zones (21). While some colonies had lost large regions of the plasticity zone, many had actually acquired DNA previously seen only in the plasticity zone of H. pylori 26695 (21). This confirmed that this region is hypervariable, and the presence of some of these orthologs on a plasmid in C. jejuni strongly suggests that both species have acquired DNA from a common ancestor or that they have exchanged DNA in the past.
Genes encoding other proteins of known function. There are only two other genes on pVir that encode proteins with similarity to proteins of known function. These are Cjp13, whose product shows similarity to single-stranded DNA-binding proteins, and Cjp28, which, although its product shows similarity to plasmid replication proteins from a number of bacterial species, contains an authentic in-frame stop codon and appears to be a pseudogene, suggesting that Cjp28 is not involved in plasmid replication. Interestingly, the only other gene encoding a protein predicted to be involved in plasmid replication was Cjp26 (see above). However, as shown below, a plasmid with a mutation in Cjp26 was stably maintained. Thus, the sequence does not reveal any clear information about plasmid replication or maintenance. The Cjp26 and Cjp28 proteins are the only pVir products that show any similarity to proteins encoded by the two large H. pylori plasmids, pHel4 and pHel5, that were recently sequenced (19).
Mutational analyses. Preliminary mutational analyses were done using selected transposon insertions generated during sequence analyses. These insertions were electroporated into 81-176 as described previously (15, 16), and putative mutants were confirmed by PCR analysis as described in Materials and Methods. These mutants, all of which were fully motile, were examined phenotypically in terms of invasion of INT407 cells to screen for potential virulence genes. We have previously described the effect of mutations in virB11 and virB10/comB3 on invasion (3). Mutations were characterized in four additional genes encoding putative components of a type IV secretion system, virB8/comB1, virB9/comB2, virD4, and virB4. Of these, only the mutant in virB9/comB2 showed a significant reduction in invasion (Table 2). It was surprising that mutation of some, but not all, of the genes encoding the components of the type IV system affected invasion in vitro. This observation may reflect, in part, the considerable variability known to exist among type IV secretion systems in diverse bacteria. Thus, the type IV secretion systems of H. pylori and Rickettsia prowazekii contain fewer components than does the prototypic type IV system of A. tumefaciens (6, 7). Moreover, Selbach et al. recently were able to distinguish VirD4-dependent and VirD4-independent functions of the H. pylori type IV secretion system (34). These workers have shown that while VirD4 is necessary for secretion of CagA through the type IV secretion machinery, induction of IL-8 release from gastric epithelial cells requires other components of the type IV system but not VirD4 (34).
Mutants with mutations in 14 other genes were screened for invasion defects. Nine of these are mutants with mutations in genes that appear to be unique to the campylobacter plasmid (ORFs 12, 15, 16, 23, 24, 29, 30, 44, and 51), four had mutations in orthologs of H. pylori genes (Cjp8, Cjp11, Cjp26, and Cjp49), and the last had a mutation in Cjp32, which is homologous to Cj0041 and FliH of NCTC 11168 (see above). Of the 14 mutants, 4 showed significant reductions in invasion. The mutant with a mutation in Cjp15, encoding a predicted soluble protein of 25.8 kDa, invaded at 53.6% of the level of wild-type 81-176 (P < 0.01). The Cjp32 mutant invaded at 34.8% of the level of wild-type 81-176 (P < 0.001). The mutant with a mutation in gene Cjp49, encoding the ortholog of HP0996, invaded at 26% the level of wild-type 81-176 (P < 0.0001). Intriguingly, the Cjp49 gene is bracketed by a perfect pair of 36-bp direct repeats (see above), which may render this gene unstable via intragenomic recombination. The fourth mutant affected in invasion had a mutation in the Cjp29 gene, which encodes a glutamine-rich protein of 44.8 kDa, which showed no significant match by BLASTP analyses. The mutant with a mutation in Cjp29 invaded at 15.2% of the level of wild-type 81-176 (P < 0.0001). There was no significant difference in the growth curves of the mutants that showed invasion defects compared to 81-176 in MH broth (data not shown). Additionally, RT-PCR was done on downstream genes, as described in Materials and Methods, to confirm that the Cmr cassette was not exerting a polar effect on downstream genes. In all cases, the gene downstream of the mutated gene was expressed (data not shown).
pVir alone is insufficient for invasion. A mutant of 81-176 insertionally inactivated in Cjp11, encoding a putative topoisomerase, invaded at wild-type levels (Tables 2 and 3). Plasmid DNA (pVir/Cjp11::Cm) was isolated from this mutant and used to electroporate NCTC 11168 to Cmr. Comparison of the restriction enzyme patterns of pVir/Cjp11::Cm isolated from an NCTC 11168 recipient and that from the 81-176 parent showed no differences (data not shown), indicating that the intact plasmid had been transferred into the recipient. Plasmid pVir/Cjp11::Cm appeared to replicate stably in NCTC 11168, and yields of plasmid DNA were comparable to yields from 81-176, suggesting similar copy numbers in the two strains. However, the presence of the plasmid in NCTC 11168 did not alter its low level of invasion into INT407 cells, as shown in Table 3. This indicates that although the plasmid is required for maximum invasion by 81-176, additional virulence factors not found in NCTC 11168 are also necessary. This is consistent with previous reports about the requirement of both the Penner 23,36 capsule and particular ganglioside mimicries in LOS for invasion of 81-176 into INT407 cells (4, 16). We have previously shown that a strain of 81-176 that has spontaneously lost pTet, the second large plasmid in this strain, is fully invasive (3), but the possibility remains that additional, novel chromosomal genes of 81-176 may contribute to invasion. The pVir plasmid appears remarkably stable in 81-176, and we have been unable, so far, to cure tagged versions (data not shown).
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ACKNOWLEDGMENTS |
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We thank Gary Majam and Isabelle Walker for technical assistance and Scarlett Goon for advice with the RT-PCR experiments.
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FOOTNOTES |
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