SPC-P1: a pathogenicity-associated prophage of Salmonella paratyphi C
- Qing-Hua Zou†1,
- Qing-Hai Li†2, 6,
- Hong-Yun Zhu1,
- Ye Feng2, 6,
- Yong-Guo Li3,
- Randal N Johnston4,
- Gui-Rong Liu2, 6Email author and
- Shu-Lin Liu1, 2, 3, 5, 6Email author
© Zou et al; licensee BioMed Central Ltd. 2010
Received: 27 January 2010
Accepted: 30 December 2010
Published: 30 December 2010
Salmonella paratyphi C is one of the few human-adapted pathogens along with S. typhi, S. paratyphi A and S. paratyphi B that cause typhoid, but it is not clear whether these bacteria cause the disease by the same or different pathogenic mechanisms. Notably, these typhoid agents have distinct sets of large genomic insertions, which may encode different pathogenicity factors. Previously we identified a novel prophage, SPC-P1, in S. paratyphi C RKS4594 and wondered whether it might be involved in pathogenicity of the bacteria.
We analyzed the sequence of SPC-P1 and found that it is an inducible phage with an overall G+C content of 47.24%, similar to that of most Salmonella phages such as P22 and ST64T but significantly lower than the 52.16% average of the RKS4594 chromosome. Electron microscopy showed short-tailed phage particles very similar to the lambdoid phage CUS-3. To evaluate its roles in pathogenicity, we lysogenized S. paratyphi C strain CN13/87, which did not have this prophage, and infected mice with the lysogenized CN13/87. Compared to the phage-free wild type CN13/87, the lysogenized CN13/87 exhibited significantly increased virulence and caused multi-organ damages in mice at considerably lower infection doses.
SPC-P1 contributes pathogenicity to S. paratyphi C in animal infection models, so it is possible that this prophage is involved in typhoid pathogenesis in humans. Genetic and functional analyses of SPC-P1 may facilitate the study of pathogenic evolution of the extant typhoid agents, providing particular help in elucidating the pathogenic determinants of the typhoid agents.
The bacterial genus Salmonella contains more than 2600 very closely related serovars, classified by the Kauffmann-White Scheme according to their differences in the somatic (O) and flagellar (H) antigens [1, 2]. Although essentially all Salmonella bacteria are pathogens, they may have different host ranges or cause different diseases.
Over 1400 Salmonella serovars may infect humans, with most of them causing self-limiting gastroenteritis. On the other hand, a few Salmonella serovars, such as Salmonella typhi, S. paratyphi A, S. paratyphi B and S. paratyphi C, are adapted to humans and cause typhoid fever, a serious and potentially fatal systemic infection . It is not clear whether these Salmonella typhoid agents use the same, similar or totally different pathogenic traits to infect the same host and cause the disease. Genomic comparisons between S. typhi and S. paratyphi A did not reveal a common genetic basis possibly responsible for human adaptation or typhoid pathogenesis [4, 5]. Notably, various Salmonella pathogenicity islands (SPIs) or prophages have been identified in the Salmonella typhoid agents, e.g., SPI-7 in S. typhi[6, 7] and S. paratyphi C , and SPA-1, SPA-2 and SPA-3 in S. paratyphi A , but their specific roles in typhoid pathogenesis have not been well established.
In a previous study, we located several insertions in the genome of S. paratyphi C strain RKS4594 by comparing it with other Salmonella genomes , including one, SPC-P1, which was a prophage present only in S. paratyphi C among all sequenced Salmonella strains . In this study, we characterized this novel prophage, predicted its possible roles in the pathogenicity of S. paratyphi C, and evaluated its potential contributions to pathogenicity in animal experiments. We found that, although no previously known pathogenicity-associated genes were identified in the prophage, SPC-P1 did increase the pathogenicity of the bacteria.
Genomic location and identification of prophage SPC-P1
We screened the complete nucleotide sequence of the S. paratyphi C RKS4594 genome (CP000857) by Phage_Finder http://phage-finder.sourceforge.net for possible prophage sequences and located five regions with typical prophage characteristics, with four of them having been reported in other Salmonella serovars and well studied previously, including Gifsy-1 and Gifsy-2 in S. typhimurium LT2  and SPA-1 and Phage SPA-3-P2 in S. paratyphi A ATCC9150 . These prophages have also been known to be present in several other Salmonella serovars, such as S. choleraesuis. The remaining genomic region corresponds to the previously mapped 39 kb insertion between genes purC and purF and has typical features of a prophage; here we designate this region SPC-P1. Sequence analysis showed that SPC-P1 lies between two adjacent genes, pgtE and yfdC, in S. paratyphi C RKS4594, whereas in fifteen other published Salmonella genomes (see their accession numbers below), we did not find DNA insertions in this region. The ends of SPC-P1 were set by two direct repeats of the sequence tggtgtcccctgcag, a typical feature for the ends of prophage DNA sequences. One of the repeat sequences begins at 109 bp upstream of SPC-P1 ORF1, and the other begins at 165 bp downstream of ORF53 and continues with an arg tRNA gene. The total length of SPC-P1 is 39,659 bp and the overall G+C content is 47.24%, which is similar to those of phage P22 (47.1%) [12, 13] and ST64T (47.5%)  and is significantly lower than the 52.16% average of the S. paratyphi C RKS4594 chromosome.
Layout and predicted products of SPC-P1 genes
Using Vector NTI 9.0 and GLIMMER3, we identified 53 ORFs in SPC-P1, designated consecutively from ORF1 through ORF53 (Additional file 1 Table S1), with the ORF encoding the terminase small unit as ORF1. As shown in Additional file 1 Table S1, the G+C contents of individual ORFs vary from 31.65% (ORF 16) to 53.89% (ORF 48), demonstrating an obvious mosaic structure.
Of the 53 ORFs, 47 had ATG and six (ORF13, ORF16, ORF21, ORF29, ORF31, ORF44) had GTG as the start codon. Functions of the SPC-P1genes were inferred based on similarities with characterized genes from other phages; some salient features of these protein-encoding genes are summarized in Additional file 1 Table S1.
Phylogenetic analysis of SPC-P1
Distribution of SPC-P1 in other Salmonella serovars and among S. paratyphi C strains
Primers used for amplification of SPC-P1
Site at SPC-P1
Product size (bp)
PCR detection of SPC-P1 in S. paratyphi C strains
Induction of SPC-P1 and morphological analysis
Since SPC-P1 seems to contain all necessary genes for a viable phage, we wondered whether it could be induced from the bacterial genome. Upon mitomycin C treatment, the culture of S. paratyphi C RKS4594 became clearer than the culture without mitomycin treatment, suggesting that phage were induced to lyse the cells.
Lysogenic conversion of CN13/87 by SPC-P1
Increased pathogenicity of lysogenized CN13/87 in mouse infection experiments
Infectiona of mice at different doses of bacteria
No. infected mice (%)
Wild type CN13/87
1 × 104
1 × 105
1 × 106
1 × 107
1 × 108
1 × 109
Bacteria evolve by accumulating mutations and incorporating laterally transferred genes, among which phages are by far the most important driving force. For example, since the divergence from E. coli about 120-160 million years ago [16–18], Salmonella have developed into a great number of distinct lineages, with more than 2600 serovars currently recognized . They all share a core genome, which is about 90% of the genes for Salmonella subgroup I serovars, with the remaining ca. 10% genes being specific to individual serovars . Genomic analyses reveal that Salmonella harbor numerous temperate bacteriophages [13, 20–23]. In fact, most of the non-core genome sequences are derived from phages, which play key roles in bacterial genome evolution and pathogenicity. In this study, we characterized a novel prophage, SPC-P1, in the genome of S. paratyphi C RKS4594 and demonstrated that this phage is present only in S. paratyphi C strains but not in any other Salmonella serovars tested. SPC-P1 exhibits typical characteristics of prophages, including a significantly lower overall G+ C content than that of the bacterial genome average, repeat sequences at the ends of its genome, and tRNA genes at the integration site. Sequence analysis showed that SPC-P1 has a substantial portion of its genome being highly related to previously characterized lambdoid phages and it has a complete set of genes to encode a viable phage. The mitomycin C induction test confirmed this.
Although prophages are widely found in bacterial genomes, most of them are defective, unable to produce viable phage particles. Sequence analysis indicated close relatedness of SPC-P1 to CUS-3 and electron microscopy also revealed morphological similarity between SPC-P1 and CUS-3. Like CUS-3, SPC-P1 also has a cosahedral head and a short tail. Since SPC-P1 could be induced from the bacterial genome and we had available the SPC-P1 sensitive strain CN13/87, we had the opportunity to propagate this phage for further studies. SPC-P1 could not only lyse CN13/87 but also lysogenize it, which allows us to study the possible roles of SPC-P1 in bacterial pathogenicity.
S. paratyphi C is one of the few Salmonella serovars that cause typhoid fever in humans, along with S. typhi, S. paratyphi A and S. paratyphi B, but it is not fully clear whether different Salmonella typhoid agents cause the disease by similar or distinct mechanisms. Prophages can contribute important biological properties to their bacterial hosts and analysis of the prophages may shed light on the evolution of their hosts. Considering that the human-adapted typhoid agents may have evolved by convergent processes , we speculate that S. paratyphi C may cause the disease by different mechanisms than those used by other Salmonella typhoid agents. As SPC-P1 is found only in S. paratyphi C, it may be involved in the pathogenesis of typhoid caused by S. paratyphi C. Taking the advantage that S. paratyphi C, unlike other human-adapted Salmonella typhoid agents, can infect hosts other than humans if large inocula are used [24, 25], we compared pathogenicity of S. paratyphi C between SPC-P1-free and SPC-P1-lysogenized isogenic strains. We found that SPC-P1 significantly increased the pathogenicity of S. paratyphi C and caused multiple organ damages in the animals (see Table 3 and Figure 7), but the molecular basis is yet to be understood.
SPC-P1 contributes pathogenicity to S. paratyphi C in animal infection models, so it is possible that this prophage is involved in typhoid pathogenesis in humans. Genetic and functional analyses of SPC-P1 may facilitate the study of evolution of the different typhoid agents, providing particular help in elucidating the pathogenic determinants of the typhoid agents.
Bacterial strains and growth conditions
Bacterial strains used in this study are listed in Table 2 and detailed information on them can be obtained from the Salmonella Genetic Stock Center http://www.ucalgary.ca/~kesander. Bacteria were grown overnight at 37°C with shaking in LB broth.
The genome sequence of RKS4594 was obtained from several pUC18 genomic shotgun libraries using dye terminator chemistry on Megabace1000 and ABI3730 automated sequencers as described previously .
ORF prediction and homology search
Open reading frames (ORF) of SPC-P1 were predicted by Vector NTI and Glimmer 3. Products of ORFs were deduced based on homologies to known proteins by the BLASTP server of NCBI. Similarity of nucleotide sequence between SPC-P1 and the other completely sequenced Salmonella prophages was evaluated by the BLASTN server of NCBI.
G+C content analysis and tRNA prediction
The G+C content of each predicted ORF was analyzed using the DNAstar software. tRNA was predicted using tRNAscan-SE software http://lowelab.ucsc.edu/tRNAscan-SE/.
Distribution of SPC-P1 in other S. paratyphi C strains
Genomic DNA of S. paratyphi C strains was isolated by the Genomic DNA Extraction Kit (TIANZE, China). LA taq polymerases were purchased from Takara. PCR reactions were performed as follows: 94°C, 1 min; 98°C, 20 sec; 68°C, 10 min, 30 cycles; 72°C, 10 min.
Induction of SPC-P1
S. paratyphi C RKS4594 was grown in LB medium at 37°C for 4 h, followed by addition of four times volume of fresh LB. At this point, mitomycin C was added to a final concentration of 0.5 μ/ml. The cultures were shaken, 120r/min, at 37°C for 14 h. Chloroform was added to a final concentration of 1% to the culture, followed by vortex of the culture for 1 min. Cell debris was removed by centrifugation at 12000 rpm for 10 min. The supernatant containing SPC-P1 was preserved at 4°C until use.
Plaque formation test
S. paratyphi C strain CN13/87 did not harbor SPC-P1 and so was used as a recipient in the test. Ten-fold serial dilutions of the bacterial lysate were made. Then 10 μl of a dilution and 100 μl of CN13/87 fresh culture (4 h) were mixed and incubated at 37°C for 20 min before addition of 3 ml 0.7% LB agar cooled to about 45°C. After mixing quickly, the 0.7% LB agar containing the lysate and bacteria was spread to a 1.5% LB agar plate, which then was cultured overnight. For identification, phage in individual plaques were picked up and propagated on CN13/87 before DNA was extracted for further PCR analysis and for transmission electron microscopy.
Lysogenization of strain CN13/87
CN13/87 was cultured at 37°C in 2 ml LB for 4 h. Then 10 μl SPC-P1 (108 pfu/ml) was added and the culture was continued at 37°C overnight. Serial 10-fold dilutions of the culture were spread onto LB agar plates and incubated overnight at 37°C. Single colonies were picked up and spread uniformly onto fresh LB plates, eight colonies per plate. A small drop of SPC-P1 (about 5 μl) was placed onto the bacterial patches. The plates were cultured overnight before inspection of SPC-P1 plaques on the bacteria.
Animal infection experiments
Female BALB/c mice, 6-8 weeks old, were divided into seven groups, twelve mice per group with six inoculated with wild type CN13/87 and six with SPC-P1-lysogenized CN13/87 orally with 0.5 ml sterile water containing no bacteria (group 1), 104 cfu/0.5 ml (group 2), 105 cfu/0.5 ml (group 3), 106 cfu/0.5 ml (group 4), 107 cfu/0.5 ml (group 5), 108 cfu/0.5 ml (group 6) or 109 cfu/0.5 ml (group 7). When the mice were sacrificed as specified, liver, lung and spleen tissues were taken for bacterial detection and histological examinations. ID50 was determined as described .
Genbank: S. typhimurium LT2 [NC_003197]; S. choleraesuis [SC-B67 NC_006905]; S. paratyphi A ATCC9150 [NC_006511]; S. typhi CT18 [NC_003198]; S. typhi Ty2 [NC_004631]; S. paratyphi C RKS4594 [CP000857]; S. schwarzengrund CVM19633 [NC_011094]; S. paratyphi A AKU_12601 [NC_011147]; S. newport SL254 [NC_011080]; S. heidelberg SL476 [NC_011083]; S. gallinarum 287/91 [NC_011274]; S. enteritidis P125109 [NC_011294]; S. dublin CT_02021853 [NC_011205]; S. agona SL483 [NC_011149]; S. arizonae 62:z4,z23:-- [NC_010067]; Enterobacteria phage ES18 [NC_006949]; Enterobacteria phage ST64T [NC_004348]; Enterobacteria phage ST104 [AB102868]; Enterobacteria phage CUS-3 [CP000711]; Enterobacteria phage HK620 [NC_002730]; Enterobacteria phage Sf6 [NC_005344]; Enterobacteria phage HK022[NC_002166]; Enterobacteria phage HK97 [NC_002167]; Enterobacteria phage lambda [NC_001416]; Bacteriophage P22[AF217253]; Salmonella typhimurium phage ST64B [AY055382]; Enterobacteria phage Min27 [NC_010237].
This work was supported by a grant of Canadian Institutes of Health Research to RNJ; a National Natural Science Foundation of China (NSFC30970078) and a grant of Natural Science Foundation of Heilongjiang Province of China to GRL; a grant from Harbin Medical University, grants of National Natural Science Foundation of China (NSFC30870098, 30970119, 81030029), a Specialized Research Fund for the Doctoral Program of Higher Education (SRFDP, 20092307110001), and a 985 Project grant of Peking University Health Science Center to SLL.
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