Letters in Applied Microbiology ISSN 0266-8254
ORIGINAL ARTICLE
Presence of T3SS2b genes in trh+ Vibrio parahaemolyticus isolated from seafood harvested along Mangalore coast, India B.K. Kumar, V.K. Deekshit, P. Rai, M. Shekar, I. Karunasagar and I. Karunasagar Department of Fisheries Microbiology, UNESCO-MIRCEN for Marine Biotechnology, Karnataka Veterinary, Animal and Fisheries Sciences University, College of Fisheries, Mangalore, India
Significance and Impact of the Study: T3SSs (a or b) are the important virulence factors of Vibrio parahaemolyticus that contribute to their pathogenicity in humans. This study demonstrated the presence of T3SS2b genes in V. parahaemolyticus isolated from the seafood harvested along Mangalore coast. RT-PCR showed that the T3SS2b genes identified in seafood isolates of V. parahaemolyticus were found to be functional. To the best of our knowledge, this is the first description of T3SS2b genes in trh+ V. parahaemolyticus isolated from seafood in India. The presence of T3SS2 along with other virulence factors such as TDH and/or TRH highlights a potential health risk for seafood consumers.
Keywords seafood, TDH-related haemolysin, thermostable direct haemolysin, type III secretion system, V. parahaemolyticus. Correspondence Dr. Indrani Karunasagar, Department of Fisheries Microbiology, UNESCO-MIRCEN for Marine Biotechnology, Karnataka Veterinary, Animal and Fisheries Sciences University, College of Fisheries, Mangalore, India. E-mail: [email protected] 2013/2176: received 30 October 2013, revised 19 December 2013 and accepted 20 December 2013 doi:10.1111/lam.12210
Abstract Vibrio parahaemolyticus is a seafood-borne pathogen autochthonous to the marine and estuarine ecosystem, responsible for gastroenteritis when contaminated raw seafood is consumed. The pathogenicity has been associated with thermostable direct haemolysin (TDH) and TDH-related haemolysin (TRH). Of late, the presence of T3SS2a and T3SS2b gene clusters has been well documented in clinical isolates of Vibrio parahaemolyticus and known to play an essential role in pathogenesis. However, reports on the presence of T3SSb genes in V. parahaemolyticus isolated from the seafood and/or environmental samples are scanty. In this study, we have identified and analysed the distribution of the T3SS2b genes in V. parahaemolyticus isolated from seafood harvested along southwest coast of India. Results showed that T3SS2b genes are solely associated with trh+ and tdh+/trh+ strains of V. parahaemolyticus. Reverse transcriptase PCR (RT-PCR) showed that the T3SS2b genes identified in trh+ V. parahaemolyticus were transcriptionally active. To our knowledge, this study appears to be the first description on the presence of T3SS2bpositive V. parahaemolyticus isolated from seafood in India. The study of T3SS2 along with other virulence factors will help in better understanding of the risk of seafood-borne illness due to V. parahaemolyticus.
Introduction Vibrio parahaemolyticus is a Gram-negative, halophilic bacterium, autochthonous to water and sediments of marine and estuarine ecosystems around the world (Nishibuchi and Kaper 1995; Deepanjali et al. 2005) and is a leading cause of seafood-borne gastroenteritis due to the consumption of raw seafood (Honda et al. 2008). The virulence properties of this organism have been associated 440
with thermostable direct haemolysin (TDH) and TDHrelated haemolysin (TRH) coded by the tdh and trh genes, respectively (Honda et al. 1987a,b; Nishibuchi and Kaper 1995). Therefore, the strains bearing tdh and/or trh genes are considered to be pathogenic (Nishibuchi and Kaper 1995). Only a very minute proportion (1–2%) of the V. parahaemolyticus isolated from the environment are known to be virulent and carry virulence genes tdh and/ or trh, whereas most of the clinical isolates (90%) are
Letters in Applied Microbiology 58, 440--446 © 2013 The Society for Applied Microbiology
T3SS2b genes in V. parahaemolyticus
B.K. Kumar et al.
found to be virulent. However, recent studies have shown that apart from tdh and trh, there are other putative pathogenicity-associated genes known to be associated with the virulence (Makino et al. 2003; Ono et al. 2006; Honda et al. 2008; Kodama et al. 2008; Hiyoshi et al. 2010). Many Gram-negative pathogens utilize type III secretion systems (T3SSs) to induce pathogenesis by translocating effector proteins into the host cells. The T3SS is composed of a needle-shaped apparatus comprising protein components and a large number of effector proteins (Cornelis 2006; Coburn et al. 2007). Upon entering the host cell, the active form of effector protein manipulates the host cell signalling pathways to disrupt host immune response. Genome mapping of a clinical isolate of V. parahaemolyticus (RIMD2210633) confirmed the existence of two sets of type III secretion system (T3SS) gene clusters, T3SS1 and T3SS2 (Makino et al. 2003). The toxins secreted by both the T3SSs are essential for cytotoxicity and enterotoxicity of these strains, thus having an apparent role in the pathogenesis (Broberg et al. 2011; Karunasagar et al. 2012). The T3SS1 is present in chromosome I of the V. parahaemolyticus isolated from clinical and environmental sources regardless of their pathogenicity, and T3SS2 is located predominantly in the chromosome II of highly virulent strains (Kodama et al. 2008; Okada et al. 2009). The gene coding for T3SS1 known to involve in cytotoxicity against eukaryotic cells and T3SS2 is responsible for the enterotoxicity in the rabbit ileal loop test (Park et al. 2004; Bhattacharjee et al. 2005, 2006; Kodama et al. 2008; Burdette et al. 2009; Zhou et al. 2009, 2010; Hiyoshi et al. 2010). Later DNA sequencing of approximately 100 kb regions that surround trh gene of V. parahaemolyticus strain, TH3996, revealed the presence of a novel T3SS, which is closely related to T3SS2 of tdh+ V. parahaemolyticus (Okada et al. 2009). After the report of novel T3SS2 in trh+ V. parahaemolyticus strain TH3996 of clinical origin, it has been further classified into two distinct phylogroups (a and b) based on the divergence in nucleotide sequences. In earlier studies, the presence of V. parahaemolyticus in seafood harvested along southwest coast of India has been well documented (Dileep et al. 2003; Deepanjali et al. 2005; Parvathi et al. 2006; Raghunath et al. 2008; Kumar et al. 2011). The study by Deepanjali et al. (2005) highlighted the prevalence (59%) of trh+ isolates among the total V. parahaemolyticus isolated from seafood harvested along the Mangalore coast, India. However, clinical cases due to these strains of V. parahaemolyticus have not been reported in this region. The aforesaid virulence attributes have been detected and well studied in the clinical strains of V. parahaemolyticus with limited reports in
isolates of environmental origin (Caburlotto et al. 2009; Noriea et al. 2010). There are no reports on the presence of T3SS2a or T3SS2b genes in V. parahaemolyticus isolated from Indian marine environment and/or seafood leading to a paucity of data. Hence, this study was principally focused to identify the presence of T3SS2 genes and their distribution in trh+ V. parahaemolyticus isolated from seafood harvested along the Mangalore coast. Results and discussion All the V. parahaemolytcus isolates used in this study were isolated from the seafood harvested along southwest coast of India during the period April 2003–March 2013 and had been identified previously using standard bacteriological methods (Raghunath et al. 2008). All the 41 V. parahaemolyticus isolates used in this study were found to be positive for toxR gene (species-specific marker), thereby further confirming the identification of V. parahaemolyticus used in this study (Kim et al. 1999). Further, the presence of genes coding for haemolysins was detected by PCR targeting tdh and trh genes (Tada et al. 1992). Based on the PCR results, isolates were categorized into four distinct groups, which were 5 tdh+, 20 trh+, 3 tdh+/trh+ and 13 tdh /trh (Table 1). The gene coding for T3SS2 (a or b) located on the chromosome II is unique to highly virulent strains of V. parahaemolyticus (Makino et al. 2003), and the toxins secreted by these T3SS machineries are believed to have a profound role in pathogenesis of the organism (Broberg et al. 2011; Karunasagar et al. 2012). Hence, it is important to understand the prevalence of genes encoding these systems in seafood and/or environmental isolates of Table 1 Distribution of T3SS2b genes in Vibrio parahaemolyticus in seafood harvested along southwest coast of India Number of seafood isolates (n = 41) Gene coding for
Target gene
tdh+/trh (n = 05)
tdh /trh+ (n = 20)
tdh+/trh+ (n = 03)
tdh /trh (n = 13)
Apparatus protein
VscC2 VscR2 VscS2 VscT2 VscU2 VcrD2 VscN2 VopB2 VopD2 VopC VopA VopL
– – – – – – – – – – – –
20 20 17 15 20 20 20 19 20 20 20 19
3 3 1 3 3 3 3 3 3 3 3 3
– – – – 2 – 2 – 1 – – –
ATPase Translocon protein Effector protein
‘–’ Means negative for the tested genes.
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V. parahaemolyticus. In this study, a PCR assay was used to identify the presence of the gene coding for apparatus (VscC2/R2/S2/T2/U2/N2, VcrD2), effector (VopA/C/L) and translocon proteins (VopB2/D2) of T3SS2b genes (Okada et al. 2009). All the 20 trh+ and 3 tdh+/trh+ isolates analysed in this study were positive for the T3SS2b genes analysed (Fig. 1). Although all the 5 tdh+ isolates analysed were negative for T3SS2b genes, they could possess the T3SS2a gene cluster. All the nonpathogenic V. parahaemolyticus isolates analysed in this study were PCR-negative for the T3SS2b gene. Two of the nonpathogenic isolates were positive for the VscU2 and VscN2 genes, while one strain was positive for VopD2 gene (Table S2). To the best of our knowledge, this is the first description of T3SS2b in trh+ V. parahaemolyticus isolated from seafood in India. The results obtained in this study showed the presence of T3SS2b in seafood isolates of V. parahaemolyticus, which is otherwise reported generally in clinical strains. The presence of the gene suggests the potential of the isolates to cause infection in humans upon ingestion of contaminated seafood. To date, a very few reports are available on the prevalence of T3SSb genes in V. parahaemolyticus isolated from the seafood and/or environmental samples worldwide (Noriea et al. 2010; Jones et al. 2012). We have also determined the transcriptional activity of VopA and VopC genes of T3SS2b. The reverse transcriptase PCR (RT-PCR) for effector genes VopC and VopA with specific primer gave prominent amplification with the expected amplicons (Fig. 2). RT-PCR analysis showed that the T3SS2 genes present in the trh+ V. parahaemolyticus isolated from the seafood were found to be functional. The gene coding for VopA and VopC is part of the T3SS2 gene cluster, was sequenced, analysed and submitted to the NCBI (GenBank accession numbers JQ996146, JQ996147). The VopC and VopA analysed in this study showed unusual base composition (G + C content of 388
M
1
2
M
3
1
2
and 386%, respectively) compared to the rest of the genome. This suggests that T3SS2 might have integrated into the V. parahaemolyticus chromosome through horizontal gene transfer. A sequence similarity search for the nucleotide and deduced amino acid sequences of VopA and VopC was performed using BLAST (Altschul et al. 1997). The gene coding for VopA shared similarity with the acetyltransferase group found in V. parahaemolyticus RIMD2210633, V. cholerae, Yersinia spp. and Aeromonas spp. Similarly, the gene coding for VopC protein analysed in this study showed sequence similarity to the catalytic domain of cytotoxic necrotizing factor (CNF) toxins with the presence of catalytic cysteine (225th) and histidine (240th) residues in the core region. The T3SS2 effector proteins are known to be involved in the intracellular survival, replication and pathogenesis of the organism, brought about by the activity of conserved catalytic amino acid residues (Orth et al. 1999; Trosky et al. 2004, 2007; Mukherjee et al. 2006). Phylogenetic trees for each of the genes were constructed using Dice correlation and unweighted pair group method with arithmetic mean (UPGMA). Analysis showed that both VopA and VopC gene sequences of V. parahaemolyticus clustered with the T3SS2b gene sequence rather than the T3SS2a gene sequence (Fig. 3), and it demonstrated that nucleotide sequences of VopA and VopC obtained in this study closely belong to the T3SS2b found in the clinical isolates of V. parahaemolyticus TH3996, AQ4037 and V. cholerae. Previous studies reported that non-O1, non-139 V. cholerae strain-1587 also possesses a T3SS2-like secretion system, which had a similarity to the T3SS2 of V. parahaemolyticus (Dziejman et al. 2005; Murphy and Boyd 2008; Zhang and Orth 2013). In conclusion, this study showed the presence of transcriptionally active T3SS2b genes in seafood isolates of V. parahaemolyticus, which is otherwise reported generally in clinical strains. The presence of the T3SS2b along with
3
M 1
2
3 1252 bp
695 bp
594 bp
(a)
(b)
(c)
Figure 1 Presence of gene coding for T3SS2b effector proteins of Vibrio parahaemolyticus. (a) VopC (594 bp), Lane M: 100-bp molecular weight marker, Lane 1: Negative control, Lanes 2 and 3: Environmental isolates of V. parahaemolyticus. (b) VopA (695 bp), Lane M: 100-bp molecular weight marker, Lane 1: Negative control, Lanes 2 and 3: Environmental isolates of V. parahaemolyticus. (c) VopL (1252 bp), Lane M: 500-bp molecular weight marker, Lane 1: Negative control, Lanes 2 and 3: Environmental isolates of V. parahaemolyticus.
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Letters in Applied Microbiology 58, 440--446 © 2013 The Society for Applied Microbiology
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M Figure 2 RT-PCR showing expression of putative virulence genes of Vibrio parahaemolyticus. cDNA (+) made from total RNA isolated from V. parahaemolyticus strain AQ4037 and VP49 was used as a template for PCR. Mock reactions ( ), which did not contain reverse transcriptase in the RT reaction, were used as controls against genomic DNA contamination of the RNA preparations. The gene rpoA is a housekeeping gene used as an internal control to ensure that RNA was present in all samples.
gDNA
AQ4037 +
–
VP49 +
–
Reverse transcriptase
197 bp
rpoA
House keeping gene
250 bp
trh
TDH-related hemolysin
289 bp
VopA Effectors
268 bp
haemolysin genes suggests the potential of the isolates to cause infection in humans upon ingestion of contaminated seafood. These isolates contain a group of virulence genes that may possibly disseminate in the marine ecosystem, and hence, they may also serve as progenitors for the other disease-causing organisms by lateral transfer of virulence genes. Further studies are considered indispensable to understand the involvement of T3SS2 in the virulence of V. parahaemolyticus isolated from the tropical marine environments. A better perspective of the virulence properties of V. parahaemolyticus isolated from seafood and/or environmental sources would be useful to consider as a potential human pathogen. Materials and methods Bacterial strains Forty-one V. parahaemolyticus isolates were revived from stock culture preserved at 80°C in a deep freezer
VopC
(Sanyo, Japan) in 30% glycerol broth at Department of Fisheries Microbiology, College of Fisheries, Mangalore. These strains were grown in 5 ml of Luria-Bertani (LB) broth (HiMedia Laboratories Pvt. Ltd, Mumbai, India) at 37°C overnight with shaking. A loopful of culture was subcultured on T1N3 agar (Tryptone 1%, NaCl 3%, Agar 15%), and obtained colonies were used for the study. Genotyping of V. parahaemolyticus Vibrio parahaemolyticus isolates were grown in 5 ml of LB broth at 37°C overnight with shaking. DNA was extracted by cetyl trimethyl ammonium bromide (CTAB)-proteinase K method (Ausubel et al. 1995) and used in the further studies. PCR assays were performed on isolates for the presence of species-specific marker toxR gene and the virulence genes tdh and trh as reported earlier (Tada et al. 1992; Kim et al. 1999). AQ4037 (tdh , trh+) was used as reference strains in all the PCR assays for genotyping studies of V. parahaemolyticus isolates.
V. parahaemolyticus F9083 V. parahaemolyticus AN 5034 V. parahaemolyticus K1533
98 V. parahaemolyticus AQ4037 93 V. parahaemolyticus10329
66 V. parahaemolyticus K5030 V. parahaemolyticus RIMD 2210633
75
V. parahaemolyticus Peru466
100
96
V. parahaemolyticus AQ3810
V. cholerae 623 100 V. cholerae 1587
V. parahaemolyticus PMC38.7 100
V. parahaemolyticus 12ETRHST4
V. parahaemolyticus TH3996
V. parahaemolyticus PMA339
V. parahaemolyticus RIMD2210633
100 V. cholerae 623 V. cholerae 1587
V. parahaemolyticus K5030
V. parahaemolyticus 12ETRHST4
100
100
66 93
V. parahaemolyticus Peru466 V. parahaemolyticus AN5034
V. parahaemolyticus TH3996
V. parahaemolyticus AQ3810
V. parahaemolyticus AQ4037
Y. pseudotuberculosis
A. Salmonicida 100
Y. Enterocolitica 0·1
VopA
E. coli
0·1
VopC
Figure 3 Phylogenetic analysis of the T3SS2 genes. Sequence information was obtained from the NCBI, and the phylogeny was estimated by maximum-likelihood method using MEGA program. Numbers indicate the percentages of bootstrap support from 1000 replicates.
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Identification of T3SS2b genes The majority of the V. parahaemolyticus strains analysed in this study were positive for trh gene, and hence, it was speculated that they may also contain T3SS2b genes. Hence, a number of PCR primers were used to identify the presence of genes coding for structural (VscC2/R2/S2/ T2/U2/N2, VcrD2), effector (VopA/C/L) and translocon proteins (VopB2/D2) of T3SS2b. Oligonucleotide primers used in the study are detailed in the supplementary table (Table S1). The PCR assay was performed in a programmable thermocycler (PTC 200; Bio-Rad, CA) as described by Okada et al. (2009). The reference strain V. parahaemolyticus AQ4037 was used as positive control, and sterile distilled water served as negative control. PCR products were electrophoresed in an agarose gel, stained with ethidium bromide (05 lg ml 1) and photographed using gel documentation system (HeroLab, Wiesloch, Germany).
Acknowledgement The financial support from the Indian Council of Medical Research, Govt. of India, is gratefully acknowledged. Conflict of interest No conflict of interest declared.
Reverse transcriptase (RT) PCR Bacterial cells were grown in 50 ml LB broth with 1% NaCl with shaking until it reached an optical density of c. 05–06 and estimated to contain 49 9 108 CFU ml 1 by plate count method. This was used for the extraction of total RNA using Qiagen RNeasy mini kit (Qiagen, Hilden, Germany). The extracted RNA was subjected to DNase I treatment (MBI Fermentas Inc, Ontario, Canada) to remove the contaminating genomic DNA. One microgram of total RNA treated with DNase I was reverse-transcribed using RevertAidTM H Minus Reverse transcriptase (Fermentas International, Inc.) by priming with 10 pmol gene-specific reverse primer. The reactions, which did not contain reverse transcriptase in the RT reaction, were used as controls against genomic DNA contamination of the RNA preparations. The gene rpoA is a housekeeping gene used as an internal control to ensure that RNA was present in all samples. The cDNA generated was used as a template for the PCR using respective primer pairs. The PCRs were carried out as mentioned above. Nucleotide sequencing and bioinformatics analysis Genomic DNA of V. parahaemolyticus was extracted using cetyl trimethyl ammonium bromide (CTAB) method (Ausubel et al. 1995). Genes coding for the T3SS2b effector proteins VopA and VopC were amplified using respective primers. The amplified products were purified using a QIAquickâ PCR Purification Kit (Qiagen), and the purified PCR products were outsourced for sequencing (M/s Eurofins Genomic India Private Limited, Bangalore, India). The nucleotide sequences obtained were analysed using Basic Local Alignment Search Tool (BLAST) blastn and blastp 444
(http://blast.ncbi.nlm.nih.gov/Blast.cgi) (Altschul et al. 1997). The presence of ORF and corresponding amino acids was deduced from the DNA sequences through a web-based java program, Molecular Tool Kit (http://www. vivo.colostate.edu/molkit). Nucleotide sequences information obtained from the NCBI was aligned with the sequences obtained during this study, and phylogenetic tree was constructed with the aid of Molecular Evolutionary Genetics Analysis (MEGA) program, ver. 5.0 (Tamura et al. 2011). The data were assessed by 1000 bootstrap replicates to determine the confidence indices within the tree.
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characterization of Vibrio parahaemolyticus isolated from seafood harvested along the southwest coast of India. Food Microbiol 25, 824–830. Tada, J., Ohashi, T., Nishimura, N., Shirasaki, Y., Ozaki, H., Fukushima, S., Takano, J., Nishibuchi, M. et al. (1992) Detection of the thermostable direct hemolysin gene (tdh) and the thermostable direct hemolysin related hemolysin gene (trh) of Vibrio parahaemolyticus by polymerase chain reaction. Mol Cell Probes 6, 477–487. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. and Kumar, S. (2011) MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28, 2731–2739. Trosky, J.E., Mukherjee, S., Burdette, D.L., Roberts, M., McCarter, L., Siegel, R.M. and Orth, K. (2004) Inhibition of MAPK signaling pathways by VopA from Vibrio parahaemolyticus. J Biol Chem 279, 51953–51957. Trosky, J.E., Li, Y., Mukherjee, S., Keitany, G., Ball, H. and Orth, K. (2007) VopA inhibits ATP binding by acetylating the catalytic loop of MAPK kinases. J Biol Chem 282, 34299–34305.
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Zhang, L. and Orth, K. (2013) Virulence determinants for Vibrio parahaemolyticus infection. Curr Opin Microbiol 16, 70–77. Zhou, X., Konkel, M.E. and Call, D.R. (2009) Type III secretion system 1 of Vibrio parahaemolyticus induces oncosis in both epithelial and monocytic cell lines. Microbiology 155, 837–851. Zhou, X., Konkel, M.E. and Call, D.R. (2010) Vp1659 is a Vibrio parahaemolyticus type III secretion system 1 protein that contributes to translocation of effector proteins needed to induce cytolysis, autophagy, and disruption of actin structure in HeLa cells. J Bacteriol 192, 3491–3502.
Supporting Information Additional Supporting Information may be found in the online version of this article: Table S1. Primers used in this study. Table S2. Distribution of T3SS2b genes in Vibrio parahaemolyticus in seafood harvested along southwest coast of India.
Letters in Applied Microbiology 58, 440--446 © 2013 The Society for Applied Microbiology
ORIGINAL ARTICLE
Presence of T3SS2b genes in trh+ Vibrio parahaemolyticus isolated from seafood harvested along Mangalore coast, India B.K. Kumar, V.K. Deekshit, P. Rai, M. Shekar, I. Karunasagar and I. Karunasagar Department of Fisheries Microbiology, UNESCO-MIRCEN for Marine Biotechnology, Karnataka Veterinary, Animal and Fisheries Sciences University, College of Fisheries, Mangalore, India
Significance and Impact of the Study: T3SSs (a or b) are the important virulence factors of Vibrio parahaemolyticus that contribute to their pathogenicity in humans. This study demonstrated the presence of T3SS2b genes in V. parahaemolyticus isolated from the seafood harvested along Mangalore coast. RT-PCR showed that the T3SS2b genes identified in seafood isolates of V. parahaemolyticus were found to be functional. To the best of our knowledge, this is the first description of T3SS2b genes in trh+ V. parahaemolyticus isolated from seafood in India. The presence of T3SS2 along with other virulence factors such as TDH and/or TRH highlights a potential health risk for seafood consumers.
Keywords seafood, TDH-related haemolysin, thermostable direct haemolysin, type III secretion system, V. parahaemolyticus. Correspondence Dr. Indrani Karunasagar, Department of Fisheries Microbiology, UNESCO-MIRCEN for Marine Biotechnology, Karnataka Veterinary, Animal and Fisheries Sciences University, College of Fisheries, Mangalore, India. E-mail: [email protected] 2013/2176: received 30 October 2013, revised 19 December 2013 and accepted 20 December 2013 doi:10.1111/lam.12210
Abstract Vibrio parahaemolyticus is a seafood-borne pathogen autochthonous to the marine and estuarine ecosystem, responsible for gastroenteritis when contaminated raw seafood is consumed. The pathogenicity has been associated with thermostable direct haemolysin (TDH) and TDH-related haemolysin (TRH). Of late, the presence of T3SS2a and T3SS2b gene clusters has been well documented in clinical isolates of Vibrio parahaemolyticus and known to play an essential role in pathogenesis. However, reports on the presence of T3SSb genes in V. parahaemolyticus isolated from the seafood and/or environmental samples are scanty. In this study, we have identified and analysed the distribution of the T3SS2b genes in V. parahaemolyticus isolated from seafood harvested along southwest coast of India. Results showed that T3SS2b genes are solely associated with trh+ and tdh+/trh+ strains of V. parahaemolyticus. Reverse transcriptase PCR (RT-PCR) showed that the T3SS2b genes identified in trh+ V. parahaemolyticus were transcriptionally active. To our knowledge, this study appears to be the first description on the presence of T3SS2bpositive V. parahaemolyticus isolated from seafood in India. The study of T3SS2 along with other virulence factors will help in better understanding of the risk of seafood-borne illness due to V. parahaemolyticus.
Introduction Vibrio parahaemolyticus is a Gram-negative, halophilic bacterium, autochthonous to water and sediments of marine and estuarine ecosystems around the world (Nishibuchi and Kaper 1995; Deepanjali et al. 2005) and is a leading cause of seafood-borne gastroenteritis due to the consumption of raw seafood (Honda et al. 2008). The virulence properties of this organism have been associated 440
with thermostable direct haemolysin (TDH) and TDHrelated haemolysin (TRH) coded by the tdh and trh genes, respectively (Honda et al. 1987a,b; Nishibuchi and Kaper 1995). Therefore, the strains bearing tdh and/or trh genes are considered to be pathogenic (Nishibuchi and Kaper 1995). Only a very minute proportion (1–2%) of the V. parahaemolyticus isolated from the environment are known to be virulent and carry virulence genes tdh and/ or trh, whereas most of the clinical isolates (90%) are
Letters in Applied Microbiology 58, 440--446 © 2013 The Society for Applied Microbiology
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found to be virulent. However, recent studies have shown that apart from tdh and trh, there are other putative pathogenicity-associated genes known to be associated with the virulence (Makino et al. 2003; Ono et al. 2006; Honda et al. 2008; Kodama et al. 2008; Hiyoshi et al. 2010). Many Gram-negative pathogens utilize type III secretion systems (T3SSs) to induce pathogenesis by translocating effector proteins into the host cells. The T3SS is composed of a needle-shaped apparatus comprising protein components and a large number of effector proteins (Cornelis 2006; Coburn et al. 2007). Upon entering the host cell, the active form of effector protein manipulates the host cell signalling pathways to disrupt host immune response. Genome mapping of a clinical isolate of V. parahaemolyticus (RIMD2210633) confirmed the existence of two sets of type III secretion system (T3SS) gene clusters, T3SS1 and T3SS2 (Makino et al. 2003). The toxins secreted by both the T3SSs are essential for cytotoxicity and enterotoxicity of these strains, thus having an apparent role in the pathogenesis (Broberg et al. 2011; Karunasagar et al. 2012). The T3SS1 is present in chromosome I of the V. parahaemolyticus isolated from clinical and environmental sources regardless of their pathogenicity, and T3SS2 is located predominantly in the chromosome II of highly virulent strains (Kodama et al. 2008; Okada et al. 2009). The gene coding for T3SS1 known to involve in cytotoxicity against eukaryotic cells and T3SS2 is responsible for the enterotoxicity in the rabbit ileal loop test (Park et al. 2004; Bhattacharjee et al. 2005, 2006; Kodama et al. 2008; Burdette et al. 2009; Zhou et al. 2009, 2010; Hiyoshi et al. 2010). Later DNA sequencing of approximately 100 kb regions that surround trh gene of V. parahaemolyticus strain, TH3996, revealed the presence of a novel T3SS, which is closely related to T3SS2 of tdh+ V. parahaemolyticus (Okada et al. 2009). After the report of novel T3SS2 in trh+ V. parahaemolyticus strain TH3996 of clinical origin, it has been further classified into two distinct phylogroups (a and b) based on the divergence in nucleotide sequences. In earlier studies, the presence of V. parahaemolyticus in seafood harvested along southwest coast of India has been well documented (Dileep et al. 2003; Deepanjali et al. 2005; Parvathi et al. 2006; Raghunath et al. 2008; Kumar et al. 2011). The study by Deepanjali et al. (2005) highlighted the prevalence (59%) of trh+ isolates among the total V. parahaemolyticus isolated from seafood harvested along the Mangalore coast, India. However, clinical cases due to these strains of V. parahaemolyticus have not been reported in this region. The aforesaid virulence attributes have been detected and well studied in the clinical strains of V. parahaemolyticus with limited reports in
isolates of environmental origin (Caburlotto et al. 2009; Noriea et al. 2010). There are no reports on the presence of T3SS2a or T3SS2b genes in V. parahaemolyticus isolated from Indian marine environment and/or seafood leading to a paucity of data. Hence, this study was principally focused to identify the presence of T3SS2 genes and their distribution in trh+ V. parahaemolyticus isolated from seafood harvested along the Mangalore coast. Results and discussion All the V. parahaemolytcus isolates used in this study were isolated from the seafood harvested along southwest coast of India during the period April 2003–March 2013 and had been identified previously using standard bacteriological methods (Raghunath et al. 2008). All the 41 V. parahaemolyticus isolates used in this study were found to be positive for toxR gene (species-specific marker), thereby further confirming the identification of V. parahaemolyticus used in this study (Kim et al. 1999). Further, the presence of genes coding for haemolysins was detected by PCR targeting tdh and trh genes (Tada et al. 1992). Based on the PCR results, isolates were categorized into four distinct groups, which were 5 tdh+, 20 trh+, 3 tdh+/trh+ and 13 tdh /trh (Table 1). The gene coding for T3SS2 (a or b) located on the chromosome II is unique to highly virulent strains of V. parahaemolyticus (Makino et al. 2003), and the toxins secreted by these T3SS machineries are believed to have a profound role in pathogenesis of the organism (Broberg et al. 2011; Karunasagar et al. 2012). Hence, it is important to understand the prevalence of genes encoding these systems in seafood and/or environmental isolates of Table 1 Distribution of T3SS2b genes in Vibrio parahaemolyticus in seafood harvested along southwest coast of India Number of seafood isolates (n = 41) Gene coding for
Target gene
tdh+/trh (n = 05)
tdh /trh+ (n = 20)
tdh+/trh+ (n = 03)
tdh /trh (n = 13)
Apparatus protein
VscC2 VscR2 VscS2 VscT2 VscU2 VcrD2 VscN2 VopB2 VopD2 VopC VopA VopL
– – – – – – – – – – – –
20 20 17 15 20 20 20 19 20 20 20 19
3 3 1 3 3 3 3 3 3 3 3 3
– – – – 2 – 2 – 1 – – –
ATPase Translocon protein Effector protein
‘–’ Means negative for the tested genes.
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V. parahaemolyticus. In this study, a PCR assay was used to identify the presence of the gene coding for apparatus (VscC2/R2/S2/T2/U2/N2, VcrD2), effector (VopA/C/L) and translocon proteins (VopB2/D2) of T3SS2b genes (Okada et al. 2009). All the 20 trh+ and 3 tdh+/trh+ isolates analysed in this study were positive for the T3SS2b genes analysed (Fig. 1). Although all the 5 tdh+ isolates analysed were negative for T3SS2b genes, they could possess the T3SS2a gene cluster. All the nonpathogenic V. parahaemolyticus isolates analysed in this study were PCR-negative for the T3SS2b gene. Two of the nonpathogenic isolates were positive for the VscU2 and VscN2 genes, while one strain was positive for VopD2 gene (Table S2). To the best of our knowledge, this is the first description of T3SS2b in trh+ V. parahaemolyticus isolated from seafood in India. The results obtained in this study showed the presence of T3SS2b in seafood isolates of V. parahaemolyticus, which is otherwise reported generally in clinical strains. The presence of the gene suggests the potential of the isolates to cause infection in humans upon ingestion of contaminated seafood. To date, a very few reports are available on the prevalence of T3SSb genes in V. parahaemolyticus isolated from the seafood and/or environmental samples worldwide (Noriea et al. 2010; Jones et al. 2012). We have also determined the transcriptional activity of VopA and VopC genes of T3SS2b. The reverse transcriptase PCR (RT-PCR) for effector genes VopC and VopA with specific primer gave prominent amplification with the expected amplicons (Fig. 2). RT-PCR analysis showed that the T3SS2 genes present in the trh+ V. parahaemolyticus isolated from the seafood were found to be functional. The gene coding for VopA and VopC is part of the T3SS2 gene cluster, was sequenced, analysed and submitted to the NCBI (GenBank accession numbers JQ996146, JQ996147). The VopC and VopA analysed in this study showed unusual base composition (G + C content of 388
M
1
2
M
3
1
2
and 386%, respectively) compared to the rest of the genome. This suggests that T3SS2 might have integrated into the V. parahaemolyticus chromosome through horizontal gene transfer. A sequence similarity search for the nucleotide and deduced amino acid sequences of VopA and VopC was performed using BLAST (Altschul et al. 1997). The gene coding for VopA shared similarity with the acetyltransferase group found in V. parahaemolyticus RIMD2210633, V. cholerae, Yersinia spp. and Aeromonas spp. Similarly, the gene coding for VopC protein analysed in this study showed sequence similarity to the catalytic domain of cytotoxic necrotizing factor (CNF) toxins with the presence of catalytic cysteine (225th) and histidine (240th) residues in the core region. The T3SS2 effector proteins are known to be involved in the intracellular survival, replication and pathogenesis of the organism, brought about by the activity of conserved catalytic amino acid residues (Orth et al. 1999; Trosky et al. 2004, 2007; Mukherjee et al. 2006). Phylogenetic trees for each of the genes were constructed using Dice correlation and unweighted pair group method with arithmetic mean (UPGMA). Analysis showed that both VopA and VopC gene sequences of V. parahaemolyticus clustered with the T3SS2b gene sequence rather than the T3SS2a gene sequence (Fig. 3), and it demonstrated that nucleotide sequences of VopA and VopC obtained in this study closely belong to the T3SS2b found in the clinical isolates of V. parahaemolyticus TH3996, AQ4037 and V. cholerae. Previous studies reported that non-O1, non-139 V. cholerae strain-1587 also possesses a T3SS2-like secretion system, which had a similarity to the T3SS2 of V. parahaemolyticus (Dziejman et al. 2005; Murphy and Boyd 2008; Zhang and Orth 2013). In conclusion, this study showed the presence of transcriptionally active T3SS2b genes in seafood isolates of V. parahaemolyticus, which is otherwise reported generally in clinical strains. The presence of the T3SS2b along with
3
M 1
2
3 1252 bp
695 bp
594 bp
(a)
(b)
(c)
Figure 1 Presence of gene coding for T3SS2b effector proteins of Vibrio parahaemolyticus. (a) VopC (594 bp), Lane M: 100-bp molecular weight marker, Lane 1: Negative control, Lanes 2 and 3: Environmental isolates of V. parahaemolyticus. (b) VopA (695 bp), Lane M: 100-bp molecular weight marker, Lane 1: Negative control, Lanes 2 and 3: Environmental isolates of V. parahaemolyticus. (c) VopL (1252 bp), Lane M: 500-bp molecular weight marker, Lane 1: Negative control, Lanes 2 and 3: Environmental isolates of V. parahaemolyticus.
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M Figure 2 RT-PCR showing expression of putative virulence genes of Vibrio parahaemolyticus. cDNA (+) made from total RNA isolated from V. parahaemolyticus strain AQ4037 and VP49 was used as a template for PCR. Mock reactions ( ), which did not contain reverse transcriptase in the RT reaction, were used as controls against genomic DNA contamination of the RNA preparations. The gene rpoA is a housekeeping gene used as an internal control to ensure that RNA was present in all samples.
gDNA
AQ4037 +
–
VP49 +
–
Reverse transcriptase
197 bp
rpoA
House keeping gene
250 bp
trh
TDH-related hemolysin
289 bp
VopA Effectors
268 bp
haemolysin genes suggests the potential of the isolates to cause infection in humans upon ingestion of contaminated seafood. These isolates contain a group of virulence genes that may possibly disseminate in the marine ecosystem, and hence, they may also serve as progenitors for the other disease-causing organisms by lateral transfer of virulence genes. Further studies are considered indispensable to understand the involvement of T3SS2 in the virulence of V. parahaemolyticus isolated from the tropical marine environments. A better perspective of the virulence properties of V. parahaemolyticus isolated from seafood and/or environmental sources would be useful to consider as a potential human pathogen. Materials and methods Bacterial strains Forty-one V. parahaemolyticus isolates were revived from stock culture preserved at 80°C in a deep freezer
VopC
(Sanyo, Japan) in 30% glycerol broth at Department of Fisheries Microbiology, College of Fisheries, Mangalore. These strains were grown in 5 ml of Luria-Bertani (LB) broth (HiMedia Laboratories Pvt. Ltd, Mumbai, India) at 37°C overnight with shaking. A loopful of culture was subcultured on T1N3 agar (Tryptone 1%, NaCl 3%, Agar 15%), and obtained colonies were used for the study. Genotyping of V. parahaemolyticus Vibrio parahaemolyticus isolates were grown in 5 ml of LB broth at 37°C overnight with shaking. DNA was extracted by cetyl trimethyl ammonium bromide (CTAB)-proteinase K method (Ausubel et al. 1995) and used in the further studies. PCR assays were performed on isolates for the presence of species-specific marker toxR gene and the virulence genes tdh and trh as reported earlier (Tada et al. 1992; Kim et al. 1999). AQ4037 (tdh , trh+) was used as reference strains in all the PCR assays for genotyping studies of V. parahaemolyticus isolates.
V. parahaemolyticus F9083 V. parahaemolyticus AN 5034 V. parahaemolyticus K1533
98 V. parahaemolyticus AQ4037 93 V. parahaemolyticus10329
66 V. parahaemolyticus K5030 V. parahaemolyticus RIMD 2210633
75
V. parahaemolyticus Peru466
100
96
V. parahaemolyticus AQ3810
V. cholerae 623 100 V. cholerae 1587
V. parahaemolyticus PMC38.7 100
V. parahaemolyticus 12ETRHST4
V. parahaemolyticus TH3996
V. parahaemolyticus PMA339
V. parahaemolyticus RIMD2210633
100 V. cholerae 623 V. cholerae 1587
V. parahaemolyticus K5030
V. parahaemolyticus 12ETRHST4
100
100
66 93
V. parahaemolyticus Peru466 V. parahaemolyticus AN5034
V. parahaemolyticus TH3996
V. parahaemolyticus AQ3810
V. parahaemolyticus AQ4037
Y. pseudotuberculosis
A. Salmonicida 100
Y. Enterocolitica 0·1
VopA
E. coli
0·1
VopC
Figure 3 Phylogenetic analysis of the T3SS2 genes. Sequence information was obtained from the NCBI, and the phylogeny was estimated by maximum-likelihood method using MEGA program. Numbers indicate the percentages of bootstrap support from 1000 replicates.
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Identification of T3SS2b genes The majority of the V. parahaemolyticus strains analysed in this study were positive for trh gene, and hence, it was speculated that they may also contain T3SS2b genes. Hence, a number of PCR primers were used to identify the presence of genes coding for structural (VscC2/R2/S2/ T2/U2/N2, VcrD2), effector (VopA/C/L) and translocon proteins (VopB2/D2) of T3SS2b. Oligonucleotide primers used in the study are detailed in the supplementary table (Table S1). The PCR assay was performed in a programmable thermocycler (PTC 200; Bio-Rad, CA) as described by Okada et al. (2009). The reference strain V. parahaemolyticus AQ4037 was used as positive control, and sterile distilled water served as negative control. PCR products were electrophoresed in an agarose gel, stained with ethidium bromide (05 lg ml 1) and photographed using gel documentation system (HeroLab, Wiesloch, Germany).
Acknowledgement The financial support from the Indian Council of Medical Research, Govt. of India, is gratefully acknowledged. Conflict of interest No conflict of interest declared.
Reverse transcriptase (RT) PCR Bacterial cells were grown in 50 ml LB broth with 1% NaCl with shaking until it reached an optical density of c. 05–06 and estimated to contain 49 9 108 CFU ml 1 by plate count method. This was used for the extraction of total RNA using Qiagen RNeasy mini kit (Qiagen, Hilden, Germany). The extracted RNA was subjected to DNase I treatment (MBI Fermentas Inc, Ontario, Canada) to remove the contaminating genomic DNA. One microgram of total RNA treated with DNase I was reverse-transcribed using RevertAidTM H Minus Reverse transcriptase (Fermentas International, Inc.) by priming with 10 pmol gene-specific reverse primer. The reactions, which did not contain reverse transcriptase in the RT reaction, were used as controls against genomic DNA contamination of the RNA preparations. The gene rpoA is a housekeeping gene used as an internal control to ensure that RNA was present in all samples. The cDNA generated was used as a template for the PCR using respective primer pairs. The PCRs were carried out as mentioned above. Nucleotide sequencing and bioinformatics analysis Genomic DNA of V. parahaemolyticus was extracted using cetyl trimethyl ammonium bromide (CTAB) method (Ausubel et al. 1995). Genes coding for the T3SS2b effector proteins VopA and VopC were amplified using respective primers. The amplified products were purified using a QIAquickâ PCR Purification Kit (Qiagen), and the purified PCR products were outsourced for sequencing (M/s Eurofins Genomic India Private Limited, Bangalore, India). The nucleotide sequences obtained were analysed using Basic Local Alignment Search Tool (BLAST) blastn and blastp 444
(http://blast.ncbi.nlm.nih.gov/Blast.cgi) (Altschul et al. 1997). The presence of ORF and corresponding amino acids was deduced from the DNA sequences through a web-based java program, Molecular Tool Kit (http://www. vivo.colostate.edu/molkit). Nucleotide sequences information obtained from the NCBI was aligned with the sequences obtained during this study, and phylogenetic tree was constructed with the aid of Molecular Evolutionary Genetics Analysis (MEGA) program, ver. 5.0 (Tamura et al. 2011). The data were assessed by 1000 bootstrap replicates to determine the confidence indices within the tree.
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Supporting Information Additional Supporting Information may be found in the online version of this article: Table S1. Primers used in this study. Table S2. Distribution of T3SS2b genes in Vibrio parahaemolyticus in seafood harvested along southwest coast of India.
Letters in Applied Microbiology 58, 440--446 © 2013 The Society for Applied Microbiology
