Bioinformatics on Unproven Secretion Systems of Vibrio Parahaemolyticus

Several investigations were made in recent years related to Type 3 Secretion System (T3SS) and Type 6 Secretion System (T6SS) of Vibrio parahaemolyticus. However, numerous pathogenicity islands in genomes of V. parahaemolyticus remain unexploited. Considering the presence of other secretion systems such as T1SS, T2SS, T4SS and T5SS, which are associated with the pathogenicity in other bacteria exploring the Whole Genome Sequence (WGS) data, we identified the presence of T1SS-specific Escherichia coli HlyA, and Morganella morganii Vibrio ferrin homologs in V. parahaemolyticus. The T2SS encodes tight adherence (tad) locus for bacterial adherence and production of biofilm in Actinobacillus actinomycetemcomitans. This gene locus was identified as pilin in V. parahaemolyticus. Presence of VCA0120 and csu, the pilus encoding gene homologs in the environmental V. parahaemolyticus strains supports the view that part of T4SS may be active in this Vibrio. This information should be explored further in the diseases progress using In vitro and In vivo models.

Vibrio parahaemolyticus is a Gram-negative, aquatic halophilic bacterium that inhabits in estuarine and marine environments. It causes three major diseases in humans; gastroenteritis, wound infections and septicemia. Among these, gastroenteritis is the most common with clinical symptoms of acute diarrhoea, abdominal cramps, nausea, vomiting, headache, and low-grade fever [1]. These symptoms are associated with seafood-borne infections, caused by V. parahaemolyticus, mostly in Southeast Asia. Based on the somatic (O) and capsular (K) antigens, several serotypes of V. parahaemolyticus have been identified. A unique serotype O3:K6 of V. parahaemolyticus has emerged in India during 1996 and spread to several countries. The other serotypes, such as O4:K68, O1: K25 and O1: KUT (untypeable) that subsequently appeared had similar virulence gene carriage and other molecular characteristics identical to that of the O3:K6 serotype. Pulsed-Field Gel Electrophoresis (PFGE), Variable Number Tandem Repeat (VNTR) and Multilocus Sequence Typing (MLST) of these serotypes displayed close genetic relationship with the pandemic O3:K6 serotype [1-3]. The clinical isolates of V. parahaemolyticus produce either the Thermostable-Direct Hemolysin (TDH) or TDH-Related Hemolysin (TRH) that are encoded in the tdh and trh genes, respectively [4]. The environmental strains on the other hand rarely have these virulence genes [5,6]. Strains devoid of any of these virulence markers are known to produce cytotoxicity and enterotoxicity In vitro. This suggests that other unknown virulence factors may have a role in the pathogenicity of V. Parahaemolyticus [7]. V. parahaemolyticus is well adopted for both natural and host environments with the ability to respond to rapid changes in the extracellular milieu [8]. Intestinal colonization of this bacterium is essential for the disease progress. Once the pathogen enters into the intestine, it induces extensive modification of intestinal microvilli by rearranging the cytoskeleton in host cells. This can lead to electrolyte imbalance and malabsorption of nutrients, which eventually cause secretory diarrhoea [9]. Using adult murine model, Vibrio-specific regulatory system ToxRS was shown to be important for colonization and survival under different stress conditions. Recently, Extra-Cytoplasmic Function (ECF) sigma factor, RpoE was identified as key cell envelope regulator for intestinal survival and colonization of vibrios [8]. During the course of infection, bacteria interact with their host and manipulate host cell response by secreting different types of protein. These proteins are located on the surface of bacteria that can be delivered into the host cells. To secrete exoproteins, bacteria utilize different machineries, which are called secretory system. Until date, six different types of secretion systems have been identified in Gram-negative bacteria, from type 1 (T1SS) to Type 6 Secretion Systems (T6SS) [10]. T3SS and T6SS are well studied and found to be associated with the pathogenesis of V. parahaemolyticus. In this article, we have shown the linkage of unreported secretion systems in V. parahaemolyticus with other pathogens by a comparative analysis with submitted gene sequences to the National Center for Biotechnology (NCBI).

Secretion systems associated proteins of (gene products) pathogens and their corresponding genes related data were from open source databases such as PubMed (http://www.ncbi.nlm.nih.gov/pubmed), PubMed Central (PMC) - (http://www.ncbi.nlm.nih.gov/pmc) and other open access journals available in the World Wide Web. The protein sequences of genome sequenced clinical V. parahaemolyticus strain RIMD 2210633 and environmental strain FDA - R31 were obtained from public domain (https://www.ncbi.nlm.nih.gov/genome). The sequence data collected from above were analysed using DNASTAR (Madison, WI, USA), Clone manager and BLAST (PubMed) search programme.

Type 1 Secretion System (T1SS)

V. parahaemolyticus colonizes in the intestinal tract and invades into enterocytes. This invasion is determined by the genes encoded in the T1SS and T3SS. In many bacteria, the gene clusters responsible for T1SS expression is located in either the chromosome or plasmids. The RTX (repeat in toxin) toxin family is a group of cytotoxins secreted by Gram-negative bacteria into extracellular via T1SS. Proteins encoding the T1SS are diverse in nature but secreted by a common mechanism [11]. Hemolysin is the prototype of the RTX exotoxin family [12]. A well-studied hemolysin is the hlyA of uropathogenic Escherichia coli [13]. The structure of E. coli hemolysin T1SS consists of three components viz. HlyB, HlyD, and TolC [14]. These components are compared with RTX produced by vibrios such as V. cholerae and V. vulnificus and found to vary in the operon arrangements with the similarity ranges from 39 to 63% [15]. In V. parahaemolyticus, T1SS gene clusters were identified in the chromosome that encodes transport substrates, export functions and genes that promote transport substrates. Based on the Whole Genome Sequence (WGS) analysis obtained from the public domain, we detected that the V. parahaemolyticus strain FDA - R31 has a complete locus of E. coli hemolysin T1SS in the small chromosome with the variation in the protein sequence from 80 to 93% (Figure 1). The Outer Membrane Protein (OMP) of TolC, a component of T1SS is a multifunctional protein involved in many cellular processes and most important in the multidrug efflux pump system in E. coli and other bacteria. Among these, Resistance Nodulation cell Division (RND) - type efflux transporters plays major role in the resistance to various antimicrobial agents and also virulence in many Gram-negative bacteria [16]. In V. parahaemolyticus, multiple and active RND - type efflux transporters has been identified in the chromosome [17]. Phylogenetic analysis of components of RND - type efflux transporters in V. parahaemolyticus, V. cholerae, V. fischeri, V. harveyi and V. Vulnificus showed the presence of VmeCD along with the other multidrug efflux transporters [17]. Multiple deletions in these efflux encoding genes have shown significant increase in the sensitivity of V. parahaemolyticus towards many antimicrobial agents including bile acid and decrease in fluid accumulation in rabbit ileal loops [17].

V. parahaemolyticus utilizes iron through their siderophore. The secreted protein Vibrioferrin, which binds with heme to form ferric vibrioferrin complex on the outer membrane, which drives iron for In vivo survival and proliferation of bacteria [18]. Vibrioferrin has 16% similarity with hemophore HasA (Table 1), which is T1SS substrate of Serratia marescens [19]. A comparative study of the heme iron utilization systems of pathogenic vibrios such as V. cholerae, V. alginolyticus and V. parahaemolyticus showed that all had similar genetic constitution [20]. At the functional level, V. parahaemolyticus had identical amino acid sequence comparable to that of V. cholera [21]. Ferric Vibrioferrin receptor encoding gene pvuA-disrupte mutant study show decreased utilization of Ferric Vibrioferrin and this gene had 48% similarity with RumA of ferric rhizoferrin receptor of Morgan ellamorganii [22]. The IutA homologue of V. parahaemolyticus encodes an outer-membrane protein, which functions as the receptor for ferric aerobactin sharing 43% genetic homology with the E. coli IutA protein. Hybridization assay revealed that these homologs are widely distributed in clinical and environmental isolates of V. Parahaemolyticus [23]. V. parahaemolyticus utilize enterobactin via PeuA receptor under iron-limiting conditions [24]. These findings supported that V. parahaemolyticus uptake heme via siderophore-dependent uptake mechanism specific to surface receptors, activated through the trans envelope, TonB–ExbB/ExbD complex than Heme acquisition system (Has). These are heme uptake routes in bacteria mediated by the T1SSs.

Type 2 Secretion System (T2SS)

T2SS is one of the most flexible systems used by Gram-negative bacteria for transport of diverse proteins including hydrolytic enzymes and virulence factors from the periplasm across the outer membrane into the extracellular space [25]. This system involves a set of 12 - 15 different proteins, many of them in multiple copies located in the complex structure of inner and outer membranes. In V. cholerae, this system is called Extra Cellular Protein (ECP) and in Enterotoxigenic E. coli (ETEc) and other bacteria this helps in the General Secretory Pathway (GSP) that spans from a regulatory ATPase in the cytoplasm to the actual pore in the outer membrane [25]. The structure of T2SS consists of the outer membrane complex, the pseudopilus, and the inner membrane platform. The outer membrane complex composed of EpsD, the pseudopilus consists of five different pseudopilins such as EpsG to EpsK. The inner membrane platform consists of bitopic membrane proteins EpsL and EpsM; the membrane-anchored EpsC; the integral membrane protein EpsF; and the membrane-associated “secretion ATPase” EpsE in the cytosol [26]. Periplasmic domain of EpsL (peri-EpsL) in V. parahaemolyticus shared 56% homology with V. cholerae and this involves in the circular permutation of ferredoxin along with the other sub-domains [25].

It was shown that the secretion system associated Tad (tight adherence) loci are essential for biosynthesis of long and bundled Flp pili, which support the bacterial adherence and biofilm formation [27,28]. The biosynthesis of Flp pilus involved in 12 - 13 genes of tad locus, in Actinobacillus actinomycetemcomitans, which share similarity to other bacteria having T2SSs, type IV pilus systems and few genes with T4SS NTPases [29]. The tad locus protein TadV functions as prepilin peptidase in maturation of the Flp pilin. In A. actinomycetemcomitans, the pre-TadE and pre-TadF inner membrane proteins are responsible for adherence and biofilm formation [29]. In A. actinomycetemcomitans and V. parahaemolyticus, these highly conserved Tad proteins act as pseudopilins [29] V.parahaemolyticus strain RIMD 2210633 has the entire Tad locus as reported in A. actinomycetemcomitans and found vary at the amino acid level (Figure 2). However, experimental evidences are needed to show the importance of Tad locus in the pathogenesis of V. parahaemolyticus.

Type 4 Secretion System (T4SS)

T4SSs is a multiprotein complex that can transport DNA, toxins and effector proteins through bacterial membranes to the extracellular milieu or directly into the cytoplasm of other cells [30]. In several bacteria, T4SS system has been established. Legionella pneumophila had shown to survive in the host cell by injecting a vast number of toxins thorough its Dot/Icm type IVB Secretion System (T4SS). T4SS effector proteins are involved three classes such as translocation channel, the pilus and ATPase proteins [31]. The T4SS effector proteins such as IcmF and IcmH (DotU) of L. pneumophila were non-essential components of this system and required for cytotoxicity of L. pneumophila toward mammalian and Dictyostelium discoideum cells [32,33] and also shared significant sequence similarity to plasmid genes involved in conjugation [34].

Protein encoding genes of icmF and icmH (dotU) shared sequence similarity with vasK (VCA010) and vasF (VCA0115) of V. cholerae. These genes are involved in regulating the motility of the pathogen and adherence to the intestinal epithelial cell line [35]. Deletion mutant of these genes in Dictyostelium sp. model has been reported as attenuation of virulence [33]. Even though the T4SS has not been recognized in V. parahaemolyticus, we identified VCA0120 protein of V. cholerae that had 52% protein sequence homology with recently sequenced Canadian V. parahaemolyticus strains [36]. This has been annotated as T6SS protein in V. parahaemolyticus, accession no. KHF17383 [36], which had 23% similarity with V. parahaemolyticus strain RIMD 2210633 locus VP1408.

Pilus is one of the sub-groups of T4SS, which is responsible for transfer of proteins to establish cell to cell contact [37]. Pilus has been recognized as one of the adherent factors of pathogenic bacteria [38]. The pili identified in different V. parahaemolyticus strains has the adhesive property in rabbit intestine [39,40]. In Acinetobacter baumannii, the csu operon was responsible for attachment and biofilms formation and this enable bacteria to persist in their natural environments or in host tissue [41]. Interestingly, a similar locus has been found in clinical and environmental strains of V. Parahaemolyticus [41,42]. V. parahaemolyticus utilizes IV-A pilus genes as sophisticated machinery for survival in natural environments. In V. parahaemolyticus RIMD 2210633, two sets of type IV-A pilus have been identified, one is chitin-regulated pilus; ChiRP, which is similar to P. aeruginosa and Vibrio spp. and the other, is similar To Mannose-Sensitive Hemagglutinin (MSHA). These two genes contribute in the biofilm formation in different pathways [43]. MSHA pilus was identified as a significant factor in bacterial-host cell adherence and pathogenic to Caco-2 cells. MSHA pilus act on glycans receptors such as asialo-GM1 ganglioside, lacto-N-fucopentaose I and lacto-N-difucohexaose I in the gastrointestinal tract and enable efficient colonization of the intestinal epithelium by V. Parahaemolyticus [44]. Type IV pili was shown to contribute persistence of V. parahaemolyticus in Pacific oysters [45].

Type 5 Secretion System (T5SS)

A protein secreted by T5SS is known as autotransporters, which comprised of autotransporter family (classical type), the Two-Partner System (TPS) and the Oligomeric Coiled coils adhesion (Oca) family. The autotransporter secretion process involves first the translocation of the precursor through the inner membrane and then its translocation through the outer membrane via a pore formed by a β-barrel [46]. TPS are translated by TpsA and TpsB proteins. TpsA is exoproteintranslocated through the outer membrane via β-barrel pore formed by TpsB [46]. The auto transporter of Oca family was identified in Yersinia enterocolitica and adhesion protein involved in this process is YadA, which is subfamily of surface-attached oligomeric auto transporters [47]. Patatin-Like Protein (PlpD) has been described in P. aeruginosa, which shared the common features of classical type of auto transporter and TpsB of TPS in one polypeptide chain [48]. More than 200 PlpD orthologues exist among pathogenic and environmental bacteria, which could secrete numerous PLPs in bacteria through this system [49].

In our analysis, we found that Patatin-like protein shared 32% homology with hypothetical protein of gene locus VP2496 of V. parahaemolyticus strain RIMD 2210633. However, this region has been annotated as serine protease in genome sequenced V. parahaemolyticus strains. As a part of T5SS, two adhesions intimin and invasin have been described in E. coli and Yersinia spp., respectively, but these proteins did not have any similarity in the V. parahaemolyticus genome.

Our study suggests that V. parahaemolyticus has other important secretion systems namely T1SS, T2SS, T4SS and T5SS, in addition to the known T3SS and T6SS, which are homologus to Gram-negative bacteria. Vibrioferrin, HlyA, Eps, Tad, pseudopilins and patatin-like lipolytic enzyme are the important secretion components found to have the virulence of V. parahaemolyticus. Understanding of these secretion systems through novel In vitro and In vivo models could create new avenues for exploration of pathogenesis and effective infection control.

The authors would like to thank Department of Pharmaceuticals, Ministry of Chemicals and Fertilizers, Government of India for their financial assistance.