Introduction

Worldwide 1.3 billion cases of acute diarrhea occur in children below 5 years annually of which more than 3 million die and 80 per cent of these deaths are in children below 2 years of age (Sur and Bhattacharya 2003). Acute diarrhea was caused by various numbers of bacterial, viral or parasitic agents. The most important bacterial agents causing outbreaks of acute diarrhoea are V. cholerae O1 and O139. Diarrheal disease outbreaks were causes of major public health emergencies in India. West Bengal located in the Gangetic delta has been hailed as the “homeland of cholera”, with frequent localized outbreaks being reported (Fule et al 1990). Vibrio cholerae O1 and V. cholerae O139 are etiological agents of epidemic cholera. However, V. cholerae O1 strains that do not produce cholera toxin, i.e., that are nontoxigenic (NT), and non-O1/non-O139 strains have also been associated with cholera, gastroenteritis, septicemia or other intestinal infections (Morris et al 1984; Mukhopadhyay et al 1995). Outbreaks of cholera were reported in Brazil during the third (1853 to 1854), fourth (1866 to 1868), and fifth (1893 to 1895) pandemics (Barua, 1992).

The computational prediction of the subcellular localization of bacterial proteins is an important step in genome annotation and in the search for novel vaccine or drug targets. V. cholerae have five major subcellular localization sites like cytoplasm, periplasm, inner membrane, outer membrane and extracellular space. The subcellular location of a protein can provide valuable information about its function. With the rapid increase of sequenced genomic data, the need for an automated and accurate tool to predict subcellular localization becomes increasingly important. Because of its simplicity, this approach can be easily extended to other organisms and should be a useful tool for the high-throughput and large-scale analysis of proteomic and genomic data. Generally, existing methods of subcellular localization developed for eukaryotic proteins like TSSub, LOCSVMPSI, ESLpred, Euk-Ploc (Guo and Lin 2006; Xie et al 2005; Bhasin and Raghava 2004; Shen et al 2007). Since, a range of bioinformatics tools were available for prediction of subcellular localization of prokaryotic proteins viz. PSORTb, PSLpred (Gardy et al 2005; Bhasin et al 2005).

Computational SCL investigation of the growing number of complete bacterial genomes or individual proteins allow researchers to screen for vaccine/drug candidates, automatically annotate gene products or select proteins for further study. The pathogenicity of V. cholerae O1 and O139 strains depends on a combination of properties, including enterotoxin (cholera toxin [CT], ctxA) and the ability to adhere to, and colonize, the small intestine (colonization factor, tcpA) (Herrington et al 1988). The major virulence-associated factors are present in clusters with at least three regions in the V. cholerae chromosome. Several factors have been associated with entero pathogenicity which, includes a El Tor-like hemolysin (hlyA) (Yamamoto et al 1984), heat-stable enterotoxin (stn/sto) (Arita et al 1986), hemagglutinins (Datta-Roy et al 1986), outer membrane protein (ompU) (Sperandio et al 1996), Shiga-like toxin (stx) (Kaper et al 1994), a ToxR regulatory protein (Miller et al 1987), and a zonula occludens toxin (zot) (Fasano et al 1991). The application of this study was to predict the sub cellular localization of putative proteins of V. cholerae O1 strain as specific subcellular localization of bacterial proteins was useful in designing the therapeutics against bacterial proteins that can not affect the host cellular activity. Subcellular localization of proteins is crucial for the pathogenesis and survival of bacteria as they might be useful for targeting drugs.

Materials And Methods

Collection of sequences

Analysis of physico chemical properties

Prediction of sub cellular localization of proteins

Results And Discussion

In this study we had selected fifty two putative protein of V. cholerae O1 and their physico chemical nature was analyzed theoretically. The molecular weight and isoelectric point of all these proteins was deduced (Table 1).

Figure 1

Table 1: Physico-chemical properties and subcellular localization of putative proteomes of O1 strain.

It indicates protein stability in a particular isoelectric point (pI). An online PSLpred server was used to predict protein subcellular localization within bacteria or targeting the host. We investigate putative proteome of V. cholerae and their specific subcellular location (Table1). V. cholerae elaborates zonula occludens toxin (Zot), a protein that increases the permeability of small intestinal mucosa by opening intercellular tight junctions. The zot gene is located, together with the genes encoding CT and Ace enterotoxins, within the genome of V. cholerae filamentous phage CTX small ef, Cyrillic. Zot localizes in the V. cholerae cell envelope with M(r); 45 kDa which is consistent with the predicted primary translation product from the first methionine of zot (44.8 kDa). A second molecule, corresponding to the 33 kDa N-terminal region of Zot, was also detected. Both molecules are exposed at the bacterial cell surface (Uzzau et al 1999). The cellular location of the haemolysin of Vibrio cholerae El Tor strain 017 has been analyzed. This protein is found both in the periplasmic space and the extracellular medium in V. cholerae (Mercurio et al 1985).

V. cholerae O1 biotype El Tor produces and secretes a 65-kDa cytolysin/hemolysin into the culture medium. We cloned the structural gene (hlyA) for the cytolysin from the total DNA of a V. cholerae O1 El Tor strain, N86. Sequence analysis of hlyA protein of 741 amino acids with a molecular weight of 81,961 was done. Consistent with this, a 79-kDa protein was identified as the product of hlyA by maxicell analysis in Escherichia coli. N-terminal amino acids of this 79-kDa HlyA protein and those of a 65-kDa El Tor cytolysin purified from V. cholerae were Asn-26 and Asn-158, respectively. The 82- and 79-kDa precursors of the 65-kDa mature cytolysin were found in V. cholerae by pulse-chase labeling and Western blot (immunoblot) analysis of hlyA products. V. cholerae, the 82-kDa preprotoxin synthesized in the cytoplasm is secreted through the membranes into the culture medium (Yamamoto et al 1990).

In previous studies thirty-nine putative proteins of Mycobacterium tuberculosis H37Rv strain were predicted for four locations viz cytoplasmic, integral membrane, secretory and protein attached to membrane by Lipid anchor in the subcellular localization (Somvanshi et al 2008).Also prediction of motif in Dengue virus serotypes polyprotein was done and several significant motifs which involves in regulation, activity and stability of virus were observed for targeting or designing the antiviral inhibitors against these motifs of Dengue virus (Somvanshi and Seth 2008).

In conclusion, we include the specified prediction of subcellular localization results in the most putative proteins of strain of V. cholerae O1. This initiative may help in annotating newly sequenced or hypothetical proteins. Thus, the search for a potential vaccine/ drug target for an important bacterial pathogen by in vitro researchers will greatly be appended by this prediction.