S. Shamya et al. / Journal of Pharmacy Research 2012,5(5),2910-2914 Research Article Available online through ISSN: 0974-6943 http://jprsolutions.info An in silico search for potential drug targets in outer membrane of Acinetobacter baumannii S. Shamya1, G. Azika1 and S. K. Sundar2,* 1 Department of Biotechnology, Noorul Islam College of Arts and Science, Kumaracoil, Tamil Nadu, India 2 Department of Microbiology, M. R. Government Arts College, Mannargudi, Tamil Nadu, India Received on:12-02-2012; Revised on: 25-03-2012; Accepted on:26-04-2012

ABSTRACT Acinetobacter baumannii is a nosocomial pathogen causing nosocomial pneumonia, bacteremia, meningitis, and urinary tract infection. It is more common in ICUs. Recent reports indicate resistance gained by the pathogen to ß-lactams, aminoglycosides, fluoroquinolones, and most notably, carbapenems. In the present study, the outer membrane protein A of A. baumannii was analysed for the presence of good target regions for drug discovery using bioinformatics tools. Physical parameters of the protein sequence retrieved from GenBank were studied followed by evaluation of conserved regions in the protein. Secondary structure analysis revealed the protein to be of ß-sheet conformation. Analysis of epitope regions and surface accessibility regions in the protein revealed positive results which suggest their choice as a good target for drugs.

Keywords: A. baumannii, antibiotic resistance, outer membrane protein, epitope, drug target INTRODUCTION Genus Acinetobacter consists of important opportunistic pathogens involved brane and to the shape of the cell and appears to function as a . OmpA in hospital- acquired infection. They cause various types of human infec- has been shown to be non-covalently associated with both the peptidoglycan tions. Of the currently known 31 Acinetobacter species, Acinetobacter and the peptidoglycan-bound Braun lipoprotein. A. baumannii OmpA are baumannii is the most prevalent in clinical specimens. A. baumannii is a known to bind to and enter host cells. The OmpA protein purified from A. pleomorphic gram- negative aerobic coccobacilli commonly isolated from the baumannii ATCC 19606T have been found to bind to the surface of host cells hospital environment and hospitalized patients. A. baumannii is a causative and are localized in both the mitochondria and nuclei, which induce the death agent of nosocomial pneumonia, bacteremia, meningitis, and urinary tract of host cells. [6] Based on the studies in various strains of A. baumannii, it has infection. [1] The main infection caused by this microorganism is nosocomial been found that the most common mechanism of resistance to ß-lactam anti- pneumonia, in particular ventilator-associated pneumonia in patients in In- biotics is by synthesis of a naturally occurring AmpC-type cephalosporinase. tensive Care Units. [2] It can cause various other infections including skin and [7] The present study would help us to develop suitable drugs that specifi- wound infections, bacteremia, and meningitis.[1] A. baumannii can survive on cally target this protein and would solve the problems caused by antibiotic the human skin or dry surfaces for weeks. It is the second most commonly resistant strains of the pathogen using bioinformatics tools. isolated non-fermenting bacteria in human specimens. A. baumannii infec- METHODS tions are uncommon but, when they occur, usually involve organ systems The physico-chemical properties of the query sequence retrieved from NCBI that have a high fluid content (e.g. respiratory tract, CSF, peritoneal fluid, were computed using Prot Param. [8] BlastP was used in the present study to urinary tract), manifesting as nosocomial pneumonia, infections associated find homologous sequences to the query sequence and the sequences were with continuous ambulatory peritoneal dialysis (CAPD), or catheter- associ- then compared with the query sequences and arranged in the ascending values ated bacteruria. Acinetobacter species are innately resistant to many classes of E. [9] The conserved regions were detected using PROSITE, Pfam and of antibiotics, including penicillin, chloramphenicol, and often aminoglycosides. CDD tools to establish functional domains and regular expression patterns. Resistance to fluoroquinolones has been reported during therapy and this has [10, 11] Finger print Analysis was done to build diagnostic signatures of the also resulted in increased resistance to other drug classes mediated through protein family membership and the fingerprints thus created were used to active drug efflux. A. baumannii has emerged as a potent nosocomial patho- identify the distant relatives of the protein in Prints database. [12] The second- gen with the recent acquisition of resistance to broad-spectrum ß-lactams, ary structure for the query protein was predicted using Jpred. The steric aminoglycosides, fluoroquinolones, and most notably, carbapenems.[3] Outer properties of amino acids were considered for the prediction of secondary membrane (Omps) of Gram-negative bacteria are key players in structure. The numbers of proline and glycine residues were taken into con- bacterial pathogenesis. OmpA of , OspC of Borrelia sideration as they exhibit reduced and complete torsional freedom respec- burgdorferi, and Opa and OpcA of Neisseria meningitidis facilitate adher- tively. The surface accessibility of the protein was analysed using Emini ence to and invasion of bacteria in host cells. [4,5] Virulence of this bacterium surface accessibility prediction tool. [13] Prediction of transmembrane regions is associated with the production of a paracrystalline outer membrane A- and orientation of the query protein was done using TMpred. [14] The hydro- layer protein. In A. baumannii, OmpA (AbOmpA) is the most abundant phobic analysis was first being carried out by computing the percentage of surface protein with a molecular mass of 38 kDa and plays a role in perme- non-polar amino acids present in the query protein. ability of small solutes. Omp A contributes to the stability of the cell mem-

RESULTS AND DISCUSSION The OmpA protein sequence of Acinetobacter baumannii with accession *Corresponding author. number ABG37059.1 was retrieved from NCBI database (Table1). The Dr. S. K. Sundar, query sequence which was a linear protein of 282 amino acid residues was Department of Microbiology, subjected to compositional analysis using various bioinformatics tools. The M. R. Government Arts College, individual amino acid composition, aliphatic and aromatic composition and Mannargudi, Tamil Nadu, India

Journal of Pharmacy Research Vol.5 Issue 5.May 2012 2910-2914 S. Shamya et al. / Journal of Pharmacy Research 2012,5(5),2910-2914 TABLE 1 – QUERY SEQUENCE (ABG37059.1) RETRIEVED FROM NCBI quences as denoted by the e-values (E-100 to E-50). They included sequences 1 mlvaaplaaa nagvtvtpll lgytfqdtqh nnggkdgelt ngpelqddlf vgaalgielt of OmpA, Omp38, outer membrane protein HMP, outer membrane porin F 61 pwlgfeaeyn qvkgdvdgla agaeykqkqi ngnfyvtsdl itknydskik pyvllgaghy precursor and OmpA/MotB derived from Acinetobacter baumannii, 121 kyeipdlsyh ndeegtlgna gvgafwrlnd alslrtearg tynfdekfwn ytalaglnvv Psychrobacter spp. and Enhydrobacter aerosaccus. Sequence with E-values 181 lgghlkpaap vvevapvept pvapqpqelt edlnmelrvf fdtnksnikd qykpeiakva in the range E-50 to E-5 included outer membrane proteins, protein F and 241 eklseypnat arieghtdnt gprklnerls laransvksa lv fibronectin-binding protein derived from other organisms (Figure 1).

TABLE 2 – PROTPARAM RESULTS OF THE QUERY SEQUENCE The Conserved Domain Search of the query sequence revealed an OprF membrane domain at the region between 108 and 185 and an OmpA-like Number of amino acids : 282 domain region lying between the amino acid residues 220 and 282 (Figure 2). Molecular weight : 30726.4 The OmpA-like domain is thought to be responsible for non-covalent inter- Theoretical pI : 4.91 [15] Total number of negatively charged residues (Asp + Glu) : 37 actions with peptidoglycan. The Pfam analysis also showed two domain Total number of positively charged residues (Arg + Lys) : 25 regions, OprF (between residues 34 and 185) and OmpA (between 220 and Ext. coefficient : 35870 M-1 cm-1 282 residues) (Table 3). The analysis made by Sundar and Nelson in Group Abs 0.1% (=1 g/l) : 1.167 The N-terminal of the sequence considered : M (Met) A Streptococci revealed domain regions in its M-protein which were pro- The estimated half-life is : 30 hours (mammalian posed to be good targets for drugs, especially in case of multidrug resistant reticulocytes, in vitro) strains. [16] >20 hours (yeast, in vivo) >10 hours (Escherichia coli, in vivo) Instability index (II) : 28.59 The query protein was then subjected to Motif analysis using PRINTS39 (This classifies the protein as stable) and Matrix Blos62. The results showed eight fingerprints, of which the Aliphatic index : 86.52 OMPA domain fingerprint had three motif regions (Table 4). The motifs Grand average of hydropathicity (GRAVY) : -0.366

FIGURE 1 – SCREENSHOT OF BLASTP ANALYSIS OF THE QUERY PROTEIN SEQUENCE hydrophobicity of the query protein were computed and the results were were drawn from conserved regions spanning the central portion of the given in Table 2. The protein possesses both acidic and basic amino acid alignment – motif 1 (221-244), motif 2 (251-266) and motif 3 (266-282) lie residues. The amino acids which provide stable secondary structures such as within the region encoded by PROSITE pattern OMPA (PS01068). These glutamic acid and leucine were present with an instability index of 28.59 and three motif regions were in the OmpA-like domain region (Figure 3). A highly therefore it was classified as a stable protein. The molecular weight of the conserved hexapeptide sequence had been identified within the C-terminal protein was found to be 30726.4 Daltons. The composition of aliphatic end of 11 known proteins from Gram positive cocci. [17] The C-terminal residues was more (86.52%) than that of aromatic residues in the query domain was shown to be immunodominant portion of the protein in experi- protein. ments using purified OmpA. [18]

The query protein was subjected to BLASTP analysis & the alignments of In the present study secondary structure of the query protein was predicted similar sequences were computed on the basis of expectation (E) values. using Jpred analysis. The query protein mainly exhibited ß-sheet conforma- Thirty seven sequences showed high level of similarity with the query se- tion (Figure 4). The three amino acids, Alanine, Glycine and Leucine, having Journal of Pharmacy Research Vol.5 Issue 5.May 2012 2910-2914 S. Shamya et al. / Journal of Pharmacy Research 2012,5(5),2910-2914

FIGURE 2 – SCREENSHOT OF CONSERVED DOMAIN DATABASE ANALYSIS OF ABG37059.1

TABLE 3 - PFAM DOMAIN ANALYSIS RESULT OF ABG37059.1 SOURCE DOMAIN START END

sig_p 1 22 low_complexity 4 12 Pfam A OprF 34 185 low_complexity 187 208 Pfam A OmpA 220 282

TABLE 4 – SCREENSHOT OF FINGERPRINT ANALYSIS OF QUERY SEQUENCE

FIGURE 3 - GRAPHICAL REPRESENTATION OF OMPA MOTIF RE- GIONS IN ABG37059.1 Since the query protein is a surface protein, computational techniques (TMpred) were used to predict the transmembrane helices. The results are presented in the (Table 5). Three helices of inside to outside orientation (2 to 21, 45 to 65 and 168 to 192) were predicted. Similarly three helices of outside to inside orientation were predicted for the query protein. Of them one inside to outside helix (2 to 21) and two outside to inside helices (1 to 25 and 168 to high propensity for ß-sheet (10.6%, 11.3%, 7.4% respectively) dominate 186) showed more orientation for helix confirmation than other regions. The the query protein, whereas the amino acids like Tryptophan and Methionine presence of transmembrane helices indicates that a protein has the membrane which have propensity for a helix were present in low quantities (1.1%, bound location. [14] This confirms that the protein lies on the 0.7% respectively). of the organism. Journal of Pharmacy Research Vol.5 Issue 5.May 2012 2910-2914 S. Shamya et al. / Journal of Pharmacy Research 2012,5(5),2910-2914

FIGURE 4 – SCREENSHOT OF SECONDARY STRUCTURE PREDICTION OF ABG37059.1

TABLE 5 – PREDICTION OF TRANSMEMBRANE HELICES IN FIGURE 5 - BEPIPRED LINEAR EPITOPE PREDICTION FOR OMPA ABG37059.1 QUERY SEQUENCE 1) Possible transmembrane helices

2) Table of correspondences

The epitope regions and the surface accessible regions of the query protein sequence were predicted using Bepipred tools. It revealed two epitopes of 14 and 24 amino acid residues at the OmpA domain region (Figure 5). It was also found that the protein was located on the surface of the organism with around ten surface accessible regions. The results are presented in Figure 6. 3) Suggested models for transmembrane topology The OmpA is a stable protein with 30 kDa molecular weight. The OmpA domain was found at the C-terminal end of the sequence and it showed three motif regions. The sequence seems to be immunogenic with two epitopes within the OmpA domain and other epitope regions outside the domain. Another result to be of importance was that the OmpA protein showed transmembrane location and also surface accessibility. All these results show that OmpA is a stable protein having conserved regions in its sequence. The organism seems to be resistant to most of the ß-lactam antibiotics and also some of the strains of the bacteria are getting resistant to more recent

Journal of Pharmacy Research Vol.5 Issue 5.May 2012 2910-2914 S. Shamya et al. / Journal of Pharmacy Research 2012,5(5),2910-2914 invasion of brain microvascular endothelial cells. Infect Immun. FIGURE 6 - EMINI SURFACE ACCESSIBILITY PREDICTION FOR 64, 1996, 146-153. OMPA QUERY SEQUENCE 6. Choi CH, Hyun SH, Lee JY, Lee JS, Lee YS, Kim SA, Chae JP, Yoo SM, Lee JC, Acinetobacter baumannii outer membrane protein A targets the nucleus and induces cytotoxicity. Cell Microbiol. 10, 2008, 309-319. 7. Bou G, Martínez-Beltrán J, Cloning, nucleotide sequencing and analysis of the gene encoding an AmpC ß-lactamase in Acinetobacter baumannii. Antimicrobial agents and Chemotherapy, 44, 2000, 428- 432. 8. Elisabeth Gasteiger, Christine Hoogland, Alexandre Gattiker, Séverine Duvaud, Marc R Wilkins, Ron D Appel, Amos Bairoch, Protein Identification and Analysis Tools on the ExPASy Server; (In) John M. Walker (ed): The Proteomics Protocols Handbook, Humana Press. 2005, pp. 571-607. 9. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ, Basic local alignment search tool. J. Mol. Biol. 215, 1990, 403-10. 10. Finn RD, Tate J, Mistry J, Coggill PC, Sammut SJ, Hotz HR, Ceric G, Forslund K, Eddy SR, Sonnhammer ELL, Bateman A, The Pfam protein families database. Nucleic Acids Research, Database Issue 36, 2008, D281-D288. 11. Marchler-Bauer A, Anderson JB, Chitsaz F, Derbyshire MK, DeWeese-Scott C, Fong JH, Geer LY, Geer RC, Gonzales NR, Gwadz M, He S, Hurwitz DI, Jackson JD, Ke Z, Lanczycki CJ, Liebert CA, Liu C, Lu F, Lu S, Marchler GH, Mullokandov M, carbapenem group of antibiotics. Considering these factors, designing a suit- Song JS, Tasneem A, Thanki N, Yamashita RA, Zhang D, Zhang N, able cost-effective drug to counter the infection of this bacterium and other Bryant SH, CDD: specific functional annotation with the Con- related Gram negative bacteria is the need of the hour. The observations made served Domain Database, Nucleic Acids Res. 37(D), 2009, 205- in the present study regarding the OmpA domain, motifs and epitopes will 10. definitely throw light in designing a new drug candidate to suitably dock 12. Attwood TK, Beck ME, Flower DR, Scordis P, Selley JN, The PRINTS protein fingerprint database in its fifth year. Nucl. Acids these regions or in constructing a recombinant vaccine using these motifs as Res. 26(1), 1998, 304-308. epitopes which will be a great leap in the treatment of dreadful diseases 13. Emini EA, Hughes JV, Perlow DS, Boger J, Induction of hepatitis caused by A. baumannii and other OmpA domain bearing Gram negative A virus-neutralizing antibody by a virus-specific synthetic pep- pathogens. tide. J Virol, 55, 1985, 836-839 14. Krogh A, Larsson B, von Heijne G, Sonnhammer EL, Predicting REFERENCES transmembrane protein topology with a hidden Markov model: 1. Bergogne-Berezin E, Towner KJ, Acinetobacter spp. as nosocomial Application to complete genomes. Journal of Molecular Biology, pathogens: microbiological, clinical, and epidemiological features. 305(3), 2001, 567-580. Clin. Microbiol. Rev. 9, 1996, 148–165. 15. Grizot S, Buchanan SK, Structure of the OmpA-like domain of 2. Marti S, Sánchez-Céspedes J, Espinal P, Vila J, In vitro activity of RmpM from Neisseria meningitidis. Mol. Microbiol, 51, 2004, ceftobiprole against Acinetobacter baumannii clinical isolates. Int J 1027-1037. Antimicrob Agents 34(3), 2009, 265-267. 16. Sundar SK, Nelson R, In silico analysis of Group A Streptococci 3. Reddy P, Zembower TR, Ison MG, Baker TA, Stosor V, virulence factor. Ind. J. Microbiol., 46(3), 2006, 223-228. Carbapenem-resistant Acinetobacter baumannii infections after 17. Fischetti VA, Pancholi V, Schneewind O, Conservation of a organ transplantation. Transplant Infectious Disease. 12(1), 2010, hexapeptide sequence in the anchor region of the surface proteins 87-93. of Gram positive cocci. Mol. Microbiol., 4, 1990, 1603- 1605. 4. Khan NA, Shin S, Chung JW, Kim KJ, Elliott S, Wang Y, Kim KS, 18. Puohiniemi R, Karvonen M, Vuopio VJ, Muotiala A, Helander Outer membrane protein A and cytotoxic necrotizing factor-1 use IM, Sarvas M, A strong antibody response to the periplasmic C- diverse signaling mechanisms for Escherichia coli K1 invasion of terminal domain of the OmpA protein of Escherichia coli is pro- human brain microvascular endothelial cells. Microb Pathol, 2003, duced by immunization with purified OmpA or with whole Es- 35, 35-42. cherichia coli or Salmonella typhimurium bacteria. Infect. Immuno., 5. Prasadarao NV, Wass CA, Weiser JN, Stins MF, Huang SH, Kim 58, 1990, 1691-1696. KS, Outer membrane protein A of Escherichia coli contributes to

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Journal of Pharmacy Research Vol.5 Issue 5.May 2012 2910-2914