Research in Veterinary Science 96 (2014) 415–421

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Research in Veterinary Science j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / r v s c

Homogeneity of VacJ outer membrane lipoproteins among Pasteurella multocida strains and heterogeneity among members of Pasteurellaceae Sathish Bhadravati Shivachandra a,*, Abhinendra Kumar a, Nihar Nalini Mohanty a, Revanaiah Yogisharadhya b, Nirmal Chacko a, K.N. Viswas c, Muthannan Andavar Ramakrishnan d a b c d

Clinical Bacteriology Laboratory, Indian Veterinary Research Institute (IVRI), Mukteswar-263138, Nainital (District), Uttarakhand (UK), India National Institute of Veterinary Epidemiology and Disease Informatics (NIVEDI), Bengaluru-560024, Karnataka, India Division of Bacteriology and Mycology, Indian Veterinary Research Institute (IVRI), Izatnagar-243122, Uttar Pradesh (UP), India Division of Virology, Indian Veterinary Research Institute (IVRI), Mukteswar-263138, Nainital (District), Uttarakhand (UK), India

A R T I C L E

I N F O

Article history: Received 31 August 2013 Accepted 25 March 2014 Keywords: Homogeneity Heterogeneity Pasteurella multocida Pasteurellaceae VacJ outer membrane lipoprotein Sequence analysis

A B S T R A C T

Outer membrane lipoproteins are widely distributed in Gram-negative bacteria which are involved in diverse mechanisms of physiology/pathogenesis. Various pathogenic bacterial strains belonging to the familyPasteurellaceae have several surface exposed virulence factors including VacJ/VacJ-like lipoproteins. In the present study, vacJ gene encoding for VacJ outer membrane lipoprotein of different Pasteurella multocida strains (n = 10) were amplified, sequenced and compared with available VacJ/VacJ-like sequences (n = 45) of Pasteurellaceae members. Comparative multiple sequence analysis at amino acid level indicated absolute homogeneity of VacJ lipoprotein among different P. multocida strains. However, heterogeneity (18.0– 89.9%) of VacJ lipoprotein was noticed among members of Pasteurellaceae. A predicted lipobox motif (L-3-[A/S/T/V]-2-[G/A]-1-C) was found to be conserved between 12-32aa residues at N-terminus among all VacJ sequences. Bioinformatic analysis indicated that VacJ is a chromosomal gene product exposed on the bacterial surface, possibly essential for either physiological or pathogenicity process of Pasteurellae and distributed widely among P. multocida serogroups. The study indicated potential possibilities of using absolutely conserved VacJ lipoprotein either as ‘signature gene/protein’ in developing diagnostic assay or as a recombinant subunit vaccine for P. multocida infections in livestock. © 2014 Elsevier Ltd. All rights reserved.

1. Introduction A majority of Gram-negative bacterial species belonging to the family-Pasteurellaceae are aerobic, coccoid- or rod shaped, nonspore forming, and non-motile (Brenner et al., 2005). They are known to play an important role as primary or opportunistic pathogens of domestic and wild animals especially in respiratory tract infections. Some species of Pasteurellaceae cause highly infectious and contagious diseases, with high morbidity and mortality rate in livestock leading to huge economic loss. Although Pasteurellaceae show ecological preferences for specific surfaces and hosts, very little is known about the factors that govern the host specificity (Bisgaard, 1993). Use of advanced molecular techniques coupled with conventional differentiation procedures led to the profound revision of

* Corresponding author. Tel.: +91 05942 286348 ext 4063 (Office) or +91 9756002533 (Mobile); fax: +91 05942 286347. E-mail address: [email protected] (S.B. Shivachandra). http://dx.doi.org/10.1016/j.rvsc.2014.03.016 0034-5288/© 2014 Elsevier Ltd. All rights reserved.

the taxonomy of Pasteurellaceae. To date, there are 73 species included under 18 genera (Kuhnert and Christensen, 2009). Out of these, most important species in relation to human and animal diseases include Pasteurella multocida, Actinobacillus pleuropneumoniae, Haemophilus parasuis, Mannheimia haemolytica, Bibersteinia trehalosi, Avibacterium paragallinarum, Haemophilus influenzae and Aggregatibacter actinomycetemcomitans. These are known to be associated with diverse disease manifestation as they have unique mechanisms of pathogenesis in susceptible host (Olsen et al., 2005). Among members of Pasteurellaceae, bacterial strains belonging to the genus ‘Pasteurella’ are of notable importance as they are involved in diverse disease manifestations in livestock. The diseases caused by strains of P. multocida belonging to capsular (A, B, D, E and F) and somatic (1–16) serotypes are known to affect wide range of domestic/wild animal and bird species worldwide with huge economic loss especially in tropical regions of Asia and Africa (Kumar et al., 2004; Shivachandra et al., 2006, 2011). Notably, diseases caused by various serotypes of P. multocida include haemorrhagic septi-

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Table 1 List of Pasteurella multocida isolates/strains and other Pasteurella species with their vacJ gene sequences used in the study. Sl. No

Isolate/strain

Host

Serogroup

Disease

Country

GenBank Accession No.

Reference

1 2 3 4 5 6 7 8 9 10

P52 IndPm176 IndPm219 IndPm167 IndPm94 IndPm111 IndPm116 IndPm113 IndPm224 IndPm115

Cattle Chicken Turkey Duck Quail Pig Goat Sheep Rabbit Buffalo

B:2 A:1 A:1 A:1 A:1 D:1 A:1 A:1 A:1 B:2

Haemorrhagic septicaemia Fowl cholera Fowl cholera Fowl cholera Fowl cholera Septicaemic pasteurellosis Septicaemic pasteurellosis Septicaemic pasteurellosis Snuffles Haemorrhagic septicaemia

India India India India India India India India India India

JX184899 JX184901 JX184902 JX184900 KJ191745 KJ191746 KJ191749 KJ191747 KJ191750 KJ191748

This study

11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34

PMTB Buffalo B:2 Anand1 Chicken A:1 Pm70 Chicken A:1 36950 Cattle A HN06 Pig D P1059 Turkey n/a X73 Chicken n/a P1062 Bovine n/a 3480 Pig n/a Anand Buffalo n/a P52VAC Buffalo B:2 1500C Bovine n/a RIIF Sheep n/a 671/90 Bovine n/a P1933 Bovine n/a 2000 Bovine n/a 1500E Bovine n/a 93002 Bovine n/a n/a n/a n/a n/a n/a n/a Pasteurella pneumotropica Pasteurella dagmatis strain ATCC 43325 Pasteurella bettyae strain CCUG 2042 Pasteurella bettyae

Haemorrhagic septicaemia Fowl cholera Fowl cholera n/a Atrophic rhinitis Fowl cholera Fowl cholera Pneumonic pasteurellosis Pneumonic pasteurellosis Haemorrhagic septicaemia Haemorrhagic septicaemia n/a n/a n/a n/a n/a n/a n/a n/a n/a

Malaysia India USA Germany China USA USA USA USA India India UK UK UK UK UK UK Sri Lanka n/a n/a

AWTD01000006 AFRR01000178 NC_002663 AET16627 NC_017027 AMBQ01000028 AMBP01000017 ASZP02000001 NC_017764 EJS87107 ALBZ01000099 AROA01000096 ARNZ01000032 ARWR01000010 ARNY01000096 ARNW01000114 AQTL01000185 ARNX01000090 WP_005718062 WP_005735535 WP_018357248 ACZR01000022 NZ_AJSX01000033 WP_005760863

Yap et al. (2013) Ahir et al. (2011) May et al. (2001) Michael et al. (2012) Liu et al. (2012) Johnson et al. (2013)

n/a

n/a, information not available; USA, United States of America; UK, United Kingdom. Note: The length of VacJ sequence in all isolates is 246 aa (741 bp) except P. bettyae and P. pneumotropica, which had 248 aa (747 bp).

caemia (HS) in cattle and buffalo (Biswas et al., 2004), septicaemic and pneumonic pasteurellosis in sheep and goat/wild animals, fowl cholera in birds, atrophic rhinitis in pigs, snuffles in rabbits, and occasional infections in humans through dog/cat bites (Harper et al., 2006; Hunt et al., 2000; Shivachandra et al., 2011). In view of intrinsic limitations of existing vaccines for pasteurellosis and a need to develop an improved broadly cross-protective novel vaccine as well as differentiating companion diagnostic assay, identification of immunogenic bacterial outer membrane proteins (OMPs) is of significant importance in recent times (Hatfaludi et al., 2010, 2012). VacJ (virulence-associated chromosome locus J), initially identified as surface lipoprotein of Shigella flexneri (Suzuki et al., 1994), was attributed for bacterial spreading and gene encoding for it was found on chromosomal locus in the NotI-J segment. Although, VacJ has been found in several Gram-negative bacterial species, the functional characterization has not been done extensively till date. The product of ORF PM1501 of avian P. multocida (Pm70) has recently been predicted to be the homolog of VacJ (Hatfaludi et al., 2010). Since many of recent bioinformatics approaches predicted prevalence of several uncharacterized lipoproteins in P. multocida strains (E-Komon et al., 2012), there is an imminent need to characterize the role of each putative lipoprotein in eliciting the immune response so that potential subunit vaccine candidate antigens for pasteurellosis can be selected. Currently, no attempts were made to analyse the VacJ lipoprotein sequences comparatively among members of Pasteurellaceae and to understand its potential implications. In the present study, for the first time, we describe the divergence of VacJ lipoprotein among members of Pasteurellaceae and absolute conservation among different serogroups of P. multocida strains.

2. Materials and methods 2.1. Bacteria, vector and primers A total of ten P. multocida strains belonging to different animal and avian hosts including one reference strain (P. multocida B:2 strain P52, an Indian HS vaccine strain) maintained in the ‘Clinical Bacteriology Laboratory’, Indian Veterinary Research Institute (IVRI), Mukteswar, Uttarakhand (UK), India, were used. All these strains were previously isolated from clinical samples collected during the natural disease outbreaks at varied time period in the respective host species belonging to different geographical regions of India (Table 1). For gene cloning, pJET1.2 (MBI-Fermentas, USA) and Escherichia coli TOP10 cells were used. A set of primers as described below were also synthesized (IDT-DNA, USA) and procured.

2.2. vacJ gene amplification, cloning and sequencing The DNAs of all the ten strains were extracted using Genomic DNA Miniprep kit (MDI, India). For vacJ gene amplification and sequencing studies, a primer set (VacJF: 5′-ATGAAAAAAACCAAACGTCT3′ and VacJR: 5′-TTAATCAATTTCATTTAAGATA-3′) targeting complete vacJ sequence (Nucleotide region-Nt 1–20 and Nt 720–741) based on its available P. multocida serogroup A:1 (Pm70) gene sequence (NC_002663) (May et al., 2001) was designed. PCR amplification of vacJ gene from respective strains was carried out using a primer set (VacJF and VacJR) and reaction conditions as described below. The PCR mixture consisted of 50 ng of template, 50 pmol of each primer, 10 mM of each dNTPs, 1x PCR buffer, 2.5 mM

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Fig. 1. Phylogentic tree based on VacJ lipoprotein sequences of genus Pasteurella. VacJ lipoprotein sequences of strains belonging to P. multocida, P. pneumotropica, P. bettyae and P. dagmatis available in GenBank as listed in Table 1, were used in phylogenetic tree construction using the Jotun Hein method (DNASTAR). Inset (in blue coloured box): PCR amplified product of vacJ gene. Lane M: DNA marker; Lanes 1–4: Representative vacJ gene products from P. multocida strains- P52, IndPm176, IndPm219 and IndPm167 sequentially. Lane C: Negative control.

MgCl2, and 1 U of Taq DNA polymerase. The volume of reaction mixture was made up to 25 µl with nuclease free water. PCR was performed with the following conditions: Initial denaturation at 94 °C for 6 min, followed by 30 cycles of denaturation at 94 °C for 45 s, annealing at 55 °C for 45 s, extension at 72 °C for 45 s and a final extension at 72 °C for 6 min. The amplified vacJ gene products were purified from the agarose gel using a gel extraction kit (Qiagen, USA) and ligated to pJET1.2 vector using CloneJET PCR Cloning Kit (MBIFermentas, USA). Following transformation to E. coli TOP10 cells and selection of recombinant clones, the extracted plasmids from respective clones using plasmid mini kit (Qiagen, USA) were sequenced using an automated DNA sequencer (ABI PRISM 3100; Perkin Elmer, Applied Biosystems, Foster City, CA, USA). The complete nucleotide sequences of vacJ gene of ten P. multocida strains were submitted to GenBank and their accession numbers are presented in Table 1. 2.3. Comparative VacJ sequence analysis VacJ lipoproteins encoded by vacJ gene belonging to Indian P. multocida strains (n = 13) as well as from other countries (n = 17) isolated from different host species and disease conditions as described in Table 1 were used in phylogenetic tree construction and comparative multiple sequence alignment of deduced VacJ lipoprotein sequences using pairwise Jotun Hein method of MegAlign (DNASTAR). The identities and divergence at nucleotide level as well as deduced amino acid sequences in these strains were also compared using DNASTAR program. For comparative analysis, vacJ/vacJlike gene sequences (n = 45) belonging to members of Pasteurellaceae as well as two representative members from Enterobacteriaceae were used in phylogenetic tree construction by Jotun Hein method analysis of MegAlign (DNASTAR).

2.4. Prediction of VacJ protein characteristics and lipobox motif The vacJ gene from P. multocida serogroup B:2 strain P52 (an Indian HS vaccine strain) was used for prediction of matured VacJ protein characteristics using the PROTEAN program (DNASTAR) as well as proteomics tools from the ExPASy website. LipoP v1.0 server was used for prediction of lipobox in VacJ/VacJ-like proteins among members of Pasteurellaceae as well as representative members of Enterobacteriaceae. 3. Results 3.1. VacJ sequence analysis among members of genus-Pasteurella All the ten P. multocida strains resulted in amplicons of ~741 bp. Following successful cloning and sequencing, their sequences were submitted to GenBank under the accession numbers as mentioned in Table 1. The sequence analysis of the coding region of the vacJ gene of P. multocida strains revealed a GC content of 39.68% in 741 bp ORF. Phylogenetic tree constructed based on VacJ protein sequence is depicted in Fig. 1. All the strains belonging to P. multocida were clustered into single group. However, P. dagmatis, P. pneumotropica and P. bettyae were branched out independently. Similar tree was also noted based on their nucleotide sequences (data not shown). On multiple sequence alignment, P. multocida strains had absolute identity at amino acid level (99.6 to 100%), whereas P. dagmatis, P. bettyae and P. pneumotropica had 14.8%, 49.8% and 49.3% divergences respectively, from P. multocida. At nucleotide sequence level also, greater homogeneity (99.1 to 100%) was noticed among P. multocida strains, which differed from P. dagmatis, and P. bettyae/ P. pneumotropica by 24.4%, 43.6%, respectively.

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Fig. 2. Phylogentic tree based on VacJ lipoprotein sequences of family Pasteurellaceae. VacJ lipoprotein sequences of strains belonging to family Pasteurellaceae and two representative members of family Enterobacteriaceae available in GenBank were used in phylogenetic tree construction using the Jotun Hein method (DNASTAR). The different clusters are labelled as I, II, III (Pasteurellaceae) and IV (Enterobacteriaceae).

3.2. VacJ sequence analysis among members of family Pasteurellaceae Phylogenetic tree based on VacJ amino acid sequences of different bacterial species is indicated in Fig. 2. All the members of Pasteurellaceae were branched out separately and grouped in to three major clusters (I, II, III). Two members of Enterobacteriaceae branched out separately forming their own cluster-IV. There was no identity/ homogeneity among the members of Pasteurellaceae. No absolute homogeneity was noticed among the species belonging to same genus. The length of VacJ/VacJ-like proteins varied from 243 to 258 amino acids. Similar divergence was also noticed at nucleotide level of vacJ gene. Overall, percentage of identity and divergence among all members of Pasteurellaceae ranged from 54.4 to 79.4% and from 18.0 to 89.9%, respectively. The percentage of identity and divergence within the different species of a particular genus (selected) under Pasteurellaceae are shown in Table 2. 3.3. Predicted characteristics of VacJ lipoprotein and prevalence of lipobox motif The predicted characteristics of full length VacJ protein sequences (246 aa with molecular weight ~27.56 kDa) using PSIPRED indicated the presence of signal peptide (1–19 aa) as well as very high antigenic index, hydrophilicity and surface probability as shown by PROTEAN (data not shown). Full length VacJ protein had a total

of 29 basic, 28 acidic, 84 hydrophobic and 65 polar residues with isoelectric point of 8.017. The secondary structure predictions revealed predominant presence of eight helix region (α1 to α8) and minor three strands (β1 to β3). The identified lipobox motifs in different members of Pasteurellaceae are mentioned in Fig. 3. The location of lipobox was found to vary among the members at 13 to 31 aa position of N-terminus. However, in genus Pasteurella, a conserved location was noticed at 17–20 aa position [L-S-G-C] with a cleavage site between 19G and 20C aa. Lysine (L) and Cysteine (C) residues were almost conserved among all lipobox regions. The variable residues at 2 (V/S/ T/A/F) and 3 (G/A) position of lipobox was noticed. The cleavage site was found to be between 3 (G/A) and 4 (C) residues position.

Table 2 Percentage identity/divergence of VacJ lipoprotein among selected members (genus) of Pasteurellaceae. Genus

Actinobacillus Aggregatibacter Haemophilus Pasteurella

At nucleotide level (%)

At amino acids level (%)

Identity

Divergence

Identity

Divergence

61.8–94.3 80.6–81.3 59.4–98.5 66.6–79.5

6.0–53.8 22.0–23.2 1.5–59.4 24.2–44.5

58.0–98.0 87.5–90.7 54.1–98.4 63.0–86.6

2.0–60.8 10.0–13.7 1.6–70.0 14.8–50.7

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Fig. 3. Predicted lipobox regions in VacJ lipoproteins of Pasteurellaceae. The lipobox residues as predicted by LipoP v1.0 are indicated by a red colour. A blue coloured square box denotes variable lipobox region among Pasteurellaceae. A dotted magenta coloured line at N-terminus denotes signal peptide region with cleavage site. An arrow denotes starting of mature VacJ lipoprotein.

4. Discussion Bioinformatic analysis of a large number of available genome sequences in the database has confirmed that lipoprotein genes are universally distributed in bacteria and constitute approximately 1–3% of their total genes (Sutcliffe et al., 2012). Molecular approaches using DNA sequence analysis are currently used for species identification of Pasteurellaceae for research activities and reference purposes, and in most cases, they allow very precise species and subspecies identification (Kuhnert and Korczak, 2006). The present study also focused on comparative analysis of vacJ gene encoded VacJ lipoprotein among members of Pasteurellaceae and its implications. Phylogenetic and multiple sequence alignments of VacJ lipoprotein sequences of P. multocida strains originating from different disease conditions, and belonging to diverse host species, showed that this gene is highly conserved. Unlike other virulence genes such as ptfA (Shivachandra et al., 2013), no change at nucleotide/protein sequences was noticed despite diverse serogroup, host, and diseases conditions. It implies that VacJ lipoprotein is having a common functional role and is an absolute requirement for the P. multocida survivability/pathogenesis. However, its accurate role needs to be further evaluated. Moreover, several P. multocida OMPs including lipoproteins as well as non-lipoproteins have recently been identified by multidisciplinary approaches including biochemical, molecular and structural or bioinformatic analysis (E-Komon et al., 2012; Hatfaludi et al., 2010; May et al., 2001; Wheeler, 2009). Recently, lipoprotein predictor programmes (Lipo and LipoP v1.0) pre-

dicted 86 and 82 proteins from the avian (Pm70) and porcine strain (3480) of P. multocida genomes respectively, on the basis of their conserved lipobox sequences. Moreover, they also predicted unique lipoproteins; 13–17 from the avian strain genome and 7–15 from the porcine strain genome (E-Komon et al., 2012). Till now, a very few lipoproteins of P. multocida such as plpB (Tabatabai and Zehr, 2004), plpE (Rimler, 2001; Wu et al., 2007), plp40 (Champlin et al., 2002), pcP and GlpQ (Lo et al., 2004), and Omp16 (Boyce et al., 2006; Kasten et al., 1997) have been characterized; and novel proteins viz., PM0442, PM0659, PM0979, PM1050, PM1064, PM1614, PM1720, PM1578, PM0576 including PM1501 (VacJ) were identified as putative lipoproteins (Al-Hasani et al., 2007; Hatfaludi et al., 2010). Moreover, majority of these lipoproteins were found to vary at amino acids sequence level among different strains of P. multocida. For example, heterogeneity of plpE gene encoding for PlpE lipoprotein among different P. multocida isolates has been reported (Singh et al., 2010). Generally, majority of the species in the family-Pasteurellaceae inhabit the mucosal membranes of the alimentary, respiratory, and genital tract of mammals, birds, and reptiles; and are known as opportunistic secondary invaders with ability to cause infections under predisposing conditions. However, a few species are primary pathogens that cause severe diseases in certain animals (Olsen et al., 2005). In our comparative VacJ sequence study, we noticed highly divergent VacJ/VacJ-like sequences among members of Pasteurellaceae. It may be due to variation in their physiology as well as pathogenesis of different disease manifestations. A previous study on the prevalence of Plp-40 in the family Pasteurellaceae showed that this

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lipoprotein is widely distributed within the family, but is not universal to all members (Champlin et al., 2002). Despite heterogeneity, presence of VacJ among all members of Pasteurellaceae reflects its absolute necessity. A majority of triacylated bacterial lipoproteins are considered to be structurally constant in each bacterium and known to play diverse, vital roles in bacterial physiology and virulence (Nakayama et al., 2012). Bioinformatics and secondary structure predictions of VacJ protein of P. multocida revealed the presence of signal peptide (119aa) followed by a lipobox at N-terminus. Lipobox motif, as predicted by LipoP v1.0, which is known to have a sensitivity of 0.964, is typically L-3-[A/S/T]-2-[G/A]-1-C+1 with the +1 cysteine absolutely conserved among all bacterial lipoproteins (Braun and Wu, 1994). A salient lipoprotein motif ‘Leu-X-Gly-Cys’ present at the C-terminal end of the signal sequence (17L-S-G-C20), strongly suggested that VacJ is a lipoprotein exposed on the bacterial surface. In bacteria, lipoproteins are synthesized with an N-terminal hydrophobic signal peptide that is cleaved from the mature polypeptide by lipoprotein signal peptidase (LSP) prior to covalent linkage of fatty acids (Hayashi and Wu, 1990). The predominant hydrophilic regions were found dispersed throughout the length of VacJ lipoprotein as evidenced by surface probability and high antigenicity indices. VacJ was found to possess predominately α-helices with a fewer (