​Cloning and Sequence Analysis of Transferrin Binding Protein Gene A of Field and Vaccine Strains of Pasteurella multocida B:2

Pasteurella multocida is one of the most important bacterial pathogen of cattle and buffaloes which causes fowl cholera in birds, atrophic rhinitis in pigs, haemorrhagic septicaemia (HS) in ungulates, enzootic pneumonia in cattle, sheep and goats, snuffles in rabbits and more rarely wound abscesses and meningitis especially in immunocompromised humans (Bisgaard, 1993., Frederiksen, 1993; Ewers et al., 2004., Muenthaisong et al., 2020). Among them, In India, the disease HS (caused by serotype B:2 in Asian countries and E:2  in African countries) is considered as the most dreaded bacterial disease and killing approximately 2048 cattle and buffaloes annually (De Alwis, 1992, Singh et al., 2014). On the basis of the distribution of disease, India is considered to be endemic for HS (Benkirane and De Alwis, 2002) as outbreaks of HS are reported in bovines mainly during rainy and winter season every year from different states of India (Jindal et al., 2002; Shivaprakash et al., 2004; Singh et al., 2014).

Iron is an important factor for the growth of bacteria and is made available by iron acquisition proteins including transferrin binding protein, lactoferrin binding protein and haemoglobin binding protein. Bacterial transferrin binding protein (Tbp) is composed of two distinct proteins, TbpA and TbpB (Gray-Owen and Schryvers, 1996; Pogoutse and Morae, 2017). TbpA, an integral outer membrane protein, mediate the transport of iron across the outer membrane efficiently without the involvement of TbpB (Schryvers and Stojiljkovic, 1999; Ogunnariwo and Schryvers, 2001). Expression of transferrin receptors in strains of P. multocida  has been correlated with HS as P. multocida isolated from HS express transferrin receptors whereas the strains of non HS serotypes failed to express this protein (Veken et al., 1996; Ogunnariwo et al., 1997; Ognnariwo and Schryvers, 2001). Iron acquisition related gene tbpA has been considered as an important epidemiological marker (Veken et al., 1994).

Although HS outbreaks occur commonly in bovines in India (Verma et al., 2004; Shome et al., 2019), there is no information about the correlation of tbpA with virulence of P. multocida. Therefore, the present study was undertaken for cloning and sequence analysis of tbpA gene of
P. multocida field and vaccine strain.
 
Highlights
 
● Nucleotide variation was recorded at two points between field isolates and vaccine strain.
● Amino acid alignment of field isolates and vaccine strain showed variation at single amino acid position.

Characterization of Pasteurella multocida
 
Two field isolates (P1 and P2) and a vaccine strain (VS) of P. multocida B: 2 (P52) cryopreserved in Department of Veterinary Microbiology, LUVAS, Hisar were used in present investigation. Both the field isolates and vaccine strain were revived on blood agar plates and characterized by culture characteristics such as morphology, Gram’s staining, capsular and bipolar characteristics and biochemical tests. P. multocida B: 2 specific PCR (Townsend et al., 1998 and Quinn et al., 1994). After confirming field isolates and vaccine strain as P. multocida B: 2, organisms were incubated in BHI broth for 24 hours at 37°C for extraction of genomic DNA.
 
DNA extraction
 
The genomic DNA from field isolates and vaccine strain were extracted from overnight incubated BHI broth culture by Gen EluteTM Bacterial Genomic DNA kit (Sigma-Aldrich) as per manufacturer’s instructions.
 
Molecular confirmation of Pasteurella multocida
 
The PCR for P. multocida was done to amplify a 620 bp fragment using specific primers (Table 1). Briefly, the PCR master-mix was made for ‘n’ number of reactions by adding nuclease free water (36.0 µl x n); 10xPCR buffer with 15 mM Mg++ (5.0 µl x n); 10 mM dNTPs mix (1.0 µl x n); 10 µM tbpA-forward primer (1.0 µl x n); 10 µM tbpA-reverse primer (1.0 µl x n); Taq DNA Polymerase (Fermentas), 5 U/µl stock (1.0 µl x n) and template DNA (5.0 µl x n).



The PCR amplification was performed with initial DNA denaturation at 94°C for 5 min, followed by 35 cycles consisting of denaturation at 94°C for 1 min, annealing at 50°C for 1 min and extension at 72°C for 1 min using XP® Gradient Cycler (Bioer Technology, PR China). Finally, the amplification was ended by a final extension at 72°C for 5 min. Furthermore, 5 μl of PCR product was analyzed by electrophoresis in 1% agarose.
 
PCR amplification and Cloning of tbpA gene (tbpA-PCR)
 
A 729-bp region of tbpA was amplified by PCR using specific primers (Table 1) and amplification conditions reported previously (Ewers et al., 2006). The amplified products of tbpA gene of isolates and vaccine strain were purified using QIAquick gel extraction kit (Qiagen, Germany). The purified products were cloned in pGEM-Teasy vector (Promega, USA). Briefly, the ligation-reaction mixture was made as per manufacturer’s instructions and incubated at 25°C for 1 hour and subsequently used for transformation of JM107 cells as described in Sambrook and Russell (2001). Transformed JM107 cells were plated on LB/amp/X-Gal/IPTG agar plates and incubated overnight at 37°C. Transformation was confirmed by tbpA specific PCR.
 
Sequencing and bioinformatics
 
For sequencing, plasmid DNA was extracted from tbpA /pGEM-Teasy vector transformed JM107 cells by the alkaline lysis method (Sambrook and Russell, 2001). Primers specific to T7 and SP6 promoter regions in pGEM-Teasy vector DNA system were used for sequencing of tbpA gene of field isolates and vaccine strain. Optimal alignment of the tbpA sequences was done using multiple sequence alignment programs in ‘CLC Bio Sequence Viewer version 6.4’ software (URL: Http://Www.Clcbio.Com/). The deduced amino acid sequence was aligned in ‘CLC Bio Sequence Viewer version 6.4’ software (URL: http://www.clcbio.com/). Three-dimensional structural model of the tbpA proteins of field isolates and vaccine strain was constructed and transferrin binding sites were located in Phyer2 protein homology/analogy recognition engine V2.0 software (http://www.sbg.bio.ic.ac.uk/phyre2).

P. multocida organism is a small, non-motile, non-spore forming, Gram-negative coccobacillary rod, facultative anaerobe and belongs to the family Pasteurellaceae (Quinn et al., 1994). The organism exhibits a characteristic bipolar staining. P. multocida causes a wide range of important diseases in domestic animals, being responsible for pneumonia in cattle and sheep (Chanter and Rutter, 1989; Frank, 1989) and HS in cattle and buffalo (Carter and de Alwis, 1989). Five serogroups (A, B, D, E and F), based on capsular antigens, were recognized in P. multocida (Harper et al., 2006). Isolation of some unusual strains of P. multocida with serotypes A:1, A:3, F:3 and F:3,4 from bovines has also been reported in India (Kumar et al., 1996). Significant associations have been observed between HS and tbpA, hgbB and pfhA genes. Similarly, aropic rhinitis was found to be in association with toxA gene (Atashpaz et al., 2009). Iron is considered to be an important factor for the growth of P. multocida. The tbpA has been found to be associated with iron acquisition mechanism.

HS can be controlled by vaccination and most countries where the disease is endemic resort to routine prophylactic vaccination (De Alwis, 1999). The case fatality rate (CFR) was comparatively-low in vaccinated animals than unvaccinated animals in buffaloes and cattle, respectively. During 1995-98, a number of outbreaks were also recorded during winter months in those areas of Haryana where routine vaccination was carried out before start of rainy season (May-June). The cause for occurrence of disease in vaccinated animals might be the low efficacy and under dosing of the available vaccines (Verma et al., 2004).

Therefore the present study was done to compare the gene and deduced amino acid sequences of tbpA gene of field isolates and vaccine strain of P. multocida B:2.
 
Characterization of both P. multocida B:2 isolate and vaccine strain
 
Both field isolates and vaccine strain formed smooth, greyish glistening translucent dew drop like non-haemolytic colonies on blood agar after 24 hours incubation at 37°C (Fig 1A). Field isolates and vaccine strain of P. multocida were Gram-negative coccobacilli (Fig 1B) when examined by Gram’s staining. The organisms showed a thick capsule on capsule staining and bipolarity on methylene blue staining (Fig 1C). Organisms were found indole, oxidase, nitrate and glucose and sucrose fermentation test positive.



Both field isolate and vaccine strain were also confirmed by P. multocida B: 2 specific PCR yielded amplified product size of 620 bp (Fig 1D).
 
Cloning, sequencing and bioinformatics of tbpA
 
Transformed clones were screened by their colour for vector insert and white colonies (Fig 2) were picked up and tested by tbpA-PCR (Fig 3). Clones having vector insert were sequenced. Nucleotide sequences were found to have 729 nucleotides. All the three sequences were blasted at NCBI and showed 99% homology with tbpA of P. multocida P52, tbpA of P. multocida D: 1 and complete genome of P. multocida 36950.





The sequences were aligned using CLC Bio Sequence Viewer version 6.4’ software programme. Alignment showed differences in nucleotides at positions 574 and 620. At 574 position nucleotide, F2 and VS have thymine while F1 has cytosine and at 620 position nucleotide, F1 and F2 have thymine while VS has cytosine. Similar to this, in a study, conducted by Shivachandra et al., (2005), comparison of tbpA of P. multocida B:2 (P52) was made with P. multocida A:1 and 98.4% homology and 1.5% divergence was reported.

The sequence of tbpA gene from F1, F2 and VS was used for prediction of matured TbpA protein characteristics using the ‘CLC Bio Sequence Viewer version 6.4’ software. Deduced amino acid sequence of 242 amino acids revealed 99% homology with TbpA of P. multocida and with a variety of other TonB-dependent receptor proteins, indicating that it belongs to the family of outer membrane receptors. This finding coincides with that of Ogunnariwo and Schryvers (2001). Deduced amino acid sequence alignment of showed a single amino acid variation at 207th position. In both field isolates Leucine was there while in vaccine strain Phenyl alanine was found. These both amino acids are hydrophobic in nature so no change in physico-chemical property of TbpA is expected. The finding of Shivachandra et al., (2005) also indicated that TbpA has a very high antigenic index, hydrophilicity and surface probability.

The amino acid variation was not found at the binding site residues (D 72, L 141, L 142), predicted by Phyer2 protein homology/analogy recognition engine V2.0 software. 3-D protein model constructed from Phyre2 software that used PDB: c2iahA as template showing 99.6% homology with TbpA protein.

Further three binding sites at amino acid residue 72 (ASP), 141 (LEU) and 142 (LEU) of TbpA protein, obtained by using Phyer2 protein homology/analogy recognition engine V2.0 software, were predicted. This indicates that the amino acid change is not found at these binding sites.

From this study it is concluded that single amino acid difference between field isolate and vaccine strain might not cause change in binding and physico-chemical property of TbpA.