Complementation of the Mycoplasma synoviae MS-H vaccine strain with wild-type obg influencing its growth characteristics

Abstract

The temperature-sensitive (ts+) Mycoplasma synoviae vaccine strain MS-H harbors a non-synonymous mutation which results in Glycine to Arginine substitution at position 123 in the highly conserved glycine-rich motif of Obg-fold in the GTP-binding protein Obg. In-silico analysis of the wild-type and mutant Obgs of M. synoviae has indicated that this amino acid substitution affects structure of the protein, potentially leading to abrogation of Obg function in vivo. Present study was conducted to develop the first expression vector for M. synoviae and to investigate the potential effect(s) of complementation of MS-H vaccine with the wild-type obg from 86079/7NS, the parent strain of MS-H. An oriC vector, pKS-VOTL, harboring the 86079/7NS obg gene, downstream of the variable lipoprotein haemagglutinin (vlhA) gene promoter, also cloned from 86079/7NS, was used to transform MS-H. The plasmid was localised at the chromosomal oriC locus of MS-H without any detectable integration at the chromosomal obg locus. Analysis of the MS-H transformants revealed abundant obg transcripts as well as Obg protein, when compared to the MS-H transformed with a similar vector, pMAS-LoriC, lacking obg coding sequence. The MS-H transformants complemented with wild-type Obg maintained their original temperature-sensitivity phenotype (consistent with MS-H vaccine) but, when compared to the MS-H transformed with pMAS-LoriC, had significantly higher (p < 0.05) growth rate and viability at the permissive (33°C) and non-permissive temperature (39.5°C), respectively. Analysis of Obg expression in MS-H and its wild-type parent strain revealed comparatively lower levels of Obg in MS-H. These results indicate that not only the mutation in Obg, but also the level of Obg expression, can confer functional abnormalities in the bacterial host. Furthermore, with the construction of first expression vector for M. synoviae, this study has set foundation for the development of recombinant vaccine(s) based on MS-H.

Fig 1. Construction of pKS-VOTL plasmid.

(A) Organisation of spo0B operon in B. subtilis and putative obg operon in M. synoviae has been shown. Putative –10 promoter region, transcription start site, ribosome binding site (RBS) of vlhA promoter region, and initiation codon for vlhA gene have been indicated. Length (bp) of each CDS is indicated inside the arrows. Identified stem loop structures in B. subtilis spo0B operon and putative stem loop in M. synoviae ‘obg operon’ have also been indicated. (B) Schematic presentation of splicing by overlap extension (SOE) PCR to join vlhA gene promoter with obg CDS. Using PCR#1 and PCR#2, vlhA promoter (solid lines) and obg CDS (dotted lines) were amplified using indicated primers. Intermediate products with overlapping fragments (shown by horizontal bars) from both PCRs were amplified by SOE-PCR (PCR#3) using primers vlhA-ExtF and obg-ExtR. (C) Agarose gel electrophoresis of amplification products of vlhA promoter region PCR, obg CDS PCR and SOE-PCR. MW, DNA molecular weight marker (PCR Marker, Sigma, Missouri, USA). (D) Final product of SOE-PCR was first cloned at T-site of pGEM-T Easy Vector and then PstI-SacII fragment containing vlhA-obg was inserted between PstI and SacII sites of pBluescript II KS (+) vector. ApaI-SalI restriction fragment containing LoriC and tetM, from pMAS-LoriC plasmid, was cloned at respective sites in pBluescript II KS (+) vector, harboring vlhA-obg, to generate pKS-VOTL plasmid.

Fig 2. Southern blot hybridisation to localise pKS-VOTL plasmid in MS-H transformants.

(A) Schematic presentation for the integration event of pKS-VOTL plasmid into genomic DNA of MS-H. A single putative homologous recombination event between the oriC copy carried by plasmid and chromosomal oriC region is represented by crossed lines. P indicates the promoter region of vlhA gene. (B, C, and D) BglII digested DNA of pKS-VOTL, untransformed MS-H and pKS-VOTL MS-H transformants MS-H-T75, MS-H-T78, MS-H-T90 (at passage 4^th and 8^th) was hybridised with DIG labeled tetM (B), oriC (C) and vlhA-obg probes (D).

Fig 3. Northern analysis of total RNA (~ 50 μg/lane) from M. synoviae 86079/7NS, MS-H, MS-H-C28, MS-H-T75, MS-H-T78, MS-H-T90, MS-H5 and 93198/1-24b.

(A) Agarose gel of total RNA from untransformed (86079/7NS, MS-H, MS-H5 and 93198/1-24b) and transformed (pMAS-LoriC transformant MS-H-C28 and pKS-VOTL transformants MS-H-T75, MS-H-T78 and MS-H-T90) M. synoviae MS-H stained with GelRed. (B, C, and D) Northern blot of the above gel hybridised with DIG-labeled vlhA-obg probe (spanning the joining site of vlhA promoter with obg CDS), obg specific probe and vlhA coding sequence specific probe, respectively. Arrowheads indicate the location of the specific bands identified by probes described above. The location of bands for the DIG-labeled RNA molecular weight marker RNA I (Roche) is indicated on the left side of all northern blots. ImageJ (1.48v; NIH, USA) was used to quantify intensity of obg transcripts.

Fig 4. Analysis of Obg expression in M. synoviae strains using SDS-PAGE and immunoblotting.

(A) SDS-PAGE (7.5%) of whole cell proteins from M. synoviae MS-H transformed with pMAS-LoriC (clone MS-H-C28) and pKS-VOTL (clones MS-H-T75, MS-H-T78 and MS-H-T90) plasmids, stained with Coomassie brilliant blue R-250 (Bio-Rad). Location of ~ 50 kDa protein in pKS-VOTL transformants is indicated with an arrow. (B) Immunostaining of untransformed 86079/7NS, MS-H, and transformed MS-H with pMAS-LoriC (clone MS-H-C28) and pKS-VOTL (clones MS-H-T75, MS-H-T78 and MS-H-T90) using polyclonal rabbit serum against M. synoviae Obg_3 peptide. Location of Obg-MBP fusion protein and overexpressed Obg is indicated with arrows on the right. (C) SDS-PAGE (8.75%) of whole cell proteins from M. synoviae 86079/7NS transformed with pMAS-LoriC (clone 7NS-C12 and 7NS-C38) and pKS-VOTL (clones 7NS-T30, 7NS-T20, 7NS-T8 and 7NS-T4) plasmids, stained with Coomassie brilliant blue R-250 (Bio-Rad). (D) Immunostaining of untransformed 86079/7NS and transformed 86079/7NS with pMAS-LoriC (clone 7NS-C12 and 7NS-C38) and pKS-VOTL (clones 7NS-T30, 7NS-T20, 7NS-8 and 7NS-T4) using polyclonal rabbit serum against M. synoviae Obg_1 peptide. Location of overexpressed Obg is indicated with an arrow on the right. (E) For comparison of Obg expression in MS-H and 86079/7NS, approximately equal amounts (88 μg/lane) of whole cell proteins from MS-H, MS-H-C28, MS-H-T75, 86079/7NS, 7NS-C38 and 7NS-T30 were separated by SDS-PAGE (8.75%) and stained with Coomassie Brilliant Blue. (F) Immunostaining at two different concentrations of whole cell proteins (i.e. 1/3 and 2/3 of that loaded onto SDS-PAGE as shown in panel E) of each strain/transformant using polyclonal rabbit serum against M. synoviae Obg_1 peptide. MW, protein marker (Precision Plus Protein, Dual Color, Bio-Rad).

Fig 5. Temperature-sensitive phenotyping, growth curve analysis and loss of viability study of MS-H and 86079/7NS transformants.

(A) Using vlhA-QPCR as described previously [29], quantification of vlhA gene copy number was performed at 33 and 39.5°C for untransformed MS-H (ts⁺ control) and 86079/7NS (ts⁻control), transformed MS-H and 86079/7NS with pMAS-LoriC (clone MS-H-C28 and 7NS-C38, respectively) and pKS-VOTL (clone MS-H-T75 and 7NS-T30, respectively). Growth was lower at non-permissive temperature (39.5°C) than permissive temperature (33°C) for all strains/transformants except 86079/7NS. Data is expressed as mean ± SD of vlhA gene copy numbers (n = 3). The 33/39.5°C vlhA gene copy number ratios, as established by Shahid et al. [29], indicated MS-H, MS-H-C28, MS-H-T75, 7NS-C38 and 7NS-T30 as ts⁺ and 86079/7NS as ts^–. (B) Growth curve analysis and loss of viability study of MS-H and 86079/7NS transformants. (a and b) For growth rate comparison, cultures of MS-H-C28 (Δ), MS-H-T75 (ϒ), 7NS-C38 (◊) and 7NS-T30 (○) transformants were grown at 33°C for 192 h. Aliquots were collected at 24 h intervals, and then titrated by incubation at 33°C for two weeks. (c and d) For loss of viability study, cultures of MS-H and 86079/7NS transformants, as indicated in panel a and b, were grown at 39.5°C for 72 h, aliquots collected at 12 h intervals, and then titrated by incubation at 33°C for two weeks. All cultures were grown in MB containing tetracycline (3 μg mL^–1). Bars indicate standard deviations of log₁₀-transformed CCU mL^–1 values from three independent cultures and "*" indicates significant differences (p < 0.05) using Student's t-test.