|Year : 2016 | Volume
| Issue : 1 | Page : 52-59
Cloning and molecular characterisation of resuscitation promoting factor-like gene from Mycobacterium avium subspecies avium
R Kavitha, R Verma
Bacteriology and Mycology Division, Mycobacteria Laboratory, Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh, india
|Date of Submission||28-Apr-2014|
|Date of Acceptance||12-Oct-2015|
|Date of Web Publication||15-Jan-2016|
Bacteriology and Mycology Division, Mycobacteria Laboratoryz, Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
Purpose: Resuscitation promoting factor (Rpf)-like gene of Mycobacterium avium subspecies paratuberculosis has been known to stimulate the growth of mycobacteria and enhances the recovery of replicating cells from non-replicating phases. The objective of the study was to produce recombinant rpf-like protein of M. avium subspecies avium protein for purification and physico-chemical characterisation. Materials and Methods: The identified rpf gene of M. avium subspecies avium was cloned, subcloned, sequenced and expressed in Escherichia coli expression system for the production of the recombinant protein. The expressed recombinant Rpf protein was confirmed by Western blot and the extract was purified to yield a pure recombinant protein. Results: An rpf-like gene of 675 bp size in the M. avium subspecies avium was identified. This gene was expressed and the recombinant Rpf weighed 65 kDa as confirmed by Western blot. The M. avium recombinant Rpf protein was extracted under denatured conditions and purified yielding a recombinant protein with >90% purity. Conclusions: Identification, cloning, sequencing and expression of a rpf-like gene from M. avium suggest that RpfA is present in this species also, which might be involved in reactivation phenomenon in this high-risk pathogen.
Keywords: Latency, Mycobacterium avium, resuscitation promoting factor
|How to cite this article:|
Kavitha R, Verma R. Cloning and molecular characterisation of resuscitation promoting factor-like gene from Mycobacterium avium subspecies avium. Indian J Med Microbiol 2016;34:52-9
|How to cite this URL:|
Kavitha R, Verma R. Cloning and molecular characterisation of resuscitation promoting factor-like gene from Mycobacterium avium subspecies avium. Indian J Med Microbiol [serial online] 2016 [cited 2019 Dec 16];34:52-9. Available from: http://www.ijmm.org/text.asp?2016/34/1/52/174102
| ~ Introduction|| |
Finding novel molecules that promote revival or recovery of dormant bacteria to become actively growing virulent cells, so as to re-establish the progressive infectious stage (reactivation phenomenon), have been an emerging area of research. One such molecule identified as resuscitation promoting factor (Rpf), a 16–17 kDa protein, originally was found to be secreted by actively growing cultures of Micrococcus luteus promoted resuscitation and growth of dormant, non-growing cells of M. luteus. Genes similar to rpf are widely distributed among the gram-positive bacteria as mycobacteria, corynebacteria and Streptomyces. Rpfs reported previously as bacterial growth factors or bacterial cytokines were shown to possess muralytic activity  and also exhibited structural similarity to C-type lysozyme and soluble lytic transglycosylases. Ravagnani et al. reported that stationary phase survival proteins of firmicutes and the actinobacterial Rpf proteins are cognate and control bacterial culturability via enzymatic modification of the bacterial cell envelope. Several homologues of rpfs such as five rpfA-E in Mycobacterium tuberculosis and M. bovis and four each in Mycobacterium avium subsp. hominissuis and M. avium subsp. paratuberculosis have also been identified. Two homologues rpf A and rpf B have been predicted to be present in M. avium subsp. avium genome (NCBI GenBank accession number NC-008595). The recombinant forms of rpfA-E have been reported to exhibit similar biological activity to Rpf of M. luteus. The products of each of the 5 rpf genes had demonstrated resuscitation activity and stimulated the growth of extended stationary phase cultures of M. bovis bacillus Calmette Guérin at picomolar concentrations. Rpf-like gene of M. avium subspecies paratuberculosis has also been cloned and expressed, whose product stimulated the growth of mycobacteria and enhanced the recovery of replicating cells from non-replicating phases.
M. avium subsp. avium belongs to M. avium complex (MAC) group of organisms which consists of both human and animal pathogens. This biotype of M. avium is common in the environment and causes opportunistic infections in AIDS patients leading to severe complications. Because the phenomena of latency and reactivation play a significant role in the pathophysiology of M. avium infection, it is important to look for such a molecule(s) that promotes recovery or resuscitation of M. avium bacilli from dormancy and subsequently explore the same as a potential drug target by designing suitable strategies for conferring protection against M. avium infection and its diagnosis. Recently, Romano et al. also reported that rpfB is the most promising candidate in terms of its immunogenicity and protective efficacy and can be used as an antigen in novel tuberculosis vaccines.
This study was initiated primarily with the objective of identifying the gene encoding rpf-like protein from M. avium subspecies avium by cloning and expressing the same in Escherichia More Details coli and its genetic characterisation both at genome and protein level based on a comprehensive sequence analysis.
| ~ Materials and Methods|| |
M. avium subspecies avium strain MTCC 1723 (NCTC 8551) was obtained from Institute of Microbial Technology, Chandigarh, India. E. coli strains JM109 and BL21 (DE3) pLysS were used for cloning and expression, respectively.
pGEM-T vector (Promega, USA) and pET-32c vector (Novagen, Merck, Germany) were used for cloning of polymerase chain reaction (PCR) amplified products and for subcloning of rpf gene and its expression in E. coli strain BL21 (DE3), respectively.
Polyclonal M. luteus-Rpf antiserum was a gift from Professors G. Mukamolova and M. Young, Institute of Biological Sciences, University of Wales Aberystwyth, UK.
Genomic DNA extraction from Mycobacterium avium subspecies avium by cetyltrimethylammonium bromide lysis method
M. avium strain MTCC1723 obtained in lyophilised form was reconstituted in sterile distilled water and genomic DNA was isolated following the protocol. An aliquot of 200 µl of this suspension was inoculated onto 15 ml of Lowenstein–Jensen medium slants prepared in McCartney bottles. The inoculated bottles were incubated at 37°C for 3–4 weeks. Colonies harvested from 25 days old cultures were used for genomic DNA isolation, which was subsequently used as a template for PCR amplification of the M. avium rpf- like gene.
Identification of region(s) with high homology to resuscitation promoting factor in Mycobacterium avium subsp avium genome
The region with high homology to rpf- like gene was identified in the M. avium genome by basic local alignment search tool (BLAST) analysis using mycobacterial and M. luteus rpf sequences with the M. avium unfinished genome sequence (TIGR database). The sequences of primers used for PCR amplification of Mycobacterium paratuberculosis rpf-like gene were also aligned with this region to define the gene as well as the open reading frame (ORF) for M. avium rpf-like protein.
Design and synthesis of oligonucleotide primers
Oligonucleotide primers were designed to amplify the M. avium rpf-like gene using DNA Star software and were synthesised from Integrated DNA Technologies, Inc., USA (Imperial Biomedics, Chandigarh, India). The sequences of the primers are: Forward primer: 5'-GGC GAA TGG GAT CAG GTA GC-3' and Reverse primer 179:5'-GGG GAA TCC GGT GGG GTT AG-3'.
Polymerase chain reaction
PCR amplification of the rpf-like gene was carried out using M. avium genomic DNA as template. Dimethyl sulfoxide (DMSO) was included in the reaction at 10% final concentration to melt the high GC containing genomic DNA, which is characteristic to mycobacterial species. The PCR conditions included initial denaturation at 95°C for 10' and 30 cycles of template denaturation at 95°C for 1', primer annealing at 63°C for 1' and primer extension at 72°C for 1', followed by final extension at 72°C for 5'.
Cloning and sequence analysis of the Mycobacterium avium rpf-like gene
The PCR amplified product of the expected size (675 bp) was gel-purified and ligated into pGEM-T vector and transformed into E. coli JM109 cells. The nucleotide sequence of the cloned product was determined at the DNA sequencing facility, Department of Biochemistry, University of Delhi South Campus, New Delhi, India. The nucleotide sequence was analysed using NCBI BLAST for similar genes. The rpf-like genes from different species were aligned using Clustal X 2.0.12. The aligned conserved domain of rpfA gene taken in Data Analysis in Molecular Biology and Evolution workbench was used for construction of phylogenetic map using UPGMA method using MEGA 4.0.2 software. The theoretical pI and instability/stability was calculated using proteomic tools (ExPASy-ProtParam tool). The RpfA was analysed by CATH (www.cathdb.info) and SCOP tools (supfam.cs.bris.ac.uk). The secondary structure prediction was done using self-optimised prediction method with alignment.
Expression of the recombinant rpf-like protein
Different sets of primers with restriction (RE) sites at their 5' end were designed and synthesised to facilitate directional cloning of PCR products into expression vector. The sequences of these primers with RE sites are: Forward primer (Bam HI and Sal I) 5'-CGC GGA TCC GTC GAC GGC GAA TGG GAT CAG GTA GC-3' and Reverse primer (Hind III and Pst I) 5'-CCC AAG CTT CTG CAG GGG GAA TCC GGT GGG GTT AG-3'. PCR amplification of the gene for rpf-like protein 210 was carried out as described previously. The gel-purified product was digested with Bam HI and Hind III RE enzymes to create cohesive ends for these enzyme sites. The purified product was ligated into pET-32c expression vector and transformed into E. coli BL21 (DE3) pLysS strain using the protocols as given by Sambrook and Russell, 2001.
Screening of recombinant clones
The transformants (white colonies) were picked randomly, and checked for the presence of insert by colony PCR. Plasmid isolation was also carried out from overnight culture of these randomly picked clones, and the presence of insert representing the gene of interest was confirmed by its release upon digestion with Bam HI and Hind III RE enzymes.
Induction of expression by isopropyl thiogalactoside
The positive and negative (control) clones were cultured overnight (~16 h) in Luria-Bertani (LB) medium containing ampicillin (100 µg/ml) and chloramphenicol (34 µg/ml) with vigorous shaking at 37°C. From the overnight culture, 30 µl was inoculated into 3 ml of LB medium (with ampicillin and chloramphenicol) and the cultures were incubated at 30°C with vigorous shaking until the cells reached mid-log phase growth (A600 of 0.5–1.0). An aliquot of 1 ml of the culture at this stage was transferred to microfuge tubes and kept separately as uninduced cultures. The remainders of each culture were then induced by adding isopropyl thiogalactoside (IPTG) to a final concentration of 1 mM and incubated further for 6 h at 30°C with aeration and vigorous shaking. Bacterial cells were then harvested by pelleting at 10,000 rpm for 2 min and the supernatant discarded. The pellets were washed with phosphate buffer saline (PBS) and stored at −85°C. These pellets were subsequently analysed for protein expression by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and/or Western blot.
Purification of His-tagged recombinant protein under denaturing conditions
The bacterial pellet was thawed on ice and to this, 4 volumes of lysis buffer (0.1M Sodium phosphate buffer, pH 8.0; Urea 8M and Imidazole 1 mM) were added and the cell suspension was stirred gently for 60 min at room temperature taking care to avoid foaming, the cell lysate was then centrifuged at 10,000 ×g for 30 min at room temperature to pellet the cell debris. The supernatant (cleared lysate) was added to pre-equilibrated nickel-agarose affinity resin and mixed gently for 60 min at room temperature. The lysate-resin mixture was then carefully loaded onto an empty column with the bottom outlet closed. After complete loading, the bottom outlet was opened, and the flow-through was collected separately and the column was simultaneously packed. The column bed was then washed with 10 volumes of wash buffer (0.1M sodium phosphate buffer, pH 6.3; urea 8M, imidazole 1 mM). The column wash was also collected separately and saved. The bound protein was then eluted with 5 ml each of 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM and 150 mM imidazole in elution buffer (0.1M Sodium phosphate buffer, pH 5.8; Urea 8M and Imidazole 1 mM) and the fractions were collected in 1 ml volume. The flow-through, wash and eluted fractions were analysed for purification pattern of His-tagged recombinant/expressed protein in SDS-PAGE and in Western blot.
Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis
SDS-PAGE of the protein samples was carried out according to the method of Laemmli in 10% uniform vertical slab gel. After completion of electrophoresis, the gels were stained with coomassie brilliant blue R- for 2 h followed by destaining in 5% glacial acetic acid.
Western blotting of proteins was done as per the protocol described by Towbin et al. The membrane was then incubated in a solution of primary antibody (M. luteus-Rpf antiserum, 1:750 dilution) for 8 h at room temperature, washed with several changes of PBS-T and incubated with secondary antibody conjugated to horse radish peroxidase (1:500 dilution) for 4 h. The membrane was washed thoroughly with PBS-T and developed with diaminobenzidine substrate solution.
| ~ Results|| |
Since the main focus of this study was to clone, sequence and express Rpf motif from M. avium subsp. avium in E. coli, the first step toward realising this target was to fish out the full length ORF of the desired gene by PCR amplification using primers specifically designed based on the known rpf nucleotide sequences from M. avium subsp. paratuberculosis and M. luteus available in the NCBI database. Accordingly, when the genomic DNA from M. avium subsp. avium was used as a template in the PCR in presence of 10% DMSO at an annealing temperature of 63°C, an amplicon of 675 bp specific to the full length of ORF of rpf was obtained as can be revealed by separation of a distinct band on the agarose gel [Figure 1]. The 675 bp PCR amplified product was also checked for its unique RE enzyme digestion pattern using XmaI that resulted in two distinct bands of 530 and 150 bp as can be evidenced from the gel picture as shown in [Figure 2]. The 675 bp PCR amplified product representing rpf of M. avium subsp. avium was cloned with pGEM-T and the complete nucleotide sequence was determined and submitted to Gene Bank database with accession number AY764128.
|Figure 1: Agarose gel electrophoresis showing specific amplification of rpf-like gene. Lanes: M, Molecular weight marker; 1–4 - polymerase chain reaction products from samples 1–4|
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|Figure 2: Agarose gel electrophoresis showing XmaI digestion pattern of amplified polymerase chain reaction product, lanes: M, molecular weight marker; 1–3 - polymerase chain reaction products digested with XmaI; 4; undigested polymerase chain reaction product (negative control)|
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Nucleotide sequencing of Mycobacterium avium rpf-like gene and sequence analysis
Single strand nucleotide sequencing of the cloned insert in pGEM-T vector was carried out and the nucleotide sequence was subsequently submitted to GenBank database of NCBI with an accession number AY764128. The cloned rpf-like gene was found to be 100% similar to rpf A of M. avium 104. BLAST similarity search using NCBI for genes of sequence similarity to M. avium rpf gene showed a number of genes with sequence identity ranging from 77% to 100% as shown in [Table 1].
|Table 1: Percentage homology of sequence similarity to M. avium rpf gene|
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In order to find the genetic relatedness of RpfA of M. avium MTCC 1723 with those of other organisms, phylogenetic tree of RpfA (2–77) obtained from the definitive alignment of conserved domain sequences of amino acids by the UPGMA method under the poisson correction model with 1000 bootstrap replicates using MEGA 4.0.2 software package was constructed and it is shown in [Figure 3]. The phylogenetic mapping analysis of M. avium rpf-like gene with other Rpfs revealed that the rpf of M. avium subspecies avium and M. avium subspecies paratuberculosis were more closely related, followed by RpfA of M. tuberculosis and M. bovis Rpf whereas M. luteus and other mycobacterial Rpfs were distantly related. The theoretical pI of RpfA of M. avium subsp. avium MTCC 1723 is 4.22 with an extinction coefficient of 32,095/M/cm. The computed instability index of 55.06 indicated that the protein is relatively unstable (ExPASy-ProtParam tool). The CATH (www.cathdb.info) showed that RpfA belongs mainly to alpha family proteins and their topology mimics lysozyme-like proteins containing transglycosylase domain. The transglycosylase domain in RpfA is located at N terminus. The SCOP analysis (supfam.cs.bris.ac.uk) also revealed that RpfA protein belongs to lysozyme and Rpf-like protein family. It can be further validated by the secondary structure prediction which also revealed that there were more of alpha sheets (23.15%), random coils (63.89%) and lesser of extended strand (6.48%). The three-dimensional structure of RpfA predicted by protein homology modelling as shown in [Figure 4] also confirms the presence of alpha sheets.
|Figure 3: Phylogenetic tree of conserved domain of RpfA (2-77) obtained from the definitive alignment by the UPGMA method|
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|Figure 4: Three-dimensional image of RpfA gene predicted by protein homology/analogy Y recognition engine V 2.0|
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Expression of Mycobacterium avium rpf-like gene
The PCR amplified product carrying likely the ORF of putative rpf-like gene and pET-32c vector were digested with Bam HI and Hind III enzymes. The concentration of the eluted PCR product and vector DNA were 85 ng/µl and 42 ng/µl as determined by densitometry. After ligation and transformation, a total of six recombinants/transformants were screened by colony PCR, of which five showed specific amplification of the product approximately of ~700 bp size. The plasmid DNA from these clones were also checked for the presence of insert by digestion with Bam HI and Hind III enzymes followed by analysis on agarose gel, which showed release of the insert DNA of ~700 bp size [Figure 5] in case of recombinant clones rpf1, rpf2, rpf3 and rpf5 except rpf4.
|Figure 5: Restriction digestion of recombinant clones with Bam HI and Hind III. Lanes: 1, rpf1; 2, rpf2; 3, rpf3; 4, 100 bp DNA ladder; 5, rpf4; 6, rpf5|
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Different concentrations of IPTG were tried for expression of M. avium rpf gene. Though there was no induction of the protein at 0.5 mM IPTG concentration, highest amount of induction was observed at 1 mM, 2 mM and 4 mM level. Overnight grown cultures of the positive recombinant rpf1 and rpf2 and negative recombinant rpf4 (religated vector without insert) were then induced with 1 mM IPTG (data not shown). However, no expression of Rpf protein was observed at 37°C while at 30°C, expression could be achieved. Hence, the induced cultures were analysed after 6 h incubation at 30°C by SDS-PAGE for protein expression. In case of rpf1 and rpf2, there was a thick protein band with apparent molecular mass of 65 kDa seen which was absent in case of rpf4 that served as negative control [Figure 6]. Further confirmation of the expressed protein was carried out by Western blot using nickel-HRP conjugate probe to check for His-tagged expressed protein that gave a band at ~65 kDa level in case of induced positive clone and no band was observed in uninduced negative control. The induction of expression of rpf-like gene was relatively more at 1, 2 and 4 mM final concentrations of IPTG and no induction occurred when 0.5 mM IPTG was used. Time course analysis of protein expression after different time intervals of induction showed start of expression at 2 h, which subsequently reached maximum at 6 h after induction (data not shown).
|Figure 6: Sodium dodecyl sulphate-polyacrylamide gel electrophoresis profile of induced and uninduced cultures for analysis of protein expression. Lanes: M - Protein molecular weight marker; 1: Induced culture from rpf4, 2: Uninduced culture from rpf1 (negative control), 3: Induced culture from rpf1, 4: Induced culture from rpf2|
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Purification of His-tagged recombinant protein
Initially, the expressed protein was checked for its solubility using lysozyme (native condition) and urea (denatured condition). The expressed protein was found to be extracted with urea when analysed by SDS-PAGE indicative of the insoluble nature of the protein. Purification of the expressed protein involved nickel-agarose affinity column fractionation of cleared bacterial lysate wherein the His-tagged expressed protein was bound to the column and eluted with 5 mM imidazole, which gave a protein preparation that was >90% pure as observed on SDS-PAGE [Figure 7].
|Figure 7: Purification of His-tagged recombinant protein by nickel affinity column chromatography - sodium dodecyl sulphate-polyacrylamide gel electrophoresis profile. Lanes: M, protein molecular weight marker; 1: Uninduced culture from rpf1 (negative control), 2: Induced culture, 3: Induced culture lysate, 4: Column flow-through, 5: 5 mM imidazole elute, 6: 10 mM imidazole elute, 7: 20 mM imidazole elute, 8: 40 mM imidazole elute|
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Further confirmation of the purified expressed protein by Western blot using specific M. luteus Rpf antiserum showed a band at 65 kDa level in case of induced culture as well as in purified protein preparation but not in uninduced culture [Figure 8].
|Figure 8: Western blot analysis of purified expressed protein using Micrococcus luteus-resuscitation promoting factor antiserum. Lanes: 1–5, Column fractions using culture form rpf1; M, protein molecular weight marker|
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| ~ Discussion|| |
M. avium subsp. avium, a key member of the MAC, is considered a major human and veterinary opportunistic environmental pathogen. As evidenced by the absence of genomic content changes following analysis of DNA sequences from mycobacteria during three decades of latent tuberculosis infections (LTBI), it is now well-recognised that the bacilli in LTBI undergo limited replication. These findings clearly point toward occurrence of very little interaction between the host immune system and the bacilli in LTBI and thus vindicates the hypothesis that the pathogen is in a genuinely dormant immune suppressive state during LTBI. Many of the Rpf-like proteins harboured a highly conserved 70 amino acid residue lysozyme-like domain , that accounted for muralytic activities in these proteins. Rpf from M. luteus was shown to hydrolyse cell wall material in vitro which was comparable to the glycoside hydrolase activity known to hydrolyse peptidoglycan displayed by Rpf B domain of M. tuberculosis,, thereby confirming that the Rpf and Rpf-like proteins are peptidoglycan hydrolases. It is by this mechanism of action through cleavage of peptidoglycan that promotes growth and more importantly permits the bacteria to exit the dormant state. However, the exact nature of the processes following peptidoglycan cleavage that leads to reactivation of dormant organisms is yet to be fully unravelled. Hence, we report here the characterisation of rpf-like genes located in the genome of M. avium subsp. avium by cloning and expressing the same in E. coli followed by a detailed bioinformatics sequence analyses at nucleotide and amino acid level to establish their fundamental mechanism of action in MAC and exploring them as potential targets for novel drug designing and diagnostics.
Our results regarding specific PCR product M. avium rpf gene were in close agreement with those of Zhu et al. who also recorded used 4% DMSO as the optimal concentration for PCR amplification of rpf gene from M. avium subsp. paratuberculosis. In our study, a 675 bp gene with homology/similarity to other rpf genes was identified in the M. avium genome, by BLAST analysis with rpf sequences from M. luteus and M. tuberculosis and M. bovis available in the database. The ORF of the rpf from M. avium had a high G+C content (77%) which was consistent with almost the similar values recorded previously for rpf from other mycobacterial strains  and M. luteus rpf. Presence of high amount of GC is a common phenomenon in species belonging to the order Actinomycetales. The nucleotide sequencing of rpf gene further revealed the presence of a unique XmaI site within the rpf ORF. The presence of unique site in the rpf gene can be explored for the detection of recombinant clones in right orientation. Nucleotide sequence analysis of M. avium MTCC 1723 rpf gene demonstrated that rpf matched completely with rpfA of M. avium 104, thereby, was classified as rpfA. The highest identity of rpfA of M. avium MTCC 1723 was with M. avium 104 (99%) and 96% with M. paratuberculosis rpf gene. The nucleotide identity between M. avium and M. paratuberculosis rpfs is due to the high degree of genetic relatedness of these two species, which are grouped within the MAC, even though they differ between each other with respect to some phenotypic properties. The amino acid sequence of M. avium Rpf was compared with corresponding reported sequences from other species, which indicated that M. avium Rpf was more closely related to RpfA of M. tuberculosis and M. bovis that is in tandem with M. paratuberculosis rpf. The sequence analysis further revealed that a 70 amino acid region was highly conserved between different Rpfs. Our results in this regard are exactly in line with those of several other workers.,, Consequently, Rpfs of one species could resuscitate dormant bacteria of other species. Some of the mycobacterial and other bacterial proteins that showed homology with M. avium Rpf, were not assigned any function in their genome annotations, and were classified as conserved hypothetical proteins. However, these proteins showed significant homology with respect to the rpf-like domain and hence they represent protein similar to RPF.
The predicted pI of RpfA of MTC 1723 was found to be 4.22 which were almost similar to that of M. avium 104 strain having pI of 4.11. The relatively high instability index deduced from the RpfA sequence indicated that RpfA could be belonging to a group of unstable protein which was consistent with the earlier observations made in respect of RpfA group of proteins from other members of Mycobacterium complex organisms unlike other orthologues of Rpf proteins namely RpfB. Both CATH and SCOP analyses revealed that RpfA of M. avium MTCC 1723 was lysozyme-like protein with a transglycosylase domain as could be supported by its predicted secondary structure. All these features along with high instability index indicate that RpfA belonged to alpha family of proteins without any beta sheet conformation which could account for their predicted instability. On the other hand, RpfB has been reported to be stable and belongs to a class of mainly alpha and beta proteins having lesser contribution of alpha helix.
The advantage of producing recombinant Rpf protein from M. avium MTCC 1723 included recovery of high better quality yields. Use of this expression system has also been reported previously for expression of other rpf genes from M. luteus, M. tuberculosis, M. bovis and M. paratuberculosis.,,, Different concentrations of IPTG were tried for expression of M. avium rpf gene. Though there was no induction of the protein at 0.5 mM IPTG concentration, highest amount of induction was observed at 1 mM, 2 mM and 4 mM level. This is in contradiction to earlier reports,, wherein only weak or poor expression of Rv0867c, Rv2389c and Av27 genes corresponding to other rpfs of mycobacteria., One noteworthy observation in this study was that M. avium rpf gene got expressed when the bacterial cultures were incubated at 30°C before and after induction but there was no significant expression when the same was actually carried out at 37°C because of slow induction or growth of cells. The apparent molecular mass of the expressed M. avium recombinant Rpf was ~65 kDa, which was greater than the predicted size of M. avium rpf gene product (24 kDa) including the fusion protein, thioredoxin (19 kDa). However, the authenticity of the M. avium rpf gene/DNA that was ligated and subsequently expressed was confirmed by nucleotide sequencing. The unexpectedly high apparent size could be attributed to the extensive series of proline (between aminoacid residues 112–200) in the protein, which was derived from the deduced amino acid sequence using the M. avium rpf gene by DNAStar programme. More or less similar observations were recorded for the recombinant Rpf proteins of M. tuberculosis RpfA and M. paratuberculosis Rpf, which was attributed to high proline, alanine and glycine content of the expressed protein., Confirmation of the expressed M. avium Rpf protein was made by Western blot (immunoblot) with M. luteus-Rpf antiserum, clearly delineating a specific and intense band at the expected size (65 kDa). This suggested that the M. avium Rpf and M. luteus Rpf should be similar, at least with respect to one or more epitopes, which might be responsible for the cross-reactivity of M. luteus-Rpf antiserum with that of recombinant Rpf of M. avium MTCC 1723. These epitopes might form the conserved 'rpf-like domain' of all Rpfs studied so far.
Recombinant M. avium Rpf protein expressed when treated with urea which indicated insoluble nature of the expressed protein which is contradictory to the outcome of a study carried out on similar lines who reported that the recombinant Rpf protein from M. paratuberculosis expressed in E. coli could be expressed in a solubilised form after urea treatment and hence exhibited biological activity.
The extracytoplasmic location and/or function(s) of this M. avium Rpf make this protein a potential target for recognition by the immune system. Hence, any strategy to interrupt the role played by this molecule by eliciting specific antibody response could help the host in combating M. avium infection at the stage of reactivated disease.
| ~ Conclusion|| |
It can be concluded that the Rpf from M. avium MTCC 1723 was successfully expressed in E. coli, characterised and resembled RpfA in this species, which may be responsible for reactivation phenomenon in this high-risk pathogen.
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Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]