Four chromosomally clustered class-C β-lactamases (blaAmpH1/2/3/4) control the cephalosporin hydrolysis inMycobacterium tuberculosisand similar genetic loci appeared as pseudogenes inMycobacterium leprae

Summary Mycobacterium tuberculosis ( Mt ) chromosomal ten PBPs and one class-A β-lactamase (blaC) were implicated in multi-drug resistance against penicillin, cephalosporin and carbapenem drugs. The E. coli Class-A PBP1A and PBP1B proteins referred as PonA1 and PonA2 in Mt with 34% homologies whereas Class-B PBP2 referred as PbpA and PbpB sub-class proteins with 27% homologies. The class-C PBPs were, PBP4, MecA_N, DacB1, DacB2 and AmpH1- AmpH4. During database search, we found that many Mt Pbp4 had no homology to E. coli PBP4 and such enzyme were found to be AmpH class with AmpC, ACC, DHA and GES β- lactamases similarities although one of such enzymes suggested as AmpH but the other referred as DacB or PBP5 in the literature. We investigated the Mt whole genomes (accession nos. AL123456, CP001642, CP054013, CP001641, CP025597) to get authentic PBP4 in Mt strain FDAARGOS_757 genome (protein id. AUP69687, 34% homology to E. coli PBP4) which was designated as conserved protein in chromosomes of Mt strains H37Rv, H37Rv-1, GG-36-11 and CCDC5180 making confusion in data analysis. Similarly, we BLASTP homology searched with plasmid-mediated 24 β-lactamases suggested that four bla AmpH genes, ( AmpH1, AmpH2, AmpH3 and AmpH4 ) predominantly control the degradation of cephalosporins in M. tuberculosis but such genes were found as pseudogenes in M. leprae . The Mt AmpH1/2/3/4 enzymes had better homologies with V. parahaemolyticus and Yersinia pekkanenii AmpH enzyme as well as with E. coli AmpH enzyme. The DacB1 (PBP6) and DacB2 (PBP7) enzymes had blaTEM similarity but no AmpH similarity indicating blaC and DacB1/B2 controlled the Penicillin hydrolysis in Mt . The blaC enzyme had 30% homology to S. aureus blaZ β-lactamase but such enzyme was also missing in M. leprae . Homology search suggested that carbapenem hydrolysis by blaOXA-23/51-like PBPA/B enzymes and blaOXA-58 related MecA_N domain β-lactamase which has 21% similarity to S. aureus mecA enzyme. The PonA1/A2 had no homology to 24 classes of β-lactamases but still were popular enzymes implicated for better penicillin hydrolysis. We designed primers for Mt PBPs to check transcription of individual genes by RT-PCR as well as to check chromosomal locus by BLASTN search after WGS. The E. coli genome had no similarities to bla AmpH1/2/3/4 genes but a 17nt (5’- CCTTGGTGCCGTCGACC-3’) sequence found in pKEC-a3c plasmid of C. fruendii with homology to bla AmpH4 gene (nt. 1236-1252) and shared a homology with IS1182-like ISKpn6 transposase of plasmids. The oligonucleotides selected many Mt chromosomes and located conserved blaAmpH4 protein in all cases. However, D435E and F495V mutations in the Mt strain 5521 blaAmpH4 were evident and frameshift deletions located in Mt stain FDAARGOS_756 bla AmpH4 gene with no protein was made. Thus, mutations, deletions and rearrangements mediated by IS-elements were the driving force to make new AmpH genes in M. tuberculosis .