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

Abstract

SummaryMycobacterium tuberculosis(Mt) chromosomal ten PBPs and one class-A β-lactamase (blaC) were implicated in multi-drug resistance against penicillin, cephalosporin and carbapenem drugs. TheE. coliClass-A PBP1A and PBP1B proteins referred as PonA1 and PonA2 inMtwith 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 manyMtPbp4 had no homology toE. coliPBP4 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 theMtwhole genomes (accession nos. AL123456, CP001642, CP054013, CP001641, CP025597) to get authentic PBP4 inMtstrain FDAARGOS_757 genome (protein id. AUP69687, 34% homology toE. coliPBP4) which was designated as conserved protein in chromosomes ofMtstrains 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 fourblaAmpH genes, (AmpH1, AmpH2, AmpH3andAmpH4) predominantly control the degradation of cephalosporins inM. tuberculosisbut such genes were found as pseudogenes inM. leprae. TheMtAmpH1/2/3/4 enzymes had better homologies withV. parahaemolyticusandYersinia pekkaneniiAmpH enzyme as well as withE. coliAmpH enzyme. The DacB1 (PBP6) and DacB2 (PBP7) enzymes had blaTEM similarity but no AmpH similarity indicating blaC and DacB1/B2 controlled the Penicillin hydrolysis inMt. The blaC enzyme had 30% homology toS. aureusblaZ β-lactamase but such enzyme was also missing inM. 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 toS. aureusmecA 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 forMtPBPs to check transcription of individual genes by RT-PCR as well as to check chromosomal locus by BLASTN search after WGS. TheE. coligenome had no similarities toblaAmpH1/2/3/4 genes but a 17nt (5’- CCTTGGTGCCGTCGACC-3’) sequence found in pKEC-a3c plasmid ofC. fruendiiwith homology toblaAmpH4 gene (nt. 1236-1252) and shared a homology with IS1182-like ISKpn6 transposase of plasmids. The oligonucleotides selected manyMtchromosomes and located conserved blaAmpH4 protein in all cases. However, D435E and F495V mutations in theMtstrain 5521 blaAmpH4 were evident and frameshift deletions located inMtstain FDAARGOS_756blaAmpH4 gene with no protein was made. Thus, mutations, deletions and rearrangements mediated by IS-elements were the driving force to make newAmpHgenes inM. tuberculosis.