Characterization of a beta-lactamase produced in Mycobacterium fortuitum D316.
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ABSTRACT:
SUBMITTER: Amicosante G
PROVIDER: S-EPMC1149623 | biostudies-other | 1990 Nov
REPOSITORIES: biostudies-other
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Project description:beta-Lactamases are the main cause of bacterial resistance to penicillins and cephalosporins. Class A beta-lactamases, the largest group of beta-lactamases, have been found in many bacterial strains, including mycobacteria, for which no beta-lactamase structure has been previously reported. The crystal structure of the class A beta-lactamase from Mycobacterium fortuitum (MFO) has been solved at 2.13-A resolution. The enzyme is a chromosomally encoded broad-spectrum beta-lactamase with low specific activity on cefotaxime. Specific features of the active site of the class A beta-lactamase from M. fortuitum are consistent with its specificity profile. Arg278 and Ser237 favor cephalosporinase activity and could explain its broad substrate activity. The MFO active site presents similarities with the CTX-M type extended-spectrum beta-lactamases but lacks a specific feature of these enzymes, the VNYN motif (residues 103 to 106), which confers on CTX-M-type extended-spectrum beta-lactamases a more efficient cefotaximase activity.
| S-EPMC1489783 | biostudies-literature
Project description:New antibiotic regimens are needed for the treatment of multidrug-resistant tuberculosis. Mycobacterium tuberculosis has a thick peptidoglycan layer, and the penicillin-binding proteins involved in its biosynthesis are inhibited by clinically relevant concentrations of beta-lactam antibiotics. beta-Lactamase production appears to be the major mechanism by which M. tuberculosis expresses beta-lactam resistance. beta-Lactamases from the broth supernatant of 3- to 4-week-old cultures of M. tuberculosis H37Ra were partially purified by sequential gel filtration chromatography and chromatofocusing. Three peaks of beta-lactamase activity with pI values of 5.1, 4.9, and 4.5, respectively, and which accounted for 10, 78, and 12% of the total postchromatofocusing beta-lactamase activity, respectively, were identified. The beta-lactamases with pI values of 5.1 and 4.9 were kinetically indistinguishable and exhibited predominant penicillinase activity. In contrast, the beta-lactamase with a pI value of 4.5 showed relatively greater cephalosporinase activity. An open reading frame in cosmid Y49 of the DNA library of M. tuberculosis H37Rv with homology to known class A beta-lactamases was amplified from chromosomal DNA of M. tuberculosis H37Ra by PCR and was overexpressed in Escherichia coli. The recombinant enzyme was kinetically similar to the pI 5.1 and 4.9 enzymes purified directly from M. tuberculosis. It exhibited predominant penicillinase activity and was especially active against azlocillin. It was inhibited by clavulanic acid and m-aminophenylboronic acid but not by EDTA. We conclude that the major beta-lactamase of M. tuberculosis is a class A beta-lactamase with predominant penicillinase activity. A second, minor beta-lactamase with relatively greater cephalosporinase activity is also present.
| S-EPMC105607 | biostudies-literature
Project description:Despite the enormous success of beta-lactams as broad-spectrum antibacterials, they have never been widely used for the treatment of tuberculosis (TB) due to intrinsic resistance that is caused by the presence of a chromosomally encoded gene (blaC) in Mycobacterium tuberculosis. Our previous studies of TB BlaC revealed that this enzyme is an extremely broad-spectrum beta-lactamase hydrolyzing all beta-lactam classes. Carbapenems are slow substrates that acylate the enzyme but are only slowly deacylated and can therefore act also as potent inhibitors of BlaC. We conducted the in vitro characterization of doripenem and ertapenem with BlaC. A steady-state kinetic burst was observed with both compounds with magnitudes proportional to the concentration of BlaC used. The results provide apparent K(m) and k(cat) values of 0.18 microM and 0.016 min(-1) for doripenem and 0.18 microM and 0.017 min(-1) for ertapenem, respectively. FTICR mass spectrometry demonstrated that the doripenem and ertapenem acyl-enzyme complexes remain stable over a time period of 90 min. The BlaC-doripenem covalent complex obtained after a 90 min soak was determined to 2.2 A, while the BlaC-ertapenem complex obtained after a 90 min soak was determined to 2.0 A. The 1.3 A diffraction data from a 10 min ertapenem-soaked crystal revealed an isomerization occurring in the BlaC-ertapenem adduct in which the original Delta(2)-pyrroline ring was tautomerized to generate the Delta(1)-pyrroline ring. The isomerization leads to the flipping of the carbapenem hydroxyethyl group to hydrogen bond to carboxyl O2 of Glu166. The hydroxyethyl flip results in both the decreased basicity of Glu166 and a significant increase in the distance between carboxyl O2 of Glu166 and the catalytic water molecule, slowing hydrolysis.
| S-EPMC2860635 | biostudies-literature
Project description:We have isolated the aph(3")-Ic gene, encoding an aminoglycoside 3"-O-phosphotransferase [APH(3")-Ic], from a genomic library of an environmental Mycobacterium fortuitum strain, selecting for streptomycin resistance. APH(3")-Ic phosphorylates and inactivates streptomycin. Similar genes have been described in Streptomyces griseus and plasmid RSF1010. It is also present in some M. fortuitum clinical isolates.
| S-EPMC1635185 | biostudies-literature
Project description:A recombinant plasmid isolated from a Mycobacterium fortuitum genomic library by selection for gentamicin and 2-N'-ethylnetilmicin resistance conferred low-level aminoglycoside and tetracycline resistance when introduced into M. smegmatis. Further characterization of this plasmid allowed the identification of the M. fortuitum tap gene. A homologous gene in the M. tuberculosis H37Rv genome has been identified. The M. tuberculosis tap gene (Rv1258 in the annotated sequence of the M. tuberculosis genome) was cloned and conferred low-level resistance to tetracycline when introduced into M. smegmatis. The sequences of the putative Tap proteins showed 20 to 30% amino acid identity to membrane efflux pumps of the major facilitator superfamily (MFS), mainly tetracycline and macrolide efflux pumps, and to other proteins of unknown function but with similar antibiotic resistance patterns. Approximately 12 transmembrane regions and different sequence motifs characteristic of the MFS proteins also were detected. In the presence of the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), the levels of resistance to antibiotics conferred by plasmids containing the tap genes were decreased. When tetracycline accumulation experiments were carried out with the M. fortuitum tap gene, the level of tetracycline accumulation was lower than that in control cells but was independent of the presence of CCCP. We conclude that the Tap proteins of the opportunistic organism M. fortuitum and the important pathogen M. tuberculosis are probably proton-dependent efflux pumps, although we cannot exclude the possibility that they act as regulatory proteins.
| S-EPMC107655 | biostudies-literature
Project description:A natural TEM variant beta-lactamase was isolated from an epidemic strain of Serratia marcescens. Nucleotide gene sequencing revealed multiple point mutations located in the 42-to-44 tripeptide and positions 145 to 146, 178, and 238. In addition, a glutamic acid 212 deletion was also found. The purified enzyme was studied from a kinetic point of view, revealing the highest catalytic efficiency (k[cat]/Km) values for ceftazidime and aztreonam compared with the TEM-1 prototype enzyme. The in vitro resistance correlated with kinetic parameters, and the enzyme also mediated resistance to some penicillins and an ampicillin-clavulanic acid combination. The mutational and kinetic changes are discussed in relation to the three-dimensional crystallographic structure of the wild-type TEM-1 enzyme.
| S-EPMC164131 | biostudies-other
Project description:A novel gene encoding an aminoglycoside 2'-N-acetyltransferase (AAC) was cloned from Mycobacterium fortuitum. DNA sequencing results identified an open reading frame that we have called aac(2')-Ib encoding a putative protein with a predicted molecular mass of 24,800 Da. The deduced AAC(2')-Ib protein showed homology to the AAC(2')-Ia from Providencia stuartii. This is the second member of a subfamily of AAC(2')-I enzymes to be identified. No homology was found with other acetyltransferases, including all of the AAC(3) and AAC(6') proteins. The aac(2')-Ib gene cloned in a mycobacterial plasmid and introduced in Mycobacterium smegmatis conferred resistance to gentamicin, tobramycin, dibekacin, netilmicin, and 6'-N-ethylnetilmicin. DNA hybridization with an intragenic probe of aac(2')-Ib showed that this gene was present in all 34 strains of M. fortuitum tested. The universal presence of the aac(2')-Ib gene in M. fortuitum was not correlated with any aminoglycoside resistance phenotype, suggesting that this gene may play a role in the secondary metabolism of the bacterium.
| S-EPMC163533 | biostudies-other
Project description:NXL104 is a novel β-lactamase inhibitor with a non-lactam structural scaffold. Our kinetic and mass spectrometric analysis demonstrates that NXL104 quantitatively inhibits BlaC, the only chromosomally encoded β-lactamase from Mycobacterium tuberculosis, by forming a carbamyl adduct with the enzyme. The inhibition efficiency (k(2)/K) of NXL104 was shown to be more than 100-fold lower than that of clavulanate, a classical β-lactamase inhibitor, which is probably caused by the bulky rings of NXL104. However, the decarbamylation rate constant (k(3)) was determined to be close to zero. The BlaC-NXL104 adduct remained stable for at least 48 h, while the hydrolysis of the BlaC-clavulanate adduct was observed after 2 days. The three-dimensional crystal structure of the BlaC--NXL104 carbamyl adduct was determined at a resolution of 2.3 Å. Interestingly, the sulfate group of NXL104 occupies the position of a phosphate ion in the structure of the BlaC-clavulanate adduct and is hydrogen bonded to residues Ser128, Thr237, and Thr239. Favorable interactions are also seen in the electrostatic potential map. We propose that these additional interactions, as well as the intrinsic stability of the carbamyl linkage, contribute to the extraordinary stability of the BlaC-NXL104 adduct.
| S-EPMC3448018 | biostudies-literature
Project description:The Capnocytophaga sputigena isolate NOR, responsible for septicemia, was resistant to amoxicillin and narrow-spectrum cephalosporins. In a cloning experiment, a new gene, bla(CSP-1), was identified; this gene encodes a novel extended-spectrum beta-lactamase (ESBL) that shares only 52% and 49% identities with the CME-1 and VEB-1 beta-lactamases, respectively. The G+C content of this gene, its genetic environment, the absence of conjugation transfer, and its detection in two reference strains suggested that it was an intrinsic resistance gene located on the chromosome.
| S-EPMC2863690 | biostudies-literature
Project description:BACKGROUND AND OBJECTIVES:The increase of infections caused by nontuberculous mycobacteria (NTM) is receiving increasing attention worldwide. Mycobacterium fortuitum is encountered with increasing frequency in clinical laboratories of Iran. MATERIALS AND METHODS:Sequence variation of 48 M. fortuitum clinical isolates, were investigated by sequence analysis of the 16S-23S Internal Transcribed Spacer. RESULTS:Twelve different sequence types (sequevar) were identified by sequence analysis of ITS region. Seven previously described sequevar including MfoA, MfoB, MfoC, MfoD, MfoE, MfoF and MfoG identified. Five novel sequevar namely MfoH, MfoI, MfoJ, MfoK and MfoL that were distinctly different from the previously described sequevar were detected among different clinical strains of M. fortuitum, from Iran. CONCLUSION:This study showed that the ITS region possesses high discriminatory power between the clinical isolates up to the clonal level. The results also suggest the possibility of the existence of predominant clone of M. fortuitum in affected patients in Iran. The data also point to the conclusion that a large variety of M. fortuitum clone can produce disease although certain clones seem to be predominant.
| S-EPMC4419039 | biostudies-literature