Discovery of the first potent and selective Mycobacterium tuberculosis Zmp1 inhibitor

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Functional characterization of the Mycobacterium tuberculosis zinc metallopeptidase Zmp1 and identification of potential substrates

Zinc metallopeptidases of bacterial pathogens are widely distributed virulence factors and represent promising pharmacological targets. In this work, we have characterized Zmp1, a zinc metallopeptidase identified as a virulence factor of Mycobacterium tuberculosis and belonging to the neprilysin (NEP; M13) family, whose X-ray structure has been recently solved. Interestingly, this enzyme shows an optimum activity toward a fluorogenic substrate at moderately acidic pH values (i.e., 6.3), which corresponds to those reported for the Mtb phagosome where this enzyme should exert its pathological activity. Substrate specificity of Zmp1 was investigated by screening a peptide library. Several sequences derived from biologically relevant proteins were identified as possible substrates, including the neuropeptides bradykinin, neurotensin, and neuropeptide FF. Further, subsequences of other small bioactive peptides were found among most frequently cleaved sites, e.g., apelin-13 and substance P. We determined the specific cleavage site within neuropeptides by mass spectrometry, observing that hydrophobic amino acids, mainly phenylalanine and isoleucine, are overrepresented at position P1′. In addition, the enzymatic mechanism of Zmp1 toward these neuropeptides has been characterized, displaying some differences with respect to the synthetic fluorogenic substrate and indicating that the enzyme adapts its enzymatic action to different substrate

Development of 2nd generation aminomethyl spectinomycins that overcome native efflux in Mycobacterium abscessus

Mycobacterium abscessus (Mab), a nontuberculous mycobacterial (NTM) species, is an emerging pathogen with high intrinsic drug resistance. Current standard-of-care therapy results in poor outcomes, demonstrating the urgent need to develop effective antimycobacterial regimens. Through synthetic modification of spectinomycin (SPC), we have identified a distinct structural subclass of N-ethylene linked aminomethyl SPCs (eAmSPCs) that are up to 64-fold more potent against Mab over the parent SPC. Mechanism of action and crystallography studies demonstrate that the eAmSPCs display a mode of ribosomal inhibition consistent with SPC. However, they exert their increased antimicrobial activity through enhanced accumulation, largely by circumventing efflux mechanisms. The N-ethylene linkage within this series plays a critical role in avoiding TetV-mediated efflux, as lead eAmSPC 2593 displays a mere fourfold susceptibility improvement against Mab ΔtetV, in contrast to the 64-fold increase for SPC. Even a minor shortening of the linkage by a single carbon, akin to 1st generation AmSPC 1950, results in a substantial increase in MICs and a 16-fold rise in susceptibility against Mab ΔtetV. These shifts suggest that longer linkages might modify the kinetics of drug expulsion by TetV, ultimately shifting the equilibrium towards heightened intracellular concentrations and enhanced antimicrobial efficacy. Furthermore, lead eAmSPCs were also shown to synergize with various classes of anti-Mab antibiotics and retain activity against clinical isolates and other mycobacterial strains. Encouraging pharmacokinetic profiles coupled with robust efficacy in Mab murine infection models suggest that eAmSPCs hold the potential to be developed into treatments for Mab and other NTM infections

Elucidation of Mycobacterium abscessus aminoglycoside and capreomycin resistance by targeted deletion of three putative resistance genes

Objectives Mycobacterium abscessus is innately resistant to a variety of drugs thereby limiting therapeutic options. Bacterial resistance to aminoglycosides (AGs) is conferred mainly by AG-modifying enzymes, which often have overlapping activities. Several putative AG-modifying enzymes are encoded in the genome of M. abscessus . The aim of this study was to investigate the molecular basis underlying AG resistance in M. abscessus . Methods M. abscessus deletion mutants deficient in one of three genes potentially involved in AG resistance, aac(2 ' ) , eis1 and eis2 , were generated by targeted gene inactivation, as were combinatorial double and triple deletion mutants. MICs were determined to study susceptibility to a variety of AG drugs and to capreomycin. Results Deletion of aac(2 ' ) increased susceptibility of M. abscessus to kanamycin B, tobramycin, dibekacin and gentamicin C. Deletion of eis2 increased susceptibility to capreomycin, hygromycin B, amikacin and kanamycin B. Deletion of eis1 did not affect drug susceptibility. Equally low MICs of apramycin, arbekacin, isepamicin and kanamycin A for WT and mutant strains indicate that these drugs are not inactivated by either AAC(2 ' ) or Eis enzymes. Conclusions M. abscessus expresses two distinct AG resistance determinants, AAC(2 ' ) and Eis2, which confer clinically relevant drug resistance

Rifabutin is inactivated by Mycobacterium abscessus Arr

Mycobacterium abscessus exhibits arr (ADP-ribosyltransferase)-dependent rifampicin (RIF) resistance. In apparent contrast, rifabutin (RBT) has demonstrated promising activity in M. abscessus infection models implying that RBT might not be inactivated by Arr. RBT susceptibility testing of M. abscessus Δarr revealed a strongly decreased minimal inhibitory concentration (MIC). Our findings therefore suggest that the efficacy of RBT might be enhanced by rendering RBT resilient to Arr-dependent modification or by blocking M. abscessus Arr activity

Lipoprotein Glycosylation by Protein-O-Mannosyltransferase (MAB_1122c) Contributes to Low Cell Envelope Permeability and Antibiotic Resistance of Mycobacterium abscessus

Lipoproteins are important components of the mycobacterial cell envelope due to their function in cell wall homeostasis and bacterial virulence. They are post-translationally modified with lipid- and glycosyl-residues in various species and interference with acylation or glycosylation leads to reduced growth and attenuated virulence in Mycobacterium tuberculosis. Lipoproteins are also expressed in the emerging and highly drug resistant pathogen Mycobacterium abscessus which frequently affects the lungs of patients with chronic pulmonary disease or cystic fibrosis. We investigated post-translational modification, acylation and glycosylation, of heterologously expressed (M. tuberculosis LppX and Mpt83) and endogenous (SodC) lipoproteins at the molecular level in M. abscessus and identified MAB_1122c as protein O-mannosyltransferase (Pmt). Both, heterologous and endogenous lipoproteins carried a characteristic lipid anchor with palmitic acid (C16), palmitoleic acid (C16:1), oleic acid (C18), or tuberculostearic acid (C19) modifications. Multiple hexose-moieties were detected in the N-terminal region of the model lipoproteins expressed in M. abscessus. Conservation of lipoprotein glycosylation in M. tuberculosis and M. abscessus was revealed and points toward the existence of an O-glycosylation motif or other regulatory mechanisms regarding this post-translational modification. Deletion of MAB_1122c prevented glycosylation and affected susceptibility to specific antibiotics which are large or target peptidoglycan synthesis and to lysozyme. Cell envelope permeability of M. abscessus Δpmt was increased and mutant bacteria showed reduced survival inside macrophages. The results provide a link between post-translational modification of lipoproteins and the permeability of the mycobacterial cell envelope which stresses the importance of lipoproteins as components of this complex structure

Intrinsic rifamycin resistance of Mycobacterium abscessus is mediated by ADP-ribosyltransferase MAB_0591

Objectives: Rifampicin, a potent first-line TB drug of the rifamycin group, shows only little activity against the emerging pathogen Mycobacterium abscessus. Reportedly, bacterial resistance to rifampicin is associated with polymorphisms in the target gene rpoB or the presence of enzymes that modify and thereby inactivate rifampicin. The aim of this study was to investigate the role of the MAB_0591 (arrMab)-encoded rifampicin ADP-ribosyltransferase (Arr_Mab) in innate high-level rifampicin resistance in M. abscessus. Methods: Recombinant Escherichia coli and Mycobacterium tuberculosis strains expressing MAB_0591 were generated, as was an M. abscessus deletion mutant deficient for MAB_0591. MIC assays were used to study susceptibility to rifampicin and C25 carbamate-modified rifamycin derivatives. Results: Heterologous expression of MAB_0591 conferred rifampicin resistance to E. coli and M. tuberculosis. Rifamycin MIC values were consistently lower for the M. abscessus ΔarrMab mutant as compared with the M. abscessus ATCC 19977 parental type strain. The rifamycin WT phenotype was restored after complementation of the M. abscessus ΔarrMab mutant with arrMab. Further MIC data demonstrated that a C25 modification increases rifamycin activity in WT M. abscessus. However, MIC studies in the M. abscessus ΔarrMab mutant suggest that C25 modified rifamycins are still subject to modification by Arr_Mab. Conclusions: Our findings identify Arr_Mab as the major innate rifamycin resistance determinant of M. abscessus. Our data also indicate that Arr_Mab-mediated rifamycin resistance in M. abscessus can only in part be overcome by C25 carbamate modification

Elucidation of Mycobacterium abscessus aminoglycoside and capreomycin resistance by targeted deletion of three putative resistance genes

Objectives: Mycobacterium abscessus is innately resistant to a variety of drugs thereby limiting therapeutic options. Bacterial resistance to aminoglycosides (AGs) is conferred mainly by AG-modifying enzymes, which often have overlapping activities. Several putative AG-modifying enzymes are encoded in the genome of M. abscessus. The aim of this study was to investigate the molecular basis underlying AG resistance in M. abscessus. Methods: M. abscessus deletion mutants deficient in one of three genes potentially involved in AG resistance, aac(2ʹ), eis1 and eis2, were generated by targeted gene inactivation, as were combinatorial double and triple deletion mutants. MICs were determined to study susceptibility to a variety of AG drugs and to capreomycin. Results: Deletion of aac(2ʹ) increased susceptibility of M. abscessus to kanamycin B, tobramycin, dibekacin and gentamicin C. Deletion of eis2 increased susceptibility to capreomycin, hygromycin B, amikacin and kanamycin B. Deletion of eis1 did not affect drug susceptibility. Equally low MICs of apramycin, arbekacin, isepamicin and kanamycin A for WT and mutant strains indicate that these drugs are not inactivated by either AAC(2ʹ) or Eis enzymes. Conclusions: M. abscessus expresses two distinct AG resistance determinants, AAC(2ʹ) and Eis2, which confer clinically relevant drug resistance