Apramycin is a structurally unique member of the 2-deoxystreptamine class of aminoglycoside antibiotics characterized by a mono-substituted 2-deoxystreptamine ring that carries an unusual bicyclic eight-carbon dialdose moiety. Because of its unusual structure apramycin is not susceptible to the most prevalent mechanisms of aminoglycoside resistance including the aminoglycoside-modifying enzymes and the ribosomal methyltransferases whose widespread presence severely compromises all aminoglycosides in current clinical practice. These attributes coupled with minimal ototoxocity in animal models combine to make apramycin an excellent starting point for the development of next-generation aminoglycoside antibiotics for the treatment

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Streptomycin, the first drug used for the treatment of tuberculosis, shows limited activity against the highly resistant pathogen We recently identified two aminoglycoside-acetylating genes [ and ] which, however, do not affect susceptibility to streptomycin. This suggests the existence of a discrete mechanism of streptomycin resistance. BLASTP analysis identified MAB_2385 as a close homologue of the 3″--phosphotransferase [APH(3″)] from the opportunistic pathogen as a putative streptomycin resistance determinant. Heterologous expression of in increased the streptomycin MIC, while the gene deletion mutant ΔMAB_2385 showed increased streptomycin susceptibility. The MICs of other aminoglycosides were not altered in ΔMAB_2385. This demonstrates that encodes a specific and prime innate streptomycin resistance determinant in We further explored the feasibility of applying-based streptomycin counterselection to generate gene deletion mutants in Spontaneous streptomycin-resistant mutants of ΔMAB_2385 were selected, and we demonstrated that the wild-type is dominant over the mutated in merodiploid strains. In a proof of concept study, we exploited this phenotype for construction of a targeted deletion mutant, thereby establishing an -based counterselection method in.

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.

Aminoglycoside antibiotics are powerful bactericidal therapeutics that are often used in the treatment of critical Gram-negative systemic infections. The emergence and global spread of antibiotic resistance, however, has compromised the clinical utility of aminoglycosides to an extent similar to that found for all other antibiotic-drug classes. Apramycin, a drug candidate currently in clinical development, was suggested as a next-generation aminoglycoside antibiotic with minimal cross-resistance to all other standard-of-care aminoglycosides. Here, we analyzed 591,140 pathogen genomes deposited in the NCBI National Database of Antibiotic Resistant Organisms (NDARO) for annotations of apramycin-resistance genes, and compared them to the genotypic prevalence of carbapenem resistance and 16S-rRNA methyltransferase (RMTase) genes. The 3-N-acetyltransferase gene aac(3)-IV was found to be the only apramycin-resistance gene of clinical relevance, at an average prevalence of 0.7%, which was four-fold lower than that of RMTase genes. In the important subpopulation of carbapenemase-positive isolates, aac(3)-IV was nine-fold less prevalent than RMTase genes. The phenotypic profiling of selected clinical isolates and recombinant strains expressing the aac(3)-IV gene confirmed resistance to not only apramycin, but also gentamicin, tobramycin, and paromomycin. Probing the structure–activity relationship of such substrate promiscuity by site-directed mutagenesis of the aminoglycoside-binding pocket in the acetyltransferase AAC(3)-IV revealed the molecular contacts to His124, Glu185, and Asp187 to be equally critical in binding to apramycin and gentamicin, whereas Asp67 was found to be a discriminating contact. Our findings suggest that aminoglycoside cross-resistance to apramycin in clinical isolates is limited to the substrate promiscuity of a single gene, rendering apramycin best-in-class for the coverage of carbapenem- and aminoglycoside-resistant bacterial infections.

With a view to facilitating prediction of the exocyclic bond to the pyranoside ring in higher carbon sugars, a model is advanced that relates the relative configuration of the three stereogenic centers comprised of the branchpoint and of the two flanking centers (C4−C5−C6 in aldoheptoses and higher and C5−C6−C7 in sialic and ulosonic acids) to that of the simple ringopened pentoses. Assignment of a given stereotriad as arabino, lxyo, ribo, or xylo by inspection of the Fischer projection formulas permits prediction of conformation of the exocyclic bond by comparison with the known solution (= crystal in all cases) conformations of the simple pentitols. More remote stereogenic centers in the side chain, as in the 8-position of N-acetylneuraminic acid, have little impact on the conformation of the exocyclic bond. On the basis of this model the conformation of the exocyclic bond in ring I of 6′-homologated 4,5-disubstituted 2-deoxystreptamine class aminoglycoside antibiotics was predicted and was borne out by NMR analysis of newly synthesized derivatives in D 2 O at pD5. The antiribosomal and antibacterial activity of these derivatives is briefly presented and discussed in terms of preorganization of the side chain for binding to the ribosomal decoding A site. It is anticipated that this predictive analysis will also find use in the prediction of the conformation of the exocyclic bonds in other 2-(1hydroxyalkyl)-3-hydroxytetrahydropyrans and tetrahydrofurans.

We describe the convergent synthesis of a 5‐O‐β‐D‐ribofuranosyl‐based apramycin derivative (apralog) that displays significantly improved antibacterial activity over the parent apramycin against wild‐type ESKAPE pathogens. In addition, the new apralog retains excellent antibacterial activity in the presence of the only aminoglycoside modifying enzyme (AAC(3)‐IV) acting on the parent, without incurring susceptibility to the APH(3’) mechanism that disables other 5‐O‐β‐D‐ribofuranosyl 2‐deoxystreptamine type aminoglycosides by phosphorylation at the ribose 5‐position. Consistent with this antibacterial activity, the new apralog has excellent 30 nM activity (IC50) for the inhibition of protein synthesis by the bacterial ribosome in a cell‐free translation assay, while retaining the excellent across‐the‐board selectivity of the parent for inhibition of bacterial over eukaryotic ribosomes. Overall, these characteristics translate into excellent in vivo efficacy against E. coli in a mouse thigh infection model and reduced ototoxicity vis à vis the parent in mouse cochlear explants.

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