Kadie Holsinger

ACS Infect Dis. 2022 Aug 12;8(8):1627-1636. doi: 10.1021/acsinfecdis.2c00229. Epub 2022 Aug 2.

ABSTRACT

The rise of antibiotic-resistant Mycobacterium tuberculosis and non-tuberculous mycobacterial infections has placed ever-increasing importance on discovering new antibiotics to treat these diseases. Recently, a new penem, T405, was discovered to have strong antimicrobial activity against M. tuberculosis and Mycobacteroides abscessus. Here, a penem library of C2 side-chain variants was synthesized, and their antimicrobial activities were evaluated against M. tuberculosis H₃₇Rv and M. abscessus ATCC 19977. Several new penems with antimicrobial activity stronger than the standard-of-care carbapenem antibiotics were identified with some candidates improving on the activity of the lead compound, T405. Moreover, many candidates showed little or no increase in the minimum inhibitory concentration in the presence of serum compared to the highly protein-bound T405. The penems with the strongest activity identified in this study were then biochemically characterized by reaction with the representative l,d-transpeptidase Ldt_Mt2 and the representative penicillin-binding protein d,d-carboxypeptidase DacB2.

PMID:35916356 | PMC:PMC10029149 | DOI:10.1021/acsinfecdis.2c00229

J Lipid Res. 2022 Sep;63(9):100262. doi: 10.1016/j.jlr.2022.100262. Epub 2022 Aug 8.

ABSTRACT

Mycobacteria share an unusually complex, multilayered cell envelope, which contributes to adaptation to changing environments. The plasma membrane is the deepest layer of the cell envelope and acts as the final permeability barrier against outside molecules. There is an obvious need to maintain the plasma membrane integrity, but the adaptive responses of the plasma membrane to stress exposure remain poorly understood. Using chemical treatment and heat stress to fluidize the membrane, we show here that phosphatidylinositol (PI)-anchored plasma membrane glycolipids known as PI mannosides (PIMs) are rapidly remodeled upon membrane fluidization in Mycobacterium smegmatis. Without membrane stress, PIMs are predominantly in a triacylated form: two acyl chains of the PI moiety plus one acyl chain modified at one of the mannose residues. Upon membrane fluidization, we determined the fourth fatty acid is added to the inositol moiety of PIMs, making them tetra-acylated variants. Additionally, we show that PIM inositol acylation is a rapid response independent of de novo protein synthesis, representing one of the fastest mass conversions of lipid molecules found in nature. Strikingly, we found that M. smegmatis is more resistant to the bactericidal effect of a cationic detergent after benzyl alcohol pre-exposure. We further demonstrate that fluidization-induced PIM inositol acylation is conserved in pathogens such as Mycobacterium tuberculosis and Mycobacterium abscessus. Our results demonstrate that mycobacteria possess a mechanism to sense plasma membrane fluidity change. We suggest that inositol acylation of PIMs is a novel membrane stress response that enables mycobacterial cells to resist membrane fluidization.

PMID:35952902 | PMC:PMC9490103 | DOI:10.1016/j.jlr.2022.100262

Microbiol Spectr. 2022 Aug 10:e0276321. doi: 10.1128/spectrum.02763-21. Online ahead of print.

ABSTRACT

Mycobacterium abscessus is an emerging human pathogen leading to significant morbidity and even mortality, intrinsically resistant to almost all the antibiotics available and so can be a nightmare. Mechanisms of its intrinsic resistance remain not fully understood. Here, we selected and confirmed an M. abscessus transposon mutant that is hypersensitive to multiple drugs including rifampin, rifabutin, vancomycin, clofazimine, linezolid, imipenem, levofloxacin, cefoxitin, and clarithromycin. The gene MAB_0189c encoding a putative arabinosyltransferase C was found to be disrupted, using a newly developed highly-efficient strategy combining next-generation sequencing and multiple PCR. Furthermore, selectable marker-free deletion of MAB_0189c recapitulated the hypersensitive phenotype. Disruption of MAB_0189c resulted in an inability to synthesize lipoarabinomannan and markedly enhanced its cell envelope permeability. Complementing MAB_0189c or M. tuberculosis embC restored the resistance phenotype. Importantly, treatment of M. abscessus with ethambutol, a first-line antituberculosis drug targeting arabinosyltransferases of M. tuberculosis, largely sensitized M. abscessus to multiple antibiotics in vitro. We finally tested activities of six selected drugs using a murine model of sustained M. abscessus infection and found that linezolid, rifabutin, and imipenem were active against the MAB_0189c deletion strain. These results identified MAB_0189 as a crucial determinant of intrinsic resistance of M. abscessus, and optimizing inhibitors targeting MAB_0189 might be a strategy to disarm the intrinsic multiple antibiotic resistance of M. abscessus. IMPORTANCE Mycobacterium abscessus is intrinsically resistant to most antibiotics, and treatment of its infections is highly challenging. The mechanisms of its intrinsic resistance remain not fully understood. Here we found a transposon mutant hypersensitive to a variety of drugs and identified the transposon inserted into the MAB_0189c (orthologous embC coding arabinosyltransferase, EmbC) gene by using a newly developed rapid and efficient approach. We further verified that the MAB_0189c gene played a significant role in its intrinsic resistance by decreasing the cell envelope permeability through affecting the production of lipoarabinomannan in its cell envelope. Lastly, we found the arabinosyltransferases inhibitor, ethambutol, increased activities of nine selected drugs in vitro. Knockout of MAB_0189c made M. abscessus become susceptible to 3 drugs in mice. These findings indicated that potential powerful M. abscessus EmbC inhibitor might be used to reverse the intrinsic resistance of M. abscessus to multiple drugs.

PMID:35946941 | DOI:10.1128/spectrum.02763-21

ACS Infect Dis. 2022 Aug 10. doi: 10.1021/acsinfecdis.2c00193. Online ahead of print.

ABSTRACT

New approaches to target antibacterial agents toward Gram-negative bacteria are key, given the rise of antibiotic resistance. Since the discovery of polymyxin B nonapeptide as a potent Gram-negative outer membrane (OM)-permeabilizing synergist in the early 1980s, a vast amount of literature on such synergists has been published. This Review addresses a range of peptide-based and small organic compounds that disrupt the OM to elicit a synergistic effect with antibiotics that are otherwise inactive toward Gram-negative bacteria, with synergy defined as a fractional inhibitory concentration index (FICI) of <0.5. Another requirement for the inclusion of the synergists here covered is their potentiation of a specific set of clinically used antibiotics: erythromycin, rifampicin, novobiocin, or vancomycin. In addition, we have focused on those synergists with reported activity against Gram-negative members of the ESKAPE family of pathogens namely, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, and/or Acinetobacter baumannii. In cases where the FICI values were not directly reported in the primary literature but could be calculated from the published data, we have done so, allowing for more direct comparison of potency with other synergists. We also address the hemolytic activity of the various OM-disrupting synergists reported in the literature, an effect that is often downplayed but is of key importance in assessing the selectivity of such compounds for Gram-negative bacteria.

PMID:35946799 | DOI:10.1021/acsinfecdis.2c00193

iScience. 2022 Jul 12;25(8):104753. doi: 10.1016/j.isci.2022.104753. eCollection 2022 Aug 19.

ABSTRACT

N-Acetylglucosamine (GlcNAc) is an essential monosaccharide required in almost all organisms. Fluorescent labeling of the peptidoglycan (PG) on N-acetylglucosamine has been poorly explored. Here, we report on the labeling of the PG with a bioorthogonal handle on the GlcNAc. We developed a facile one-step synthesis of uridine diphosphate N-azidoacetylglucosamine (UDP-GlcNAz) using the glycosyltransferase OleD, followed by in vitro incorporation of GlcNAz into the peptidoglycan precursor Lipid II and fluorescent labeling of the azido group via click chemistry. In a PG synthesis assay, fluorescent GlcNAz-labeled Lipid II was incorporated into peptidoglycan by the DD-transpeptidase activity of bifunctional class A penicillin-binding proteins. We further demonstrate the incorporation of GlcNAz into the PG layer of OleD-expressed bacteria by feeding with 2-chloro-4-nitrophenyl GlcNAz (GlcNAz-CNP). Hence, our labeling method using the heterologous expression of OleD is useful to study PG synthesis and possibly other biological processes involving GlcNAc metabolism in vivo.

PMID:35942089 | PMC:PMC9356107 | DOI:10.1016/j.isci.2022.104753

Front Mol Biosci. 2022 Jul 20;9:889825. doi: 10.3389/fmolb.2022.889825. eCollection 2022.

ABSTRACT

Peptidoglycan is a cross-linked polymer responsible for maintaining the bacterial cell wall integrity and morphology in Gram-negative and Gram-positive bacteria. The peptidoglycan pathway consists of the enzymatic reactions held in three steps: cytoplasmic, membrane-associated, and periplasmic. The Mur enzymes (MurA-MurF) are involved in a cytoplasmic stage. The UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) enzyme is responsible for transferring the enolpyruvate group from phosphoenolpyruvate (PEP) to UDP-N-acetylglucosamine (UNAG) to form UDP-N-acetylglucosamine enolpyruvate (EP-UNAG). Fosfomycin is a natural product analogous to PEP that acts on the MurA target enzyme via binding covalently to the key cysteine residue in the active site. Similar to fosfomycin, other MurA covalent inhibitors have been described with a warhead in their structure that forms a covalent bond with the molecular target. In MurA, the nucleophilic thiolate of Cys115 is pointed as the main group involved in the warhead binding. Thus, in this minireview, we briefly describe the main recent advances in the design of MurA covalent inhibitors.

PMID:35936791 | PMC:PMC9346081 | DOI:10.3389/fmolb.2022.889825

Bioorg Med Chem Lett. 2022 Jul 11;73:128887. doi: 10.1016/j.bmcl.2022.128887. Online ahead of print.

ABSTRACT

A ring-closing metathesis (RCM) - peptide coupling - ruthenium-catalyzed azide alkyne cycloaddition (RuAAC) strategy was developed to synthesize a tricyclic hexapeptide in which the side chain to side chain connectivity pattern resulted in a mimic with a topology that effectively mimics the bioactivity of vancomycin as a potent binder of the bacterial cell wall D-Ala-D-Ala dipeptide sequence and more importantly being an effective inhibitor of bacterial growth.

PMID:35835378 | DOI:10.1016/j.bmcl.2022.128887

FASEB J. 2022 Aug;36(8):e22446. doi: 10.1096/fj.202101595R.

ABSTRACT

d-alanine (d-Ala) and several other d-amino acids (d-AAs) act as hormones and neuromodulators in nervous and endocrine systems. Unlike the endogenously synthesized d-serine in animals, d-Ala may be from exogenous sources, e.g., diet and intestinal microorganisms. However, it is unclear if the capability to produce d-Ala and other d-AAs varies among different microbial strains in the gut. We isolated individual microorganisms of rat gut microbiota and profiled their d-AA production in vitro, focusing on d-Ala. Serial dilutions of intestinal contents from adult male rats were plated on agar to obtain clonal cultures. Using MALDI-TOF MS for rapid strain typing, we identified 38 unique isolates, grouped into 11 species based on 16S rRNA gene sequences. We then used two-tier screening to profile bacterial d-AA production, combining a d-amino acid oxidase-based enzymatic assay for rapid assessment of non-acidic d-AA amount and chiral LC-MS/MS to quantify individual d-AAs, revealing 19 out of the 38 isolated strains as d-AA producers. LC-MS/MS analysis of the eight top d-AA producers showed high levels of d-Ala in all strains tested, with substantial inter- and intra-species variations. Though results from the enzymatic assay and LC-MS/MS analysis aligned well, LC-MS/MS further revealed the existence of d-glutamate and d-aspartate, which are poor substrates for this enzymatic assay. We observed large inter- and intra-species variation of d-AA production profiles from rat gut microbiome species, demonstrating the importance of chemical profiling of gut microbiota in addition to sequencing, furthering the idea that microbial metabolites modulate host physiology.

PMID:35816159 | DOI:10.1096/fj.202101595R

Bioorg Med Chem. 2022 Sep 1;69:116896. doi: 10.1016/j.bmc.2022.116896. Epub 2022 Jun 23.

ABSTRACT

There is a dearth of tuberculosis (TB) drug development activity as current therapeutic treatments are inadequate due to the appearance of drug-resistant TB. The enzyme UDP-galactopyranose mutase (UGM) is involved in the biosynthesis of galactan which is essential for cell wall integrity and bacterial viability. Its inhibition has thus been featured as profitable strategy for anti-TB drug discovery. In this study, we report on the synthesis of amides derived from rosmarinic acid, their inhibitory effect towards purified UGM using three distinct biochemical assays: FP, HPLC and SPR. The rosmarinic amides generally showed a significantly higher affinity for UGM than the corresponding rosmarinic ester. In particular, compound 5h displayed interesting binding affinity values (K_d = 58 ± 7, 63 ± 9 µM towards KpUGM and MtUGM respectively). Furthermore, a new UGM SPR assay was established and confirmed that 5h binds to UGM with a dissociation constant of 104.8 ± 6.5 μM. Collectively, this study validates the amide bioisosteric strategy which has been successfully implemented to develop UGM inhibitors from rosmarinic acid, providing a substantial basis for further design of novel UGM inhibitors and anti-mycobacterial agents.

PMID:35777270 | DOI:10.1016/j.bmc.2022.116896

Sci Rep. 2022 Jun 30;12(1):11061. doi: 10.1038/s41598-022-15324-1.

ABSTRACT

Peptidoglycan (PG) is the exoskeleton of bacterial cells and is required for their viability, growth, and cell division. Unlike most bacteria, mycobacteria possess an atypical PG characterized by a high degree of unique linkages and chemical modifications which most likely serve as important determinants of virulence and pathogenesis in mycobacterial diseases. Despite this important role, the chemical composition and molecular architecture of mycobacterial PG have yet to be fully determined. Here we determined the chemical composition of PG from Mycobacterium smegmatis using high-resolution liquid chromatography-mass spectrometry. Purified cell walls from the stationary phase were digested with mutanolysin and compositional analysis was performed on 130 muropeptide ions that were identified using an in silico PG library. The relative abundance for each muropeptide ion was measured by integrating the extracted-ion chromatogram. The percentage of crosslink per PG subunit was measured at 45%. While both 3→3 and 4→3 transpeptide cross-linkages were found in PG dimers, a high abundance of 3→3 linkages was found associated with the trimers. Approximately 43% of disaccharides in the PG of M. smegmatis showed modifications by acetylation or deacetylation. A significant number of PG trimers are found with a loss of 41.00 amu that is consistent with N-deacetylation, whereas the dimers show a gain of 42.01 amu corresponding to O-acetylation of the PG disaccharides. This suggests a possible role of PG acetylation in the regulation of cell wall homeostasis in M. smegmatis. Collectively, these data report important novel insights into the ultrastructure of mycobacterial PG.

PMID:35773428 | PMC:PMC9247062 | DOI:10.1038/s41598-022-15324-1

Nature. 2022 Jun;606(7916):866-867. doi: 10.1038/d41586-022-01739-3.

NO ABSTRACT

PMID:35760964 | DOI:10.1038/d41586-022-01739-3

Antimicrob Agents Chemother. 2022 Jun 16:e0224721. doi: 10.1128/aac.02247-21. Online ahead of print.

ABSTRACT

In the time of antimicrobial resistance, phage therapy is frequently suggested as a possible solution for such difficult-to-treat infections. Vancomycin-intermediate Staphylococcus aureus (VISA) remains a relatively rare yet increasing occurrence in the clinic for which phage therapy may be an option. However, the data presented herein suggest a potential cross-resistance mechanism to phage following vancomycin exposure in VISA strains. When comparing genetically similar strains differing in their susceptibility to vancomycin, those with intermediate levels of vancomycin resistance displayed decreased sensitivity to phage in solid and liquid assays. Serial passaging with vancomycin induced both reduced vancomycin susceptibility and phage sensitivity. As a consequence, the process of phage infection was shown to be interrupted after DNA ejection from adsorbed phage but prior to phage DNA replication, as demonstrated through adsorption assays, lysostaphin sensitivity assays, electron microscopy, and quantitative PCR (qPCR). At a time when phage products are being used for experimental treatments and tested in clinical trials, it is important to understand possible interference between mechanisms underlying antibiotic and phage resistance in order to design effective therapeutic regimens.

PMID:35708333 | DOI:10.1128/aac.02247-21