Supramolecular assemblies are fundamental to cellular biochemical processes, relying on their dynamic nature to perform essential functions. The protease ClpP1P2, in association with ATPase partners ClpC1 or ClpX, is critical for the survival of Mycobacterium tuberculosis (Mtb). While the ClpP1P2 complex requires activation by specific N-blocked dipeptides to exhibit proteolytic activity in vitro, the mechanism of in vivo activation remains unclear. In this study, we use cryo-electron microscopy (cryo-EM) to determine the structure of the ClpC1P1P2 complex, revealing a highly asymmetric architecture with ClpC1 bound to the ClpP1P2 protease barrel. The activator dipeptide is observed only in the ClpP2 active site, while the ClpP1 entry pore remains closed. Molecular crowding agents promote the formation of larger ClpXP1P2 and ClpC1P1P2 complexes, enhancing structural stability and enzymatic activity. These findings suggest that molecular crowding stabilizes these complexes and promotes their activation, providing new insights into ClpC1P1P2 structural dynamics and function.

Mycobacterium abscessus has emerged as a significant pulmonary pathogen characterized by its resistance to most first-line antimycobacterial drugs. Recent investigations have highlighted the clinical efficacy of including the oxazolidinone antibiotic linezolid in M. abscessus combination therapies, despite moderate resistance frequently being observed in patient isolates. Even with the potential usefulness of linezolid, the mechanisms that drive linezolid resistance in M. abscessus remain poorly understood. In several bacterial pathogens, including Mycobacterium tuberculosis, ATP-binding cassette (ABC) family proteins of the F subtype (ABC-F) have been found to confer antibiotic resistance to ribosome-targeting antibiotics, including linezolid. Here, we identified an M. abscessus ABC-F protein, MAB_2736c, that causes specific resistance to antibiotics that bind the 50S ribosomal subunit, including linezolid, macrolides, and chloramphenicol. These results demonstrate that targeting ABC-F proteins could help combat intrinsic resistance to several ribosome-targeting antibiotics in mycobacteria.

Controlled human infection models (CHIMs) can accelerate vaccine development for infectious diseases. Mycobacterium tuberculosis is a human-adapted pathogen that is the leading infectious cause of death worldwide. M tuberculosis infection results in a spectrum of clinical outcomes that are incompletely modelled in animals. To date, the risks of infection, prolonged treatment, and sequelae related to CHIMs with M tuberculosis have been considered ethically unacceptable. However, recent advances in bacterial engineering have resulted in safe strains that could permit M tuberculosis CHIM studies with reduced risks. In this Personal View, we address the practical considerations for conducting a pulmonary M tuberculosis CHIM study. We summarise the ethical issues of M tuberculosis CHIM studies in tuberculosis-endemic and non-endemic settings; describe safety considerations, such as optimising the challenge dose and minimising risks to third parties; and outline and prioritise clinical, microbiological, immunological, and radiological endpoints that would render such a model useful for vaccine development.

Mycobacterium abscessus (Mab) is a highly drug-resistant non-tuberculous mycobacterium that presents major treatment challenges, particularly in individuals with structural lung disease. Although historically considered ineffective, β-lactam antibiotics have gained renewed attention due to advances in β-lactamase inhibition and cell wall biology. This review synthesizes more than a decade of work, including in vitro susceptibility studies, biochemical characterization of Mab's β-lactamase (BlaMab) and peptidoglycans synthesis, and published clinical cases supporting the potential role of β-lactam-based regimens. We detail the enzymatic pathways involved in peptidoglycan cross-linking and the dual inhibition of D,D- and L,D-transpeptidases by select β-lactams, as well as the functional impact of inhibiting BlaMab. Novel β-lactamase inhibitors such as durlobactam may further enhance β-lactam efficacy. By integrating laboratory insights with clinical experience, this review provides a comprehensive perspective and informs ongoing efforts to design clinical trials repurposing β-lactam/β-lactamase inhibitor combinations.

Tuberculosis (TB) continues to pose a global public health threat, exacerbated by rising drug-resistant strains of Mycobacterium tuberculosis (Mtb). DacB1, a D,D-carboxypeptidase critical in Mtb peptidoglycan biosynthesis, is a promising target for β-lactam antibiotics (BLs), which remain underutilized in TB treatment. Dual BL therapy may enhance efficacy by inactivating multiple targets within the peptidoglyan synthesis pathway. Minimum inhibitory concentrations (MICs) for β-lactams and β-lactamase inhibitors against Mtb H37Ra, H37Rv, and clinical isolates showed that imipenem, meropenem, or tebipenem MICs were reduced when combined with amoxicillin or ceftriaxone or β-lactamase inhibitors such as clavulanate or durlobactam. Timed electrospray ionization mass spectrometry (ESI-MS) captured acyl-enzyme adducts between DacB1 and BLs, revealing binding interactions with carbapenems (imipenem, meropenem, and tebipenem) but not most penicillins or cephalosporins except cloxacillin and cefoxitin. Differential scanning fluorimetry (DSF) combined with circular dichroism (CD) confirmed physical and structural changes in DacB1 upon BL binding despite no alteration in melting temperature. Carbapenem-DacB1 interactions were notably faster with imipenem, likely due to reduced steric hindrance compared to meropenem and tebipenem. Molecular modeling revealed conserved penicillin-binding protein motifs within the active site of DacB1: S121XXK124, S176XN178, and K282TG284 (PDB ID # 4PPR). Building on this, molecular docking suggested favorable interactions between these motifs and the carbapenems: the carbapenem carbonyl group aids in positioning within DacB1's oxyanion hole, ready for acylation, while hydrophobic interactions with the cyclic R2 side chains and C1 methyl groups in meropenem and tebipenem contribute to steric hindrance hence slow acyl-enzyme formation. These findings enhance our understanding of DacB1 inhibition and suggest that carbapenems, particularly in combination therapies, hold promise as effective TB treatments. IMPORTANCE:TB remains a significant public health threat, particularly due to the rising prevalence of drug-resistant Mtb strains. Current treatment options for drug-resistant TB are costly, toxic, and often ineffective, necessitating the exploration of alternative therapeutic strategies. This study is of critical importance as it investigates the potential of β-lactam antibiotics (BLs), a class historically considered ineffective against Mtb, for repurposing in TB treatment. By targeting DacB1, a key enzyme in Mtb peptidoglycan biosynthesis, this research provides new insights into the mechanism of β-lactam interactions and their potential to disrupt cell wall synthesis. The findings demonstrate that dual β-lactam therapy and β-lactam/β-lactamase inhibitor combinations enhance antibiotic efficacy, suggesting a promising avenue for combating drug-resistant TB. Furthermore, structural and molecular analyses confirm that carbapenems, particularly imipenem, meropenem, and tebipenem, effectively bind to DacB1, paving the way for optimized treatment strategies. Given the challenges in developing new TB drugs, repurposing β-lactams offers a cost-effective and readily implementable solution to address antimicrobial resistance. This study contributes valuable knowledge that could accelerate the development of novel TB therapies, improve treatment success rates, and ultimately reduce TB-related mortality worldwide.

Mycobacterium abscessus (Mab) presents significant clinical challenges. This study evaluated the synergistic effects of a beta-lactam and beta-lactamase inhibitor combination against Mab and explored the underlying mechanisms. Synergy was assessed through MIC tests and time-kill studies, and binding affinities of nine beta-lactams and BLIs to eight target receptors (L,D-transpeptidases [LDT] 1-5, D,D-carboxypeptidase, penicillin-binding protein [PBP] B, and PBP-lipo) were assessed using mass spectrometry and kinetic studies. Thermal stability and morphological changes were determined. Imipenem demonstrated high binding affinity to LDTs and PBPs, with extremely low inhibition constants (K-i,K-app; <= 0.002 mg/L for LDT1-2, <= 0.6 mg/L for PBPs), while cephalosporins, sulopenem, tebipenem, and amoxicillin exhibited moderate to low binding affinity. Durlobactam inactivated Bla(Mab) and LDT/PBPs more potently than avibactam. The K(i,app)s of durlobactam for PBP B, PBP-lipo, and LDT2 were below clinically achievable unbound concentrations, while avibactam's K-i,K-app for LDT/PBPs exceeded the clinical concentrations. Single beta-lactam treatments resulted in minimal killing (similar to 1 log(10) reduction). Although avibactam yielded no effect, combinations with avibactam showed a significant reduction (similar to 4 log(10) CFU/mL). Durlobactam alone showed similar to 2 log(10) reduction, and when combined with imipenem or two beta-lactams, durlobactam achieved near-eradication of Mab, surpassing the current therapy (amikacin + clarithromycin + imipenem/cefoxitin). Inactivation of PBP-lipo by sulopenem, imipenem, durlobactam, and amoxicillin (with avibactam) led to morphological changes, showing filaments. This study demonstrates the mechanistic basis of combinations therapy, particularly imipenem + durlobactam, in overcoming beta-lactam resistance in Mab.

Structure-guided optimization was applied to develop a potent and selective inhibitor of the aminotransferase BioA, a key enzyme in bacterial biotin biosynthesis. Strategic modifications of a screening hit enhanced potency and pharmacokinetics, yielding C48, which binds BioA with a Ki of 200 pM and displays sub-micromolar MICs against Mycobacterium tuberculosis (Mtb) and nontuberculous mycobacteria. Biochemical, structural, and genetic studies confirmed C48's mechanism of action. In vitro, C48 induced cell envelope stress and membrane remodeling, mimicking biotin starvation. Pharmacokinetic profiling revealed excellent oral bioavailability resulting in over 39,000-fold improved exposure versus the parent compound. To address the discrepancy in biotin levels between humans and mice, we developed a low-biotin mouse model that recapitulates human biotin physiology. In this model C48 reduced Mtb burden in lungs and spleen, providing the first in vivo proof-of-concept for targeting biotin biosynthesis as a therapeutic strategy against tuberculosis.

Mycobacterium abscessus (Mab) poses significant clinical challenges and underscores the urgent need for safer and more effective treatments, including β-lactams. Among currently available carbapenems, imipenem is widely used to treat Mab infections by combining with other antibiotics. Commercial carbapenems share a common scaffold with C2 modifications, whereas this study focuses on novel carbapenem candidates with C5α modifications. We evaluated their antibacterial activity against Mab ATCC 19977 and clinical isolates, as well as their acylation of peptidoglycan target receptors (L,D-transpeptidases [LDTs] and penicillin-binding proteins [PBPs]) and the β-lactamase enzyme BlaMab. In vitro studies of two C5α-modified carbapenems, JDB/NA-1-157 and JDB/NA-1-208, revealed distinct antibacterial effects. JDB/NA-1-157 demonstrated potent bacterial killing with low minimum inhibitory concentrations (MICs; 0.125-8 mg/L) and near-complete eradication within 5 days, surpassing the efficacy of the standard-of-care regimen (amikacin + clarithromycin + imipenem). In contrast, JDB/NA-1-208 exhibited poor bacterial killing, with high MICs (16-256 mg/L) and limited efficacy in time-kill studies. However, JDB/NA-1-208 showed synergistic killing when combined with other β-lactams. Mechanistically, JDB/NA-1-208 is not a substrate for BlaMab, while JDB/NA-1-157 is, albeit with low catalytic efficiency. This is supported by the observation that the addition of avibactam did not enhance synergy with JDB/NA-1-157. The substantial bacterial killing effect of JDB/NA-1-157 is attributed to its high binding affinity for PBP-B, PBP-lipo, PonA2, D,D-carboxypeptidase, and LDT1-2. These findings highlight the potential of novel C5α-modified carbapenems, particularly JDB/NA-1-157, as promising therapeutic candidates for Mab infections.

Human challenge experiments could accelerate tuberculosis vaccine development. This requires a safe Mycobacterium tuberculosis (Mtb) strain that can both replicate in the host and be reliably cleared. Here we genetically engineered Mtb strains encoding up to three kill switches: two mycobacteriophage lysin operons negatively regulated by tetracycline and a degron domain–NadE fusion, which induces ClpC1-dependent degradation of the essential enzyme NadE, negatively regulated by trimethoprim. The triple-kill-switch (TKS) strain showed similar growth kinetics and antibiotic susceptibilities to wild-type Mtb under permissive conditions but was rapidly killed in vitro without trimethoprim and doxycycline. It established infection in mice receiving antibiotics but was rapidly cleared upon cessation of treatment, and no relapse was observed in infected severe combined immunodeficiency mice or Rag−/− mice. The TKS strain had an escape mutation rate of less than 10−10 per genome per generation. These findings suggest that the TKS strain could be a safe, effective candidate for a human challenge model. Engineered kill-switch-encoding Mycobacterium tuberculosis infects, elicits immune responses and is cleared from immunocompetent and immunocompromised mice, providing a model of controlled tuberculosis infection.

Abstract Background Mycobacterium abscessus (Mab) poses significant clinical challenges, leading to chronic pulmonary disease in immunocompromised patients. Current treatment involve amikacin (AMK), which is toxic, highlighting the imperative for safer options. This study continues our evaluation of the synergistic effects of a β-lactam and β-lactamase inhibitor (BL/BLI) against Mab and to elucidate the underlying mechanism. The inhibition constant (Ki,app) Methods The binding affinities and chemical interactions between target receptors (LDT1-5, DDC, PBP B, and PBP-lipo) and BL/BLI were determined via kinetics, mass spectrometry, DSF, flow cytometry and microscopy. The synergistic effects of BL/BLI were evaluated in time-kill studies, conducted over 10 days, using ATCC 19977 producing BlaMab. Time-kill curves of monotherapy or double β-lactams (A, D, and G) and their combinations with avibactam (B, E, and H) or durlobactam+sulbactam (C, F, and I). Results Imipenem (IPM) showed high binding affinities for both LDTs and PBPs, with an extremely low inhibition constant (Ki,app; Tab. 1). Cephalosporins exhibited moderate binding affinity for both LDTs and PBPs, whereas amoxicillin (AMX) selectively targeted PBPs. The inactivation of BlaMab and LDTs/PBPs by durlobactam (DUR) exhibited greater efficacy compared to avibactam (AVI), aligning with their respective bactericidal effects. DUR showed a 75-fold lower Ki,app for BlaMab in comparison to AVI. The Ki,app of DUR for PBP B, PBP-lipo, and LDT2 fell below the clinical concentration, whereas those of AVI did not. Single β-lactam treatment resulted in minimal killing (∼1 log10 reduction), but adding AVI significantly enhanced killing by inhibition of β-lactamase (∼4 log10 reduction for IPM+AVI and ∼2 log10 reduction for others; Fig. 1). DUR alone showed 2 log10 reduction, and when combined with IPM or dual BL, it achieved near-eradication, surpassing the SOC (AMK + Clarithromycin + IPM or cefoxitin). Inhibition of PBP-lipo by sulopenem, IPM, DUR, and AMX changed the cell morphology to filaments. In accordance with the binding affinity to LDTs and PBPs, alterations in melting temperature were detected. Conclusion IPM + DUR + SUL showed the most killing of Mab with limited regrowth. This outcome can be attributed to β-lactamase inhibition and the inactivation of multiple targets, not only LDTs but also PBPs. This combination strategy holds promise for enhanced efficacy of Mab treatment, necessitating further study in clinical trials. Disclosures All Authors: No reported disclosures

Understanding the functional impact of bacterial genetic diversity is crucial for linking pathogen variants to clinical outcomes. Here, we introduce a high-throughput cytological profiling pipeline optimized for Mycobacterium tuberculosis (Mtb) clinical strains, integrating OD-calibrated feature analysis and high-content microscopy. Our system quantifies single-bacterium morphological and physiological traits related to DNA replication, redox state, carbon metabolism, and cell envelope dynamics. Applied to 64 Mtb clinical isolates from lineages 1, 2, and 4, the approach revealed that cytological phenotypes recapitulate genetic relationships and exhibit both lineage- and density-dependent dynamics. Notably, we identified a link between a convergent "small cell" phenotype and a convergent ino1 mutation that is associated with the presence of an antisense transcript, suggesting a potential non-canonical regulatory mechanism under selection. In summary, we present a resource-efficient approach for mapping Mtb's phenotypic landscape, uncovering cellular traits that underlie its evolution and providing new insights into the functional consequences of bacterial genetic diversity. IMPORTANCE:Understanding how genetic variation in Mycobacterium tuberculosis (Mtb) shapes its physical traits is essential to unraveling the evolution of this global pathogen. Here, we introduce a systematically optimized, high-throughput imaging platform for the comprehensive characterization of Mtb clinical strains. We demonstrate that Mtb's phenotypic manifestation is shaped by both genetic background and culture density. By accounting for these factors, our analysis linked distinct cellular dynamics to specific lineages, sublineages, and even single nucleotide variations. Notably, we linked a recurring mutation to a unique cell-shortening phenotype, finding that it potentially acts by creating a cryptic antisense transcript. This platform provides a powerful framework for systematically dissecting the physiological dynamics underlying Mtb evolution and identifying new therapeutic vulnerabilities of this deadly pathogen.

Mycobacterium abscessus is a pulmonary pathogen that exhibits intrinsic resistance to antibiotics, but the factors driving this resistance are incompletely understood. Insufficient intracellular drug accumulation could explain broad-spectrum resistance, but whether antibiotics fail to accumulate in M. abscessus and the mechanisms required for drug exclusion remain poorly understood. We measured antibiotic accumulation in M. abscessus using mass spectrometry and found a wide range of drug accumulation across clinically relevant antibiotics. Of these compounds, linezolid accumulates the least, suggesting that inadequate uptake impacts its efficacy. We utilized transposon mutagenesis screening to identify genes that cause linezolid resistance and found multiple transporters that promote membrane permeability or efflux, including an uncharacterized protein that effluxes linezolid and several chemically related antibiotics. This demonstrates that membrane permeability and drug efflux are critical mechanisms of antibiotic resistance in M. abscessus and suggests that targeting membrane transporters could potentiate the efficacy of certain antibiotics.

Rifampin is a frontline antibiotic that inhibits the RNA polymerase of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB). Unlike most antibiotics, rifampin has an unusual ability to shorten the duration of treatment needed to cure TB that is not simply explained by its antimicrobial potency. We sought specific secondary effects of rifampin's inhibition of Mtb RNA polymerase that may mediate this activity. We discovered that rifampin elicited a cell division arrest that was mediated through its inhibition of RNA polymerase. This arrest resulted in a downstream inhibition of the MtrAB two-component regulatory system, a mediator of intrinsic antibiotic resistance in Mtb. This inhibition is broadly conserved in other bacteria and represents a novel form of antimicrobial activity, termed adjunctive sensitization, that can mediate synergy and may contribute to rifampin's unusual treatment shortening activity.

In an era of increasing resistance, new and effective strategies are needed for antibiotic discovery. Whole-cell active screens yield candidate compounds lacking mechanism-of-action (MOA) information and thus do not provide biological insight for prioritization. We previously reported PROSPECT ( PR imary screening O f S trains to P rioritize E xpanded C hemistry and T argets), an antimicrobial discovery strategy that measures chemical-genetic interactions between small molecules and a pool of Mycobacterium tuberculosis mutants, each depleted of a different essential protein target. PROSPECT facilitates efficient hit prioritization by simultaneously identifying whole-cell active compounds with high sensitivity and providing early insights into their MOA. Here, we report a reference-based approach to infer MOA from often complex PROSPECT data. For this aim, we curated a reference set of 437 compounds with published, annotated MOA and known or suspected antitubercular activity, and applied PROSPECT to it. We then developed P erturbagen CL ass (PCL) analysis, a computational method that predicts MOA by comparing chemical-genetic interaction profiles of unknown compounds to those of this reference set. In leave-one-out cross-validation, PCL analysis correctly predicted MOA with 70% sensitivity and 75% precision. When applied to 75 antitubercular leads with known MOA previously reported by GlaxoSmithKline (GSK), PCL analysis similarly achieved 69% sensitivity and 87% precision. We also analyzed 98 GSK compounds lacking MOA information, predicting 60 of them to act via a reference MOA, and followed up with functional validation of 29 compounds predicted to target respiration-related MOAs. Finally, we applied PROSPECT and PCL analysis to ~5,000 compounds from larger unbiased libraries that had not been preselected for antitubercular activity. PCL analysis identified a novel scaffold lacking wild-type activity but predicted to inhibit respiration via QcrB, and we confirmed this prediction while chemically optimizing this scaffold to achieve wild-type activity. PCL analysis of PROSPECT data thus enables rapid MOA assignment and hit prioritization, advancing the discovery of new, potent antitubercular compounds. ### Competing Interest Statement The authors have declared no competing interest.