Biology subjects

Lamichhane, G.

Publications and source records attributed to Lamichhane, G..

15 recordsLinked to original sources

bioRxiv2026

Carbon starvation of Mycobacterium abscessus induces a non-replicating state with extensive proteomic remodeling

Mycobacterium abscessus (Mab) is an opportunistic pathogen that can cause chronic, debilitating lung disease. Mab is intrinsically resistant to most antibiotics, making Mab infections challenging to manage and frequently incurable. During infection, Mab adapts to survive various stresses, including hypoxia and nutrient starvation. In vitro, these conditions drive Mab into a drug-tolerant, non-replicating state. Changes in the Mab proteome that result from entering a non-replicating state have been minimally described despite the clinical importance of this physiological state. Using Mab reference strain ATCC 19977, we collected proteomic data comparing replicating to non-replicating states using a carbon starvation (CS) model of persistence. We identified 2251 proteins overall (46% proteome coverage), and 17% of these proteins were found in only one of the two conditions. A third of identified proteins were significantly changed in abundance, indicating an extensive proteomic response to CS. The response regulator DosR and many DosRS responsive proteins were significantly more abundant under CS, suggesting that the DosRS stress response regulator plays a key role in CS-induced Mab persistence. Many aspects of cell wall biosynthesis were changed, including changes in glycolipid abundance under CS. Proteins involved in other key cellular processes such as secretion, oxidative phosphorylation, and nutrient metabolism were altered under CS. The proteomic analysis presented provides new insights and clarity into how the Mab proteome is regulated during non-replicating persistence, a key consideration for understanding Mab pathophysiology.

microbiology↗

bioRxiv2025

Chemoproteomic elucidation of β-lactam drug targets in Mycobacterium abscessus

The pathogen Mycobacterium abscessus (Mab) can cause severe and difficult to treat chronic lung infections. Despite the rising incidence and clinical concern of Mab infections, treatment options are limited and often ineffective. Treatment is complicated by Mabs ability to persist in a non-replicative, drug-resistant state. Several {beta}-lactam antibiotics are potently bactericidal against Mab but are underutilized because their molecular mechanisms of action against Mab are incompletely understood. In the current study, we used {beta}-lactam-derived activity-based probes and chemoproteomics to report the first comprehensive list of enzymes in Mab targeted by {beta}-lactams. We compared {beta}-lactam targets across two Mab subspecies in actively replicating and non-replicative cultures, using a new carbon starvation model of persistence. We identified 17 targets that were active in every condition tested, seven of which were previously unknown to bind {beta}-lactams. Lastly, we characterized the {beta}-lactamase activity and {beta}-lactam inhibition profiles of nine Mab enzymes, demonstrating that imipenem inhibits these targets more effectively than cefoxitin. These findings provide clarity on the mechanisms of action of clinically relevant {beta}-lactams in Mab, a crucial step toward fully realizing their potential for treating infections caused by this opportunistic pathogen.

microbiology↗

bioRxiv2025

Expanding the scope of Mycobacterium abscessus reference strains to improve pulmonary disease modeling

Mycobacterium abscessus (Mab) pulmonary disease is an emerging clinical challenge, particularly among individuals with immunosuppression or underlying structural lung conditions. There are currently no FDA-approved therapies for Mab disease. Existing treatment strategies using repurposed drugs are prolonged, complex, and yield low cure rates (30-50%), underscoring the urgent need for more effective therapeutics. Developing new treatments requires preclinical disease models that faithfully replicate human disease, and the choice of Mab strain is a key determinant of model relevance. The commonly used reference strain, ATCC 19977, was isolated from a non-pulmonary source but became the default due to its early availability. To evaluate its relevance for pulmonary disease modeling, we compared ATCC 19977 with 15 clinical Mab isolates derived from lung infections across diverse regions of the United States. In both in vitro assays and a validated mouse lung infection model, ATCC 19977 behavior differed from the clinical isolates for key traits including rapid systemic dissemination, failure to develop robust lung granulomas, and early mortality. In contrast, clinical isolates demonstrated greater pulmonary tropism and reduced dissemination, with several producing robust lung pathology. Based on these findings, we propose a set of pulmonary clinical isolates representing the major Mab subspecies for use in lung infection research. These isolates more accurately recapitulate the pathological features of human Mab lung disease and are expected to enhance the translational value of future mechanistic and therapeutic studies. SUMMARY STATEMENTUsing a mouse model, this study identified Mycobacterium abscessus clinical isolates whose infection profiles more closely resemble human pulmonary disease, establishing them as superior reference strains to ATCC 19977 for future translational and therapeutic research.

microbiology↗

bioRxiv2025

Efficacies of sequenced monotherapies of Mycobacterium avium lung infection in mouse

BackgroundThe incidence of non-tuberculous mycobacterial (NTM) infections has been rising and now exceeds tuberculosis in several countries. Among NTMs, Mycobacterium avium is the most common cause of chronic lung disease. Current guidelines recommend simultaneous administration of three or more antibiotics, modeled after tuberculosis treatment, but these regimens are limited by toxicity, poor adherence, and low cure rates. Importantly, unlike M. tuberculosis, M. avium is acquired from the environment rather than transmitted between humans, weakening the rationale for multidrug therapy as a strategy to suppress resistance at the population level. MethodsTo test an alternative treatment approach, we evaluated sequential monotherapy in a validated murine model of chronic M. avium lung infection. Mice were treated with either the standard triple-drug regimen of clarithromycin, ethambutol, and rifampicin or with sequential monotherapy: clarithromycin, bedaquiline, and clofazimine, with only one drug administered at a time for four-week intervals. Lung and spleen bacterial burdens were quantified, and minimum inhibitory concentrations (MICs) were determined for isolates recovered during treatment to assess resistance emergence. ResultsSequential monotherapy achieved reductions in lung bacterial burden equivalent to those of the standard multidrug regimen and prevented extrapulmonary dissemination. Notably, no increase in MICs was observed for clarithromycin, bedaquiline, or clofazimine across treatment phases, indicating that sequential monotherapy did not select for resistant clones. ConclusionsThese findings provide the first experimental evidence that sequential monotherapy can deliver efficacy comparable to multidrug therapy for M. avium disease without promoting resistance. This proof-of-concept supports further investigation of sequencing strategies as a potentially more tolerable alternative to current regimens.

microbiology↗

bioRxiv2025

Predicting Mycobacterium abscessus proteins with atypical amino acid composition essential for human infections

Mycobacterium abscessus is an emerging opportunistic pathogen that causes chronic, difficult-to-treat lung infections, particularly in individuals with underlying lung disease or immune suppression. Despite its clinical significance, the fundamental biological systems of M. abscessus remain poorly understood due to limited research on this organism. Proteins that are unique to an organism are likely to contribute to the organisms distinctive phenotypic traits. Therefore, we initiated this study by identifying proteins with unique features, hypothesizing that such proteins could be critical for M. abscessus pathogenesis. To identify these proteins, we analyzed the genome sequence of the laboratory reference strain using bioinformatics tools to detect proteins with unusual amino acid compositions. We then examined the genomes of a large collection of patient-derived M. abscessus isolates to predict proteins essential for the pathogens ability to cause disease in humans. Our analysis identified 10 proteins--MAB_0010, MAB_0039, MAB_1134, MAB_1602, MAB_1657, MAB_3052, MAB_3131, MAB_3413, MAB_4263, and MAB_4537--that exhibit restricted evolutionary variation in human infections, similar to five known essential proteins DnaA, DnaN, RpoA, RpoB and RpoC which comprise proteins involved in DNA and RNA synthesis. A majority of these proteins lack sequence homology with proteins of known function and are currently annotated as proteins of unknown function. The unique amino acid compositions of these proteins, their limited capacity to tolerate mutations, and their apparent exclusivity to M. abscessus suggest that they play essential roles in the pathogens ability to establish and maintain infection in humans. These findings highlight potentially promising targets for future drug development aimed at combating M. abscessus infections.

microbiology↗

bioRxiv2025

Comprehensive identification of β-lactam antibiotic polypharmacology in Mycobacterium tuberculosis

Infections with Mycobacterium tuberculosis (Mtb) cause tuberculosis (TB), which requires at least six months of treatment with multiple antibiotics. There is emergent interest in using {beta}-lactam antibiotics to improve treatment outcomes for patients. These drugs target cell wall biosynthesis, but a comprehensive list of enzymes inhibited by {beta}-lactams in Mtb is lacking. In the current study, we sought to identify and characterize Mtb enzymes inhibited by {beta}-lactam antibiotics using physiological conditions representative of both acute and chronic TB disease. We used new activity-based probes based on the {beta}-lactam antibiotic meropenem due to its approval by the World Health Organization for TB treatment. Activity-based probes label enzymes based on both substrate specificity and catalytic mechanism, enabling precise identification of drug targets. We identified previously undiscovered targets of meropenem in addition to known cell wall biosynthetic enzymes. We validated {beta}-lactam binding and hydrolysis for six newly identified targets: Rv1723, Rv2257c, Rv0309, DapE (Rv1202), MurI (Rv1338), and LipD (Rv1923). Our results demonstrate that there are at least 30 enzymes in Mtb vulnerable to inhibition by meropenem. This is many more {beta}-lactam targets than historically described, suggesting that efficacy in Mtb is a direct result of polypharmacology.

biochemistry↗

bioRxiv2025

Combinations comprising dual β-lactams and a β-lactamase inhibitor achieve optimal synergistic inhibition of Mycobacterium abscessus growth

The historical model, which posits that {beta}-lactams inhibit bacterial growth while {beta}-lactamase inhibitors (BLIs) merely protect {beta}-lactams from enzymatic degradation, fails to fully explain their activity against Mycobacterium abscessus (Mab). This study demonstrates that synergistic effects extend beyond the traditional one {beta}-lactam+one BLI paradigm, refuting the oversimplified mechanistic framework. First, {beta}-lactam-based BLIs such as clavulanic acid, sulbactam, and tazobactam exhibit intrinsic antibacterial activity against Mab. These agents synergized not only with {beta}-lactams but also with one another, undermining their historical classification as mere {beta}-lactamase inhibitors. The data indicate that their activity is not limited to inhibiting {beta}-lactamases but extends to directly targeting critical bacterial processes. Second, dual {beta}-lactam combinations exhibit synergism against Mab even in the absence of BLIs. For example, despite being rapidly hydrolyzed by the native {beta}-lactamase BlaMab, amoxicillin demonstrates strong synergism with {beta}-lactams such as imipenem or ceftaroline. This suggests that the second {beta}-lactam either acts as a functional BLI surrogate or targets complementary pathways. Supporting this, experiments using penicillin- and carbapenem-based probes revealed that {beta}-lactams bind to multiple Mab proteins simultaneously, reinforcing the idea that their synergy arises from targeting complementary essential proteins. Finally, triple combinations comprising dual {beta}-lactam and one BLI, such as amoxicillin + ceftaroline + avibactam, achieved very high synergy, underscoring the complementary roles of dual {beta}-lactams and BLIs. The evidence in this study necessitates a revised model that can more accurately explain the activities of {beta}-lactams and BLIs and underscores the potential for optimizing {beta}-lactam/BLI regimens against Mab. IMPORTANCEThis research challenges old assumptions about how antibiotics fight bacteria, particularly Mycobacterium abscessus (Mab), a tough-to-treat infection. Traditionally, {beta}-lactam antibiotics were thought to stop bacterial growth, while {beta}-lactamase inhibitors (BLIs) just protected them from breakdown. However, this study reveals that BLIs like clavulanic acid can work together with another BLI or {beta}-lactam antibiotics for stronger effects. Surprisingly, even combinations comprising two BLIs can be highly effective, showing they target multiple critical bacterial processes simultaneously. Triple combinations--two {beta}-lactams and one BLI--proved especially powerful. These findings overturn outdated ideas, offering a smarter way to use these drugs to combat difficult infections and save lives.

microbiology↗

bioRxiv2024

Regimen comprising clarithromycin, clofazimine and bedaquiline is more efficacious than monotherapy in a mouse model of chronic Mycobacterium avium lung infection

Mycobacterium avium, a leading non-tuberculous mycobacterium (NTM) pathogen, causes chronic pulmonary infections, particularly in individuals with underlying lung conditions or immunosuppression. Current treatments involve prolonged multi-drug regimens with poor outcomes and significant side effects, highlighting the urgent need for improved therapies. Using a BALB/c mouse model of chronic M. avium pulmonary disease, we evaluated the efficacy of individual antibiotics-- clarithromycin, clofazimine, and rifabutin--and combination regimens including clarithromycin+bedaquiline and clarithromycin+clofazimine+bedaquiline. Clarithromycin demonstrated potent bactericidal activity, reducing lung bacterial burden by 2.2 log10 CFU, while clofazimine transitioned from bacteriostatic to bactericidal, achieving a 1.7 log10 CFU reduction. Rifabutin was bacteriostatic against M. avium MAC 101 but ineffective against MAC 104. The triple-drug regimen of clarithromycin+clofazimine+bedaquiline was the most effective, achieving a 3.3 log10 CFU reduction in bacterial load, with 98% clearance within the first week and continued efficacy over eight weeks. Gross pathology confirmed these results, with granulomatous lesions observed only in untreated or rifabutin-treated mice. Combination therapy demonstrated enhanced efficacy compared to monotherapy. The findings underscore the potential of oral clarithromycin+clofazimine+bedaquiline or clarithromycin+clofazimine regimen as a promising therapeutic strategy for M. avium pulmonary disease.

microbiology↗

bioRxiv2024

Proteomic characterization of Mycobacterium tuberculosis subjected to carbon starvation

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (TB), the leading cause of infectious-disease related deaths worldwide. TB infections present as a spectrum from active to latent disease. In the human host, Mtb faces hostile environments, such as nutrient deprivation, hypoxia, and low pH. Under these conditions, Mtb can enter a dormant, but viable, state characterized by a lack of cell replication and increased resistance to antibiotics. These dormant Mtb pose a major challenge to curing infections and eradicating TB globally. In the current study, we subjected Mtb to carbon starvation (CS), a culture condition that induces growth stasis and mimics nutrient-starved conditions associated with dormancy in vivo. We provide a detailed analysis of the proteome in CS compared to replicating samples. We observed extensive proteomic reprogramming, with 36% of identified proteins significantly altered in CS. Many enzymes involved in oxidative phosphorylation and lipid metabolism were retained or upregulated in CS. The cell wall biosynthetic machinery was present in CS, although numerous changes in the abundance of peptidoglycan, arabinogalactan, and mycolic acid biosynthetic enzymes likely result in pronounced remodeling of the cell wall. Many clinically approved anti-TB drugs target cell wall biosynthesis, and we found that these enzymes were largely retained in CS. Lastly, we compared our results to those of other dormancy models and propose that CS produces a physiologically-distinct state of stasis compared to hypoxia in Mtb.

microbiology↗

bioRxiv2021

Allosteric cooperation in β-lactam binding to a non-classical transpeptidase

Mycobacterium tuberculosis peptidoglycan (PG) is atypical as its synthesis involves a new enzyme class, L,D-transpeptidases. Prior studies of L,D-transpeptidases have identified only the catalytic site that binds to peptide moiety of the PG substrate or {beta}-lactam antibiotics. This insight was leveraged to develop mechanism of its activity and inhibition by {beta}-lactams. Here we report identification of an allosteric site at a distance of 21 [A] from the catalytic site that binds the sugar moiety of PG substrates (hereafter referred to as the S-pocket). This site also binds a second {beta}-lactam molecule and influences binding at the catalytic site. We provide evidence that two {beta}-lactam molecules bind co-operatively to this enzyme, one non-covalently at the S-site and one covalently at the catalytic site. This dual {beta}-lactam binding phenomenon is previously unknown and is an observation that may offer novel approaches for the structure-based design of new {beta}-lactam antibiotics for M. tuberculosis.

biochemistry↗

bioRxiv2021

Glby, is a PBP with β-lactamase activity and is required for in vivo viability of M. abscessus

The prevalence of Mycobacteroides abscessus, Mab, (also known as Mycobacterium abscessus) has been increasing steadily globally. Patients with structural lung conditions such as bronchiectasis, cystic fibrosis and chronic obstructive pulmonary disease are at high risk of developing pulmonary Mab disease. The disease is often recurrent as the current treatment regimen is considered sub-efficacious. The cell wall peptidoglycan of bacteria is required for their viability and its biosynthetic pathway is enriched in proteins whose inhibition is the basis for two of the most widely used classes of antibiotics to treat bacterial infections. The peptidoglycan of Mab is distinct from that of most bacteria as its synthesis involves penicillin binding proteins (PBP) and L,D-transpeptidases. Here, we demonstrate that Mab gene locus MAB_3167c encodes a PBP (hereafter referred to as Glby) and is required for normal planktonic growth in liquid broth. Glby exhibits a strong {beta}-lactamase activity and is sensitive to {beta}-lactamase inhibitors. In a mouse model of pulmonary Mab disease, mutant lacking this gene was unable to proliferate, gradually cleared and undetectable after three weeks. In a collection of 1.046 Mab clinical isolates, there is evidence that changes in amino acid sequence that compromise Glby function are not favored. These evidences suggest that an agent that can inhibit Glby in vivo has the potential to be an efficacious treatment against Mab disease.

microbiology↗

bioRxiv2020

In Silico Analysis of Secondary Metabolites that Modulates Enzymes of Cholesterol Target

Hypercholesterolemia has posed a serious threat of heart diseases and stroke worldwide. Xanthine oxidase (XO), the rate-limiting enzyme in uric acid biosynthesis, is regarded as the root of reactive oxygen species (ROS) that generates atherosclerosis and cholesterol crystals. {beta}-Hydroxy {beta}-methylglutaryl-coenzyme A reductase (HMGR) is a rate-limiting enzyme in cholesterol biosynthesis. Although some commercially available enzyme inhibiting drugs have effectively reduced the cholesterol level, most of them have failed to meet the requirements of being apt drug candidates. Here, we have carried out an in-silico analysis of secondary metabolites that have already shown good inhibitory activity against XO and HMGR. Out of 118 secondary metabolites reviewed, sixteen molecules inhibiting XO and HMGR were taken based on IC50 values reported in vitro assays. Further, receptor-based virtual screening was carried out against secondary metabolites using GOLD Protein-Ligand Docking Software, combined with subsequent post-docking, to study the binding affinities of ligands to the enzymes. In-Silico ADMET analysis was carried out to study their pharmacokinetic properties, followed by toxicity prediction through ProTox-II. The molecular docking of amentoflavone (1) (GOLD score 70.54), and ganomycin I (9) (GOLD score 59.61) evinced that the drug has effectively bind at the competitive site of XO and HMGR, respectively. Besides, 6-paradol (3) and selgin (4) could be potential drug candidates to inhibit XO. Likewise, n-octadecanyl-O--D-glucopyranosyl(6[->]1")-O--D-glucopyranoside (10) could be potential drug candidates to maintain serum cholesterol. In-silico ADMET analysis showed that the sixteen metabolites were optimal within the categorical range in comparison to commercially available XO and HMGR inhibitors, respectively. Toxicity analysis through Protox-II revealed that 6-gingerol (2), ganoleucoin K (11), and ganoleucoin Z (12) are toxic for human use. This computational analysis supports earlier experimental evidence towards the inhibition of XO and HMGR by natural products. Further study is necessary to explore the clinical efficacy of these secondary molecules, which might be alternatives for the treatment of hypercholesterolemia. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=163 HEIGHT=200 SRC="FIGDIR/small/219998v2_ufig1.gif" ALT="Figure 1"> View larger version (59K): org.highwire.dtl.DTLVardef@48d66borg.highwire.dtl.DTLVardef@1f51741org.highwire.dtl.DTLVardef@1c9d87dorg.highwire.dtl.DTLVardef@a41e70_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2019

Select β-lactam combinations exhibit synergy against Mycobacterium abscessus in vitro

Mycobacterium abscessus (Mab) is a nontuberculous mycobacterium that causes invasive pulmonary infections in patients with structural lung disease. Mab is intrinsically resistant to several classes of antibiotics and an increasing number of strains isolated from patients exhibit resistance to most antibiotics considered for treatment of Mab infections. Therefore, there is an unmet need for new regimens with improved efficacy to treat this disease. Synthesis of the essential cell wall peptidoglycan in Mab is achieved via two enzyme classes, L,D- and D-D-transpeptidases, with each class preferentially inhibited by different subclasses of {beta}-lactam antibiotics. We hypothesized that a combination of two {beta}-lactams that comprehensively inhibit the two enzyme classes will exhibit synergy in killing Mab. Paired combinations of antibiotics tested for in vitro synergy against Mab included dual {beta}-lactams, a {beta}-lactam and a {beta}-lactamase inhibitor, and a {beta}-lactam and a rifamycin. Of the initial 206 combinations screened, 24 pairs exhibited synergy. 13/24 pairs were combinations of two {beta}-lactams. 12/24 pairs brought the minimum inhibitory concentrations of both drugs to within the therapeutic range. Additionally, synergistic drug pairs significantly reduced the frequency of selection of spontaneous resistant mutants. These novel combinations of currently-available antibiotics may offer viable immediate treatment options against highly-resistant Mab infections.

microbiology↗

bioRxiv2018

In vitro activity of the new β-lactamase inhibitors relebactam and vaborbactam in combination with β-lactams against Mycobacterium abscessus complex clinical isolates

Pulmonary disease due to infection with Mycobacterium abscessus complex (MABC) is notoriously difficult to treat, in large part due to MABCs intrinsic resistance to most antibiotics, including {beta}-lactams. MABC organisms express a broad-spectrum {beta}-lactamase that is resistant to traditional {beta}-lactam-based {beta}-lactamase inhibitors but inhibited by a newer non-{beta}-lactam-based {beta}-lactamase inhibitor, avibactam. Consequently, the susceptibility of MABC to some {beta}-lactams is increased in the presence of avibactam. Therefore, we hypothesized that two new non-{beta}-lactam-based {beta}-lactamase inhibitors, relebactam and vaborbactam, would also increase susceptibility of MABC to {beta}-lactams. The objective of the present study was to evaluate the in vitro activity of various marketed {beta}-lactams alone and in combination with either relebactam or vaborbactam against multidrug-resistant MABC clinical isolates. Our data demonstrate that both {beta}-lactamase inhibitors significantly improved the anti-MABC activity of many carbapenems (including imipenem and meropenem) and cephalosporins (including cefepime, ceftaroline, and cefuroxime). As a meropenem/vaborbactam combination is now marketed and an imipenem/relebactam combination is currently in phase III trials, these fixed combinations may become the {beta}-lactams of choice for the treatment of MABC infections. Furthermore, given the evolving interest in dual {beta}-lactam regimens, our results identify select cephalosporins, such as cefuroxime, with superior activity in the presence of a {beta}-lactamase inhibitor, deserving of further evaluation in combination with these carbapenem/{beta}-lactamase inhibitor products.

microbiology↗

bioRxiv2018

Progressive Mycobacterium abscessus lung infection in C3HeB/FeJ mice associated with corticosteroid administration

Mycobacterium abscessus (Mab) is a rapidly-growing nontuberculous mycobacterium that is a growing health concern among both immunocompetent and immunocompromised patient populations. It most commonly causes skin and soft tissue or pulmonary infection. As an emerging health issue there is much that still needs to be understood about the infection and its progression to disease. In the context of pulmonary infection, an in vivo system of Mab infection that permits investigation of host-microbe interactions that result in Mab infection and the transition to pathogenesis, and also the evaluation of treatments, is an essential tool that is currently lacking. Here, we describe a system of pulmonary Mab infection in the C3HeB/FeJ mouse strain under corticosteroid immunosuppressive therapy that progresses to pathology.

microbiology↗