Biology subjects

Rimal, B.

Publications and source records attributed to Rimal, B..

7 recordsLinked to original sources

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

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↗

bioRxiv2023

A Taxonomically-informed Mass Spectrometry Search Tool for Microbial Metabolomics Data

MicrobeMASST, a taxonomically-informed mass spectrometry (MS) search tool, tackles limited microbial metabolite annotation in untargeted metabolomics experiments. Leveraging a curated database of >60,000 microbial monocultures, users can search known and unknown MS/MS spectra and link them to their respective microbial producers via MS/MS fragmentation patterns. Identification of microbial-derived metabolites and relative producers, without a priori knowledge, will vastly enhance the understanding of microorganisms role in ecology and human health.

microbiology↗

bioRxiv2018

A proteolytic complex targets multiple cell wall hydrolases in Pseudomonas aeruginosa

Carboxy-terminal processing proteases (CTPs) occur in all three domains of life. In bacteria some of them have been associated with virulence. However, the precise roles of bacterial CTPs are poorly understood and few direct proteolytic substrates have been identified. One bacterial CTP is the CtpA protease of Pseudomonas aeruginosa, which is required for type III secretion system function, and for virulence in a mouse model of acute pneumonia. Here, we have investigated the function of CtpA in P. aeruginosa and identified some of the proteins it cleaves. We discovered that CtpA forms a complex with a previously uncharacterized protein, which we have named LbcA (lipoprotein binding partner of CtpA). LbcA is required for CtpA activity in vivo and promotes its activity in vitro. We have also identified four proteolytic substrates of CtpA, all of which are uncharacterized proteins predicted to cleave the peptide cross-links within peptidoglycan. Consistent with this, a ctpA null mutant was found to have fewer peptidoglycan cross-links than the wild type and grew slowly in salt-free medium. Intriguingly, the accumulation of just one of the CtpA substrates was required for some {Delta}ctpA mutant phenotypes, including the defective T3SS. We propose that LbcA*CtpA is a proteolytic complex in the P. aeruginosa cell envelope, which controls the activity of several peptidoglycan cross-link hydrolases by degrading them. Furthermore, based on these and other findings we suggest that many bacterial CTPs might be similarly controlled by partner proteins as part of a widespread mechanism to control peptidoglycan hydrolase activity.\n\nIMPORTANCEBacterial carboxy-terminal processing proteases (CTPs) are widely conserved and have been associated with the virulence of several species. However, their roles are poorly understood and few direct substrates have been identified in any species. Pseudomonas aeruginosa is an important human pathogen in which one CTP, known as CtpA, is required for type III secretion system function, and for virulence. This work provides an important advance by showing that CtpA works with a previously uncharacterized binding partner to degrade four substrates. These substrates are all predicted to hydrolyze peptidoglycan cross-links, suggesting that the CtpA complex is an important control mechanism for peptidoglycan hydrolysis. This is likely to emerge as a widespread mechanism used by diverse bacteria to control some of their peptidoglycan hydrolases. This is significant, given the links between CTPs and virulence in several pathogens, and the importance of peptidoglycan remodeling to almost all bacterial cells.

microbiology↗