Biology subjects

Yusuf, B.

Publications and source records attributed to Yusuf, B..

10 recordsLinked to original sources

bioRxiv2026

A synonymous mutation in MSMEG_4729 occurs at a high frequency in spontaneous D29-resistant mutants of Mycobacterium smegmatis

Compassionate use of mycobacteriophage therapy highlights the promising potential of phage therapy as an alternative treatment option for antibiotic-resistant infections when conventional treatments fail. However, realizing the full potential of phage therapy requires addressing key challenges, including host immune responses, the limited arsenal of therapeutically-useful mycobacteriophages, and the emergence of phage resistance. Dissecting the mechanisms of phage resistance is critical for ensuring the effectiveness and sustainability of phage therapy. In this study, we demonstrate that exposure to the lytic mycobacteriophage D29 triggers diverse genetic changes in Mycobacterium smegmatis. A synonymous mutation in MSMEG_4729 arises frequently but is insufficient to confer D29 resistance on its own. Instead, we identified possible Lsr2-independent activation of the lipooligosaccharide (LOS) biosynthesis cluster in a D29-resistant mutant harboring this mutation. We have also detected the possible activity of MSMEG_3213, a type II methyltransferase associated with m6A modifications in M. smegmatis. Finally, we isolated defense escape mutants (DEMs) of D29 capable of overcoming resistance in a strain with the MSMEG_4729 synonymous mutation. This profiling of M. smegmatiss likely defensive arsenal against the therapeutically-useful mycobacteriophage D29 provides a roadmap for further investigations and rational engineering of next-generation mycobacteriophages to combat drug-resistant mycobacterial infections. Impact statementInterest in phage therapy has been gaining traction recently, which is largely due to the serious threat of antimicrobial resistance. However, the efficacy and sustainability of phage therapy is threatened by certain challenges, which includes the ever existent threat of phage resistance. In this study, we identified several likely factors involved in D29 interaction with the model mycobacterium M. smegmatis. These findings set a roadmap for future investigations that would guide rational phage engineering to expand the currently limited arsenal of therapeutically useful mycobacteriophages as well as improve the efficiency of existing ones.

microbiology↗

bioRxiv2026

Likely role of promoter reconstitution in Mpr-mediated D29 resistance by Mycobacterium smegmatis

The multi-copy phage resistance gene (mpr) of M. smegmatis is a major factor in resistance to the lytic mycobacteriophage D29. Mpr is a membrane-bound exonuclease that cleaves phage DNA post injection, hence blocking downstream stages in the phage infection cycle. The mechanism of resistance allows for adsorption, is non-abortive and independent of any mutation in the gene. Rather, it depends on overexpression of a wild type copy of the gene. However, the underlying factor behind mpr overexpression in spontaneous D29-resistant mutants of M. smegmatis remained elusive. Here, we report that D29 infection triggers insertion sequence (IS) rearrangements, including the transposition and integration of IS6120 directly upstream of mpr. Mutants with IS6120 integration upstream of mpr show highly elevated Mpr expression. Whole genome sequence analysis reveals that IS6120 introduced a putative transcription factor-binding site and a canonical -35 promoter element at the integration site, hence reconstituting a fuller promoter (rcp) than the original promoter (wtp) at the site. Promoter reporter assays suggest that rcp is a far stronger promoter than wtp, implying that elevated mpr expression in D29-resistant mutants with this transposition event could be due to promoter reconstitution. While strains with this transposition event appear to grow normally, rcp-driven, vector-borne Mpr overexpression appears to be toxic as it barely allows for colony formation on agar plates. This study reports a previously unknown factor likely behind mpr regulation in M. smegmatis, adding to the existing knowledge of mycobacterial anti-phage defense mechanisms and guiding rational phage engineering efforts for therapeutic applications.

microbiology↗

bioRxiv2025

A division-associated envelope protein, MAB_2363, drives intrinsic resistance and virulence in Mycobacterium abscessus

Mycobacterium abscessus exhibits intrinsic resistance to conventional antibiotics, significantly limiting treatment options. Our previous studies established that MAB_2362 (SteA) is a key regulator of cell division that contributes to intrinsic resistance and virulence. Considering that SteA-like proteins often act alongside SteB counterparts, we hypothesized that the adjacent gene MAB_2363 encodes the corresponding SteB-like division regulator. In this study, we found that deletion of MAB_2363 significantly increased susceptibility to multiple antibiotics and disrupted cell wall permeability. Microscopy revealed striking cell division defects in the mutant, including elongated cell morphology and multiple septa. Subcellular localization of a GFP-MAB_2363 fusion protein demonstrated its enrichment at division septa, confirming its direct involvement in cell division. Deletion of MAB_2363 resulted in attenuated virulence, as evidenced by reduced bacterial survival in macrophages and murine infection models. To assess its functional relation with MAB_2362, we compared the single-deletion mutant of MAB_2363 with the single-deletion mutant of MAB_2362 and the double-deletion mutant of MAB_2362-MAB_2363. Notably, the phenotypes of the MAB_2363 mutant, including cell division defects, antibiotic susceptibility, and virulence, were markedly milder than those of the other two mutants. Collectively, these findings indicate that MAB_2363 functions as a secondary but essential division-associated factor that operates during cell division, thereby influencing intrinsic resistance and virulence in M. abscessus.

microbiology↗

bioRxiv2025

Targeting homologous recombination repair to potentiate fluoroquinolone efficacy in Mycobacterium abscessus

Synthetic antibiotics, fluoroquinolones (FQs), are extensively important treatment options for fast growing mycobacterial infections. However, their widespread use has led to decreased efficacy due to intrinsic or acquired resistance. FQs exhibit antimicrobial activity by stabilizing type II topoisomerases-generated DNA breaks, thereby inducing lethal double-strand breaks (DSBs) and leading to bacterial death. Escalating resistance among mycobacterial pathogens highlights the need to elucidate resistance mechanisms and identify novel strategies to restore or enhance the drug activity. Here, we revealed homologous recombination (HR) as a key determinant of FQ tolerance in Mycobacterium abscessus, a notoriously drug-resistant pathogen. Through a transposon mutagenesis screen, we identified that disruption of adnB - an HR-associated gene, markedly sensitized M. abscessus to FQs. This finding prompted a systematic dissection of DSB repair pathways in M. abscessus. Targeted deletion of HR core components (adnB, recO, recA, recR, ruvB) significantly increased FQ susceptibility in vitro, while inactivation of alternative DSB repair pathways - single-strand annealing (SSA) and non-homologous end joining (NHEJ) - had no effect, revealing a unique reliance on HR for FQ tolerance. Furthermore, the complementation of gene knock-out M. abscessus strains {Delta}adnB, {Delta}recO, {Delta}recA, {Delta}recR or {Delta}ruvB with the corresponding genes from M. abscessus, as well as homologous genes from Mycobacterium tuberculosis restored the resistance phenotype, indicating a conserved HR-dependent tolerance mechanism in M. abscessuss. In a murine infection model, genetic abrogation of HR significantly improved the therapeutic efficacy of FQs against M. abscessus, demonstrating translational relevance. Collectively, our findings position HR as a conserved and actionable vulnerability in M. abscessus, and provide a compelling rationale for developing HR-targeted adjuvant therapies to resensitize drug-refractory strains to FQ treatment. Impact statementFluoroquinolone (FQ) resistance poses a major challenge in the treatment of Mycobacterium abscessus infections. This study identifies homologous recombination (HR) as a key defense mechanism that limits FQ efficacy across multiple mycobacterial species. Genetic inhibition of HR not only sensitizes M. abscessus to FQs in vitro but also enhances antibiotic activity in vivo. These results highlight HR repair as a promising therapeutic target to potentiate FQ activity and combat drug resistance in M. abscessus infections.

molecular biology↗

bioRxiv2025

The stress response factor SigH mediates intrinsic resistance to multiple antibiotics in Mycobacterium abscessus

Mycobacterium abscessus (Mab) causes pulmonary diseases with limited treatment options due to its high level of intrinsic resistance to available drugs. Mab possesses complex and poorly understood drug resistance mechanisms. Identifying new drug targets and gaining a deeper understanding of drug resistance mechanisms are essential for discovering novel therapeutic alternatives. Here, we investigated the role of a putative sigma factor SigH in intrinsic multi-drug resistance in Mab. Mab SigH shares an 84% peptide sequence identity with Mycobacterium tuberculosis (Mtb) SigH, a well-known stress response protein and global transcriptional regulator. We constructed a sigH gene deletion strain of Mab ({Delta}sigH) and complemented strains by expressing either Mab sigH (CPMabsigH) or Mtb sigH (CPMtbsigH) in {Delta}sigH. The {Delta}sigH strain exhibited hypersensitivity to a broad range of antibiotics, including levofloxacin, moxifloxacin, tigecycline, tetracycline, amikacin, vancomycin, and rifabutin and all complemented strains restored the drug resistance phenotype. Additionally, {Delta}sigH showed increased sensitivity to oxidative and heat stress compared to the wild-type Mab and complemented strains. Transcriptomic analysis revealed that deletion of sigH disrupted the balance of gene expression, primarily elevating the expression of genes encoding YrbE and MCE family proteins and downregulating genes expressing ABC-type transporters, sigma and anti-sigma factors and other genes associated with antimicrobial resistance. Collectively, our findings indicate that SigH is a key regulator of global gene expression in response to environmental stresses, including antimicrobial treatment, and is crucial for the intrinsic drug resistance of Mab. SigH represents a promising target for the development of novel therapeutic strategies against Mab infections.

molecular biology↗

bioRxiv2024

MtrAB two-component system is crucial for the intrinsic resistance and virulence of Mycobacterium abscessus

Mycobacterium abscessus (Mab) poses serious therapeutic challenges, principally due to its intrinsic resistance to a wide array of antibiotics. The pressing issue of drug resistance has spurred an urgent need to explore novel targets and develop new therapeutic agents against Mab. The MtrAB two-component system, conserved among Actinobacteria, is pivotal for regulating various metabolic processes. Nevertheless, the role of MtrAB in Mab remains elusive. In this study, we uncovered that Mab strains with disrupted mtrA, mtrB or both exhibited heightened susceptibility to a variety of antibiotics with diverse mechanisms of action, in contrast to the wild-type strain. In a murine model, rifabutin, bedaquiline, and amikacin, which were inactive against the wild-type Mab strain, demonstrated efficacy against all the mtrA, mtrB and mtrAB knockout strains, significantly reducing pulmonary bacterial burdens compared to vehicle controls after ten days of treatment. Notably, the virulence of all the mtrA, mtrB, and mtrAB knockout strains was highly diminished in the murine model, as evidenced by a substantial decrease in bacterial load in the lungs of mice after 16 days. We observed that all three knockout strains exhibited a significantly reduced growth rate compared to the wild-type strain. We discovered that cells lacking mtrA, mtrB or both exhibited an elongated cell length and had multiple septa, suggesting that both MtrA and MtrB regulate cell division of Mab. Subsequently, an ethidium bromide accumulation assay disclosed that the absence of either mtrA or mtrB or both significantly increased cell envelope permeability. In summary, this study suggests that mtrA and mtrB play an important role in the intrinsic resistance and virulence of Mab by affecting cell division and altering cell permeability. Consequently, MtrA and MtrB represent promising targets for the discovery of anti-Mab drugs. HIGHLIGHTSO_LIKnockout of mtrA, mtrB or mtrAB leads to increased sensitivity of M. abscessus in vitro and in vivo. C_LIO_LIThe mtrA, mtrB or mtrAB knockout M. abscessus strains exhibit highly reduced virulence. C_LIO_LIMtrA and MtrB are potential targets for anti-M. abscessus drug discovery. C_LIO_LIKnockout of mtrA, mtrB or mtrAB results in defective cell division in M. abscessus. C_LI

microbiology↗

bioRxiv2024

High-throughput Screening of Compounds against Autoluminescent Nonreplicating Mycobacterium tuberculosis under Diverse Conditions

The screening of new anti-mycobacterial chemicals is primarily focused on inhibiting the active growing bacteria. However, a major challenge in tuberculosis control is the ability of Mycobacterium tuberculosis to enter a nonreplicating state for extended periods, rendering it resistant to many clinical drugs and complicating eradication efforts. Existing low-oxygen-recovery assays designed for screening compounds targeting nonreplicating M. tuberculosis have limitations, including the colony-forming unit counting for non-luminous M. tuberculosis and the instability of the free plasmid carrying luxAB genes in luminescent M. tuberculosis, along with exogenous substrate requirements for light producing. Moreover, these assays fail to accurately replicate the growth conditions of nonreplicating M. tuberculosis in vitro, thus resulting in less convincing results. To address these challenges, we have developed an autoluminescence-based, cholesterol-enriched culture evaluation model to assess 17 anti-tuberculosis drugs of different classes against nonreplicating M. tuberculosis. Our findings indicate that the relative light unit, measured in real-time, serves as a reliable surrogate marker for colony-forming unit, which typically becomes available one month later. This suggests the utility of our model for the rapid determination of drug susceptibility dynamically. The autoluminescent M. tuberculosis, harbouring luxCDABE gene cluster within its genome, can emit blue-green light stably and autonomously without requiring an external substrate supplement. The minimal inhibitory concentrations of all the drugs tested under anaerobic conditions are significantly different from that detected in aerobic environment. Our model allows for rapid, precise, and efficient assessment of drug activity under anaerobic conditions, thereby enabling a more comprehensive evaluation of anti-mycobacterial efficacy. Overall, our model represents a significant advancement in anti-tuberculosis drug discovery and pharmaceutical development.

microbiology↗

bioRxiv2021

Endothelial integration of mechanosensory signals by the spectrin cytoskeleton

Physiological blood flow induces the secretion of vasoactive compounds, notably NO, and promotes endothelial cell elongation and reorientation parallel to the direction of applied shear. How shear is sensed and relayed to intracellular effectors is incompletely understood. We demonstrate that an apical spectrin network is essential to convey the force imposed by shear to endothelial mechanosensors. By anchoring CD44, spectrin modulates the cell surface density of hyaluronan, which senses and translates shear into changes in plasma membrane tension. Spectrins also regulate the stability of apical caveolae, where the mechanosensitive Piezo1 channels are thought to reside. Accordingly, shear-induced Piezo1 activation and the associated calcium influx were absent in spectrin-deficient cells. As a result, cell realignment and flow-induced eNOS stimulation were similarly dependent on spectrin. We concluded that the apical spectrin network is not only required for shear sensing, but transmits and distributes the resulting tensile forces to mechanosensors that elicit protective and vasoactive responses.

cell biology↗

bioRxiv2021

Sterilizing Effects of Novel Regimens Containing TB47, Clofazimine and Linezolid in a Murine Model of Tuberculosis.

Toxicity and inconvenience associated with the use of injectable drug-containing regimens for tuberculosis (TB) have made all-oral regimens a preferred alternative. Widespread resistance to fluoroquinolones and pyrazinamide makes it essential to identify new drug candidates and study their effects on current regimens for TB. TB47 is a pyrazolo[1,5-a]pyridine-3-carboxamide with powerful synergistic in vitro and in vivo activities against mycobacteria, especially with clofazimine. Here, we investigated the bactericidal and sterilizing activities of novel oral regimens containing TB47 + clofazimine + linezolid, and the potential roles of levofloxacin and/or pyrazinamide in such drug combinations. Using a well-established mouse model, we assessed the effect of these regimens on bacterial burden in the lung during treatment and relapse (4 months after stopping treatment + immunosuppression). Our findings indicate that the TB47 + clofazimine + linezolid + pyrazinamide, with/without levofloxacin, regimens had fast-acting (4 months) sterilizing activity and no relapse was observed. When pyrazinamide was excluded from the regimen, treatment times were longer (5-6 months) to achieve sterilizing conditions. We propose that TB47 + clofazimine + linezolid can form a highly sterilizing block for use as an alternative pan-TB regimen.

microbiology↗

bioRxiv2021

A recombinant selective drug-resistant M. bovis BCG enhances the bactericidal activity of a second-line tuberculosis regimen

Drug-resistant tuberculosis (DR-TB) results from infection by Mycobacterium tuberculosis strains resistant to at least rifampin or isoniazid. To improve the treatment outcome in DR-TB, therapeutic vaccines are considered an ideal choice as they can enhance pathogen clearance and minimize disease sequelae. To date, there is no therapeutic vaccine reported to be effective when combined with a chemotherapy regimen against DR-TB. The only available TB vaccine, the M. bovis BCG (BCG) is susceptible to several anti-TB drugs hence not a perfect option for therapeutic vaccination. Herein, we developed a recombinant BCG (RdrBCG) overexpressing Ag85B and Rv2628 with resistance to selected anti-TB drugs. When administered three times adjunct to a second-line anti-TB regimen in a classical murine model of DR-TB, the RdrBCG lowered lung M. tuberculosis colony-forming units by 1 log10. Furthermore, vaccination with the RdrBCG adjunct to TB chemotherapy minimized lung tissue pathology in mice. Most importantly, the RdrBCG maintained the exogenously inserted genes and showed almost the same virulence as its parent BCG Tice strain in severe combined immune-deficient mice. All these suggested that the RdrBCG was stable, safe and effective. Hence, the "recombinant" plus "drug-resistant" BCG strategy could be a useful concept for developing therapeutic vaccines against DR-TB.

microbiology↗