Biology subjects

Maziero, M.

Publications and source records attributed to Maziero, M..

4 recordsLinked to original sources

bioRxiv2026

The porin MspA is a phage-specific determinant of mycobacteriophage infection

Mycobacteriophages are being developed as therapeutics for mycobacterial infections, but their highly variable host ranges limit this application and the bacterial factors that determine whether infection succeeds remain poorly defined. The determinants characterized so far are dominated by cell envelope lipids and glycolipids. Here, we performed a genome-wide CRISPRi screen in Mycobacterium smegmatis to identify host functions associated with susceptibility to BPs{Delta}33HTH HRM10. After prolonged phage challenge, the outer membrane porin genes mspA and mspC were among the most strongly enriched hits, whereas the closely related paralogs mspB and mspD were not enriched despite sharing high amino-acid identity across the family. Deletion of mspA or mspC reduced BPs{Delta}33HTH HRM10 plaque formation and produced smaller, more turbid plaques, with the stronger phenotype following loss of mspA, the major outer membrane porin of M. smegmatis. Across a panel of 25 mycobacteriophages, 11 showed either a more than tenfold reduction in titer or loss of discrete plaque formation on {Delta}mspA, while the remainder were largely unaffected. These findings identify outer membrane porins as phage-dependent determinants of mycobacterial susceptibility, revealing an additional layer of specificity in phage infection beyond previously characterized cell envelope lipids and glycolipids.

microbiology↗

bioRxiv2026

Development of inducible genetic systems to finely tune efficacy of InhA-targeting anti-tuberculous drugs

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a leading global health threat, especially due to multidrug-resistant strains. The first-line drug isoniazid (INH) and second-line drugs ethionamide (ETH) and prothionamide (PTH), are prodrugs that target the essential enoyl-ACP reductase InhA, a key enzyme involved in mycolic acid biosynthesis. Resistance to these drugs primarily arises from genetic mutations that inactivate the enzymatic function of their respective bioactivating enzymes. While the recent development of chemical boosters has shown promising results in restoring drug susceptibility in these mutants, genetic tools to dynamically modulate these pathways and fine-tune resistance could be extremely valuable to develop the next-generation of anti-InhA drugs. In this context, we leverage CRISPR interference (CRISPRi) to develop inducible and reversible genetic systems to fine-tune antibiotic susceptibility in Mycobacterium smegmatis (Msmeg) and Mtb. By using our CRISPRi-based approach we first generated an inducible inhA hypomorph, an essential target required for mycolic acid production and bacterial growth, therefore confirming that our method is fully functional. Then, we developed genetic tools to selectively target katG, ethA, and the transcriptional repressor ethR, in order to generate conditional hypomorphs with altered antibiotic susceptibility profiles. Antibiotic susceptibility assays confirmed that the conditional hypomorphs selectively displayed high-level resistance or hypervulnerability to INH or ETH upon CRISPRi induction. Altogether, this work establishes CRISPRi as a powerful genetic platform for selectively fine-tuning mycobacterial drug susceptibility, with potential applications in developing next-generation anti-TB therapies.

microbiology↗

bioRxiv2025

Mycobacterial cell division arrest and smooth-to-rough envelope transition using CRISPRi-mediated genetic repression systems

The genetic basis underlying non-tuberculous mycobacteria (NTM) pathogenesis remains poorly understood. This gap in knowledge has been partially filled over the years through the generation of novel and efficient genetic tools, including the recently developed CRISPR interference (CRISPRi) technology. Our group recently capitalized on the well-established mycobacteria-optimized dCas9Sth1-mediated gene knockdown system to develop a new subset of fluorescence-based CRISPRi vectors that enable simultaneous controlled genetic repression and fluorescence imaging. In this Research Protocol, we use the model organism Mycobacterium smegmatis (M. smegmatis) as surrogate for NTM species and provide simple procedures to assess CRISPRi effectiveness. We describe how to evaluate the efficacy of gene-silencing when targeting essential genes but also genes involved in smooth-to-rough envelope transition, a critical feature in NTM pathogenesis. This protocol will have a broad utility for mycobacterial functional genomics and phenotypic assays in NTM species.

microbiology↗

bioRxiv2023

Fever-like temperature bursts promote competence development via an HtrA-dependent pathway in Streptococcus pneumoniae.

Streptococcus pneumoniae (the pneumococcus) is well known for its ability to develop competence for natural DNA transformation. Competence development is regulated by an autocatalytic loop driven by variations in the basal level of transcription of the comCDE and comAB operons. These genes are part of the early gene regulon that controls expression of the late competence genes known to encode the apparatus of transformation. Several stressful conditions are known to promote competence development, although the induction pathways are remain poorly understood so far. To date there have been no reports of the response of the pneumococcal competence regulon to changes in the physical conditions of the environment, oxidative potential and temperature. Here we demonstrate that transient temperature elevation induces an immediate increase in the basal expression level of the comCDE operon and early genes that, in turn, stimulates its full induction, including that of the late competence regulon. This thermal regulation depends on the HtrA chaperone/protease and its proteolytic activity. We find that certain other competence induction stimuli are not conveyed by the HtrA-dependent pathway. This finding strongly supports that competence can be induced by any of several independent pathways and thus reinforces the view that competence is a general stress response system in the pneumococcus. Authors summaryStreptococcus pneumoniae is a commensal bacterium and an opportunistic pathogen of humans. Certain environmental stimuli, such as a variety of antibiotics targeting distinct cellular functions, trigger the induction of the distinct physiological state of competence, in which cells can, among other things, import and integrate external DNA. Competence is thus considered a general stress response in this highly adaptable species. To understand the role of competence in pneumococcal interaction with its host and in pathogenicity, we attempted to decipher the pathways that enable appropriate reactions to environmental stress, and have focused here on induction of competence by elevation of temperature to levels similar to that of a host in fever. We found that elevated temperature raises the basal expression level of the competence control operon, and thus lowers the threshold of transition to full competence induction. By genetic characterisation of the thermal induction pathway of competence, we demonstrated that the chaperone/protease HtrA is essential to relaying of the thermal signal but is not involved in transmitting other stimuli such as those arising from the presence of certain antibiotics. Our work supports the view that competence can be induced through various pathways in response to distinct aggressions, but also by fever-like bursts of temperature that the pneumococcus could face in its natural habitat.

microbiology↗