Deciphering the Role of <i>pafBC</i> in Mycobacteriophage Resistance and Biofilm Formation

Tuberculosis (TB) remains the world's deadliest bacterial infection, with 8.2 million newly notified cases and an estimated 1.25 million deaths in 2023. Alarmingly, ∼19% of multidrug- or rifampicin-resistant (MDR/RR) strains already meet the World Health Organization (WHO) definition of pre-XDR-TB because they are resistant to at least one fluoroquinolone (FQ). Although <b>gyrA/gyrB</b> target-site mutations dominate clinical FQ resistance, <i>Mycobacteria</i> also rely on transcriptional networks that help them withstand the oxidative and DNA strand-breaking stress caused by these drugs. Central to this response is the heterodimeric transcription factor <i>pafBC</i><b>,</b> whose WYL domain binds to single-stranded DNA and redirects RNA polymerase to a dedicated promoter set, thereby orchestrating a LexA-independent DNA-damage response (DDR). Up-regulation of <b><i>pafBC</i></b> has been linked to enhanced intracellular survival of <i>M. tuberculosis</i> and nontuberculous mycobacteria after FQ exposure, yet the downstream phenotypes and their connection to drug or phage resistance have remained unclear. Here, we demonstrate that deletion of <b><i>pafBC</i></b> in <i>Mycobacterium smegmatis</i> profoundly remodels the cell envelope, as evidenced by altered colony rugosity, reduced sliding motility, enhanced aggregation, and a three- to 5-fold decline in quantitative biofilm biomass. Untargeted lipid profiling revealed the selective depletion of long-chain trehalose polyphosphates and other apolar glycolipids that normally decorate the outer membrane─lipid classes that have recently been shown in other studies to serve as essential receptors for therapeutic mycobacteriophages such as BPs and Muddy. Consistent with this lipid deficit, the <i>pafBC</i> mutant exhibited markedly reduced phage adsorption and plaque formation; ectopic expression of <b><i>RecA</i></b> restored adsorption efficiency, implicating DDR envelope crosstalk in antiphage defense. Complementation with wild-type <b><i>pafBC</i></b> rescued lipid composition, biofilm mass, and phage resistance, whereas a WYL-domain mutant that cannot bind single-stranded DNA failed to do so, underscoring the necessity of canonical <i>pafBC</i> activation for envelope homeostasis. Immunoprofiling in THP-1 macrophages further showed that <i>pafBC</i><b>-proficient</b> bacilli induce significantly higher secretion of <i>IL-1</i>β, <i>TNF-</i>α, and <i>IL-6</i> compared to their isogenic mutant. This effect correlated with the presence of intact surface glycolipids, molecules known to interact with scavenger and Toll-like receptors on phagocytes and to enhance opsonizing antibody deposition at the host-pathogen interface. Overall, our findings connect the molecular mechanisms of the <i>pafBC</i> DDR with observable phenotypes such as fluoroquinolone tolerance, biofilm structure, phage resistance, and host immune recognition, by highlighting cell-envelope remodeling as the central factor.