A new role for lipoproteins LpqZ and FecB in orchestrating mycobacterial cell envelope biogenesis

Accounting for more deaths than any other bacterial species, <i>Mycobacterium tuberculosis</i> (Mtb) represents a critical threat to public health worldwide. A key factor contributing to Mtb's virulence is its unique cell envelope, which acts as a protective barrier. Among the components of this envelope, lipoproteins represent a critical but understudied group of proteins. In this study, we focused on 79 conserved putative lipoproteins, shared between Mtb and the closely related <i>M. marinum</i>. Leveraging the CRISPR/Cas9 gene editing system for Mycobacteria, we generated frameshift mutations, targeting one conserved lipoprotein-coding gene at a time. We identified two mutants, <i>lpqZ</i> and <i>fecB</i>, that exhibited increased susceptibility to all tested antibiotics, suggesting critical roles in cell envelope biogenesis. Interestingly, despite having homology to periplasmic substrate-binding proteins (SBPs), neither protein is associated with any inner membrane transporter complex. Instead, co-immunoprecipitation experiments revealed that LpqZ interacts with AftA and FecB interacts with AftB. Both these interaction partners are essential enzymes involved in arabinogalactan and lipoarabinomannan synthesis. Accordingly, we observed alterations for both glycoconjugates in <i>lpqZ</i> and <i>fecB</i> mutants. Together, these findings show that orphaned SBP-like proteins have been repurposed in mycobacteria to aid key enzymes involved in cell envelope biosynthesis.IMPORTANCETuberculosis, caused by <i>Mycobacterium tuberculosis</i> (Mtb), remains the world's deadliest bacterial infection, in part because the bacterium's unique cell envelope makes it highly resistant to antibiotics. Understanding how this protective barrier is built is essential for developing better treatments. In this study, we discovered that two previously uncharacterized lipoproteins help maintain the integrity of the mycobacterial cell envelope and contribute to drug resistance. Surprisingly, instead of acting as transport proteins as expected by structural similarity, these molecules regulate enzymes that assemble the bacterial envelope. This discovery highlights a previously unrecognized layer of control in envelope construction and opens new directions for targeting Mtb's defenses with future therapies.