Impairment of two non-essential LytR-cpsA-psr (Lcp) cell wall ligases decelerate cell wall assembly in Mycolicibacterium smegmatis

Background: The LytR-CpsA-Psr (Lcp) family of proteins are highly abundant amongst actinomycetes and Gram-positive bacteria, and known to exist as multiple paralogs in an organism. Several reports demonstrated the essential LcpA to play a crucial role in coupling cell wall teichoic acids (arabinogalactan in mycobacteria) to peptidoglycan, making it an attractive drug target against tuberculosis (TB). However, apart from LcpB that accommodates capsular biosynthesis, role of the non-essential Lcp paralogs in mycobacteria largely remains elusive. Therefore, we made a comprehensive approach in characterizing the role of these non-essential Lcp proteins in mycobacterial cell wall assembly. Additionally, we used homology and interactive models as an alternate approach to X-ray crystallography, to identify hotspot residues for Lcp inhibition and subsequent bactericidal activity. Results: Single and double knockout strains of each of the three Mycolicibacterium smegmatis (M. smegmatis) non-essential lcp genes were generated, out of which ΔlcpBΔlcpC exhibited severely compromised cell envelope with impeding cell division, altered morphology, reduced biofilms, enhanced susceptibility to stress, loss of capsular ⍺-glucans and exposed Galf residues. The higher pyrophosphatase activity of LcpB and LcpC, their interaction in native conditions and reduced AG/PG markers in ΔlcpBΔlcpC, substantiates their role in AG-PG ligation. Higher expression of lcpB/lcpC irrespective of external stress indicates tight regulation and coping mechanism conferred by these two homologs on the mycobacterial cell. Cell wall targeting anti-TB drugs in combination with two Lcp inhibitory compounds, Entrectinib and Sorafenib, proved bactericidal. Conclusion: This study highlights the primary role of LcpB/LcpC alongside LcpA in maintaining structural integrity of the M. smegmatis cell envelope, and combined anti-Lcp drug strategy as a novel approach to inhibit mycobacterial replication.