Functional characterization of novel mycobacterial zinc metalloprotease MSMEG3019 and its role in bacterial physiology.

Abstract

Tuberculosis (TB), the infectious disease caused by the pathogenic bacteria Mycobacterium tuberculosis (Mtb), remains amongst the ten leading causes of death worldwide. Although TB is preventable and curable, approximately 10 million people are diagnosed with TB and 1.5 million TB fatalities are reported annually globally. Complete eradication of TB remains a challenge due to its ability to establish a latent infection and its highly effective virulence mechanisms which facilitates manipulation and colonization of the host, as well as cause subsequent suppression and evasion of the host’s immune system. Furthermore, current TB treatment strategies face numerous limitations, such as socio-economic barriers and the emergence of drug resistant TB strains. The pathogenic success of Mtb can be attributed to its stellar virulence factors, one of which are zinc metalloproteases, which are proteases that catalyze the hydrolysis of proteins into peptides by the use of an indispensable zinc ion. In mycobacteria, zinc metalloproteases play essential roles in the intracellular survival of Mtb in host macrophages. Therefore, investigating other novel zinc metalloproteases, such as Rv2568c, is of significant interest. In this study, the Rv2568c ortholog MSMEG3019 was investigated in M. smegmatis mc2155, which is a model organism for TB research. The characterization of MSMEG3019 involved the creation of a deletion mutant strain, named MΔ3019, which harbored a non-functional version of MSMEG3019. The gene deletion method utilized in this study was the two-step allelic exchange method, this was followed by analysis and comparison of the resultant phenotype, to the wild type and complementation strains. Bioinformatics analyses revealed the presence of zinc metalloprotease and zinc ribbon domains inMSMEG3019 and Rv2568c, in addition to predicting protein-protein interactions with transglutaminase genes directly upstream. Bioinformatics were also utilized to identify proteins with structural homology to the target genes. These homologs were involved in pathogenesis of their respective species, which indicates Rv2568c’s involvement in Mtb virulence. MΔ3019 exhibited a reduced capacity to support the exponential phase of mycobacterial growth. Additionally, MΔ3019 cells displayed increased lengths and decreased widths, when compared to mc2155. MSMEG3019 was also discovered to be implicated in mycobacterial translocation, as evidenced by MΔ3019’s impaired sliding capability. Furthermore, MΔ3019 presented increased susceptibility to the peptidoglycan-targeting drug, Vancomycin. These phenotypic attributes were correlated to the disruption of peptidoglycan synthesis/regulation as the result of MSMEG3019 deletion. The observations made in this study suggests that MSMEG3019 is implicated in peptidoglycan-mediated mycobacterial growth, proliferation and dissemination, which represents Rv2568c’s contribution to pathogenesis in Mtb. The findings of this study demonstrated the importance of zinc metalloproteases to mycobacterial viability and physiology, thereby further corroborating that zinc metalloproteases remain an excellent reservoir of drug targets for TB drug development.