Determining the functionality of the putative twin-arginine signal peptides of rhomboid proteases from Mycobacterium tuberculosis using the beta-lactamase reporter system

Abstract

Rhomboid proteases are a group of integral membrane proteases present in several organisms. Because of their roles in many medically important processes, rhomboid proteases are potential drug targets. In Mycobacteria species, rhomboid proteases have been highlighted to play a role in drug resistance. Besides, bioinformatics analyses revealed the presence of putative twin-arginine translocation (TAT) signal peptides within this family of rhomboid proteases. Thus, there was a clear need to understand rhomboid secretion pathways with the view of their utilization for Tuberculosis control. To determine the functionality of these signal peptides, this study employed a β-lactamase reporter system. Here, the partial DNA sequences encoding for the two putative TAT signal peptides were separately fused in-frame to the ‘BlaC gene, and the ability to restore β-lactamase phenotype in ΔblaS and ΔblaSΔtatA M. smegmatis mutants was determined. It was revealed that both rhomboid protease TAT signal peptide-’BlaC fusions were each capable of restoring the β-lactamase phenotype in the ΔblaS M. smegmatis mutants. However, the same fusions failed to restore the β-lactamase phenotype when expressed in the ΔblaSΔtatA M. smegmatis mutants. Although very sensitive, the β-lactamase phenotype represents a qualitative way to detect export. To have quantitative measurement, the β-lactamase activity on the transformed M. smegmatis mutants was performed using nitrocefin. It was further noted that both rhomboid protease TAT signal peptides efficiently restored the β-lactamase activity relative to the non-transformed ΔblaS M. smegmatis mutants (p≤ 0.05). Furthermore, it was observed that the rhomboid protease II TAT signal peptide-’BlaC fusion complemented the β-lactamase activity more efficiently relative to the rhomboid protease I TAT signal peptide-’BlaC fusion. However, the difference was not statistically significant. This study shows that the two putative rhomboid protease TAT signal peptides are indeed functional. Thus, rhomboid proteases I and II use these signal peptides to mediate their export across the cytoplasmic membrane via the TAT pathway in M. tuberculosis. However, their precise role once they reach their site of action is still unclear.