Collagen-like Motifs of SasG: A Novel Fold for Protein Mechanical Strength.

The Staphylococcus aureus surface protein G (SasG) is associated with host colonisation and biofilm formation. As colonisation occurs at the liquid-substrate interface bacteria are subject to a myriad of external forces and, presumably as a consequence, SasG displays extreme mechanical strength. This mechanical phenotype arises from the B-domain; a repetitive region composed of alternating E and G5 subdomains. These subdomains have an unusual structure comprising collagen-like regions capped by triple-stranded ß-sheets. To identify the determinants of SasG mechanical strength, we characterised the mechanical phenotype and thermodynamic stability of 18 single substitution variants of a pseudo-wildtype protein. Visualising the mechanically-induced transition state at a residue-level by ϕ-value analysis reveals that the main force-bearing regions are the N- and C-terminal 'Mechanical Clamps' and their side-chain interactions. This is tailored by contacts at the pseudo-hydrophobic core interface. We also describe a novel mechanical motif - the collagen-like region and show that glycine to alanine substitutions, analogous to those found in Osteogenesis Imperfecta (brittle bone disease), result in a significantly reduced mechanical strength.

Mechanism of catalysis and inhibition of Mycobacterium tuberculosis SapM, implications for the development of novel antivirulence drugs.

Mycobacterium tuberculosis (Mtb) SapM is a secreted virulence factor critical for intracellular survival of the pathogen. The role of SapM in phagosome maturation arrest in host macrophages suggests its potential as a drug target to assist in the clearance of tuberculosis infection. However, the mechanism of action of SapM at the molecular level remains unknown. In this study, we provide new insights into the mechanism of catalysis, substrate specificity and inhibition of SapM, and we identify the critical residues for catalysis and substrate binding. Our findings demonstrate that SapM is an atypical monoester alkaline phosphatase, with a serine-based mechanism of catalysis probably metal-dependent. Particularly relevant to SapM function and pathogenesis, is its activity towards PI(4,5)P2 and PI3P, two phosphoinositides that function at the early stages of microbial phagocytosis and phagosome formation. This suggests that SapM may have a pleiotropic role with a wider importance on Mtb infection than initially thought. Finally, we have identified two inhibitors of SapM, L-ascorbic acid and 2-phospho-L-ascorbic, which define two different mechanisms by which the catalytic activity of this phosphatase could be regulated. Critically, we demonstrate that 2-phospho-L-ascorbic reduces mycobacterial survival in macrophage infections, hence confirming the potential of SapM as a therapeutic drug target.