Autolysin-mediated peptidoglycan hydrolysis is required for the surface display of Staphylococcus aureus cell wall-anchored proteins.

Peptidoglycan hydrolases, or autolysins, play a critical role in cell wall remodeling and degradation, facilitating bacterial growth, cell division, and cell separation. In Staphylococcus aureus, the so-called "major" autolysin, Atl, has long been associated with host adhesion; however, the molecular basis underlying this phenomenon remains understudied. To investigate, we used the type V glycopeptide antibiotic complestatin, which binds to peptidoglycan and blocks the activity of autolysins, as a chemical probe of autolysin function. We also generated a chromosomally encoded, catalytically inactive variant of the Atl enzyme. Autolysin-mediated peptidoglycan hydrolysis, in particular Atl-mediated daughter cell separation, was shown to be critical for maintaining optimal surface levels of S. aureus cell wall-anchored proteins, including the fibronectin-binding proteins (FnBPs) and protein A (Spa). As such, disrupting autolysin function reduced the affinity of S. aureus for host cell ligands, and negatively impacted early stages of bacterial colonization in a systemic model of S. aureus infection. Phenotypic studies revealed that Spa was sequestered at the septum of complestatin-treated cells, highlighting that autolysins are required to liberate Spa during cell division. In summary, we reveal the hydrolytic activities of autolysins are associated with the surface display of S. aureus cell wall-anchored proteins. We demonstrate that by blocking autolysin function, type V glycopeptide antibiotics are promising antivirulence agents for the development of strategies to control S. aureus infections.

A genetic platform to investigate the functions of bacterial drug efflux pumps.

Efflux pumps are a serious challenge for the development of antibacterial agents. Overcoming efflux requires an in-depth understanding of efflux pump functions, specificities and the development of inhibitors. However, the complexities of efflux networks have limited such studies. To address these challenges, we generated Efflux KnockOut-35 (EKO-35), a highly susceptible Escherichia coli strain lacking 35 efflux pumps. We demonstrate the use of this strain by constructing an efflux platform comprising EKO-35 strains individually producing efflux pumps forming tripartite complexes with TolC. This platform was profiled against a curated diverse compound collection, which enabled us to define physicochemical properties that contribute to transport. We also show the E. coli drug efflux network is conditionally essential for growth, and that the platform can be used to investigate efflux pump inhibitor specificities and efflux pump interplay. We believe EKO-35 and the efflux platform will have widespread application for the study of drug efflux.

A Key Glycine in Bacterial Steroid-Degrading Acyl-CoA Dehydrogenases Allows Flavin-Ring Repositioning and Modulates Substrate Side Chain Specificity.

A wide variety of steroid metabolites synthesized by eukaryotes are all ultimately catabolized by bacteria; while generally saprophytic, pathogenic Mycobacteria have repurposed these pathways to utilize host intracellular cholesterol pools. Steroid degradation is complex, but a recurring theme is that cycles of ß-oxidation are used to iteratively remove acetyl- or propanoyl-CoA groups. These ß-oxidation cycles are initiated by the FAD-dependent oxidation of acyl groups, catalyzed by acyl-CoA dehydrogenases (ACADs). We show here that the tcur3481 and tcur3483 genes of Thermomonospora curvata encode subunits of a single ACAD that degrades steroid side chains with a preference for three-carbon over five-carbon substituents. The structure confirms that this enzyme is heterotetrameric, with active sites only in the Tcur3483 subunits. In comparison with the steroid ACAD FadE26-FadE27 from Mycobacterium tuberculosis, the active site is narrower and closed at the steroid-binding end, suggesting that Tcur3481-Tcur3483 is in a catalytically productive state, while FadE26-FadE27 is opened up to allow substrate entry. The flavin rings in Tcur3481-Tcur3483 sit in an unusual pocket created by Gly363, a residue conserved as Ala in steroid ACADs narrowly specific for five-carbon side chains, including FadE34. A Gly363Ala variant of Tcur3481-Tcur3483 prefers five-carbon side chains, while an inverse Ala691Gly FadE34 variant enables three-carbon side chain steroid oxidation. We determined the structure of the Tcur3483 Gly363Ala variant, showing that the flavin rings shift into the more conventional position. Modeling suggests that the shifted flavin position made possible by Gly363 is required to allow the bulky, inflexible three-carbon steroid to bind productively in the active site.

The steroid side-chain-cleaving aldolase Ltp2-ChsH2DUF35 is a thiolase superfamily member with a radically repurposed active site.

An aldolase from the bile acid-degrading actinobacterium Thermomonospora curvata catalyzes the C-C bond cleavage of an isopropyl-CoA side chain from the D-ring of the steroid metabolite 17-hydroxy-3-oxo-4-pregnene-20-carboxyl-CoA (17-HOPC-CoA). Like its homolog from Mycobacterium tuberculosis, the T. curvata aldolase is a protein complex of Ltp2 with a DUF35 domain derived from the C-terminal domain of a hydratase (ChsH2DUF35) that catalyzes the preceding step in the pathway. We determined the structure of the Ltp2-ChsH2DUF35 complex at 1.7 Å resolution using zinc-single anomalous diffraction. The enzyme adopts an αßßα organization, with the two Ltp2 protomers forming a central dimer, and the two ChsH2DUF35 protomers being at the periphery. Docking experiments suggested that Ltp2 forms a tight complex with the hydratase but that each enzyme retains an independent CoA-binding site. Ltp2 adopted a fold similar to those in thiolases; however, instead of forming a deep tunnel, the Ltp2 active site formed an elongated cleft large enough to accommodate 17-HOPC-CoA. The active site lacked the two cysteines that served as the nucleophile and general base in thiolases and replaced a pair of oxyanion-hole histidine residues with Tyr-246 and Tyr-344. Phenylalanine replacement of either of these residues decreased aldolase catalytic activity at least 400-fold. On the basis of a 17-HOPC-CoA -docked model, we propose a catalytic mechanism where Tyr-294 acts as the general base abstracting a proton from the D-ring hydroxyl of 17-HOPC-CoA and Tyr-344 as the general acid that protonates the propionyl-CoA anion following C-C bond cleavage.