Identifying components required for OMP biogenesis as novel targets for antiinfective drugs.

The emergence of multiresistant Gram-negative bacteria requires new therapies for combating bacterial infections. Targeting the biogenesis of virulence factors could be an alternative strategy instead of killing bacteria with antibiotics. The outer membrane (OM) of Gram-negative bacteria acts as a physical barrier. At the same time it facilitates the exchange of molecules and harbors a multitude of proteins associated with virulence. In order to insert proteins into the OM, an essential oligomeric membrane-associated protein complex, the ß-barrel assembly machinery (BAM) is required. Being essential for the biogenesis of outer membrane proteins (OMPs) the BAM and also periplasmic chaperones may serve as attractive targets to develop novel antiinfective agents. Herein, we aimed to elucidate which proteins belonging to the OMP biogenesis machinery have the most important function in granting bacterial fitness, OM barrier function, facilitating biogenesis of dedicated virulence factors and determination of overall virulence. To this end we used the enteropathogen Yersinia enterocolitica as a model system. We individually knocked out all non-essential components of the BAM (BamB, C and E) as well as the periplasmic chaperones DegP, SurA and Skp. In summary, we found that the most profound phenotypes were produced by the loss of BamB or SurA with both knockouts resulting in significant attenuation or even avirulence of Ye in a mouse infection model. Thus, we assume that both BamB and SurA are promising targets for the development of new antiinfective drugs in the future.

Identification and characterization of a novel periplasmic polygalacturonic acid binding protein from Yersinia enterolitica.

A number of bacteria in the family Enterobacteriaceae harbor the genes comprising well-developed pectinolytic pathways (e.g. Erwinia sp.) or abridged versions of this pathway (e.g. Yersinia sp.). One of the most enigmatic components present in some of these pathways is a small gene that encodes a predicted secreted protein of approximately 160 amino acid residues with unknown function. This protein shows distant amino acid sequence similarity over its entire length to galactose-specific family 32 carbohydrate-binding modules (CBMs). Here we demonstrate the ability of the Yersinia enterocolitica example, here called YeCBM32, to bind polygalacturonic acid containing components of pectin. This binding is selective for highly polymerized galacturonic acid and shows a complex mode of polysaccharide recognition. The high resolution X-ray crystal structure (1.35 A) shows YeCBM32s overall structural similarity to galactose specific CBMs and conserved binding site location but reveals a substantially different binding site topology, which likely reflects its unique polymeric and acidic ligand. The results suggest the possibility of a unique role for YeCBM32 in polygalacturonic acid transport.