Wangshu Jiang

Proteus mirabilis, a Gram-negative uropathogen, is a major causative agent in catheter-associated urinary tract infections (CAUTI). Mannose-resistant Proteus-like fimbriae (MR/P) are crucially important for P. mirabilis infectivity and are required for biofilm formation and auto-aggregation, as well as for bladder and kidney colonization. Here, the X-ray crystal structure of the MR/P tip adhesin, MrpH, is reported. The structure has a fold not previously described and contains a transition metal center with Zn²⁺ coordinated by three conserved histidine residues and a ligand. Using biofilm assays, chelation, metal complementation, and site-directed mutagenesis of the three histidines, we show that an intact metal binding site occupied by zinc is essential for MR/P fimbria-mediated biofilm formation, and furthermore, that P. mirabilis biofilm formation is reversible in a zinc-dependent manner. Zinc is also required for MR/P-dependent agglutination of erythrocytes, and mutation of the metal binding site renders P. mirabilis unfit in a mouse model of UTI. The studies presented here provide important clues as to the mechanism of MR/P-mediated biofilm formation and serve as a starting point for identifying the physiological MR/P fimbrial receptor. Many bacteria use fimbriae to adhere to surfaces, and this function is often essential for pathogens to gain a foothold in the host. In this study, we examine the major virulence-associated fimbrial protein, MrpH, of the bacterial urinary tract pathogen Proteus mirabilis. This species is particularly known for causing catheter-associated urinary tract infections, in which it forms damaging urinary stones and crystalline biofilms that can block the flow of urine through indwelling catheters. MrpH resides at the tip of mannose-resistant Proteus-like (MR/P) fimbriae and is required for MR/P-dependent adherence to surfaces. Although MR/P belongs to a well-known class of adhesive fimbriae encoded by the chaperone-usher pathway, we found that MrpH has a dramatically different structure compared with other tip-located adhesins in this family. Unexpectedly, MrpH was found to bind a zinc cation, which we show is essential for MR/P-mediated biofilm formation and adherence to red blood cells. Furthermore, MR/P-mediated adherence can be modified by controlling zinc levels. These findings have the potential to aid development of better anti-biofilm urinary catheters or other methods to prevent P. mirabilis infection of the urinary tract.

Spider silk is a biomaterial with exceptional mechanical toughness, and there is great interest in developing biomimetic methods to produce engineered spider silk-based materials. However, the mechanisms that regulate the conversion of spider silk proteins (spidroins) from highly soluble dope into silk are not completely understood. The N-terminal domain (NT) of Euprosthenops australis dragline silk protein undergoes conformational and quaternary-structure changes from a monomer at a pH above 7 to a homodimer at lower pH values. Conversion from the monomer to the dimer requires the protonation of three conserved glutamic acid residues, resulting in a low-pH `locked' dimer stabilized by symmetric electrostatic interactions at the poles of the dimer. The detailed molecular events during this transition are still unresolved. Here, a 2.1 Å resolution crystal structure of an NT T61A mutant in an alternative, asymmetric, dimer form in which the electrostatic interactions at one of the poles are dramatically different from those in symmetrical dimers is presented. A similar asymmetric dimer structure from dragline silk of Nephila clavipes has previously been described. It is suggested that asymmetric dimers represent a conserved intermediate state in spider silk formation, and a revised `lock-and-trigger' mechanism for spider silk formation is presented.

The important uropathogenProteus mirabilisencodes a record number of chaperone/usher-pathway adhesive fimbriae. Such fimbriae, which are used for adhesion to cell surfaces/tissues and for biofilm formation, are typically important virulence factors in bacterial pathogenesis. Here, the structures of the receptor-binding domains of the tip-located two-domain adhesins UcaD (1.5 Å resolution) and AtfE (1.58 Å resolution) from twoP. mirabilisfimbriae (UCA/NAF and ATF) are presented. The structures of UcaD and AtfE are both similar to the F17G type of tip-located fimbrial receptor-binding domains, and the structures are very similar despite having only limited sequence similarity. These structures represent an important step towards a molecular-level understanding ofP. mirabilisfimbrial adhesins and their roles in the complex pathogenesis of urinary-tract infections.