Identification of critical amino acid residues in the plague biofilm Hms proteins

Abstract

Yersinia pestis biofilm formation causes massive adsorption of haemin or Congo red in vitro as well as colonization and eventual blockage of the flea proventriculus in vivo. This blockage allows effective transmission of plague from some fleas, like the oriental rat flea, to mammals. Four Hms proteins, HmsH, HmsF, HmsR and HmsS, are essential for biofilm formation, with HmsT and HmsP acting as positive and negative regulators, respectively. HmsH has a β-barrel structure with a large periplasmic domain while HmsF possesses polysaccharide deacetylase and COG1649 domains. HmsR is a putative glycosyltransferase while HmsS has no recognized domains. In this study, specific amino acids within conserved domains or within regions of high similarity in HmsH, HmsF, HmsR and HmsS proteins were selected for site-directed mutagenesis. Some but not all of the substitutions in HmsS and within the periplasmic domain of HmsH were critical for protein function. Substitutions within the glycosyltransferase domain of HmsR and the deacetylase domain of HmsF abolished biofilm formation in Y. pestis. Surprisingly, substitution of highly conserved residues within COG1649 did not affect HmsF function.

Yersinia pestis Biofilm in the Flea Vector and Its Role in the Transmission of Plague

“…As in other bacteria, the Hms-dependent ECM is required for Congo red binding (pigmentation), the binding of other ligands with polysaccharide affinity such as calcofluor ) and certain lectins (Tan and Darby 2004), as well as for Yersinia biofilm formation (Darby et al 2002;Forman et al 2006;Jarrett et al 2004;Joshua et al 2003).…”

“…The hms locus is required for Y. pestis and Y. pseudotuberculosis biofilm growth in 96-well polystyrene microtiter plates and on the surface of glass continuous-flow (flowcell) chambers (Fig. 3) (Erickson et al 2006;Forman et al 2006;Jarrett et al 2004;Kirillina et al 2004). Key evidence for the biofilm model of plague transmission came from in vivo studies using the rat flea X. cheopis as an infection model, in which the Y. pestis hms genes were shown to be required to produce a transmissible infection in the flea proventriculus (Hinnebusch et al 1996;Jarrett et al 2004).…”

“…Given the membrane localization and similar domains of the Ica, Pga, and Hms gene products, ECM biosynthesis in E. coli and Yersinia species probably proceeds by an analogous mechanism. Notably, all four of the Y. pestis hmsHFRS gene products are required for pigmentation and biofilm phenotype, and site-directed mutagenesis of the periplasmic, deacetylase, and glycosyl transferase domains of HmsH, F, and R, respectively, severely diminished Congo red binding and in vitro biofilm formation (Forman et al 2006). …”

The Structure- and Metal-dependent Activity of Escherichia coli PgaB Provides Insight into the Partial De-N-acetylation of Poly-β-1,6-N-acetyl-d-glucosamine

“…The exact function of the C-terminal domain of PgaB is unknown; however, it is required for efficient de-N-acetylation of PNAG in vivo (17). The domain shows sequence homology to COG1649 (27), a domain common to TIM barrel glycoside hydrolases (28), and has been suggested to play a role in PNAG binding and/or hydrolysis (17). PgaA is a two-domain outer membrane protein that is predicted to contain a C-terminal 16-stranded ␤-barrel domain that likely acts as the porin for exporting dPNAG, and an N-terminal periplasmic domain that carries multiple copies of the tetratricopeptide repeat motif that has been implicated in protein-protein and protein-ligand interactions (17, 29 -35).…”

Formation and regulation of Yersinia biofilms

“…HmsH has a β-barrel structure with a large periplasmic domain. HmsF possesses polysaccharide deacetylase and glycosyl hydrolase (COG1649) domains whereas HmsR is a putative glycosyl transferase, and HmsS has no recognized domains (Forman et al, 2006;Abu Khweek et al, 2010). Some but not all of the substitutions within the periplasmic domain of HmsH and HmsS are critical for their protein function.…”