Structural insights into the regulatory mechanism of the Pseudomonas aeruginosa YfiBNR system

Abstract

YfiBNR is a recently identified bis-(3’-5’)-cyclic dimeric GMP (c-di-GMP) signaling system in opportunistic pathogens. It is a key regulator of biofilm formation, which is correlated with prolonged persistence of infection and antibiotic drug resistance. In response to cell stress, YfiB in the outer membrane can sequester the periplasmic protein YfiR, releasing its inhibition of YfiN on the inner membrane and thus provoking the diguanylate cyclase activity of YfiN to induce c-di-GMP production. However, the detailed regulatory mechanism remains elusive. Here, we report the crystal structures of YfiB alone and of an active mutant YfiBL43P complexed with YfiR with 2:2 stoichiometry. Structural analyses revealed that in contrast to the compact conformation of the dimeric YfiB alone, YfiBL43P adopts a stretched conformation allowing activated YfiB to penetrate the peptidoglycan (PG) layer and access YfiR. YfiBL43P shows a more compact PG-binding pocket and much higher PG binding affinity than wild-type YfiB, suggesting a tight correlation between PG binding and YfiB activation. In addition, our crystallographic analyses revealed that YfiR binds Vitamin B6 (VB6) or L-Trp at a YfiB-binding site and that both VB6 and L-Trp are able to reduce YfiBL43P-induced biofilm formation. Based on the structural and biochemical data, we propose an updated regulatory model of the YfiBNR system.

Predicting mutational routes to new adaptive phenotypes

“…The 2.3 kb aws operon contains three genes transcribed from a single promoter ( awsXRO ). Homologous genes in Pseudomonas aeruginosa ( yfiRNB, PA1121-1119) have been characterised in detail (Malone et al, 2010; Malone et al, 2012; Xu et al, 2016). The outer membrane lipoprotein AwsO (PFLU5209) has an OmpA domain, a signal peptide and binds to peptidoglycan.…”

“…This is also supported by data from Pseudomonas aeruginosa in which mutations in nine different positions in this region lead to a small colony variant phenotype similar to WS (Malone et al, 2012). A functional model based on the YfiBNR in P. aeruginosa (Malone et al, 2012; Xu et al, 2016), suggest that AwsO sequesters AwsX at the outer membrane and that mutations in the N-terminal part of the protein lead to constitutive activation and increased binding of AwsX. This would correspond to an increase in r 2 in Figure 4B, which would relieve negative regulation of AwsR.Mws pathway: The MwsR pathway (comprising just a single gene) harbours mutations in both DGC and phosphodiesterase (PDE) domains.…”

Evaluation of Annexin A1 Protein in an Infectious Keratitis Model: Therapeutic Perspectives

“…Due to the persistence of P. aeruginosa and its ability to acquire resistance [3,14,15] a wide variety of broadspectrum antibiotic eye drops have been employed in bacterial keratitis, such as ciprofloxacin, tobramycin, gentamicin and cephalosporins [16,17]. Although topical antibiotics are the best treatment for bacterial keratitis [1], high-dose steroid treatment was significantly associated with better visual outcomes in patients with culturepositive bacterial keratitis [18].…”

Temperature-Dependent Gene Expression in Yersinia ruckeri: Tracking Specific Genes by Bioluminescence During in Vivo Colonization

“…One group of mutants presented transposon insertions in genes related to response to environmental changes. Among them, the yfiN gene encoding the diguanylate cyclase (OEU26487.1) involved in the synthesis of cyclic di-GMP, a bacterial second messenger related to the control of many bacterial cellular functions such as changes from the transition between sessile and motile bacterial states ( Römbling et al, 2013 ) and virulence ( Raterman et al, 2013 ; Xu et al, 2016 ). Two other mutants presented transposon insertion in a putative operon of two genes encoding for a glycosyltransferase (OEU24749.1) and a SAM-dependent methyltransferase (OEU24750.1).…”