The YfiBNR Signal Transduction Mechanism Reveals Novel Targets for the Evolution of Persistent Pseudomonas aeruginosa in Cystic Fibrosis Airways

Abstract

The genetic adaptation of pathogens in host tissue plays a key role in the establishment of chronic infections. While whole genome sequencing has opened up the analysis of genetic changes occurring during long-term infections, the identification and characterization of adaptive traits is often obscured by a lack of knowledge of the underlying molecular processes. Our research addresses the role of Pseudomonas aeruginosa small colony variant (SCV) morphotypes in long-term infections. In the lungs of cystic fibrosis patients, the appearance of SCVs correlates with a prolonged persistence of infection and poor lung function. Formation of P. aeruginosa SCVs is linked to increased levels of the second messenger c-di-GMP. Our previous work identified the YfiBNR system as a key regulator of the SCV phenotype. The effector of this tripartite signaling module is the membrane bound diguanylate cyclase YfiN. Through a combination of genetic and biochemical analyses we first outline the mechanistic principles of YfiN regulation in detail. In particular, we identify a number of activating mutations in all three components of the Yfi regulatory system. YfiBNR is shown to function via tightly controlled competition between allosteric binding sites on the three Yfi proteins; a novel regulatory mechanism that is apparently widespread among periplasmic signaling systems in bacteria. We then show that during long-term lung infections of CF patients, activating mutations invade the population, driving SCV formation in vivo . The identification of mutational “scars” in the yfi genes of clinical isolates suggests that Yfi activity is both under positive and negative selection in vivo and that continuous adaptation of the c-di-GMP network contributes to the in vivo fitness of P. aeruginosa during chronic lung infections. These experiments uncover an important new principle of in vivo persistence, and identify the c-di-GMP network as a valid target for novel anti-infectives directed against chronic infections.

Genetic Analysis of the Role of yfiR in the Ability of Escherichia coli CFT073 To Control Cellular Cyclic Dimeric GMP Levels and To Persist in the Urinary Tract

“…Recent reports on P. aeruginosa identified the YfiBNR system as a regulatory complex that plays a role in the development of the SCV phenotype important for persistence in a subcutaneous catheter model. YfiN was identified as an active membrane-associated DGC, and YfiR was identified as a periplasmic regulator of YfiN activity (46,47). The authors proposed that YfiB sequesters YfiR to the outer membrane, which, in turn, relieves repression of YfiN activity (47).…”

“…YfiN was identified as an active membrane-associated DGC, and YfiR was identified as a periplasmic regulator of YfiN activity (46,47). The authors proposed that YfiB sequesters YfiR to the outer membrane, which, in turn, relieves repression of YfiN activity (47). In the analogous operon, E. coli contains an additional gene, yfiL, that has no predicted function (Fig.…”

Evolutionary convergence in experimental Pseudomonas populations

“…Such modular organisation, combined with a flexible regulatory network, might confer a degree of evolvability on Pseudomonas that underpins its capacity to adapt to novel conditions and invade new ecological niches (Spiers et al, 2000;Rainey and Cooper, 2004). In this context, this work adds to the increasing number of studies that show how model microbial populations can contribute understanding of the mechanistic bases of biofilm formation and biofilm community evolution with particular relevance to the establishment of chronic infections (Smith et al, 2006;Malone et al, 2012) and more generally to the challenge of surface colonisation (Martin et al, 2016). Selection is a potent evolutionary force.…”

Cyclic di-GMP: the First 25 Years of a Universal Bacterial Second Messenger

“…For example, in X. campestris and Xanthomonas axonopodis, not only do some GGDEF domain DGCs specifically interact with the HD-GYP domain PDE, RpfG, but these interactions proved important for regulating a subset of downstream processes (98,192). The periplasmic protein YfiR (PA1121) from P. aeruginosa inhibits the activity of the DGC TpbB (YfiN; PA1120), which prevents the formation of small-colony variants of this species (193). The H-NOX domain, a selective NO sensor, interacts as a free-standing protein with DGCs to affect c-di-GMP metabolism (188,189).…”

“…These variants are characterized by enhanced biofilm formation, high fimbrial expression, repression of flagellar genes, resistance to phagocytosis, and enhanced antibiotic resistance (96,97,206,245). Small-colony variants generated in vitro as well as obtained from clinical isolates contained mutations that upregulate the activity of the DGC TpbB (YfiN), suggesting a key role of this enzyme (193,245). The importance of c-di-GMP for enhanced persistence of P. aeruginosa has also been demonstrated in a chinchilla model of middle ear infection (355).…”