Crystal structure and oligomeric state of the RetS signaling kinase sensory domain

The opportunistic pathogen Pseudomonas aeruginosa may cause both acute and chronic‐persistent infections in predisposed individuals. Acute infections require the presence of a functional type III secretion system (T3SS), whereas chronic P. aeruginosa infections are characterized by the formation of drug‐resistant biofilms. The T3SS and biofilm formation are reciprocally regulated by the signaling kinases LadS, RetS, and GacS. RetS downregulates biofilm formation and upregulates expression of the T3SS through a unique mechanism. RetS forms a heterodimeric complex with GacS and thus prevents GacS autophosphorylation and downstream signaling. The signals that regulate RetS are not known but RetS possesses a distinctive periplasmic sensor domain that is believed to serve as receptor for the regulatory ligand. We have determined the crystal structure of the RetS sensory domain at 2.0 Å resolution. The structure closely resembles those of carbohydrate binding modules of other proteins, suggesting that the elusive ligands are likely carbohydrate moieties. In addition to the conserved beta‐sandwich structure, the sensory domain features two alpha helices which create a unique surface topology. Protein–protein crosslinking and fluorescence energy transfer experiments also revealed that the sensory domain dimerizes with a dissociation constant of K_d = 580 ± 50 nM, a result with interesting implications for our understanding of the underlying signaling mechanism. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Hybrid sensor kinase PA1611 in Pseudomonas aeruginosa regulates transitions between acute and chronic infection through direct interaction with RetS

Pseudomonas aeruginosa causes serious acute and chronic infections in humans. Major differences exist in disease pathogenesis, clinical treatment and outcomes between acute and chronic infections. P. aeruginosa acute infection characteristically involves the type III secretion systems (T3SS) while chronic infection is often associated with the formation of biofilms, a major cause of difficulties to eradicate chronic infections. The choice between acute and chronic infection or the switch between them by P. aeruginosa is controlled by regulatory pathways that control major virulence factors and genes associated with biofilm formation. In this study, we characterized a hybrid sensor kinase PA1611 that controls the expression of genes associated with acute and chronic infections in P. aeruginosa PAO1. Expression of PA1611 completely repressed T3SS and swarming motility while it promoted biofilm formation. The protein PA1611 regulates two small RNAs (sRNAs), rsmY and rsmZ which in turn control RsmA. Independent of phosphate relay, PA1611 interacts directly with RetS in vivo. The positive effect of RetS on factors associated with acute infection could presumably be restrained by PA1611 when chronic infection conditions are present. This RetS–PA1611 interaction, together with the known RetS–GacS interaction, may control disease progression and the lifestyle choice of P. aeruginosa.     

Regulatory RNAs and the HptB/RetS signalling pathways fine‐tune Pseudomonas aeruginosa pathogenesis

Bacterial pathogenesis often depends on regulatory networks, two‐component systems and small RNAs (sRNAs). In Pseudomonas aeruginosa, the RetS sensor pathway downregulates expression of two sRNAs, rsmY and rsmZ. Consequently, biofilm and the Type Six Secretion System (T6SS) are repressed, whereas the Type III Secretion System (T3SS) is activated. We show that the HptB signalling pathway controls biofilm and T3SS, and fine‐tunes P. aeruginosa pathogenesis. We demonstrate that RetS and HptB intersect at the GacA response regulator, which directly controls sRNAs production. Importantly, RetS controls both sRNAs, whereas HptB exclusively regulates rsmY expression. We reveal that HptB signalling is a complex regulatory cascade. This cascade involves a response regulator, with an output domain belonging to the phosphatase 2C family, and likely an anti‐anti‐σ factor. This reveals that the initial input in the Gac system comes from several signalling pathways, and the final output is adjusted by a differential control on rsmY and rsmZ. This is exemplified by the RetS‐dependent but HptB‐independent control on T6SS. We also demonstrate a redundant action of the two sRNAs on T3SS gene expression, while the impact on pel gene expression is additive. These features underpin a novel mechanism in the fine‐tuned regulation of gene expression.     

The Pseudomonas aeruginosa sensor RetS switches type III and type VI secretion via c-di-GMP signalling

Acute bacterial infections are associated with motility and cytotoxicity via the type III secretion system (T3SS), while chronic infections are linked to biofilm formation and reduced virulence. In Pseudomonas aeruginosa, the transition between motility and sessility involves regulatory networks including the RetS/GacS sensors, as well as the second messenger c-di-GMP. The RetS/GacS signalling cascade converges on small RNAs, RsmY and RsmZ, which control a range of functions via RsmA. A retS mutation induces biofilm formation, and high levels of c-di-GMP produce a similar response. In this study, we connect RetS and c-di-GMP pathways by showing that the retS mutant displays high levels of c-di-GMP. Furthermore, a retS mutation leads to repression of the T3SS, but also upregulates the type VI secretion system (T6SS), which is associated with chronic infections. Strikingly, production of the T3SS and T6SS can be switched by artificially modulating c-di-GMP levels. We show that the diguanylate cyclase WspR is specifically involved in the T3SS/T6SS switch and that RsmY and RsmZ are required for the c-di-GMP-dependent response. These results provide a firm link between the RetS/GacS and the c-di-GMP pathways, which coordinate bacterial lifestyles, as well as secretion systems that determine the infection strategy of P. aeruginosa.     

A Potent Anti-SpuE Antibody Allosterically Inhibits Type III Secretion System and Attenuates Virulence of Pseudomonas Aeruginosa

Multidrug-resistant gram-negative bacteria infection is particularly severe within the designated ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species), which underscores the urgent need to explore alternative therapeutic strategies. The type III secretion system (T3SS) is considered to be a key virulence factor in many gram-negative bacteria, and T3SS is in turn regulated by SpuE in P. aeruginosa, which is a spermidine binding protein from an ATP-binding cassette transporter family and highly conserved within ESKAPE pathogens. Here, we identified a potent anti-SpuE antagonistic antibody that allosterically inhibits the expression of T3SS and attenuates virulence of P. aeruginosa. X-ray crystallography and molecular dynamics simulations revealed that binding of antibody to SpuE induces a change in the dynamics of SpuE, which in turn may reduce spermidine uptake by P. aeruginosa. The antibody could serve as a template for developing novel biologics to target a broad spectrum of gram-negative bacteria.