RetS inhibits Pseudomonas aeruginosa biofilm formation by disrupting the canonical histidine kinase dimerization interface of GacS

Bacterial signaling histidine kinases (HKs) have long been postulated to function exclusively through linear signal transduction chains. However, several HKs have recently been shown to form complex multikinase networks (MKNs). The most prominent MKN, involving the enzymes RetS and GacS, controls the switch between the motile and biofilm lifestyles in the pathogenic bacterium Pseudomonas aeruginosa. While GacS promotes biofilm formation, RetS counteracts GacS using three distinct mechanisms. Two are dephosphorylating mechanisms. The third, a direct binding between the RetS and GacS HK regions, blocks GacS autophosphorylation. Focusing on the third mechanism, we determined the crystal structure of a cocomplex between the HK region of RetS and the dimerization and histidine phosphotransfer (DHp) domain of GacS. This is the first reported structure of a complex between two distinct bacterial signaling HKs. In the complex, the canonical HK homodimerization interface is replaced by a strikingly similar heterodimeric interface between RetS and GacS. We further demonstrate that GacS autophosphorylates in trans, thus explaining why the formation of a RetS-GacS complex inhibits GacS autophosphorylation. Using mutational analysis in conjunction with bacterial two-hybrid and biofilm assays, we not only corroborate the biological role of the observed RetS-GacS interactions, but also identify a residue critical for the equilibrium between the RetS-GacS complex and the respective RetS and GacS homodimers. Collectively, our findings suggest that RetS and GacS form a domain-swapped hetero-oligomer during the planktonic growth phase of P. aeruginosa before unknown signals cause its dissociation and a relief of GacS inhibition to promote biofilm formation.     

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.     

The Hybrid Histidine Kinase LadS Forms a Multicomponent Signal Transduction System with the GacS/GacA Two-Component System in Pseudomonas aeruginosa

In response to environmental changes, Pseudomonas aeruginosa is able to switch from a planktonic (free swimming) to a sessile (biofilm) lifestyle. The two-component system (TCS) GacS/GacA activates the production of two small non-coding RNAs, RsmY and RsmZ, but four histidine kinases (HKs), RetS, GacS, LadS and PA1611, are instrumental in this process. RetS hybrid HK blocks GacS unorthodox HK autophosphorylation through the formation of a heterodimer. PA1611 hybrid HK, which is structurally related to GacS, interacts with RetS in P. aeruginosa in a very similar manner to GacS. LadS hybrid HK phenotypically antagonizes the function of RetS by a mechanism that has never been investigated. The four sensors are found in most Pseudomonas species but their characteristics and mode of signaling may differ from one species to another. Here, we demonstrated in P. aeruginosa that LadS controls both rsmY and rsmZ gene expression and that this regulation occurs through the GacS/GacA TCS. We additionally evidenced that in contrast to RetS, LadS signals through GacS/GacA without forming heterodimers, either with GacS or with RetS. Instead, we demonstrated that LadS is involved in a genuine phosphorelay, which requires both transmitter and receiver LadS domains. LadS signaling ultimately requires the alternative histidine-phosphotransfer domain of GacS, which is here used as an Hpt relay by the hybrid kinase. LadS HK thus forms, with the GacS/GacA TCS, a multicomponent signal transduction system with an original phosphorelay cascade, i.e. H1_LadS→D1_LadS→H2_GacS→D2_GacA. This highlights an original strategy in which a unique output, i.e. the modulation of sRNA levels, is controlled by a complex multi-sensing network to fine-tune an adapted biofilm and virulence response.     

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.     

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 reference strain PA14 displays increased virulence due to a mutation in ladS

Pseudomonas aeruginosa is a pathogen that causes acute and chronic infections in a variety of hosts. The pathogenic potential of P. aeruginosa is strain-dependent. PA14 is a highly virulent strain that causes disease in a wide range of organisms, whereas PAO1 is moderately virulent. Although PA14 carries pathogenicity islands that are absent in PAO1, the presence or absence of specific gene clusters is not predictive of virulence. Here, we show that the virulent strain PA14 has an acquired mutation in the ladS gene. This mutation has a deleterious impact on biofilm, while it results in elevated type III secretion system (T3SS) activity and increased cytotoxicity towards mammalian cells. These phenotypes can be reverted by repairing the ladS mutation on the PA14 genome. The RetS/LadS/GacS signaling cascade is associated with virulence and the switch between acute and chronic infections. RetS is a sensor that down-regulates biofilm formation and up-regulates the T3SS. Mutations in retS are acquired in strains isolated from chronically infected cystic fibrosis patients and lead to hyperbiofilm formation and reduced cytotoxicity. Conversely, the LadS sensor promotes biofilm formation and represses the T3SS. We conclude that the ladS mutation is partly responsible for the high cytotoxicity of PA14, and our findings corroborate the central role of RetS and LadS in the switch between acute and chronic infections. Given the extensive use of the reference strain PA14 in infection and virulence models, the bias caused by the ladS mutation on the observed phenotypes will be crucial to consider in future research.     

The Diguanylate Cyclase SadC Is a Central Player in Gac/Rsm-Mediated Biofilm Formation in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a Gram-negative opportunistic human pathogen and a threat for immunocompromised and cystic fibrosis patients. It is responsible for acute and chronic infections and can switch between these lifestyles upon taking an informed decision involving complex regulatory networks. The RetS/LadS/Gac/Rsm network and the cyclic-di-GMP (c-di-GMP) signaling pathways are both central to this phenomenon redirecting the P. aeruginosa population toward a biofilm mode of growth, which is associated with chronic infections. While these two pathways were traditionally studied independently from each other, we recently showed that cellular levels of c-di-GMP are increased in the hyperbiofilm retS mutant. Here, we have formally established the link between the two networks by showing that the SadC diguanylate cyclase is central to the Gac/Rsm-associated phenotypes, notably, biofilm formation. Importantly, SadC is involved in the signaling that converges onto the RsmA translational repressor either via RetS/LadS or via HptB/HsbR. Although the level of expression of the sadC gene does not seem to be impacted by the regulatory cascade, the production of the SadC protein is tightly repressed by RsmA. This adds to the growing complexity of the signaling network associated with c-di-GMP in P. aeruginosa. While this organism possesses more than 40 c-di-GMP-related enzymes, it remains unclear how signaling specificity is maintained within the c-di-GMP network. The finding that SadC but no other diguanylate cyclase is related to the formation of biofilm governed by the Gac/Rsm pathway further contributes to understanding of this insulation mechanism.     

铜绿假单胞菌T6SS的组装、分泌、功能和调控

作为一种对抗真核细胞和原核细胞的强有力细菌武器,Ⅵ型分泌系统(type Ⅵ secretion system,T6SS)广泛存在于革兰氏阴性菌中。铜绿假单胞菌是一种对多种抗生素具有耐药性并能够在人体引发急性和慢性感染的条件致病菌,它编码3套独立的T6SS,分别为H1-、H2-和H3-T6SS。T6SS通过介导细菌间竞争、生物被膜的形成、金属离子的摄取以及与真核宿主细胞之间的相互作用,对铜绿假单胞菌在毒力和适应环境方面发挥重要作用。本文主要对铜绿假单胞菌T6SS的组装、效应蛋白的分泌、功能及调控机制展开综述,旨在为T6SS的研究提供一定的参考,并为铜绿假单胞菌感染的预防和治疗提供一定的指导。     

A revised model for the role of GacS/GacA in regulating type III secretion by Pseudomonas syringae pv. tomato DC3000

GacS/GacA is a conserved two-component system that functions as a master regulator of virulence-associated traits in many bacterial pathogens, including Pseudomonas spp., that collectively infect both plant and animal hosts. Among many GacS/GacA-regulated traits, type III secretion of effector proteins into host cells plays a critical role in bacterial virulence. In the opportunistic plant and animal pathogen Pseudomonas aeruginosa, GacS/GacA negatively regulates the expression of type III secretion system (T3SS)-encoding genes. However, in the plant pathogenic bacterium Pseudomonas syringae, strain-to-strain variation exists in the requirement of GacS/GacA for T3SS deployment, and this variability has limited the development of predictive models of how GacS/GacA functions in this species. In this work we re-evaluated the function of GacA in P. syringae pv. tomato DC3000. Contrary to previous reports, we discovered that GacA negatively regulates the expression of T3SS genes in DC3000, and that GacA is not required for DC3000 virulence inside Arabidopsis leaf tissue. However, our results show that GacA is required for full virulence of leaf surface-inoculated bacteria. These data significantly revise current understanding of GacS/GacA in regulating P. syringae virulence.     

Global Regulator MorA Affects Virulence-Associated Protease Secretion in Pseudomonas aeruginosa PAO1

Bacterial invasion plays a critical role in the establishment of Pseudomonas aeruginosa infection and is aided by two major virulence factors – surface appendages and secreted proteases. The second messenger cyclic diguanylate (c-di-GMP) is known to affect bacterial attachment to surfaces, biofilm formation and related virulence phenomena. Here we report that MorA, a global regulator with GGDEF and EAL domains that was previously reported to affect virulence factors, negatively regulates protease secretion via the type II secretion system (T2SS) in P. aeruginosa PAO1. Infection assays with mutant strains carrying gene deletion and domain mutants show that host cell invasion is dependent on the active domain function of MorA. Further investigations suggest that the MorA-mediated c-di-GMP signaling affects protease secretion largely at a post-translational level. We thus report c-di-GMP second messenger system as a novel regulator of T2SS function in P. aeruginosa. Given that T2SS is a central and constitutive pump, and the secreted proteases are involved in interactions with the microbial surroundings, our data broadens the significance of c-di-GMP signaling in P. aeruginosa pathogenesis and ecological fitness.     

Unique Biofilm Signature,Drug Susceptibility and Decreased Virulence in Drosophila through the Pseudomonas aeruginosa Two-Component System PprAB

Bacterial biofilm is considered as a particular lifestyle helping cells to survive hostile environments triggered by a variety of signals sensed and integrated through adequate regulatory pathways. Pseudomonas aeruginosa, a Gram-negative bacterium causing severe infections in humans, forms biofilms and is a fantastic example for fine-tuning of the transition between planktonic and community lifestyles through two-component systems (TCS). Here we decipher the regulon of the P. aeruginosa response regulator PprB of the TCS PprAB. We identified genes under the control of this TCS and once this pathway is activated, analyzed and dissected at the molecular level the PprB-dependent phenotypes in various models. The TCS PprAB triggers a hyper-biofilm phenotype with a unique adhesive signature made of BapA adhesin, a Type 1 secretion system (T1SS) substrate, CupE CU fimbriae, Flp Type IVb pili and eDNA without EPS involvement. This unique signature is associated with drug hyper-susceptibility, decreased virulence in acutely infected flies and cytotoxicity toward various cell types linked to decreased Type III secretion (T3SS). Moreover, once the PprB pathway is activated, decreased virulence in orally infected flies associated with enhanced biofilm formation and dissemination defect from the intestinal lumen toward the hemolymph compartment is reported. PprB may thus represent a key bacterial adaptation checkpoint of multicellular and aggregative behavior triggering the production of a unique matrix associated with peculiar antibiotic susceptibility and attenuated virulence, a particular interesting breach for therapeutic intervention to consider in view of possible eradication of P. aeruginosa biofilm-associated infections.     

Binding mode analysis of a major T3SS translocator protein PopB with its chaperone PcrH from Pseudomonas aeruginosa

Pseudomonas aeruginosa, a Gram‐negative pathogen uses a specialized set of Type III secretion system (T3SS) translocator proteins to establish virulence in the host cell. An understanding of the factors that govern translocation by the translocator protein–chaperone complex is thus of immense importance. In this work, experimental and computational techniques were used to probe into the structure of the major translocator protein PopB from P. aeruginosa and to identify the important regions involved in functioning of the translocator protein. This study reveals that the binding sites of the common chaperone PcrH, needed for maintenance of the translocator PopB within the bacterial cytoplasm, which are primarily localized within the N‐terminal domain. However, disordered and flexible residues located both at the N‐ and C‐terminal domains are also observed to be involved in association with the chaperone. This intrinsic disorderliness of the terminal domains is conserved for all the major T3SS translocator proteins and is functionally important to maintain the intrinsically disordered state of the translocators. Our experimental and computational analyses suggest that a “disorder‐to‐order” transition of PopB protein might take place upon PcrH binding. The long helical coiled‐coil part of PopB protein perhaps helps in pore formation while the flexible apical region is involved in chaperone interaction. Thus, our computational model of translocator protein PopB and its binding analyses provide crucial functional insights into the T3SS translocation mechanism. Proteins 2014; 82:3273–3285. © 2014 Wiley Periodicals, Inc.     

The archetype Pseudomonas aeruginosa proteins TssB and TagJ form a novel subcomplex in the bacterial type VI secretion system

In Pseudomonas aeruginosa three type VI secretion systems (T6SSs) coexist, called H1‐ to H3‐T6SSs. Several T6SS components are proposed to be part of a macromolecular complex resembling the bacteriophage tail. The T6SS protein HsiE1 (TagJ) is unique to the H1‐T6SS and absent from the H2‐ and H3‐T6SSs. We demonstrate that HsiE1 interacts with a predicted N‐terminal α‐helix in HsiB1 (TssB) thus forming a novel subcomplex of the T6SS. HsiB1 is homologous to the Vibrio cholerae VipA component, which contributes to the formation of a bacteriophage tail sheath‐like structure. We show that the interaction between HsiE1 and HsiB1 is specific and does not occur between HsiE1 and HsiB2. Proteins of the TssB family encoded in T6SS clusters lacking a gene encoding a TagJ‐like component are often devoid of the predicted N‐terminal helical region, which suggests co‐evolution. We observe that a synthetic peptide corresponding to the N‐terminal 20 amino acids of HsiB1 interacts with purified HsiE1 protein. This interaction is a common feature to other bacterial T6SSs that display a TagJ homologue as shown here with Serratia marcescens. We further show that hsiE1 is a non‐essential gene for the T6SS and suggest that HsiE1 may modulate incorporation of HsiB1 into the T6SS.     

Effect of the proton motive force inhibitor carbonyl cyanide‐m‐chlorophenylhydrazone (CCCP) on Pseudomonas aeruginosa biofilm development

Aims: Proton motive force (PMF) inhibition enhances the intracellular accumulation of autoinducers possibly interfering with biofilm formation. We evaluated the effect of the PMF inhibitor carbonyl cyanide‐m‐chlorophenylhydrazone (CCCP) on Pseudomonas aeruginosa biofilm development. Methods and Results: Four epidemiologically unrelated P. aeruginosa isolates were studied. A MexAB‐oprM overproducing strain was used as control. Expression of gene mexB was examined and biofilm formation after incubation with 0, 12·5 and 25 μmol l^?1 of CCCP was investigated. Mean values of optical density were analysed with one‐way analysis of variance and t‐test. Two isolates subexpressed mexB gene and only 25 μmol l^?1 of CCCP affected biofilm formation. Biofilms of the other two isolates and control strain PA140 exhibited significantly lower absorbance (P ranging from <0·01 to <0·05) with either 12·5 or 25 μmol l^?1 of CCCP. Conclusions: The PMF inhibitor CCCP effect was correlated with the expression of MexAB‐OprM efflux system and found to compromise biofilm formation in P. aeruginosa. Significance and Impact of the Study: These data suggest that inhibition of PMF‐dependent trasporters might decrease biofilm formation in P. aeruginosa.     

PrgB promotes aggregation,biofilm formation,and conjugation through DNA binding and compaction

Gram‐positive bacteria deploy type IV secretion systems (T4SSs) to facilitate horizontal gene transfer. The T4SSs of Gram‐positive bacteria rely on surface adhesins as opposed to conjugative pili to facilitate mating. Enterococcus faecalis PrgB is a surface adhesin that promotes mating pair formation and robust biofilm development in an extracellular DNA (eDNA) dependent manner. Here, we report the structure of the adhesin domain of PrgB. The adhesin domain binds and compacts DNA in vitro. In vivo PrgB deleted of its adhesin domain does not support cellular aggregation, biofilm development and conjugative DNA transfer. PrgB also binds lipoteichoic acid (LTA), which competes with DNA binding. We propose that PrgB binding and compaction of eDNA facilitates cell aggregation and plays an important role in establishment of early biofilms in mono‐ or polyspecies settings. Within these biofilms, PrgB mediates formation and stabilization of direct cell‐cell contacts through alternative binding of cell‐bound LTA, which in turn promotes establishment of productive mating junctions and efficient intra‐ or inter‐species T4SS‐mediated gene transfer.