The Host Plant Metabolite Glucose Is the Precursor of Diffusible Signal Factor (DSF) Family Signals in Xanthomonas campestris

Plant pathogen Xanthomonas campestris pv. campestris produces cis-11-methyl-2-dodecenoic acid (diffusible signal factor [DSF]) as a cell-cell communication signal to regulate biofilm dispersal and virulence factor production. Previous studies have demonstrated that DSF biosynthesis is dependent on the presence of RpfF, an enoyl-coenzyme A (CoA) hydratase, but the DSF synthetic mechanism and the influence of the host plant on DSF biosynthesis are still not clear. We show here that exogenous addition of host plant juice or ethanol extract to the growth medium of X. campestris pv. campestris could significantly boost DSF family signal production. It was subsequently revealed that X. campestris pv. campestris produces not only DSF but also BDSF (cis-2-dodecenoic acid) and another novel DSF family signal, which was designated DSF-II. BDSF was originally identified in Burkholderia cenocepacia to be involved in regulation of motility, biofilm formation, and virulence in B. cenocepacia. Functional analysis suggested that DSF-II plays a role equal to that of DSF in regulation of biofilm dispersion and virulence factor production in X. campestris pv. campestris. Furthermore, chromatographic separation led to identification of glucose as a specific molecule stimulating DSF family signal biosynthesis in X. campestris pv. campestris. ¹³C-labeling experiments demonstrated that glucose acts as a substrate to provide a carbon element for DSF biosynthesis. The results of this study indicate that X. campestris pv. campestris could utilize a common metabolite of the host plant to enhance DSF family signal synthesis and therefore promote virulence.     

Phenotypic and Genotypic Characterisation of Burkholderia cenocepacia J2315 Mutants Affected in Homoserine Lactone and Diffusible Signal Factor-Based Quorum Sensing Systems Suggests Interplay between Both Types of Systems

Many putative virulence factors of Burkholderia cenocepacia are controlled by various quorum sensing (QS) circuits. These QS systems either use N-acyl homoserine lactones (AHL) or cis-2-dodecenoic acid (“Burkholderia diffusible signal factor”, BDSF) as signalling molecules. Previous work suggested that there is little cross-talk between both types of systems. We constructed mutants in B. cenocepacia strain J2315, in which genes encoding CepI (BCAM1870), CciI (BCAM0239a) and the BDSF synthase (BCAM0581) were inactivated, and also constructed double (ΔcepIΔBCAM0581, ΔcciIΔBCAM0581 and ΔcepIΔcciI) mutants and a triple (ΔcepIΔcciIΔBCAM0581) mutant. Subsequently we investigated phenotypic properties (antibiotic susceptibility, biofilm formation, production of AHL and BDSF, protease activity and virulence in Caenorhabditis elegans) and measured gene expression in these mutants, and this in the presence and absence of added BDSF, AHL or both. The triple mutant was significantly more affected in biofilm formation, antimicrobial susceptibility, virulence in C. elegans, and protease production than either the single or double mutants. The ΔBCAM0581 mutant and the ΔcepIΔBCAM0581 and ΔcciIΔBCAM0581 double mutants produced significantly less AHL compared to the WT strain and the ΔcepI and ΔcciI single mutant, respectively. The expression of cepI and cciI in ΔBCAM0581, was approximately 3-fold and 7-fold (p<0.05) lower than in the WT, respectively. The observed differences in AHL production, expression of cepI and cciI and QS-controlled phenotypes in the ΔBCAM0581 mutant could (at least partially) be restored by addition of BDSF. Our data suggest that, in B. cenocepacia J2315, AHL and BDSF-based QS systems co-regulate the same set of genes, regulate different sets of genes that are involved in the same phenotypes and/or that the BDSF system controls the AHL-based QS system. As the expression of the gene encoding the C6-HSL synthase CciI (and to a lesser extent the C8-HSL synthase CepI) is partially controlled by BDSF, it seems likely that the BDSF QS systems controls AHL production through this system.     

Diffusible Signal Factor (DSF) Synthase RpfF of Xylella fastidiosa Is a Multifunction Protein Also Required for Response to DSF

Xylella fastidiosa, like related Xanthomonas species, employs an Rpf cell-cell communication system consisting of a diffusible signal factor (DSF) synthase, RpfF, and a DSF sensor, RpfC, to coordinate expression of virulence genes. While phenotypes of a ΔrpfF strain in Xanthomonas campestris could be complemented by its own DSF, the DSF produced by X. fastidiosa (XfDSF) did not restore expression of the XfDSF-dependent genes hxfA and hxfB to a ΔrpfF strain of X. fastidiosa, suggesting that RpfF is involved in XfDSF sensing or XfDSF-dependent signaling. To test this conjecture, rpfC and rpfF of X. campestris were replaced by those of X. fastidiosa, and the contribution of each gene to the induction of a X. campestris DSF-dependent gene was assessed. As in X. fastidiosa, XfDSF-dependent signaling required both X. fastidiosa proteins RpfF and RpfC. RpfF repressed RpfC signaling activity, which in turn was derepressed by XfDSF. A mutated X. fastidiosa RpfF protein with two substitutions of glutamate to alanine in its active site was incapable of XfDSF production yet enabled a response to XfDSF, indicating that XfDSF production and the response to XfDSF are two separate functions in which RpfF is involved. This mutant was also hypervirulent to grape, demonstrating the antivirulence effects of XfDSF itself in X. fastidiosa. The Rpf system of X. fastidiosa is thus a novel example of a quorum-sensing signal synthase that is also involved in the response to the signal molecule that it synthesizes.     

Dual signaling functions of the hybrid sensor kinase RpfC of Xanthomonas campestris involve either phosphorelay or receiver domain-protein interaction

The hybrid sensor kinase RpfC positively regulates the expression of a range of virulent genes and negatively modulates the synthesis of the quorum sensing signal diffusible signal factor (DSF) in Xanthomonas campestris. Three conserved amino acid residues of RpfC implicated in phosphorelay (His(198) in the histidine kinase domain, Asp(512) in the receiver domain, and His(657) in the histidine phosphotransfer domain) were essential for activation of the production of extracellular enzymes and extracellular polysaccharide (EPS) virulence factors but were not essential for repression of DSF biosynthesis. Domain deletion and subsequent in trans expression analysis revealed that the receiver domain of RpfC alone was sufficient to repress DSF overproduction in an rpfC deletion mutant. Further deletion and alanine scanning mutagenesis analyses identified a peptide of 107 amino acids and three amino acid residues (Gln(496), Glu(504), and Ile(552)) involved in modulating DSF production. Co-immunoprecipitation and far Western blot analyses suggested an interaction between the receiver domain and RpfF, the enzyme involved in DSF biosynthesis. These data support a model in which RpfC modulates two different functions (virulence factor synthesis and DSF synthesis) by utilization of a conserved phosphorelay system and a novel domain-specific protein-protein interaction mechanism, respectively. This latter mechanism represents an added dimension to conventional two-component signaling paradigms.     

The HD-GYP domain, cyclic di-GMP signaling, and bacterial virulence to plants

Cyclic di-GMP is an almost ubiquitous second messenger in bacteria that was first described as an allosteric activator of cellulose synthase but is now known to regulate a range of functions, including virulence in human and animal pathogens. Two protein domains, GGDEF and EAL, are implicated in the synthesis and degradation, respectively, of cyclic di-GMP. These domains are widely distributed in bacteria, including plant pathogens. The majority of proteins with GGDEF and EAL domains contain additional signal input domains, suggesting that their activities are responsive to environmental cues. Recent studies have demonstrated that a third domain, HD-GYP, is also active in cyclic di-GMP degradation. In the plant pathogen Xanthomonas campestris pv. campestris, a two-component signal transduction system comprising the HD-GYP domain regulatory protein RpfG and cognate sensor RpfC positively controls virulence. The signals recognized by RpfC may include the cell-cell signal DSF, which also acts to regulate virulence in X. campestris pv. campestris. Here, we review these recent advances in our understanding of cyclic di-GMP signaling with particular reference to one or more roles in the bacterial pathogenesis of plants.     

Xanthomonas campestris RpfB is a fatty Acyl‐CoA ligase required to counteract the thioesterase activity of the RpfF diffusible signal factor (DSF) synthase

In Xanthomonas campestris pv. campestris (Xcc), the proteins encoded by the rpf (regulator of pathogenicity factor) gene cluster produce and sense a fatty acid signal molecule called diffusible signalling factor (DSF, 2(Z)‐11‐methyldodecenoic acid). RpfB was reported to be involved in DSF processing and was predicted to encode an acyl‐CoA ligase. We report that RpfB activates a wide range of fatty acids to their CoA esters in vitro. Moreover, RpfB can functionally replace the paradigm bacterial acyl‐CoA ligase, Escherichia coli FadD, in the E. coli ß‐oxidation pathway and deletion of RpfB from the Xcc genome results in a strain unable to utilize fatty acids as carbon sources. An essential RpfB function in the pathogenicity factor pathway was demonstrated by the properties of a strain deleted for both the rpfB and rpfC genes. The ΔrpfB ΔrpfC strain grew poorly and lysed upon entering stationary phase. Deletion of rpfF, the gene encoding the DSF synthetic enzyme, restored normal growth to this strain. RpfF is a dual function enzyme that synthesizes DSF by dehydration of a 3‐hydroxyacyl‐acyl carrier protein (ACP) fatty acid synthetic intermediate and also cleaves the thioester bond linking DSF to ACP. However, the RpfF thioesterase activity is of broad specificity and upon elimination of its RpfC inhibitor RpfF attains maximal activity and its thioesterase activity proceeds to block membrane lipid synthesis by cleavage of acyl‐ACP intermediates. This resulted in release of the nascent acyl chains to the medium as free fatty acids. This lack of acyl chains for phospholipid synthesis results in cell lysis unless RpfB is present to counteract the RpfF thioesterase activity by catalysing uptake and activation of the free fatty acids to give acyl‐CoAs that can be utilized to restore membrane lipid synthesis. Heterologous expression of a different fatty acid activating enzyme, the Vibrio harveyi acyl‐ACP synthetase, replaced RpfB in counteracting the effects of high level RpfF thioesterase activity indicating that the essential role of RpfB is uptake and activation of free fatty acids.     

Diffusible signal factor‐dependent quorum sensing in pathogenic bacteria and its exploitation for disease control

Cell‐to‐cell signals of the diffusible signal factor (DSF) family are cis‐2‐unsaturated fatty acids of differing chain length and branching pattern. DSF signalling has been described in diverse bacteria to include plant and human pathogens where it acts to regulate functions such as biofilm formation, antibiotic tolerance and the production of virulence factors. DSF family signals can also participate in interspecies signalling with other bacteria and interkingdom signalling such as with the yeast Candida albicans. Interference with DSF signalling may afford new opportunities for the control of bacterial disease. Such strategies will depend in part on detailed knowledge of the molecular mechanisms underlying the processes of signal synthesis, perception and turnover. Here, I review both recent progress in understanding DSF signalling at the molecular level and prospects for translating this knowledge into approaches for disease control.     

A two-component system involving an HD-GYP domain protein links cell-cell signalling to pathogenicity gene expression in Xanthomonas campestris

The synthesis of extracellular enzymes and extracellular polysaccharide (EPS) in Xanthomonas campestris pv. campestris (Xcc) is regulated by a cluster of genes called rpf (for regulation of pathogenicity factors). Two of the genes, rpfF and rpfB, have previously been implicated in the synthesis of a diffusible regulatory molecule, DSF. Here, we describe a screen of transposon insertion mutants of Xcc that identified two DSF-overproducing strains. In each mutant, the gene disrupted is rpfC, which encodes a hybrid two-component regulatory protein in which the sensor and regulator domains are fused and which contains an additional C-terminal phosphorelay (HPt) domain. We show that rpfC is in an operon with rpfH and rpfG. The predicted protein RpfG has a regulatory input domain attached to a specialized version of an HD domain, previously suggested to function in signal transduction. The predicted protein RpfH is structurally related to the sensory input domain of RpfC. We show that RpfC and RpfG act positively to regulate the synthesis of extracellular enzymes and EPS, but that RpfC acts negatively to regulate the synthesis of DSF. We propose that RpfGHC is a signal transduction system that couples the synthesis of pathogenicity factors to sensing of environmental signals that may include DSF itself.     

Single‐molecule atomic force microscopy unravels the binding mechanism of a Burkholderia cenocepacia trimeric autotransporter adhesin

Trimeric autotransporter adhesins (TAAs) are bacterial surface proteins that fulfil important functions in pathogenic Gram‐negative bacteria. Prominent examples of TAAs are found in Burkholderia cepacia complex, a group of bacterial species causing severe infections in patients with cystic fibrosis. While there is strong evidence that Burkholderia cenocepacia TAAs mediate adhesion, aggregation and colonization of the respiratory epithelium, we still know very little about the molecular mechanisms behind these interactions. Here, we use single‐molecule atomic force microscopy to unravel the binding mechanism of BCAM0224, a prototype TAA from B. cenocepacia K56‐2. We show that the adhesin forms homophilic trans‐interactions engaged in bacterial aggregation, and that it behaves as a spring capable to withstand high forces. We also find that BCAM0224 binds collagen, a major extracellular component of host epithelia. Both homophilic and heterophilic interactions display low binding affinity, which could be important for epithelium colonization. We then demonstrate that BCAM0224 recognizes receptors on living pneumocytes, and leads to the formation of membrane tethers that may play a role in promoting adhesion. Collectively, our results show that BCAM0224 is a multifunctional adhesin endowed with remarkable binding properties, which may represent a general mechanism among TAAs for strengthening bacterial adhesion.     

Interspecies signalling via the Stenotrophomonas maltophilia diffusible signal factor influences biofilm formation and polymyxin tolerance in Pseudomonas aeruginosa

Interspecies signalling through the action of diffusible signal molecules can influence the behaviour of organisms growing in polymicrobial communities. Stenotrophomonas maltophilia and Pseudomonas aeruginosa occur ubiquitously in the environment and can be found together in diverse niches including the rhizosphere of plants and the cystic fibrosis lung. In mixed species biofilms, S. maltophilia substantially influenced the architecture of P. aeruginosa structures, which developed as extended filaments. This effect depended upon the synthesis of the diffusible signal factor (DSF) by S. maltophilia and could be mimicked by the addition of synthetic DSF. This response of P. aeruginosa to DSF required PA1396, a sensor kinase with an input domain of related amino acid sequence to the sensory input domain of RpfC, which is responsible for DSF perception in xanthomonads. Mutation of PA1396 or addition of DSF to P. aeruginosa led to increased levels of a number of proteins with roles in bacterial stress tolerance, including those implicated in resistance to cationic antimicrobial peptides. This effect was associated with increased tolerance to polymyxins. Homologues of PA1396 occur in a number of phytopathogenic and plant-associated pseudomonads, suggesting that modulation of bacterial behaviour through DSF-mediated interspecies signalling with xanthomonads is a phenomenon that occurs widely.     

Virulence and in planta movement of Xanthomonas hortorum pv. pelargonii are affected by the diffusible signal factor (DSF)‐dependent quorum sensing system

Xanthomonas hortorum pv. pelargonii (Xhp), the causal agent of bacterial blight in pelargonium, is the most threatening bacterial disease of this ornamental worldwide. To gain an insight into the regulation of virulence in Xhp, we have disrupted the quorum sensing (QS) genes, which mediate the biosynthesis and sensing of the diffusible signal factor (DSF). Mutations in rpfF (encoding the DSF synthase) and rpfC (encoding the histidine sensor kinase of the two‐component system RfpC/RpfG) and overexpression of rpfF showed a significant reduction in incidence and severity of the disease on pelargonium. Confocal laser scanning microscopy images of inoculated plants with a green fluorescent protein (GFP)‐labelled wild‐type strain showed that the pathogen is homogeneously dispersed in the lumen of xylem vessels, reaching the apex and invading the intercellular spaces of the leaf mesophyll tissue within 21 days. In contrast, the rpfF and rpfC knockout mutants, as well as the rpfF‐overexpressing strain, remained confined to the vicinity of the inoculation site. The rpfF and rpfC mutants formed large incoherent aggregates in the xylem vessels that might interfere with upward movement of the bacterium within the plant. Both mutants also formed extended aggregates under in vitro conditions, whereas the wild‐type strain formed microcolonies. Expression levels of putative virulence genes in planta were substantially reduced within 48 h after inoculation with the QS mutants when compared with the wild‐type. The results presented indicate that an optimal DSF concentration is crucial for successful colonization and virulence of Xhp in pelargonium.     

Phenotypic characterization of trimeric autotransporter adhesin-defective bcaC mutant of Burkholderia cenocepacia: cross-talk towards the histidine kinase BCAM0218

Burkholderia cenocepacia is a virulent species belonging to the Burkholderia cepacia complex (Bcc) and one of the most problematic agents of chronic lung infection in cystic fibrosis patients. B. cenocepacia possesses a large panel of virulence traits that include trimeric autotransporter adhesins (TAAs). Such proteins are obligate homotrimeric anchored in the outer membrane. They are players in the adhesion events that occur between bacteria and biotic/abiotic surfaces. In this study, we constructed two insertional-mutants for TAA bcaC and Histidine kinase (HK) BCAM0218 genes, which are clustered together within the B. cenocepacia K56-2 TAA cluster. The bcaC-mutant affects B. cenocepacia adhesion to extracellular matrix proteins and red blood cells hemagglutination. BcaC contributes to enhancing B. cenocepacia K56-2 adhesion to bronchial epithelial cells. The expression of bcaC seems to affect biofilm formation negatively. Due to a BCAM0218 disruption, the bcaC expression increases significantly, indicating that they are functionally linked. The overexpression of bcaC in the BCAM0218-mutant background rescues at least part of the BcaC functions. Altogether, these findings reveal the multifunctionality of BcaC as a novel B. cenocepacia K56-2 virulence factor and postulate the involvement of a sensor HK (BCAM0218) in the control of this TAA gene.     

Characterization of BCAM0224, a Multifunctional Trimeric Autotransporter from the Human Pathogen Burkholderia cenocepacia

Members of the trimeric autotransporter adhesin (TAA) family play a crucial role in adhesion of Gram-negative pathogens to host cells. Moreover, these proteins are multifunctional virulence factors involved in several other biological traits, including invasion into host cells and evasion of the host immune system. In cystic fibrosis epidemic Burkholderia cenocepacia strain J2315, we identified a unique TAA (BCAM0224)-encoding gene, previously described as being implicated in virulence. Here, we characterized this multifunctional protein, trying to establish its role in B. cenocepacia pathogenicity. We show that BCAM0224 occurs on the bacterial surface and adopts a trimeric conformation. Furthermore, we demonstrated that BCAM0224 is needed for earlier stages of biofilm formation and is required for swarming motility. In addition, BCAM0224 plays an important role in evasion of the human innate immune system, providing resistance against the bactericidal activity of serum via the complement classical pathway. Finally, BCAM0224 mediates bacterial adhesion to and invasion of cultured human bronchial epithelial cells. Together, these data reveal the high versatility of the BCAM0224 protein as a virulence factor in the pathogenic bacterium B. cenocepacia.