Xanthomonas campestris pv. campestris possesses a single gluconeogenic pathway that is required for virulence

Disruption of ppsA, a key gene in gluconeogenesis, of Xanthomonas campestris pv. campestris resulted in the failure of the pathogen to grow in medium with pyruvate or C4-dicarboxylates as the sole carbon source and a significant reduction in virulence, indicating that X. campestris pv. campestris possesses only the malic enzyme-PpsA route in gluconeogenesis, which is required for virulence.     

DsbB is required for the pathogenesis process of Xanthomonas campestris pv. campestris

The DsbA/DsbB oxidation pathway is one of the two pathways that catalyze disulfide bond formation of proteins in the periplasm of gram-negative bacteria. It has been demonstrated that DsbA is essential for multiple virulence factors of several animal bacterial pathogens. In this article, we present genetic evidence to show that the open reading frame XC_3314 encodes a DsbB protein that is involved in disulfide bond formation in periplasm of Xanthomonas campestris pv. campestris, the causative agent of crucifer black rot disease. The dsbB mutant of X. campestris pv. campestris exhibited attenuation in virulence, hypersensitive response, cell motility, and bacterial growth in planta. Furthermore, mutation in the dsbB gene resulted in ineffective type II and type III secretion systems as well as flagellar assembly. These findings reveal that DsbB is required for the pathogenesis process of X. campestris pv. campestris.     

Pga26 mediates filamentation and biofilm formation and is required for virulence in Candida albicans

The Candida albicans gene PGA26 encodes a small cell wall protein and is upregulated during de novo wall synthesis in protoplasts. Disruption of PGA26 caused hypersensitivity to cell wall-perturbing compounds (Calcofluor white and Congo red) and to zymolyase, which degrades the cell wall β-1,3-glucan network. However, susceptibility to caspofungin, an inhibitor of β-1,3-glucan synthesis, was decreased. In addition, pga26Δ mutants show increased susceptibility to antifungals (fluconazol, posaconazol or amphotericin B) that target the plasma membrane and have altered sensitivities to environmental (heat, osmotic and oxidative) stresses. Except for a threefold increase in β-1,6-glucan and a slightly widened outer mannoprotein layer, the cell wall composition and structure was largely unaltered. Therefore, Pga26 is important for proper cell wall integrity, but does not seem to be directly involved in the synthesis of cell wall components. Deletion of PGA26 further leads to hyperfilamentation, increased biofilm formation and reduced virulence in a mouse model of disseminated candidiasis. We propose that deletion of PGA26 may cause an imbalance in the morphological switching ability of Candida, leading to attenuated dissemination and infection.     

The zinc uptake regulator Zur is essential for the full virulence of Xanthomonas campestris pv. campestris

Zur is a regulator of the high-affinity zinc uptake system in many bacteria. In Xanthomonas campestris pv. campestris 8004, a putative protein encoded by the open reading frame designated as XC1430 shows 42% amino acid similarity with the Zur of Escherichia coli. An XC1430-disrupted mutant 1430nk was constructed by homologous suicide plasmid integration. 1430nk failed to grow in rich medium supplemented with Zn2+ at a concentration of 400 microM and in nonrich medium supplemented with Zn2+ at a concentration of 110 microM, whereas the wild-type strain grew well in the same conditions. In rich medium with 400 microM Zn2+, 1430nk accumulated significantly more Zn2+ than the wild-type strain. 1430nk showed a reduction in virulence on the host plant Chinese radish (Raphanus sativus L. var. radiculus Pers.) and produced less extracellular polysaccharide (EPS) than did the wild-type strain in the absence of added zinc. These results revealed that XC1430 is a functional member of the Zur regulator family that controls zinc homeostasis, EPS production, and virulence in X. campestris pv. campestris.     

Quorum sensing and virulence regulation in Xanthomonas campestris

It is now clear that cell–cell communication, often referred to as quorum sensing (QS), is the norm in the prokaryotic kingdom and this community-wide genetic regulatory mechanism has been adopted for regulation of many important biological functions. Since the 1980s, several types of QS signals have been identified, which are associated commonly with different types of QS mechanisms. Among them, the diffusible signal factor (DSF)-dependent QS system, originally discovered from bacterial pathogen Xanthomonas campestris pv. campestris , is a relatively new regulatory mechanism. The rapid research progress over the last few years has identified the chemical structure of the QS signal DSF, established the DSF regulon, and unveiled the general signaling pathways and mechanisms. Particular noteworthy are that DSF biosynthesis is modulated by a novel posttranslational autoinduction mechanism involving protein–protein interaction between the DSF synthase RpfF and the sensor RpfC, and that QS signal sensing is coupled to intracellular regulatory networks through a second messenger cyclic-di-GMP and a global regulator Clp. Genomic and genetic analyses show that the DSF QS-signaling pathway regulates diverse biological functions including virulence, biofilm dispersal, and ecological competence. Moreover, evidence is emerging that the DSF QS system is conserved in a range of plant and human bacterial pathogens.     

A novel regulatory system required for pathogenicity of Xanthomonas campestris is mediated by a small diffusible signal molecule

Mutations in the seven clustered rpf genes cause downregulated synthesis of extracellular enzymes and reduced virulence of Xanthomonas campestris pathovar campestris ( Xcc ). The phenotype of mutants in one of the genes, rpfF , can be restored by a diffusible extracellular factor (DSF) produced by all Xcc strains tested, apart from rpfF and rpfB mutants. DSF accumulates in early stationary phase (when synthesis of enzymes is maximal), but levels decline subsequently. Addition of DSF to exponentially-growing wild-type bacteria does not cause precocious enzyme synthesis. rpfB and rpfF are expressed throughout growth, but the rate increases in early stationary phase. RpfB is predicted to be a long-chain fatty acyl CoA ligase, and RpfF shows some relatedness to enoyl CoA hydratases. The properties of DSF suggest that it may be a fatty-acid derivative, and certain lipid preparations possess DSF activity at higher concentrations. These include lipid extracts and acid-hydrolysed lipopolysaccharide and lipid A from Xcc , and purified dodecanoic and hydroxydodecanoic acid. DSF production is confined to certain xanthomonads. We propose a model for the DSF system, which represents a novel mechanism for regulating virulence factor synthesis in response to physiological or environmental changes.     

Biofilm formation and dispersal in Xanthomonas campestris

Xanthomonas campestris pathovar campestris is the causal agent of black rot disease of cruciferous plants. A cell-cell signalling system encoded by genes within the rpf cluster is required for the full virulence of this plant pathogen. This system has recently been implicated in regulation of the formation and dispersal of Xanthomonas biofilms.     

Genome scale analysis of diffusible signal factor regulon in Xanthomonas campestris pv. campestris: identification of novel cell-cell communication-dependent genes and functions

The bacterial pathogen Xanthomonas campestris pv. campestris (Xcc) recruits a diffusible signal factor (DSF), which has recently been structurally characterized as cis-11-methyl-2-dodecenoic acid, as a cell-cell communication signal to synchronize virulence gene expression and biofilm dispersal. In this study, we showed that despite the existance of phenotype variations in different Xcc isolates, the DSF-mediated functions were in general conserved. To investigate the genomic profiles of DSF regulation, we designed and conducted oligomicroarray analysis by comparison of the gene expression patterns of wild-type strain XC1 and its DSF-deficient mutant XC1dF, as well as those of XC1dF in the presence or absence of DSF signals. The analyses led to identification of 165 genes, whose expression was significantly influenced by DSF signals. These genes encode proteins and enzymes belonging to at least 12 functional groups. In addition to those previously known DSF-dependent activities such as production of extracellular enzymes and extracellular polysaccharides, microarray analyses also revealed new functions mediated by DSF, such as flagellum synthesis, resistance to toxins and oxidative stress, and aerobic respiration. Phenotype analyses confirmed that DSF signalling contributed to resistance to toxin acriflavin and hydrogen peroxide, and to the survival of bacterial cells at different temperatures. We conclude that DSF cell-cell signalling is not only essential for co-ordinating the expression of virulence genes but also plays a vital role in keeping up the general competence of the pathogen in ecosystems.     

Requirement of a mip-like gene for virulence in the phytopathogenic bacterium Xanthomonas campestris pv. campestris

Macrophage infectivity potentiators (Mips) are FKBP domain-containing proteins reported as virulence factors in several human pathogens, such as members of genera Legionella, Salmonella and Chlamydia. The putative peptidylprolyl cis-trans isomerase (PPIase) encoded by XC2699 of the plant bacterial pathogen Xanthomonas campestris pv. campestris 8004 exhibits a 49% similarity at the amino-acid level to the Mip protein of Legionella pneumophila. This mip-like gene, XC2699, was overexpressed in Escherichia coli and the purified (His)6-tagged Mip-like protein encoded by XC2699 exhibited a PPIase activity specifically inhibited by FK-506. A mutation in the mip-like gene XC2699 led to significant reductions in virulence and replication capacity in the host plant Chinese radish (Raphanus sativus L. var. radiculus Pers.). Furthermore, the production of exopolysaccharide and the activity of extracellular proteases, virulence factors of X. campestris pv. campestris, were significantly decreased in the mip-like mutant. These results reveal that the mip-like gene is involved in the pathogenesis of X. campestris pv. campestris through an effect on the production of these virulence factors.     

Alpha-toxin is required for biofilm formation by Staphylococcus aureus

Staphylococcus aureus is a common pathogen associated with nosocomial infections. It can persist in clinical settings and gain increased resistance to antimicrobial agents through biofilm formation. We have found that alpha-toxin, a secreted, multimeric, hemolytic toxin encoded by the hla gene, plays an integral role in biofilm formation. The hla mutant was unable to fully colonize plastic surfaces under both static and flow conditions. Based on microscopy studies, we propose that alpha-hemolysin is required for cell-to-cell interactions during biofilm formation.     

The role of PilZ domain proteins in the virulence of Xanthomonas campestris pv. campestris

Cyclic di‐GMP [(bis‐(3′–5′)‐cyclic di‐guanosine monophosphate)] is an almost ubiquitous second messenger in bacteria that is implicated in the regulation of a range of functions that include developmental transitions, aggregative behaviour, adhesion, biofilm formation and virulence. Comparatively little is known about the mechanism(s) by which cyclic di‐GMP exerts these various regulatory effects. PilZ has been identified as a cyclic di‐GMP binding protein domain; proteins with this domain are involved in regulation of specific cellular processes, including the virulence of animal pathogens. Here we have examined the role of PilZ domain proteins in virulence and the regulation of virulence factor synthesis in Xanthomonas campestris pv. campestris (Xcc), the causal agent of black rot of crucifers. The Xcc genome encodes four proteins (XC0965, XC2249, XC2317 and XC3221) that have a PilZ domain. Mutation of XC0965, XC2249 and XC3221 led to a significant reduction of virulence in Chinese radish. Mutation of XC2249 and XC3221 led to a reduction in motility whereas mutation of XC2249 and XC0965 affected extracellular enzyme production. All mutant strains were unaffected in biofilm formation in vitro. The reduction of virulence following mutation of XC3221 could not be wholly attributed to an effect on motility as mutation of pilA, which abolishes motility, has a lesser effect on virulence.     

Xanthomonas citri ssp. citri requires the outer membrane porin OprB for maximal virulence and biofilm formation

Xanthomonas citri ssp. citri (Xcc) causes canker disease in citrus, and biofilm formation is critical for the disease cycle. OprB (Outer membrane protein B) has been shown previously to be more abundant in Xcc biofilms compared with the planktonic state. In this work, we showed that the loss of OprB in an oprB mutant abolishes bacterial biofilm formation and adherence to the host, and also compromises virulence and efficient epiphytic survival of the bacteria. Moreover, the oprB mutant is impaired in bacterial stress resistance. OprB belongs to a family of carbohydrate transport proteins, and the uptake of glucose is decreased in the mutant strain, indicating that OprB transports glucose. Loss of OprB leads to increased production of xanthan exopolysaccharide, and the carbohydrate intermediates of xanthan biosynthesis are also elevated in the mutant. The xanthan produced by the mutant has a higher viscosity and, unlike wild‐type xanthan, completely lacks pyruvylation. Overall, these results suggest that Xcc reprogrammes its carbon metabolism when it senses a shortage of glucose input. The participation of OprB in the process of biofilm formation and virulence, as well as in metabolic changes to redirect the carbon flux, is discussed. Our results demonstrate the importance of environmental nutrient supply and glucose uptake via OprB for Xcc virulence.     

Regulation of the synthesis of cyclic glucan in Xanthomonas campestris by a diffusible signal molecule

The rpf gene cluster of Xanthomonas campestris pv. campestris is involved in the co-ordinate positive regulation of the production of extracellular enzymes and the extracellular polysaccharide xanthan. Several of the rpf genes are involved in a regulatory system involving the small diffusible molecule DSF (for diffusible signal factor). Synthesis of DSF requires RpfF, and a two-component sensory transduction system involving RpfC has been implicated in the perception of the signal and signal transduction. Here we show that mutations in both rpfF and rpfC lead to reductions in the levels of cyclic glucan. The levels of cyclic glucan synthetase in membrane preparations from rpfF and rpfC mutants were, however, unaltered from the wild-type. Similar alterations in the level of cyclic glucan without changes in cyclic glucan synthetase activity were seen when wild-type bacteria were exposed to osmotic stress. These results extend the range of cellular functions subject to regulation by the rpf genes and DSF system.     

Cyclic di-GMP signalling in the virulence and environmental adaptation of Xanthomonas campestris

Cyclic di-GMP is a second messenger with a role in regulation of a range of cellular functions in diverse bacteria including the virulence of pathogens. Cellular levels of cyclic di-GMP are controlled through synthesis, catalysed by the GGDEF protein domain, and degradation by EAL or HD-GYP domains. Here we report a comprehensive study of cyclic di-GMP signalling in bacterial disease in which we examine the contribution of all proteins with GGDEF, EAL or HD-GYP domains to virulence and virulence factor production in the phytopathogen Xanthomonas campestris pathovar campestris (Xcc). Genes with significant roles in virulence to plants included those encoding proteins whose probable function is in cyclic-di-GMP synthesis as well as others (including the HD-GYP domain regulator RpfG) implicated in cyclic di-GMP degradation. Furthermore, RpfG controlled expression of a subset of these genes. A partially overlapping set of elements controlled the production of virulence factors in vitro. Other GGDEF-EAL domain proteins had no effect on virulence factor synthesis but did influence motility. These findings indicate the existence of a regulatory network that may allow Xcc to integrate information from diverse environmental inputs to modulate virulence factor synthesis as well as of cyclic di-GMP signalling systems dedicated to other specific tasks.