Many animal and plant pathogenic bacteria employ a type three secretion system (T3SS) to deliver type three effector proteins (T3Es) into host cells. Efficient secretion of many T3Es in the plant ...pathogen Xanthomonas campestris pv. campestris (Xcc) relies on the global chaperone HpaB. However, how the domain of HpaB itself affects effector translocation/secretion is poorly understood. Here, we used genetic and biochemical approaches to identify a novel domain at the C-terminal end of HpaB (amino acid residues 137-160) that contributes to virulence and hypersensitive response (HR). Both in vitro secretion assay and in planta translocation assay showed that the secretion and translocation of T3E proteins depend on the C-terminal region of HpaB. Deletion of the C-terminal region of HpaB did not affect binding to T3Es, self-association or interaction with T3SS components. However, the deletion of C-terminal region sharply reduced the mounts of free T3Es liberated from the complex of HpaB with the T3Es, a reaction catalyzed in an ATP-dependent manner by the T3SS-associated ATPase HrcN. Our findings demonstrate the C-terminal domain of HpaB contributes to disassembly of chaperone-effector complex and reveal a potential molecular mechanism underpinning the involvement of HpaB in secretion of T3Es in Xcc.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The Gram-negative bacterium Xanthomonas oryzae pv. oryzicola (Xoc) is the causal agent of rice bacterial leaf streak (BLS), one of the most destructive diseases of rice (Oryza sativa L.) that is the ...important staple crop. Xoc can invade host leaves via stomata and wounds and its type three secretion system (T3SS) is pivotal to its pathogenic lifestyle. In this study, using a novel dual RNA-seq approach, we examined transcriptomes of rice and Xoc in samples inoculated with wild type Xoc GX01 and its T3SS defective strain (T3SD), to investigate the global transcriptional changes in both organisms. Compared with T3SD strain, rice inoculated with wild type Xoc GX01 resulted in significant expression changes of a series of plant defence related genes, including ones altered in plant signalling pathway, and downregulated in phenylalanine metabolism, flavonoid and momilactone biosynthesis, suggesting repression of plant defence response and reduction in both callose deposition and phytoalexin accumulation. Also, some known transcription activator-like effector (TALE) targets were induced by Xoc GX01, e.g. OsSultr3;6 which contributes to rice susceptibility. Some cell elongation related genes, including several expansin genes, were induced by GX01 too, suggesting that Xoc may exploit this pathway to weaken cell wall strength, beneficial for bacterial infection. On the other hand, compared with wild type, the T3SD strain transcriptome in planta was characterized by downregulation of ATP, protein and polysaccharide synthesis, and upregulation of antioxidation and detoxification related genes, revealing that T3SD strain faced serious starvation and oxidation stresses in planta without a functional T3SS. In addition, comparative global transcript profiles of Xoc in planta and in medium revealed an upregulation of virulence factor synthesis and secretion in planta in favour of bacterial infection. Collectively, this study provides a comprehensive representation of cross talk between the host and bacterial pathogen, revealing insights into the Xoc-rice pathogenic dynamic and reveals novel strategies exploited by this important pathogen to cause disease.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
α-Synucleinopathies are characterized by autonomic dysfunction and motor impairments. In the pure autonomic failure (PAF), α-synuclein (α-Syn) pathology is confined within the autonomic nervous ...system with no motor features, but mouse models recapitulating PAF without motor dysfunction are lacking. Here, we show that in TgM83
mice, inoculation of α-Syn preformed fibrils (PFFs) into the stellate and celiac ganglia induces spreading of α-Syn pathology only through the autonomic pathway to both the central nervous system (CNS) and the autonomic innervation of peripheral organs bidirectionally. In parallel, the mice develop autonomic dysfunction, featured by orthostatic hypotension, constipation, hypohidrosis and hyposmia, without motor dysfunction. Thus, we have generated a mouse model of pure autonomic dysfunction caused by α-Syn pathology. This model may help define the mechanistic link between transmission of pathological α-Syn and the cardinal features of autonomic dysfunction in α-synucleinopathy.
The bacterial phytopathogen Xanthomonas campestris pv. campestris (Xcc) relies on the hrp (hypersensitive response and pathogenicity) genes to cause disease and induce hypersensitive response (HR). ...The hrp genes of bacterial phytopathogens are divided into two groups. Xcc hrp genes belong to group II. It has long been known that the group II hrp genes are activated by an AraC‐type transcriptional regulator whose expression is controlled by a two‐component system (TCS) response regulator (named HrpG in Xcc). However, no cognate sensor kinase has yet been identified. Here, we present evidence showing that the Xcc open‐reading frame XC_3670 encodes a TCS sensor kinase (named HpaS). Mutation of hpaS almost completely abolished the HR induction and virulence. Bacterial two‐hybrid and protein pull‐down assays revealed that HpaS physically interacted with HrpG. Phos‐tag™ SDS‐PAGE analysis showed that mutation in hpaS reduced markedly the phosphorylation of HrpG in vivo. These data suggest that HpaS and HrpG are most likely to form a TCS. We also showed that XC_3669 (named hpaR2), which is adjacent to hpaS and encodes a putative TCS response regulator, is required for full virulence but not HR induction. HpaR2 also physically interacted with HpaS, suggesting that HpaS may also form another TCS with HpaR2.
The Gram-negative phytopathogenic bacterium Xanthomonas campestris pv. campestris recruits the hrp/T3SS system to inject pathogenicity effector proteins into host cells and uses the rpf/DSF cell-cell ...signaling system to regulate the expression of virulence factors such as extracellular enzymes and polysaccharide. Whether these two systems have any connection is unknown.
Positive regulator candidates affecting hrpX expression were identified by sacB strategy. The transcriptional expression was determined by qRT-PCR and GUS activity analysis. Transcriptome analysis was performed by RNA deep-sequencing. The hypersensitive response (HR) was determined in the nonhost plant pepper ECW-10R and electrolyte leakage assay.
Mutation of the gene encoding the sensor RpfC of the rpf/DSF system significantly reduced the expression of hrpX, the key regulator of the hrp/T3SS system, all of the genes in the hrp cluster and most reported type III effector genes. Mutation of rpfG did not affect the expression of hrpX. The rpfC mutant showed a delayed and weakened HR induction.
RpfC positively regulates the expression of hrpX independent of RpfG, showing a complex regulatory network linking the rpf/DSF and hrp/T3SS systems.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Summary
Molecules of the diffusible signal factor (DSF)‐family are a class of quorum sensing (QS) signals used by the phytopathogens Xanthomonas. Studies during the last decade have outlined how ...Xanthomonas cells enter the QS phase. However, information on the mechanism underlying its exit from the QS phase is limited. RpfB has recently been reported as a fatty acyl‐CoA ligase (FCL) that activates a wide range of fatty acids to their CoA esters in vitro. Here, we establish an improved quantification assay for DSF‐family signals using liquid chromatography‐mass spectrometry in X. campestris pv. campestris (Xcc). We first demonstrated that RpfB represents a naturally occurring DSF‐family signal turnover system. RpfB effectively turns over DSF‐family signals DSF and BDSF in vivo. RpfB FCL enzymatic activity is required for DSF and BDSF turnover. Deletion of rpfB slightly increased Xcc virulence in the Chinese radish and overexpression of rpfB significantly decreased virulence. We further showed that the expression of rpfB is growth phase‐dependent, and its expression is significantly enhanced when Xcc cells enter the stationary phase. DSF regulates rpfB expression in a concentration‐dependent manner. rpfB expression is also negatively regulated by the DSF signalling components RpfC, RpfG and Clp. The global transcription factor Clp directly binds to the AATGC‐tgctgc‐GCATC motif in the promoter region of rpfB to repress its expression. Finally, RpfB‐dependent signal turnover system was detected in a wide range of bacterial species, suggesting that it is a conserved mechanism.
Summary
The essential stages of bacterial cell separation are described as the synthesis and hydrolysis of septal peptidoglycan (PG). The amidase, AmiC, which cleaves the peptide side‐chains linked ...to the glycan strands, contributes critically to this process and has been studied extensively in model strains of Escherichia coli. However, insights into the contribution of this protein to other processes in the bacterial cell have been limited. Xanthomonas campestris pv. campestris (Xcc) is a phytopathogen that causes black rot disease in many economically important plants. We investigated how AmiC and LytM family regulators, NlpD and EnvC, contribute to virulence and cell separation in this organism. Biochemical analyses of purified AmiC demonstrated that it could hydrolyse PG and its activity could be potentiated by the presence of the regulator NlpD. We also established that deletion of the genes encoding amiC1 or nlpD led to a reduction in virulence as well as effects on colony‐forming units and cell morphology. Moreover, further genetic and biochemical evidence showed that AmiC1 and NlpD affect the secretion of type III effector XC3176 and hypersensitive response (HR) induction in planta. These findings indicate that, in addition to their well‐studied role(s) in cell separation, AmiC and NlpD make an important contribution to the type III secretion (T3S) and virulence regulation in this important plant pathogen.
Background Observational studies suggest that patients with immunoglobulin A nephropathy (IgAN) with active proliferative lesions show a good response to immunosuppressive treatment. Study Design ...Multicenter, prospective, randomized, controlled trial. Setting & Participants 176 patients with IgAN with active proliferative lesions (cellular and fibrocellular crescents, endocapillary hypercellularity, or necrosis), proteinuria with protein excretion ≥ 1.0 g/24 h, and estimated glomerular filtration rate > 30 mL/min/1.73 m2. Intervention Mycophenolate mofetil (MMF) group: MMF, 1.5 g/d, for 6 months and prednisone, 0.4 to 0.6 mg/kg/d, for 2 months and then tapered by 20% per month for the next 4 months; prednisone group: prednisone, 0.8 to 1.0 mg/kg/d, for 2 months and then tapered by 20% per month for the next 4 months. All patients were followed up for another 6 months. Outcomes The primary end point was complete remission rate at 6 and 12 months. Results At baseline, median estimated glomerular filtration rates were 90.2 and 94.3 mL/min/1.73 m2 and mean proteinuria was protein excretion of 2.37 and 2.47 g/24 h in the MMF and prednisone groups, respectively. At 6 months, complete remission rates were 37% (32 of 86 patients) and 38% (33 of 88 patients); the between-group difference was not statistically significant ( P = 0.9). At 12 months, complete remission rates were 48% (35 of 73 patients) and 53% (38 of 72 patients) in the MMF and prednisone groups, respectively; the between-group difference was not statistically significant ( P = 0.6). Incidences of Cushing syndrome and newly diagnosed diabetes mellitus were lower in the MMF group than in the prednisone group. Limitations Not all participants were treated with renin-angiotensin system blockers, relatively short follow-up. Conclusions MMF plus prednisone versus full-dose prednisone did not differ in reducing proteinuria, but patients treated with the former had fewer adverse events in patients with IgAN with active proliferative lesions.
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.
Two types of Zn–Ln nanoclusters Nd 2 Zn 2 L 2 (L′) 2 (OAc) 2 (NO 3 ) 2 (H 2 O) 2 ( 1 ) and Ln 6 Zn 6 L 2 (L′′) 2 O 2 (OAc) 18 (Ln = Nd ( 2 ) and Sm ( 3 )) were prepared using one long Schiff base ...ligand (H 2 L) with a naphthyl backbone and two short polydentate ligands (H 2 L′ and H 2 L′′). 1 and 2–3 show linear and planar structures with sizes of approximately 10 × 10 × 30 Å and 10 × 15 × 22 Å, respectively. The chromogenic Zn/ligand components can act as efficient sensitizers for lanthanide luminescence, and thus 1–3 show visible and NIR emissions. In these clusters, the Schiff base ligands display a “U-shaped” configuration, which is suitable to form π⋯π interactions and hydrogen bonds with nitro aromatic compounds. Interestingly, Zn–Sm cluster 3 shows visible and NIR luminescence sensing of nitro explosives and exhibits high sensitivity to 1,4-DNB and TNT at the ppb level.
PDF
