Distinct Molecular Features of NleG Type 3 Secreted Effectors Allow for Different Roles during Citrobacter rodentium Infection in Mice.

The NleGs are the largest family of type 3 secreted effectors in attaching and effacing (A/E) pathogens, such as enterohemorrhagic Escherichia coli (EHEC), enteropathogenic E. coli, and Citrobacter rodentium. NleG effectors contain a conserved C-terminal U-box domain acting as a ubiquitin protein ligase and target host proteins via a variable N-terminal portion. The specific roles of these effectors during infection remain uncertain. Here, we demonstrate that the three NleG effectors-NleG1Cr, NleG7Cr, and NleG8Cr-encoded by C. rodentium DBS100 play distinct roles during infection in mice. Using individual nleGCr knockout strains, we show that NleG7Cr contributes to bacterial survival during enteric infection while NleG1Cr promotes the expression of diarrheal symptoms and NleG8Cr contributes to accelerated lethality in susceptible mice. Furthermore, the NleG8Cr effector contains a C-terminal PDZ domain binding motif that enables interaction with the host protein GOPC. Both the PDZ domain binding motif and the ability to engage with host ubiquitination machinery via the intact U-box domain proved to be necessary for NleG8Cr function, contributing to the observed phenotype during infection. We also establish that the PTZ binding motif in the EHEC NleG8 (NleG8Ec) effector, which shares 60% identity with NleG8Cr, is engaged in interactions with human GOPC. The crystal structure of the NleG8Ec C-terminal peptide in complex with the GOPC PDZ domain, determined to 1.85 Å, revealed a conserved interaction mode similar to that observed between GOPC and eukaryotic PDZ domain binding motifs. Despite these common features, nleG8Ec does not complement the ΔnleG8Cr phenotype during infection, revealing functional diversification between these NleG effectors.

Structure of the full-length Serratia marcescens acetyltransferase AAC(3)-Ia in complex with coenzyme A.

Acyl-coenzyme A-dependent N-acetyltransferases (AACs) catalyze the modification of aminoglycosides rendering the bacteria carrying such enzymes resistant to this class of antibiotics. Here we present the crystal structure of AAC(3)-Ia enzyme from Serratia marcescens in complex with coenzyme A determined to 1.8 Å resolution. This enzyme served as an architype for the AAC enzymes targeting the amino group at Position 3 of aminoglycoside main aminocyclitol ring. The structure of this enzyme has been previously determined only in truncated form and was interpreted as distinct from subsequently characterized AACs. The reason for the unusual arrangement of secondary structure elements of AAC(3)-Ia was not further investigated. By determining the full-length structure of AAC(3)-Ia we establish that this enzyme adopts the canonical AAC fold conserved across this family and it does not undergo through significant rearrangement of secondary structure elements upon ligand binding as was proposed previously. In addition, our results suggest that the C-terminal tail in AAC(3)-Ia monomer forms intramolecular hydrogen bonds that contributes to formation of stable dimer, representing the predominant oligomeric state for this enzyme.

Mai1 Protein Acts Between Host Recognition of Pathogen Effectors and Mitogen-Activated Protein Kinase Signaling.

The molecular mechanisms acting between host recognition of pathogen effectors by nucleotide-binding leucine-rich repeat receptor (NLR) proteins and mitogen-activated protein kinase (MAPK) signaling cascades are unknown. MAPKKKα (M3Kα) activates MAPK signaling leading to programmed cell death (PCD) associated with NLR-triggered immunity. We identified a tomato M3Kα-interacting protein, SlMai1, that has 80% amino acid identity with Arabidopsis brassinosteroid kinase 1 (AtBsk1). SlMai1 has a protein kinase domain and a C-terminal tetratricopeptide repeat domain that interacts with the kinase domain of M3Kα. Virus-induced gene silencing of Mai1 homologs in Nicotiana benthamiana increased susceptibility to Pseudomonas syringae and compromised PCD induced by four NLR proteins. PCD was restored by expression of a synthetic SlMai1 gene that resists silencing. Expression of AtBsk1 did not restore PCD in Mai1-silenced plants, suggesting SlMai1 is functionally divergent from AtBsk1. PCD caused by overexpression of M3Kα or MKK2 was unaffected by Mai1 silencing, suggesting Mai1 acts upstream of these proteins. Coexpression of Mai1 with M3Kα in leaves enhanced MAPK phosphorylation and accelerated PCD. These findings suggest Mai1 is a molecular link acting between host recognition of pathogens and MAPK signaling.

Complete Genome Sequence of Citrobacter rodentium Strain DBS100.

Citrobacter rodentium strain DBS100 causes an infection of the intestines in mice. It provides an important model for human gastrointestinal pathogens, such as enteropathogenic and enterohemorrhagic Escherichia coli, which cause life-threatening infections. To identify the genetic determinants that are common across the enteropathogenic bacteria, we sequenced the DBS100 genome.

Effector Gene xopAE of Xanthomonas euvesicatoria 85-10 Is Part of an Operon and Encodes an E3 Ubiquitin Ligase.

The type III effector XopAE from the Xanthomonas euvesicatoria strain 85-10 was previously shown to inhibit plant immunity and enhance pathogen-induced disease symptoms. Evolutionary analysis of 60 xopAE alleles (AEal) revealed that the xopAE locus is conserved in multiple Xanthomonas species. The majority of xopAE alleles (55 out of 60) comprise a single open reading frame (ORF) (xopAE), while in 5 alleles, including AEal 37 of the X. euvesicatoria 85-10 strain, a frameshift splits the locus into two ORFs (hpaF and a truncated xopAE). To test whether the second ORF of AEal 37 (xopAE85-10 ) is translated, we examined expression of yellow fluorescent protein (YFP) fused downstream to truncated or mutant forms of the locus in Xanthomonas bacteria. YFP fluorescence was detected at maximal levels when the reporter was in proximity to an internal ribosome binding site upstream of a rare ATT start codon in the xopAE85-10 ORF but was severely reduced when these elements were abolished. In agreement with the notion that xopAE85-10 is a functional gene, its protein product was translocated into plant cells by the type III secretion system, and translocation was dependent on its upstream ORF, hpaF Homology modeling predicted that XopAE85-10 contains an E3 ligase XL box domain at the C terminus, and in vitro assays demonstrated that this domain displays monoubiquitination activity. Remarkably, the XL box was essential for XopAE85-10 to inhibit pathogen-associated molecular pattern (PAMP)-induced gene expression in Arabidopsis protoplasts. Together, these results indicate that the xopAE85-10 gene resides in a functional operon, which utilizes the alternative start codon ATT and encodes a novel XL box E3 ligase.IMPORTANCEXanthomonas bacteria utilize a type III secretion system to cause disease in many crops. This study provides insights into the evolution, translocation, and biochemical function of the XopAE type III secreted effector, contributing to the understanding of Xanthomonas-host interactions. We establish XopAE as a core effector of seven Xanthomonas species and elucidate the evolution of the Xanthomonas euvesicatoriaxopAE locus, which contains an operon encoding a truncated effector. Our findings indicate that this operon evolved from the split of a multidomain gene into two ORFs that conserved the original domain function. Analysis of xopAE85-10 translation provides the first evidence for translation initiation from an ATT codon in Xanthomonas Our data demonstrate that XopAE85-10 is an XL box E3 ubiquitin ligase and provide insights into the structure and function of this effector family.

The Xanthomonas euvesicatoria type III effector XopAU is an active protein kinase that manipulates plant MAP kinase signaling.

The Gram-negative bacterium Xanthomonas euvesicatoria (Xe) is the causal agent of bacterial spot disease of pepper and tomato. Xe delivers effector proteins into host cells through the type III secretion system to promote disease. Here, we show that the Xe effector XopAU, which is conserved in numerous Xanthomonas species, is a catalytically active protein kinase and contributes to the development of disease symptoms in pepper plants. Agrobacterium-mediated expression of XopAU in host and non-host plants activated typical defense responses, including MAP kinase phosphorylation, accumulation of pathogenesis-related (PR) proteins and elicitation of cell death, that were dependent on the kinase activity of the effector. XopAU-mediated cell death was not dependent on early signaling components of effector-triggered immunity and was also observed when the effector was delivered into pepper leaves by Xanthomonas campestris pv. campestris, but not by Xe. Protein-protein interaction studies in yeast and in planta revealed that XopAU physically interacts with components of plant immunity-associated MAP kinase cascades. Remarkably, XopAU directly phosphorylated MKK2 in vitro and enhanced its phosphorylation at multiple sites in planta. Consistent with the notion that MKK2 is a target of XopAU, silencing of the MKK2 homolog or overexpression of the catalytically inactive mutant MKK2K99R in N. benthamiana plants reduced XopAU-mediated cell death and MAPK phosphorylation. Furthermore, yeast co-expressing XopAU and MKK2 displayed reduced growth and this phenotype was dependent on the kinase activity of both proteins. Together, our results support the conclusion that XopAU contributes to Xe disease symptoms in pepper plants and manipulates host MAPK signaling through phosphorylation and activation of MKK2.

Assessing the Ability of Salmonella enterica to Translocate Type III Effectors Into Plant Cells.

Salmonella enterica serovar Typhimurium, a human enteric pathogen, has the ability to multiply and survive endophytically in plants. Genes encoding the type III secretion system (T3SS) or its effectors (T3Es) may contribute to its colonization. Two reporter plasmids for T3E translocation into plant cells that are based on hypersensitive response domains of avirulence proteins from the Pantoea agglomerans-beet and Xanthomonas euvesicatoria-pepper pathosystems were employed in this study to investigate the role of T3Es in the interaction of Salmonella ser. Typhimurium 14028 with plants. The T3Es of Salmonella ser. Typhimurium, SipB and SifA, which are translocated into animal cells, could not be delivered by Salmonella ser. Typhimurium into cells of beet roots or pepper leaves. In contrast, these effectors were translocated into plant cells by the phytopathogenic bacteria P. agglomerans pv. betae, Erwinia amylovora, and X. euvesicatoria. Similarly, HsvG, a T3E of P. agglomerans pv. gypsophilae, and XopAU of X. euvesicatoria could be translocated into beet roots and pepper leaves, respectively, by the plant pathogens but not by Salmonella ser. Typhimurium. Mutations in Salmonella ser. Typhimurium T3SS genes invA, ssaV, sipB, or sifA, did not affect its endophytic colonization of lettuce leaves, supporting the notion that S. enterica cannot translocate T3Es into plant cells.

Multiple Xanthomonas euvesicatoria Type III Effectors Inhibit flg22-Triggered Immunity.

Xanthomonas euvesicatoria is the causal agent of bacterial spot disease in pepper and tomato. X. euvesicatoria bacteria interfere with plant cellular processes by injecting effector proteins into host cells through the type III secretion (T3S) system. About 35 T3S effectors have been identified in X. euvesicatoria 85-10, and a few of them were implicated in suppression of pattern-triggered immunity (PTI). We used an Arabidopsis thaliana pathogen-free protoplast-based assay to identify X. euvesicatoria 85-10 effectors that interfere with PTI signaling induced by the bacterial peptide flg22. Of 33 tested effectors, 17 inhibited activation of a PTI-inducible promoter. Among them, nine effectors also interfered with activation of an abscisic acid-inducible promoter. However, effectors that inhibited flg22-induced signaling did not affect phosphorylation of mitogen-activated protein (MAP) kinases acting downstream of flg22 perception. Further investigation of selected effectors revealed that XopAJ, XopE2, and XopF2 inhibited activation of a PTI-inducible promoter by the bacterial peptide elf18 in Arabidopsis protoplasts and by flg22 in tomato protoplasts. The effectors XopF2, XopE2, XopAP, XopAE, XopH, and XopAJ inhibited flg22-induced callose deposition in planta and enhanced disease symptoms caused by attenuated Pseudomonas syringae bacteria. Finally, selected effectors were found to localize to various plant subcellular compartments. These results indicate that X. euvesicatoria bacteria utilize multiple T3S effectors to suppress flg22-induced signaling acting downstream or in parallel to MAP kinase cascades and suggest they act through different molecular mechanisms.

Social and clinical determinants of quality of life in Parkinson's disease in a Russian cohort study.

Parkinson's disease (PD) is a chronic neurodegenerative disorder that has a major impact on health and longevity in Eastern countries. Studies investigating health-related quality of life (HRQoL) in Eastern European and Asian countries are scarce. The objective of this cross-sectional survey was to assess HRQoL in Russian patients with PD and identify its social and clinical determinants. The study included 100 outpatients with idiopathic PD and 100 controls. Patients were consecutively recruited from the neurological department of the Russian Medical State University in Moscow between October 2004 and December 2005. Regional healthy controls were matched for age and sex. The evaluation of HRQoL was performed using the EuroQol instrument (EQ-5D and EQ VAS). Disease severity was assessed using the Unified Parkinson's Disease Rating Scale (UPDRS). Multivariate regression analyses were used to identify independent determinants of HRQoL. HRQoL was more notably decreased in PD patients than in controls (98% versus 74% of individuals with moderate or severe problems in at least one dimension of the EQ-5D (p < 0.001), respectively). As compared to patients, the controls reported a higher mean EQ VAS score (74.0 +/- 16.0 versus 47.7 +/- 16.7, p < 0.001). Social and clinical determinants of HRQoL were age, disease severity, dystonia, depression, dementia and social support. While the HRQoL of patients with PD in Western countries is predominately affected by clinical parameters, social factors play an important role in Eastern countries. Our data should be considered in the development of national healthcare programs that seek to provide better social services support for patients with PD.

Costs of illness in a Russian cohort of patients with Parkinson's disease.

BACKGROUND: The economic burden associated with Parkinson's disease (PD) is increasing as the worldwide population ages. While cost-of-illness studies for PD from developed countries have recently been published, data for Eastern Europe and Asia are still lacking. OBJECTIVE: To prospectively evaluate direct and indirect costs in a cohort of Russian patients with PD in order to identify cost-driving factors. METHODS AND PATIENTS: We recruited 100 patients with idiopathic PD who visited the outpatient department for movement disorders of the Russian Medical State University in Moscow between October 2004 and December 2005. The Unified Parkinson's Disease Rating Scale was used to evaluate clinical status. Economic data were collected in a 'bottom-up' approach and evaluated from the societal perspective. Indirect costs were estimated using a human capital approach. Russian currency was converted into euro, year 2005 values, using the purchasing power parity. All costs were then inflated to euro, year 2008 values, using the Medical Care Component of the Consumer Price Index. Independent cost predictors were identified by means of multivariate regression analyses. RESULTS: From the societal perspective, total costs per patient over 6 months amounted to euro2620 (95% CI 2050, 3200), with direct costs accounting for 67% and indirect costs for 33% of the total. Patients' expenditures accounted for 43% of their private income. The primary burden on patients was due to informal care and drugs. Only 10% of home care was provided by the formal service sector. Costs for the nation are estimated at euro1.1 billion per year. CONCLUSION: The economic burden of PD in Russia is considerable, especially when taking into account low private incomes. Further development of a formal care system and better reimbursement systems for drugs are necessary in Russia.