Sap Transporter Mediated Import and Subsequent Degradation of Antimicrobial Peptides in Haemophilus

Antimicrobial peptides (AMPs) contribute to host innate immune defense and are a critical component to control bacterial infection. Nontypeable Haemophilus influenzae (NTHI) is a commensal inhabitant of the human nasopharyngeal mucosa, yet is commonly associated with opportunistic infections of the upper and lower respiratory tracts. An important aspect of NTHI virulence is the ability to avert bactericidal effects of host-derived antimicrobial peptides (AMPs). The Sap (sensitivity to antimicrobial peptides) ABC transporter equips NTHI to resist AMPs, although the mechanism of this resistance has remained undefined. We previously determined that the periplasmic binding protein SapA bound AMPs and was required for NTHI virulence in vivo. We now demonstrate, by antibody-mediated neutralization of AMP in vivo, that SapA functions to directly counter AMP lethality during NTHI infection. We hypothesized that SapA would deliver AMPs to the Sap inner membrane complex for transport into the bacterial cytoplasm. We observed that AMPs localize to the bacterial cytoplasm of the parental NTHI strain and were susceptible to cytoplasmic peptidase activity. In striking contrast, AMPs accumulated in the periplasm of bacteria lacking a functional Sap permease complex. These data support a mechanism of Sap mediated import of AMPs, a novel strategy to reduce periplasmic and inner membrane accumulation of these host defense peptides

Type III Secretion System Genes of Dickeya dadantii 3937 Are Induced by Plant Phenolic Acids

Background: Dickeya dadantii is a broad-host range phytopathogen. D. dadantii 3937 (Ech3937) possesses a type III secretion system (T3SS), a major virulence factor secretion system in many Gram-negative pathogens of plants and animals. In Ech3937, the T3SS is regulated by two major regulatory pathways, HrpX/HrpY-HrpS-HrpL and GacS/GacA-rsmB-RsmA pathways. Although the plant apoplast environment, low pH, low temperature, and absence of complex nitrogen sources in media have been associated with the induction of T3SS genes of phytobacteria, no specific inducer has yet been identified. Methodology/Principal Findings: In this work, we identified two novel plant phenolic compounds, o-coumaric acid (OCA) and t-cinnamic acid (TCA), that induced the expression of T3SS genes dspE (a T3SS effector), hrpA (a structural protein of the T3SS pilus), and hrpN (a T3SS harpin) in vitro. Assays by qRT-PCR showed higher amounts of mRNA of hrpL (a T3SS alternative sigma factor) and rsmB (an untranslated regulatory RNA), but not hrpS (a s 54-enhancer binding protein) of Ech3937 when these two plant compounds were supplemented into minimal medium (MM). However, promoter activity assays using flow cytometry showed similar promoter activities of hrpN in rsmB mutant Ech148 grown in MM and MM supplemented with these phenolic compounds. Compared with MM alone, only slightly higher promoter activities of hrpL were observed in bacterial cells grown in MM supplemented with OCA/TCA. Conclusion/Significance: The induction of T3SS expression by OCA and TCA is moderated through the rsmB-Rsm

Genome-Wide Identification of HrpL-Regulated Genes in the Necrotrophic Phytopathogen Dickeya dadantii 3937

BACKGROUND: Dickeya dadantii is a necrotrophic pathogen causing disease in many plants. Previous studies have demonstrated that the type III secretion system (T3SS) of D. dadantii is required for full virulence. HrpL is an alternative sigma factor that binds to the hrp box promoter sequence of T3SS genes to up-regulate their expression. METHODOLOGY/PRINCIPAL FINDINGS: To explore the inventory of HrpL-regulated genes of D. dadantii 3937 (3937), transcriptome profiles of wild-type 3937 and a hrpL mutant grown in a T3SS-inducing medium were examined. Using a cut-off value of 1.5, significant differential expression was observed in sixty-three genes, which are involved in various cellular functions such as type III secretion, chemotaxis, metabolism, regulation, and stress response. A hidden Markov model (HMM) was used to predict candidate hrp box binding sites in the intergenic regions of 3937, including the promoter regions of HrpL-regulated genes identified in the microarray assay. In contrast to biotrophic phytopathgens such as Pseudomonas syringae, among the HrpL up-regulated genes in 3937 only those within the T3SS were found to contain a hrp box sequence. Moreover, direct binding of purified HrpL protein to the hrp box was demonstrated for hrp box-containing DNA fragments of hrpA and hrpN using the electrophoretic mobility shift assay (EMSA). In this study, a putative T3SS effector DspA/E was also identified as a HrpL-upregulated gene, and shown to be translocated into plant cells in a T3SS-dependent manner. CONCLUSION/SIGNIFICANCES: We provide the genome-wide study of HrpL-regulated genes in a necrotrophic phytopathogen (D. dadantii 3937) through a combination of transcriptomics and bioinformatics, which led to identification of several effectors. Our study indicates the extent of differences for T3SS effector protein inventory requirements between necrotrophic and biotrophic pathogens, and may allow the development of different strategies for disease control for these different groups of pathogens

Nitrous oxide and methane emissions from a vetch cropping season are changed by long-term tillage practices in a Mediterranean agroecosystem

Lower greenhouse gas (GHG) emissions from legume-based cropping systems have encouraged their use to deliver mitigation in agricultural systems. Considerable uncertainties remain about the interaction of legumes with long-term tillage systems on GHG emissions under rainfed agroecosystems. In this context, a field experiment was undertaken under a rainfed vetch crop to evaluate the effect of three long-term tillage systems (i.e. no tillage (NT), minimum tillage (MT) and conventional tillage (CT)) on nitrous oxide (N2O) and methane (CH4) emissions for 1 year. Different N2O flux patterns were observed among tillage systems during the growth period of vetch, which depended on the soil conditions favouring nitrification and denitrification. The NT system maintained a higher sink for N2O than MT and CT from January to mid-April, which significantly reduced N2O emissions at this stage. In this period, denitrification capacity and nirK gene numbers were higher for MT than NT and CT. Additionally, an increase in soil NO− 3 content and more favourable denitrification conditions in MT and NT than in CT for the last crop period increased N2O emissions in conservation tillage systems. Total annual N2O losses were significantly higher in MT (124.2 g N2O–N ha−1) than NT (51.1 g N2O–N ha−1) and CT (54 g N2O–N ha−1) in a vetch crop. Low net uptake of CH4 was observed for all tillage systems. These results suggested that long-term NT may be a better option thanMT to mitigate GHG emissions in rainfed legume-cereal rotation. © Springer-Verlag Berlin Heidelberg 2014

Annotation and overview of the Pseudomonas savastanoi pv. savastanoi NCPPB 3335 draft genome reveals the virulence gene complement of a tumour-inducing pathogen of woody hosts

Pseudomonas savastanoi pv. savastanoi is a tumour-inducing pathogen of Olea europaea L. causing oliveknot disease. Bioinformatic analysis of the draftgenome sequence of strain NCPPB 3335, whichencodes 5232 predicted coding genes on a totallength of 5856 998 bp and a 57.12% G + C, revealed alarge degree of conservation with Pseudomonassyringae pv. phaseolicola 1448A and P. syringae pv.tabaci 11528. However, NCPPB 3335 contains twelvevariable genomic regions, which are absent in all pre-viously sequenced P. syringae strains. Various fea-tures that could contribute to the ability of this strainto survive in a woody host were identified, includingbroad catabolic and transport capabilities for degrad-ing plant-derived aromatic compounds, the duplica-tion of sequences related to the biosynthesis of thephytohormone indoleacetic acid (iaaM, iaaH) and itsamino acid conjugate indoleacetic acid-lysine (iaaLgene), and the repertoire of strain-specific putativetype III secretion system effectors. Access to thisseventh genome sequence belonging to the ‘P. syrin-gae complex’ allowed us to identify 73 predictedcoding genes that are NCPPB 3335-specific. Resultsshown here provide the basis for detailed functionalanalysis of a tumour-inducing pathogen of woodyhosts and for the study of specific adaptations of a P.savastanoi pathovar

Identification of Proteus mirabilis Mutants with Increased Sensitivity to Antimicrobial Peptides

Antimicrobial peptides (APs) are important components of the innate defenses of animals, plants, and microorganisms. However, some bacterial pathogens are resistant to the action of APs. For example, Proteus mirabilis is highly resistant to the action of APs, such as polymyxin B (PM), protegrin, and the synthetic protegrin analog IB-367. To better understand this resistance, a transposon mutagenesis approach was used to generate P. mirabilis mutants sensitive to APs. Four unique PM-sensitive mutants of P. mirabilis were identified (these mutants were >2 to >128 times more sensitive than the wild type). Two of these mutants were also sensitive to IB-367 (16 and 128 times more sensitive than the wild type). Lipopolysaccharide (LPS) profiles of the PM- and protegrin-sensitive mutants demonstrated marked differences in both the lipid A and O-antigen regions, while the PM-sensitive mutants appeared to have alterations of either lipid A or O antigen. Matrix-assisted laser desorption ionization–time of flight mass spectrometry analysis of the wild-type and PM-sensitive mutant lipid A showed species with one or two aminoarabinose groups, while lipid A from the PM- and protegrin-sensitive mutants was devoid of aminoarabinose. When the mutants were streaked on an agar-containing medium, the swarming motility of the PM- and protegrin-sensitive mutants was completely inhibited and the swarming motility of the mutants sensitive to only PM was markedly decreased. DNA sequence analysis of the mutagenized loci revealed similarities to an O-acetyltransferase (PM and protegrin sensitive) and ATP synthase and sap loci (PM sensitive). These data further support the role of LPS modifications as an elaborate mechanism in the resistance of certain bacterial species to APs and suggest that LPS surface charge alterations may play a role in P. mirabilis swarming motility

A type III effector ADP-ribosylates RNA-binding proteins and quells plant immunity

Fu ZQ, Guo M, Jeong B-R, et al. A type III effector ADP-ribosylates RNA-binding proteins and quells plant immunity. Nature. 2007;447(7142):284-288.The bacterial plant pathogen Pseudomonas syringae injects effector proteins into host cells through a type III protein secretion system to cause disease. The enzymatic activities of most of P. syringae effectors and their targets remain obscure. Here we show that the type III effector HopU1 is a mono-ADP-ribosyltransferase (ADP-RT). HopU1 suppresses plant innate immunity in a manner dependent on its ADP-RT active site. The HopU1 substrates in Arabidopsis thaliana extracts were RNA-binding proteins that possess RNA-recognition motifs (RRMs). A. thaliana knockout lines defective in the glycine-rich RNA-binding protein GRP7 ( also known as AtGRP7), a HopU1 substrate, were more susceptible than wild-type plants to P. syringae. The ADP-ribosylation of GRP7 by HopU1 required two arginines within the RRM, indicating that this modification may interfere with GRP7' s ability to bind RNA. Our results suggest a pathogenic strategy where the ADP-ribosylation of RNA-binding proteins quells host immunity by affecting RNA metabolism and the plant defence transcriptome