Comparative genomic analysis of Pectobacterium carotovorum subsp. brasiliense SX309 provides novel insights into its genetic and phenotypic features.

BACKGROUND: Pectobacterium carotovorum subsp. brasiliense is a broad host range bacterial pathogen, which causes blackleg of potatoes and bacterial soft rot of vegetables worldwide. Production of plant cell wall degrading enzymes is usually critical for Pectobacterium infection. However, other virulence factors and the mechanisms of genetic adaptation still need to be studied in detail. RESULTS: In this study, the complete genome of P. carotovorum subsp. brasiliense strain SX309 isolated from cucumber was compared with eight other pathogenic bacteria belonging to the Pectobacterium genus, which were isolated from various host plants. Genome comparison revealed that most virulence genes are highly conserved in the Pectobacterium strains, especially for the key virulence determinants involved in the biosynthesis of extracellular enzymes and others including the type II and III secretion systems, quorum sensing system, flagellar and chemotactic genes. Nevertheless, some variable regions of the T6SS and the CRISP-Cas immune system are unique for P. carotovorum subsp. brasiliense. CONCLUSIONS: The extensive comparative genomics analysis revealed highly conserved virulence genes in the Pectobacterium strains. However, several variable regions of type VI secretion system and two subtype Cas mechanism-Cas immune systems possibly contribute to the process of Pectobacterium infection and adaptive immunity.

Cytosolic and Secreted Peptidoglycan-Degrading Enzymes in Drosophila Respectively Control Local and Systemic Immune Responses to Microbiota.

Gut-associated bacteria produce metabolites that both have a local influence on the intestinal tract and act at a distance on remote organs. In Drosophila, bacteria-derived peptidoglycan (PGN) displays such a dual role. PGN triggers local antimicrobial peptide production by enterocytes; it also activates systemic immune responses in fat-body cells and modulates fly behavior by acting on neurons. How these responses to a single microbiota-derived compound are simultaneously coordinated is not understood. We show here that the PGRP-LB locus generates both cytosolic and secreted PGN-cleaving enzymes. Through genetic analysis, we demonstrate that the cytosolic PGRP-LB isoforms cell-autonomously control the intensity of NF-κB activation in enterocytes, whereas the secreted isoform prevents massive and detrimental gut-derived PGN dissemination throughout the organism. This study explains how Drosophila are able to uncouple the modulation of local versus systemic responses to a single gut-bacteria-derived product by using isoform-specific enzymes.

Oligopeptide Transporters of the SLC15 Family Are Dispensable for Peptidoglycan Sensing and Transport in Drosophila.

Peptidoglycan (PGN) detection by PGN recognition proteins (PGRP) is the main trigger of the antibacterial immune response in Drosophila. Depending on the type of immune cell, PGN can be sensed either at the cell membrane by PGRP-LC or inside the cell by PGRP-LE, which plays a role similar to that of Nod2 in mammals. Previous work, mainly in cell cultures, has shown that oligopeptide transporters of the SLC15 family are essential for the delivery of PGN for Nod2 detection inside of the cells, and that this function might be conserved in flies. By generating and analyzing the immune phenotypes of loss-of-function mutations in 3 SLC15 Drosophila family members, we tested their role in mediating PGRP-LE-dependent PGN activation. Our results show that Yin, CG2930, and CG9444 are required neither for PGRP-LE activation by PGN nor for PGN transport from the gut lumen to the insect blood. These data show that, while intracellular PGN detection is an essential step of the antibacterial response in both insects and mammals, the types of PGN transporters and sensors are different in these animals.

The regulatory isoform rPGRP-LC induces immune resolution via endosomal degradation of receptors.

The innate immune system needs to distinguish between harmful and innocuous stimuli to adapt its activation to the level of threat. How Drosophila mounts differential immune responses to dead and live Gram-negative bacteria using the single peptidoglycan receptor PGRP-LC is unknown. Here we describe rPGRP-LC, an alternative splice variant of PGRP-LC that selectively dampens immune response activation in response to dead bacteria. rPGRP-LC-deficient flies cannot resolve immune activation after Gram-negative infection and die prematurely. The alternative exon in the encoding gene, here called rPGRP-LC, encodes an adaptor module that targets rPGRP-LC to membrane microdomains and interacts with the negative regulator Pirk and the ubiquitin ligase DIAP2. We find that rPGRP-LC-mediated resolution of an efficient immune response requires degradation of activating and regulatory receptors via endosomal ESCRT sorting. We propose that rPGRP-LC selectively responds to peptidoglycans from dead bacteria to tailor the immune response to the level of threat.

Plant immunity triggered by engineered in vivo release of oligogalacturonides, damage-associated molecular patterns.

Oligogalacturonides (OGs) are fragments of pectin that activate plant innate immunity by functioning as damage-associated molecular patterns (DAMPs). We set out to test the hypothesis that OGs are generated in planta by partial inhibition of pathogen-encoded polygalacturonases (PGs). A gene encoding a fungal PG was fused with a gene encoding a plant polygalacturonase-inhibiting protein (PGIP) and expressed in transgenic Arabidopsis plants. We show that expression of the PGIP-PG chimera results in the in vivo production of OGs that can be detected by mass spectrometric analysis. Transgenic plants expressing the chimera under control of a pathogen-inducible promoter are more resistant to the phytopathogens Botrytis cinerea, Pectobacterium carotovorum, and Pseudomonas syringae. These data provide strong evidence for the hypothesis that OGs released in vivo act as a DAMP signal to trigger plant immunity and suggest that controlled release of these molecules upon infection may be a valuable tool to protect plants against infectious diseases. On the other hand, elevated levels of expression of the chimera cause the accumulation of salicylic acid, reduced growth, and eventually lead to plant death, consistent with the current notion that trade-off occurs between growth and defense.

One shot-two pathogens blocked: exposure of Arabidopsis to hexadecane, a long chain volatile organic compound, confers induced resistance against both Pectobacterium carotovorum and Pseudomonas syringae.

Bacteria and plant derived volatile organic compounds have been reported as the chemical triggers that elicit induced resistance in plants. Previously, volatile organic compounds (VOCs), including acetoin and 2,3-butanediol, were found to be emitted from plant growth-promoting rhizobacteria (PGPR) Bacillus subtilis GB03, which had been shown to elicit ISR and plant growth promotion. More recently, we reported data that stronger induced resistance could be elicited against Pseudomonas syringae pv maculicola ES4326 in plants exposed to C13 VOC from another PGPR Paenibacillus polymyxa E681 compared with that of strain GB03. Here, we assessed whether another long hydrocarbon C16 hexadecane (HD) conferred protection to Arabidopsis from infection of a biotrophic pathogen, P. syringae pv maculicola and a necrotrophic pathogen, Pectobacterium carotovorum subsp carotovorum. Collectively, long-chain VOCs can be linked to a plant resistance activator for protecting plants against both biotrophic and necrotrophic pathogens at the same time.

JNK/FOXO mediated PeroxiredoxinV expression regulates redox homeostasis during Drosophila melanogaster gut infection.

Innate immunity plays an important role in combating microbial infection in animals. During bacterial infection in Drosophila melanogaster gut, Dual oxidase (Duox) generates reactive oxygen species (ROS) to fight against the infected microbes. Concurrently, antioxidant systems eliminate residual ROS and protect the hosts. Here we found that Drosophila melanogaster Peroxiredoxin V (dPrxV) is an immune-related antioxidant enzyme which maintains intestinal redox homeostasis. dPrxV was highly expressed in gut and induced by the oral infection of Erwinia carotovora carotovora. dPrxV expression was increased by the gut-specific Duox overexpression but decreased by Duox inhibition. Moreover, dPrxV expression was mediated by the JNK/FOXO signaling and dPrxV mutant reduced survival after gut infection. These results suggest that JNK/FOXO mediated dPrxV expression plays a critical role in Drosophila melanogaster gut during bacterial infection in protecting the host gut epithelial cells from oxidative damage.

Genetic evidence for a protective role of the peritrophic matrix against intestinal bacterial infection in Drosophila melanogaster.

The peritrophic matrix (PM) forms a layer composed of chitin and glycoproteins that lines the insect intestinal lumen. This physical barrier plays a role analogous to that of mucous secretions of the vertebrate digestive tract and is thought to protect the midgut epithelium from abrasive food particles and microbes. Almost nothing is known about PM functions in Drosophila, and its function as an immune barrier has never been addressed by a genetic approach. Here we show that the Drosocrystallin (Dcy) protein, a putative component of the eye lens of Drosophila, contributes to adult PM formation. A loss-of-function mutation in the dcy gene results in a reduction of PM width and an increase of its permeability. Upon bacterial ingestion a higher level of expression of antibacterial peptides was observed in dcy mutants, pointing to an influence of this matrix on bacteria sensing by the Imd immune pathway. Moreover, dcy-deficient flies show an increased susceptibility to oral infections with the entomopathogenic bacteria Pseudomonas entomophila and Serratia marcescens. Dcy mutant flies also succumb faster than wild type upon ingestion of a P. entomophila toxic extract. We show that this lethality is due in part to an increased deleterious action of Monalysin, a pore-forming toxin produced by P. entomophila. Collectively, our analysis of the dcy immune phenotype indicates that the PM plays an important role in Drosophila host defense against enteric pathogens, preventing the damaging action of pore-forming toxins on intestinal cells.

Long-range activation of systemic immunity through peptidoglycan diffusion in Drosophila.

The systemic immune response of Drosophila is known to be induced both by septic injury and by oral infection with certain bacteria, and is characterized by the secretion of antimicrobial peptides (AMPs) into the haemolymph. To investigate other possible routes of bacterial infection, we deposited Erwinia carotovora (Ecc15) on various sites of the cuticle and monitored the immune response via expression of the AMP gene Diptericin. A strong response was observed to deposition on the genital plate of males (up to 20% of a septic injury response), but not females. We show that the principal response to genital infection is systemic, but that some AMPs, particularly Defensin, are induced locally in the genital tract. At late time points we detected bacteria in the haemolymph of immune deficient Relish(E20) flies, indicating that the genital plate can be a route of entry for pathogens, and that the immune response protects flies against the progression of genital infection. The protective role of the immune response is further illustrated by our observation that Relish(E20) flies exhibit significant lethality in response to genital Ecc15 infections. We next show that a systemic immune response can be induced by deposition of the bacterial elicitor peptidoglycan (PGN), or its terminal monomer tracheal cytotoxin (TCT), on the genital plate. This immune response is downregulated by PGRP-LB and Pirk, known regulators of the Imd pathway, and can be suppressed by the overexpression of PGRP-LB in the haemolymph compartment. Finally, we provide strong evidence that TCT can activate a systemic response by crossing epithelia, by showing that radiolabelled TCT deposited on the genital plate can subsequently be detected in the haemolymph. Genital infection is thus an intriguing new model for studying the systemic immune response to local epithelial infections and a potential route of entry for naturally occurring pathogens of Drosophila.

Drosophila intestinal response to bacterial infection: activation of host defense and stem cell proliferation.

Although Drosophila systemic immunity is extensively studied, little is known about the fly's intestine-specific responses to bacterial infection. Global gene expression analysis of Drosophila intestinal tissue to oral infection with the Gram-negative bacterium Erwinia carotovora revealed that immune responses in the gut are regulated by the Imd and JAK-STAT pathways, but not the Toll pathway. Ingestion of bacteria had a dramatic impact on the physiology of the gut that included modulation of stress response and increased stem cell proliferation and epithelial renewal. Our data suggest that gut homeostasis is maintained through a balance between cell damage due to the collateral effects of bacteria killing and epithelial repair by stem cell division. The Drosophila gut provides a powerful model to study the integration of stress and immunity with pathways associated with stem cell control, and this study should prove to be a useful resource for such further studies.

Antiviral immunity in Drosophila requires systemic RNA interference spread.

Multicellular organisms evolved sophisticated defence systems to confer protection against pathogens. An important characteristic of these immune systems is their ability to act both locally at the site of infection and at distal uninfected locations. In insects, such as Drosophila melanogaster, RNA interference (RNAi) mediates antiviral immunity. However, the antiviral RNAi defence in flies seems to be a local, cell-autonomous process, as flies are thought to be unable to generate a systemic RNAi response. Here we show that a recently defined double-stranded RNA (dsRNA) uptake pathway is essential for effective antiviral RNAi immunity in adult flies. Mutant flies defective in this dsRNA uptake pathway were hypersensitive to infection with Drosophila C virus and Sindbis virus. Mortality in dsRNA-uptake-defective flies was accompanied by 100-to 10(5)-fold increases in viral titres and higher levels of viral RNA. Furthermore, inoculating naked dsRNA into flies elicited a sequence-specific antiviral immune response that required an intact dsRNA uptake pathway. These findings suggest that spread of dsRNA to uninfected sites is essential for effective antiviral immunity. Notably, infection with green fluorescent protein (GFP)-tagged Sindbis virus suppressed expression of host-encoded GFP at a distal site. Thus, similar to protein-based immunity in vertebrates, the antiviral RNAi response in flies also relies on the systemic spread of a virus-specific immunity signal.

Rudra interrupts receptor signaling complexes to negatively regulate the IMD pathway.

Insects rely primarily on innate immune responses to fight pathogens. In Drosophila, antimicrobial peptides are key contributors to host defense. Antimicrobial peptide gene expression is regulated by the IMD and Toll pathways. Bacterial peptidoglycans trigger these pathways, through recognition by peptidoglycan recognition proteins (PGRPs). DAP-type peptidoglycan triggers the IMD pathway via PGRP-LC and PGRP-LE, while lysine-type peptidoglycan is an agonist for the Toll pathway through PGRP-SA and PGRP-SD. Recent work has shown that the intensity and duration of the immune responses initiating with these receptors is tightly regulated at multiple levels, by a series of negative regulators. Through two-hybrid screening with PGRP-LC, we identified Rudra, a new regulator of the IMD pathway, and demonstrate that it is a critical feedback inhibitor of peptidoglycan receptor signaling. Following stimulation of the IMD pathway, rudra expression was rapidly induced. In cells, RNAi targeting of rudra caused a marked up-regulation of antimicrobial peptide gene expression. rudra mutant flies also hyper-activated antimicrobial peptide genes and were more resistant to infection with the insect pathogen Erwinia carotovora carotovora. Molecularly, Rudra was found to bind and interfere with both PGRP-LC and PGRP-LE, disrupting their signaling complex. These results show that Rudra is a critical component in a negative feedback loop, whereby immune-induced gene expression rapidly produces a potent inhibitor that binds and inhibits pattern recognition receptors.

Endophytic actinobacteria induce defense pathways in Arabidopsis thaliana.

Endophytic actinobacteria, isolated from healthy wheat tissue, which are capable of suppressing a number wheat fungal pathogens both in vitro and in planta, were investigated for the ability to activate key genes in the systemic acquired resistance (SAR) or the jasmonate/ethylene (JA/ET) pathways in Arabidopsis thaliana. Inoculation of A. thaliana (Col-0) with selected endophytic strains induced a low level of SAR and JA/ET gene expression, measured using quantitative polymerase chain reaction. Upon pathogen challenge, endophyte-treated plants demonstrated a higher abundance of defense gene expression compared with the non-endophyte-treated controls. Resistance to the bacterial pathogen Erwinia carotovora subsp. carotovora required the JA/ET pathway. On the other hand, resistance to the fungal pathogen Fusarium oxysporum involved primarily the SAR pathway. The endophytic actinobacteria appear to be able to "prime" both the SAR and JA/ET pathways, upregulating genes in either pathway depending on the infecting pathogen. Culture filtrates of the endophytic actinobacteria were investigated for the ability to also activate defense pathways. The culture filtrate of Micromonospora sp. strain EN43 grown in a minimal medium resulted in the induction of the SAR pathway; however, when grown in a complex medium, the JA/ET pathway was activated. Further analysis using Streptomyces sp. strain EN27 and defense-compromised mutants of A. thaliana indicated that resistance to E. carotovora subsp. carotovora occurred via an NPR1-independent pathway and required salicylic acid whereas the JA/ET signaling molecules were not essential. In contrast, resistance to F. oxysporum mediated by Streptomyces sp. strain EN27 occurred via an NPR1-dependent pathway but also required salicylic acid and was JA/ET independent.

[Functional organization of prophage and lysogeny in Erwinia carotovora with participation of a temperate bacteriophage ZF40].

Functional organization of a prophage of the temperate bacteriophage ZF40 of Erwinia carotovora subsp. carotovora which includes its immunity and inducibility as well as its effect on the host phenotype. It was established that the prophage ZF40 forms several different states in E. carotovora which are distinguished by the indices of spontaneous and lysogenic induction. In contrast to other prophages, including the lambdoid ones, the prophage ZF40 is capable to establish cytoplasmic overimmunity which protects the lysogenic system from superinfection by virulent mutants or other homoimmune bacteriophages. An increase of sensitivity of ZF40-lysogens to killing activity of colicino-like carotovoricin (CCTV) and destabilization of defective lysogeny, or resistant MCTV-prophages are related to the phenomenon of the phage lysogenic conversion of E. carotovora.

Nitric oxide contributes to induction of innate immune responses to gram-negative bacteria in Drosophila.

Studies in mammals uncovered important signaling roles of nitric oxide (NO), and contributions to innate immunity. Suggestions of conservation led us to explore the involvement of NO in Drosophila innate immunity. Inhibition of nitric oxide synthase (NOS) increased larval sensitivity to gram-negative bacterial infection, and abrogated induction of the antimicrobial peptide Diptericin. NOS was up-regulated after infection. Antimicrobial peptide reporters revealed that NO triggered an immune response in uninfected larvae. NO induction of Diptericin reporters in the fat body required immune deficiency (imd) and domino. These findings show that NOS activity is required for a robust innate immune response to gram-negative bacteria, NOS is induced by infection, and NO is sufficient to trigger response in the absence of infection. We propose that NO mediates an early step of the signal transduction pathway, inducing the innate immune response upon natural infection with gram-negative bacteria.

Altered lignin structure and resistance to pathogens in spi 2-expressing tobacco plants.

The physiological role of the Norway spruce [ Picea abies (L.) Karst.] spi 2 gene, encoding a defense-related cationic peroxidase was examined in transgenic tobacco (Nicotiana tabacum L.). Expression of spi 2, under control of the 35S promoter, in tobacco plants resulted in higher total peroxidase activities. The phenotype of the spi 2-transformed lines was normal. The spi 2-transformed lines displayed lignin levels similar to levels in the control line, but with some alteration in lignin histochemistry and structure. These changes were associated with reduced flexibility of the tobacco stems. The defense against pathogenic microorganisms was altered in the transgenic tobacco plants compared with control plants. High peroxidase activities increased the susceptibility to the pathogenic oomycete Phytophthora parasitica var. nicotianae, but increased the ability of the tobacco plants to suppress growth of the pathogenic bacterium Erwinia carotovora.

Enhanced disease resistance conferred by expression of an antimicrobial magainin analog in transgenic tobacco.

Magainins are a group of short peptides originally isolated from frog skin and thought to function as a natural defense mechanism against infection due to their antimicrobial properties. The engineered magainin analog peptide Myp30 was found to inhibit spore germination of the oomycete, Peronospora tabacina (Adam) in vitro, and the growth of a bacterial pathogen Erwinia carotovora subsp. carotovora (Jones). Transgenic tobacco (Nicotiana tabacum L.) plants expressing Myp30 were evaluated for resistance to these pathogens. The expression of the peptide only to an extracellular location resulted in significant reduction in sporulation and lesion size due to P. tabacina infection. A significant increase in resistance to the bacterial pathogen was also observed regardless of the targeting location of the peptide.

The effect of polysialylation on the immunogenicity and antigenicity of asparaginase: implication in its pharmacokinetics.

Erwinia carotovora L-asparaginase was conjugated via the epsilon-amino groups of its lysine residues with colominic acid (CA) (polysialic acid) of average molecular mass of 10 kDa by reductive amination in the presence of NaCNBH3. Polysialylation using 50-, 100- and 250-fold molar excess CA relative to the enzyme led to an increasing proportion of the enzyme's in-amino groups (5.8, 7.6 and 11.3%, respectively) being conjugated to CA. Polysialylated and native (intact) asparaginase were used to immunize mice intravenously. Results (total IgG immune responses) indicate that all preparations elicited antibody production against the enzyme moiety but not against the CA of the conjugates. Moreover, antibody titres appeared highest for the native enzyme and were generally reduced as the degree of polysialylation increased. In other experiments mice pre-immunized with native or polysialylated asparaginase, with anti-asparaginase antibodies in their blood, were injected intravenously with the corresponding enzyme preparations. Results revealed that polysialylation reduces the antigenicity of asparaginase thus leading to circulatory half-lives (t 1/2 beta) that were 3-4-fold greater than that of the native enzyme, and similar to those observed in naive, non-immunized mice. Our data suggest that polysialylation of therapeutic enzymes and other proteins may be useful in maintaining their pharmacokinetics in individuals with antibodies to the therapeutic proteins as a result of chronic treatment.

Immunomagnetic separation of Erwinia carotovora subsp. atroseptica from potato peel extracts to improve detection sensitivity on a crystal violet pectate medium or by PCR.

Immunomagnetic separation (IMS) procedures for the selective separation of Erwinia carotovora subsp. atroseptica from potato peel extract were optimized for the recovery of target and removal of non-target bacteria. A streptomycin-resistant strain of Erw. carotovora subsp. atroseptica was used in combination with a crystal violet pectate (CVP) medium supplemented with 100 micrograms ml-1 of streptomycin to determine the recovery level of the target bacterium. Recovery obtained with a polyclonal antiserum against Erw. carotovora subsp. atroseptica at a concentration of 6 micrograms IgG ml-1 was greater than that obtained with two monoclonal antibodies against lipopolysaccharides of Erw. carotovora subsp. atroseptica at a concentration of 10 micrograms IgG ml-1. A linear relationship was found between particle concentration ranging from 12 to 200 micrograms ml-1 and recovery level. When the Advanced Magnetics (AM) protein A and anti-rabbit IgG particles in the AM separation system and the Dynal anti-rabbit IgG particles in the Dynal separation system were examined, the highest recovery level per microgram of particles (66%) was obtained with the Advanced Magnetics protein A particles, followed by AM anti-rabbit particles (37%). Without IMS, detection of Erw. carotovora subsp. atroseptica in tuber peel extracts on a CVP-medium without streptomycin was impossible when the ratio of Erw. carotovora subsp. carotovora to Erw. carotovora subsp. atroseptica was greater than 100 or when large numbers of other saprophytic bacteria were present, because of overcrowding. IMS, using the AM anti-rabbit IgG particles, ensured that Erw. carotovora subsp. atroseptica could be enumerated in tuber peel extract consistently, to a detection level of 100 cells ml-1. Similarly, the IMS procedure lowered the detection level of Erw. carotovora subsp. atroseptica in a twofold diluted peel extract by PCR to ca 2.0 x 10(3) cells ml-1 or 50 cells per reaction tube. In contrast, positive results in PCR without IMS were obtained only when the peel extract was diluted 100 times and when the concentration of Erw. carotovora subsp. atroseptica was at least 10(5) cell ml-1.

Characterization of monoclonal antibodies against Erwinia carotovora subsp. atroseptica serogroup I: specificity and epitope analysis.

The characteristics of two monoclonal antibodies (Mabs), A23/1221.59.44.d.3 (1221) and A23/1239.36.64.e.2 (1239), against Erwinia carotovora subsp. atroseptica serogroup I produced in this study were compared with those of two other independently obtained Mabs, 4G4 in Spain and 4F6 in Canada, using different strains as immunogen and different screening procedures. The reaction pattern of Mabs 1221 and 1239 determined by indirect ELISA on over 200 bacterial strains including five E.c. atroseptica and 36 E.c. carotovora serogroups, seven Erw. chrysanthemi biovars, 23 other plant bacterial pathogens and 33 saprophytic bacteria from potato was similar to that of 4G4. Specificity for E.c. atroseptica serogroup I was improved, especially when skimmed milk (Marvel) was used instead of bovine serum albumin as blocking agent. Mabs 1221, 1239 and 4G4 reacted positively with all 22 E.c. atroseptica serogroup I, the dominant E.c. atroseptica serogroup on potato, strains tested and only with two out of five E.c. atroseptica serogroup XXII strains, one E.c. carotovora serogroup XXI strain and one strain of a saprophytic bacterium, Comamonas sp. Essentially similar results were obtained when examined by immunofluorescence. Characterization of the four Mabs showed that they were IgG3 and SDS-PAGE/immunoblot results suggested that they were probably against the O-side chain of bacterial cell wall lipopolysaccharides. In competition ELISA between biotin-labelled and unlabelled Mabs, the competition pattern of the four Mabs was similar.(ABSTRACT TRUNCATED AT 250 WORDS)