Characterization and expression analysis of Staphylococcus aureus pathogenicity island 3 - Implications for the evolution of staphylococcal pathogenicity islands

We describe the complete sequence of the 15.9-kb staphylococcal pathogenicity island 3 encoding staphylococcal enterotoxin serotypes B, K, and Q. The island, which meets the generally accepted definition of pathogenicity islands, contains 24 open reading frames potentially encoding proteins of more than 50 amino acids, including an apparently functional integrase. The element is bordered by two 17-bp direct repeats identical to those found flanking staphylococcal pathogenicity island 1. The island has extensive regions of homology to previously described pathogenicity islands, particularly staphylococcal pathogenicity islands 1 and bov. The expression of 22 of the 24 open reading frames contained on staphylococcal pathogenicity island 3 was detected either in vitro during growth in a laboratory medium or serum or in vivo in a rabbit model of toxic shock syndrome using DNA microarrays. The effect of oxygen tension on staphylococcal pathogenicity island 3 gene expression was also examined. By comparison with the known staphylococcal pathogenicity islands in the context of gene expression described here, we propose a model of pathogenicity island origin and evolution involving specialized transduction events and addition, deletion, or recombination of pathogenicity island "modules.

Hepatocyte-derived IL-10 plays a crucial role in attenuating pathogenicity during the chronic phase of T. congolense infection

Bovine African Trypanosomosis is an infectious parasitic disease affecting livestock productivity and thereby impairing the economic development of Sub-Saharan Africa. The most important trypanosome species implicated is T. congolense, causing anemia as most important pathological feature. Using murine models, it was shown that due to the parasite's efficient immune evasion mechanisms, including (i) antigenic variation of the variable surface glycoprotein (VSG) coat, (ii) induction of polyclonal B cell activation, (iii) loss of B cell memory and (iv) T cell mediated immunosuppression, disease prevention through vaccination has so far been impossible. In trypanotolerant models a strong, early pro-inflammatory immune response involving IFN-gamma, TNF and NO, combined with a strong humoral anti-VSG response, ensures early parasitemia control. This potent protective inflammatory response is counterbalanced by the production of the anti-inflammatory cytokine IL-10, which in turn prevents early death of the host from uncontrolled hyper-inflammation-mediated immunopathologies. Though at this stage different hematopoietic cells, such as NK cells, T cells and B cells as well as myeloid cells (i.e. alternatively activated myeloid cells (M2) or Ly6c(-) monocytes), were found to produce IL-10, the contribution of non-hematopoietic cells as potential IL-10 source during experimental T. congolense infection has not been addressed. Here, we report for the first time that during the chronic stage of T. congolense infection non-hematopoietic cells constitute an important source of IL-10. Our data shows that hepatocyte-derived IL-10 is mandatory for host survival and is crucial for the control of trypanosomosis-induced inflammation and associated immunopathologies such as anemia, hepatosplenomegaly and excessive tissue injury. Author summary Bovine African Trypanosomosis is a parasitic disease of veterinary importance that adversely affects the public health and economic development of sub-Saharan Africa. The most important trypanosome species implicated is T. congolense, causing anemia as most important pathological feature and major cause of death. Using murine models, it was shown that the disease is characterized by a well-timed and balanced production of pro-inflammatory cytokine promoting factors followed by an anti-inflammatory response, involving IL-10. The latter is required to attenuate infection-associated pathogenicity and to prevent early host death from uncontrolled hyper-inflammation mediated immunopathologies. However, the cellular source of IL-10 in vivo and the window within which these cells exert their function during the course of African trypanosomiasis remain poorly understood, which hampers the design of effective therapeutic strategies. Using a T. congolense infection mouse model, relevant for bovine trypanosomosis, we demonstrate that during the chronic stage of infection hepatocyte-derived IL-10, but not myeloid cell-derived IL-10, regulates the main infection-associated immunopathologies and ultimately mediates host survival. Hence, strategies that tilt the balance of hepatocyte cytokine production in favor of IL-10 could majorly impact the wellbeing and survival of T. congolense-infected animals. Given the unmet medical need for this parasite infection, our findings offer promise for improved treatment protocols in the field

Qualitative comparison of cassiicolin in four strains of Corynespora cassiicola

Corynespora cassiicola is a necrotrophic ascomycete fungus affecting a wide range of plants. In rubber tree, C. cassiicola causes the Corynespora Leaf Fall (CLF) disease, responsible for sporadic but often severe epidemics in rubber plantations in most Asian and African producing countries. Divergence in the susceptibility of rubber clones to CLFD in various locations revealed the existence of different physiological races. A toxin secreted by the fungus (cassiicolin) has been identified as the primary determinant of C. cassiicola pathogenicity. An optimized purification protocol allowed the preparation of highly purified toxin in sufficient amount to fulfil its molecular characterization. Cassiicolin was shown to be a 27 amino acids glycosylated protein with 3 disulfide bounds. To test whether qualitative differences in the toxin may influence the pathogenicity, four strains of various geographic origins and with different virulence profiles were analysed comparatively. Biochemical purification of the toxin followed by mass spectrometry was attempted from all four strains, as well as cloning and expression analysis of the full cassiicolin-encoding gene, when detected. The results are discussed with respect to the pathogenicity of the selected strains. (Résumé d'auteur

Basal rot of narcissus : understanding pathogenicity in fusarium oxysporum f. sp. narcissi

Fusarium oxysporum is a globally distributed soilborne fungal pathogen causing root rots, bulb rots, crown rots and vascular wilts on a range of horticultural plants. Pathogenic F. oxysporum isolates are highly host specific and are classified as formae speciales. Narcissus is an important ornamental crop and both the quality and yield of flowers and bulbs can be severely affected by a basal rot caused by F. oxysporum f. sp. narcissi (FON); 154 Fusarium isolates were obtained from different locations and Narcissus cultivars in the United Kingdom, representing a valuable resource. A subset of 30 F. oxysporum isolates were all found to be pathogenic and were therefore identified as FON. Molecular characterisation of isolates through sequencing of three housekeeping genes, suggested a monophyletic origin with little divergence. PCR detection of 14 Secreted in Xylem (SIX) genes, previously shown to be associated with pathogenicity in other F. oxysporum f. spp., revealed different complements of SIX7, SIX9, SIX10, SIX12 and SIX13 within FON isolates which may suggest a race structure. SIX gene sequences were unique to FON and SIX10 was present in all isolates, allowing for molecular identification of FON for the first time. The genome of a highly pathogenic isolate was sequenced and lineage specific (LS) regions identified which harboured putative effectors including the SIX genes. Real-time RT-PCR, showed that SIX genes and selected putative effectors were expressed in planta with many significantly upregulated during infection. This is the first study to characterise molecular variation in FON and provide an analysis of the FON genome. Identification of expressed genes potentially associated with virulence provides the basis for future functional studies and new targets for molecular diagnostics

Development of a pathogenicity testing system for Dothistroma pini infection of Pinus radiata : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University

Dothistroma pini is a fungal pathogen of pine species around the world and can be found in most parts of New Zealand. Infection by D. pini causes a disease commonly known as Dothistroma needle blight. Dothistroma needle blight has a significant financial impact on New Zealand's forestry industry. Although control of infection by D. pini is currently very successful there is a possibility that a new strain introduced from another country could be a lot more damaging and overcome current control measures. In recent years both the incidence and severity of the disease have increased in the northern hemisphere and other parts of the world. A distinctive characteristic of Dothistroma needle blight is the production in the infected needle of a toxic red pigment called dothistromin. Dothistromin is produced as a secondary metabolite by D. pini and has known phytotoxic properties as well as clastogenic and mutagenic properties towards human cells. Purified dothistromin toxin injected into pine needles has been shown to reproduce symptoms similar to those observed during D. pini infection. Because of this production, dothistromin is thought to play an important role in the infection process. Mutants of D. pini that are deficient in dothistromin production have been made recently that will allow this role to be investigated. The aim of this study was to develop a pathogenicity testing system under PC2 containment (required for dothistromin deficient mutant) and to develop microscopy methods required to monitor both epiphytic and endophytic growth of the fungus on the needle D. pini requires high light intensity, continuous leaf moisture and a specific temperature range in order to infect pine needles. Progress was made towards developing a robust pathogenicity testing system. This study has also developed several microscopy techniques for the visualisation of epiphytic growth including a fluorescent microscopy technique. Other bright field and fluorescent staining techniques were investigated with some success. Staining techniques were not successful for the visualisation of endophytic D. pini growth but a green fluorescent protein (sgfp) reporter construct was obtained and two gfp plasmid contracts were developed for the transformation of D. pini for use as biomarkers. Successful introduction of the gfp constructs into D. pini will allow in situ visualisation of endophytic and epiphytic D. pini growth. The work done in this study will be useful for the further investigation into the role of dothistromin toxin, which may lead to new or more efficient methods of controlling D. pini as well as possibly providing information about other polyketide molecules of economic or medical significance

Generation and analysis of expressed sequence tags from Botrytis cinerea

http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0716-97602006000200018&lng=es&nrm=isoBotrytis cinerea is a filamentous plant pathogen of a wide range of plant species, and its infection may cause enormous damage both during plant growth and in the post-harvest phase. We have constructed a cDNA library from an isolate of B. cinerea and have sequenced 11,482 expressed sequence tags that were assembled into 1,003 contigs sequences and 3,032 singletons. Approximately 81% of the unigenes showed significant similarity to genes coding for proteins with known functions: more than 50% of the sequences code for genes involved in cellular metabolism, 12% for transport of metabolites, and approximately 10% for cellular organization. Other functional categories include responses to biotic and abiotic stimuli, cell communication, cell homeostasis, and cell development. We carried out pair-wise comparisons with fungal databases to determine the B. cinerea unisequence set with relevant similarity to genes in other fungal pathogenic counterparts. Among the 4,035 non-redundant B. cinerea unigenes, 1,338 (23%) have significant homology with Fusarium verticillioides unigenes. Similar values were obtained for Saccharomyces cerevisiae and Aspergillus nidulans (22% and 24%, respectively). The lower percentages of homology were with Magnaporthe grisae and Neurospora crassa (13% and 19%, respectively). Several genes involved in putative and known fungal virulence and general pathogenicity were identified. The results provide important information for future research on this fungal pathogen

Mutations in the Schmallenberg virus Gc glycoprotein facilitate cellular protein synthesis shutoff and restore pathogenicity of NSs deletion mutants in mice

Serial passage of viruses in cell culture has been traditionally used to attenuate virulence and identify determinants of viral pathogenesis. In a previous study, we found that a strain of Schmallenberg virus (SBV) serially passaged in tissue culture (termed SBVp32) unexpectedly displayed increased pathogenicity in suckling mice compared to wild type SBV. In this study, we mapped the determinants of SBVp32 virulence to the viral genome M segment. SBVp32 virulence is associated with the capacity of this virus to reach higher titers in the brains of experimentally infected suckling mice. We also found that the Gc glycoprotein, encoded by the M segment of SBVp32, facilitates host cell protein shutoff in vitro. Interestingly, while the M segment of SBVp32 is a virulence factor, we found that the S segment of the same virus confers by itself an attenuated phenotype to wild type SBV as has lost the ability to block the innate immune system of the host. Single mutations present in the Gc glycoprotein of SBVp32 are sufficient to compensate both the attenuated phenotype of the SBVp32 S segment and the attenuated phenotype of NSs deletion mutants. Our data also indicate that the SBVp32 M segment does not act as an IFN antagonist. Therefore SBV mutants can retain pathogenicity even when they are unable to fully control the production of IFN by the infected cells. Overall, this study suggests that the viral glycoprotein of orthobunyaviruses can compensate, at least in part, the function of NSs. In addition, we also provide evidence that the induction of total cellular protein shutoff by SBV is determined by multiple viral proteins while the ability to control the production of IFN maps to the NSs protein. Importance The identification of viral determinants of pathogenesis is key to the development of prophylactic and interventions measures. In this study we found that the bunyavirus Gc glycoprotein is a virulence factor. Importantly, we show that mutations in the Gc glycoprotein can restore pathogenicity of attenuated mutants resulting from deletions or mutations in the non-structural protein NSs. Our findings highlight the fact that careful consideration should be taken when designing live attenuated vaccines based on deletions of non-structural proteins since single mutations in the viral glycoproteins appear to revert attenuated mutants to virulent phenotypes

Role of Jasmonic Acid Pathway in Tomato Plant-Pseudomonas syringae Interaction

The jasmonic acid pathway has been considered as the backbone of the response against necrotrophic pathogens. However, a hemi-biotrophic pathogen, such as Pseudomonas syringae, has taken advantage of the crosstalk between the different plant hormones in order to manipulate the responses for its own interest. Despite that, the way in which Pseudomonas syringae releases coronatine to activate jasmonic acid-derived responses and block the activation of salicylic acid-mediated responses is widely known. However, the implication of the jasmonic intermediates in the plant-Pseudomonas interaction is not studied yet. In this work, we analyzed the response of both, plant and bacteria using SiOPR3 tomato plants. Interestingly, SiOPR3 plants are more resistant to infection with Pseudomonas. The gene expression of bacteria showed that, in SiOPR3 plants, the activation of pathogenicity is repressed in comparison to wild type plants, suggesting that the jasmonic acid pathway might play a role in the pathogenicity of the bacteria. Moreover, treatments with JA restore the susceptibility as well as activate the expression of bacterial pathogenicity genes. The observed results suggest that a complete jasmonic acid pathway is necessary for the susceptibility of tomato plants to Pseudomonas syringae

Occurrence and diversity of Xanthomonas campestris pv. campestris in vegetable brassica fields in Nepal

Black rot caused by Xanthomonas campestris pv. campestris was found in 28 sampled cabbage fields in five major cabbage-growing districts in Nepal in 2001 and in four cauliflower fields in two districts and a leaf mustard seed bed in 2003. Pathogenic X. campestris pv. campestris strains were obtained from 39 cabbage plants, 4 cauliflower plants, and 1 leaf mustard plant with typical lesions. Repetitive DNA polymerase chain reaction-based fingerprinting (rep-PCR) using repetitive extragenic palindromic, enterobacterial repetitive intergenic consensus, and BOX primers was used to assess the genetic diversity. Strains were also race typed using a differential series of Brassica spp. Cabbage strains belonged to five races (races 1, 4, 5, 6, and 7), with races 4, 1, and 6 the most common. All cauliflower strains were race 4 and the leaf mustard strain was race 6. A dendrogram derived from the combined rep-PCR profiles showed that the Nepalese X. campestris pv. campestris strains clustered separately from other Xanthomonas spp. and pathovars. Race 1 strains clustered together and strains of races 4, 5, and 6 were each split into at least two clusters. The presence of different races and the genetic variability of the pathogen should be considered when resistant cultivars are bred and introduced into regions in Nepal to control black rot of brassicas