Uncovering oomycete metabolism using systems biology

Cell metabolism is a network of biochemical reactions that allows organisms to assimilate nutrients from their environment into their building blocks of life. There are many variables that influence this system, and this makes metabolism hard to study. Systems biology is a relatively new scientific discipline that tries to encapsulate biological phenomena into mathematical models, as a means to generate new, testable hypotheses. In this research we used systems biology to study the interactions that oomycetes can undergo with their hosts. Oomycetes are parasites of plants and animals, and especially their metabolism is hardly studied thus far. We performed large-scale comparative studies on the metabolic gene content of oomycetes, and model the metabolism of the late blight pathogen Phytophthora infestans. We integrate this model with a metabolic model of tomato, to predict the metabolic exchanges that take place during infection. We also study the repertoire of metalloproteases in oomycetes, a class of enzymes that have potential roles in host infection

Profile hidden Markov models trained on aligned KEGG Orthology sequences for enzyme annotation

Profile hidden Markov models trained on aligned KEGG Orthology sequences for enzyme annotation. These HMMs were used to reconstruct metabolic networks for the manuscript: The genome of Peronospora belbahrii reveals high heterozygosity, a low number of canonical effectors and CT-rich promoter

Uncovering the Role of Metabolism in Oomycete–Host Interactions Using Genome-Scale Metabolic Models

Metabolism is the set of biochemical reactions of an organism that enables it to assimilate nutrients from its environment and to generate building blocks for growth and proliferation. It forms a complex network that is intertwined with the many molecular and cellular processes that take place within cells. Systems biology aims to capture the complexity of cells, organisms, or communities by reconstructing models based on information gathered by high-throughput analyses (omics data) and prior knowledge. One type of model is a genome-scale metabolic model (GEM) that allows studying the distributions of metabolic fluxes, i.e., the “mass-flow” through the network of biochemical reactions. GEMs are nowadays widely applied and have been reconstructed for various microbial pathogens, either in a free-living state or in interaction with their hosts, with the aim to gain insight into mechanisms of pathogenicity. In this review, we first introduce the principles of systems biology and GEMs. We then describe how metabolic modeling can contribute to unraveling microbial pathogenesis and host–pathogen interactions, with a specific focus on oomycete plant pathogens and in particular Phytophthora infestans. Subsequently, we review achievements obtained so far and identify and discuss potential pitfalls of current models. Finally, we propose a workflow for reconstructing high-quality GEMs and elaborate on the resources needed to advance a system biology approach aimed at untangling the intimate interactions between plants and pathogens

Metabolic Model of the Phytophthora infestans-Tomato Interaction Reveals Metabolic Switches during Host Colonization

The oomycete pathogen Phytophthora infestans causes potato and tomato late blight, a disease that is a serious threat to agriculture. P. infestans is a hemibiotrophic pathogen, and during infection, it scavenges nutrients from living host cells for its own proliferation. To date, the nutrient flux from host to pathogen during infection has hardly been studied, and the interlinked metabolisms of the pathogen and host remain poorly understood. Here, we reconstructed an integrated metabolic model of P. infestans and tomato (Solanum lycopersicum) by integrating two previously published models for both species. We used this integrated model to simulate metabolic fluxes from host to pathogen and explored the topology of the model to study the dependencies of the metabolism of P. infestans on that of tomato. This showed, for example, that P. infestans, a thiamine auxotroph, depends on certain metabolic reactions of the tomato thiamine biosynthesis. We also exploited dual-transcriptome data of a time course of a full late blight infection cycle on tomato leaves and integrated the expression of metabolic enzymes in the model. This revealed profound changes in pathogen-host metabolism during infection. As infection progresses, P. infestans performs less de novo synthesis of metabolites and scavenges more metabolites from tomato. This integrated metabolic model for the P. infestans-tomato interaction provides a framework to integrate data and generate hypotheses about in planta nutrition of P. infestans throughout its infection cycle.IMPORTANCE Late blight disease caused by the oomycete pathogen Phytophthora infestans leads to extensive yield losses in tomato and potato cultivation worldwide. To effectively control this pathogen, a thorough understanding of the mechanisms shaping the interaction with its hosts is paramount. While considerable work has focused on exploring host defense mechanisms and identifying P. infestans proteins contributing to virulence and pathogenicity, the nutritional strategies of the pathogen are mostly unresolved. Genome-scale metabolic models (GEMs) can be used to simulate metabolic fluxes and help in unravelling the complex nature of metabolism. We integrated a GEM of tomato with a GEM of P. infestans to simulate the metabolic fluxes that occur during infection. This yields insights into the nutrients that P. infestans obtains during different phases of the infection cycle and helps in generating hypotheses about nutrition in planta.</p

Mining oomycete proteomes for metalloproteases leads to identification of candidate virulence factors in Phytophthora infestans

Pathogens deploy a wide range of pathogenicity factors, including a plethora of proteases, to modify host tissue or manipulate host defences. Metalloproteases (MPs) have been implicated in virulence in several animal and plant pathogens. Here we investigated the repertoire of MPs in 46 stramenopile species including 37 oomycetes, 5 diatoms, and 4 brown algae. Screening their complete proteomes using hidden Markov models (HMMs) trained for MP detection resulted in over 4,000 MPs, with most species having between 65 and 100 putative MPs. Classification in clans and families according to the MEROPS database showed a highly diverse MP repertoire in each species. Analyses of domain composition, orthologous groups, distribution, and abundance within the stramenopile lineage revealed a few oomycete-specific MPs and MPs potentially related to lifestyle. In-depth analyses of MPs in the plant pathogen Phytophthora infestans revealed 91 MPs, divided over 21 protein families, including 25 MPs with a predicted signal peptide or signal anchor. Expression profiling showed different patterns of MP gene expression during pre-infection and infection stages. When expressed in leaves of Nicotiana benthamiana, 12 MPs changed the sizes of lesions caused by inoculation with P. infestans; with 9 MPs the lesions were larger, suggesting a positive effect on the virulence of P. infestans, while 3 MPs had a negative effect, resulting in smaller lesions. To the best of our knowledge, this is the first systematic inventory of MPs in oomycetes and the first study pinpointing MPs as potential pathogenicity factors in Phytophthora

Mining oomycete proteomes for metalloproteases leads to identification of candidate virulence factors in Phytophthora infestans

Pathogens deploy a wide range of pathogenicity factors, including a plethora of proteases, to modify host tissue or manipulate host defences. Metalloproteases (MPs) have been implicated in virulence in several animal and plant pathogens. Here we investigated the repertoire of MPs in 46 stramenopile species including 37 oomycetes, 5 diatoms, and 4 brown algae. Screening their complete proteomes using hidden Markov models (HMMs) trained for MP detection resulted in over 4,000 MPs, with most species having between 65 and 100 putative MPs. Classification in clans and families according to the MEROPS database showed a highly diverse MP repertoire in each species. Analyses of domain composition, orthologous groups, distribution, and abundance within the stramenopile lineage revealed a few oomycete-specific MPs and MPs potentially related to lifestyle. In-depth analyses of MPs in the plant pathogen Phytophthora infestans revealed 91 MPs, divided over 21 protein families, including 25 MPs with a predicted signal peptide or signal anchor. Expression profiling showed different patterns of MP gene expression during pre-infection and infection stages. When expressed in leaves of Nicotiana benthamiana, 12 MPs changed the sizes of lesions caused by inoculation with P. infestans; with 9 MPs the lesions were larger, suggesting a positive effect on the virulence of P. infestans, while 3 MPs had a negative effect, resulting in smaller lesions. To the best of our knowledge, this is the first systematic inventory of MPs in oomycetes and the first study pinpointing MPs as potential pathogenicity factors in Phytophthora

Estudo comparativo da análise citológica e histopatológica de massas cutâneas e subcutâneas em cães e gatos

Dissertação de Mestrado Integrado em Medicina VeterináriaA análise citológica consiste num exame complementar que pode ser fonte de informação diagnóstica relevante para qualquer clínico de pequenos animais. De baixo custo e de relativa facilidade de execução, a análise citológica pode facilmente ser integrada nos exames de diagnóstico proporcionados por qualquer centro veterinário. A avaliação microscópica das amostras citológicas pode ser feita diretamente pelo clínico, sendo requerida alguma dedicação e prática, ou serem enviadas para laboratórios externos onde são avaliadas por profissionais experientes. Por outro lado, a análise histopatológica é considerada análise de eleição para o diagnóstico de massas tumorais, incluindo as cutâneas e subcutâneas. Embora a análise histopatológica consiga na grande maioria dos casos identificar claramente a origem celular da neoplasia, fornecendo informações indispensáveis para o correto seguimento clínico, tratamento e prognóstico, trata-se de um exame que necessita de uma colheita de material realizada de forma mais invasiva e de um maior processamento da amostra. No caso da citologia a colheita e a visualização da lâmina são muito rápidos. O objetivo deste estudo retrospetivo foi então compreender a correlação entre as análises citológica e histopatológica, comparando os diversos diagnósticos alcançados através das duas análises. A amostra consiste num total de 65 lesões, provenientes de 41 (89,4%) cães e 5 (10,6%) gatos, recolhidas entre 2009 e 2013. Destas 65 lesões apenas 60 (92,3%) foram incluídas no estudo, já que 5 (7,7%) foram consideradas inconclusivas e sem valor de diagnóstico. Todas as amostras depois de colhidas por punção aspirativa por agulha fina, foram depois avaliadas no Hospital Veterinário do Oeste. Já as análises histopatológicas foram enviadas para os laboratórios INNO® e Segalab® após exérese cirúrgica das massas. Os resultados foram depois analisados, concluindo que as neoplasias mais diagnosticadas foram as de células redondas, com destaque para os mastocitomas, neoplasia mais comumente diagnosticada, representado cerca de 33,3% do total de lesões analisadas. A neoplasia benigna mais comumente diagnosticada foi o adenoma das glândulas perianais com 25,0% do total de de neoplasias. Na verificação dos diagnósticos obtidos, a concordância entre as duas análises ficou provada em 84,6% dos casos.ABSTRACT - Comparative Study of the Cytologic and Hystopatologic Analyses of Cutaneous and Subcutaneuos Masses from Cats and Dogs - The cytologic analysis is a diagnostic procedure that can be a valuable source of information for Veterinarians. This analysis can easily be done in every veterinary center due to its low cost and moderate difficulty. With some dedication and technique the cytologic specimens can be microscopicly evaluated by the veterinarian or sent to an external laboratory to be evaluated by experienced professionals. Despite its numerous advantages (easiness of sample collection, rapidity of results and low cost), to achieve a definitive diagnosis, the cytologic analysis is not the most reliable of the complementary exams. Thus, for cutaneous and subcutaneous masses, the histopathology is considered the golden method to achieve a definitive diagnosis. Although the histopathologic exam can, in most of the cases, identify the cellular origin of the mass and provide crucial information for a correct clinical follow-up, treatment and prognostic, the sample collection is more aggressive and requires a longer processing time. As far as the cytologic examination is concerned, the collection and evaluation of samples is almost immediate. This retrospective study aimed to evaluate the correlation between cytologic and histopathologic analysis.The study was based in 65 lesions (41 from dogs (89,4%) and 5 from cats (10,6%)) collected between 2009 and 2013. However, only 60 lesions (92,3%) were taken in consideration as the remaining 5 (7,7%) were unsatisfactory and therefore excluded from the study. After proceeding with the fine-needle aspiration biopsy the samples were sent to and processed in the laboratory of the Hospital Veterinario do Oeste. After surgical excision, the histopathologic samples were sent to external laboratories: INNO® and Segalab®. The round cell tumors were the most diagnosed ones with 36,4%, where mastocitomas represented 33,3% of all lesions. The perianal gland tumor was the most beningn neoplasia diagnosed, representing 25% of all lesions. The correlation of both analyses was proven in 84,6% of the cases studied

The genome of Peronospora belbahrii reveals high heterozygosity, a low number of canonical effectors and CT-rich promoters

Along with Plasmopara destructor, Peronosopora belbahrii has arguably been the economically most important newly emerging downy mildew pathogen of the past two decades. Originating from Africa, it has started devastating basil production throughout the world, most likely due to the distribution of infested seed material. Here we present the genome of this pathogen and results from comparisons of its genomic features to other oomycetes. The assembly of the nuclear genome was ca. 35.4 Mbp in length, with an N50 scaffold length of ca. 248 kbp and an L50 scaffold count of 46. The circular mitochondrial genome consisted of ca. 40.1 kbp. From the repeat-masked genome 9049 protein-coding genes were predicted, out of which 335 were predicted to have extracellular functions, representing the smallest secretome so far found in peronosporalean oomycetes. About 16 % of the genome consists of repetitive sequences, and based on simple sequence repeat regions, we provide a set of microsatellites that could be used for population genetic studies of Pe. belbahrii. Peronospora belbahrii has undergone a high degree of convergent evolution, reflecting its obligate biotrophic lifestyle. Features of its secretome, signalling networks, and promoters are presented, and some patterns are hypothesised to reflect the high degree of host specificity in Peronospora species. In addition, we suggest the presence of additional virulence factors apart from classical effector classes that are promising candidates for future functional studies

Comparative genomics including the basal pathogen Peronospora belbahrii reveal common evolutionary patterns and the monophyly of downy mildews in a paraphyletic Phytophthora

The obligate biotrophic downy mildew constitute the most species rich group of oomycetes. So far only handful of genomes of this group of pathogens has been sequenced. Most likely due to low taxon sampling, until now phylogenomic studies with few taxa were in stark contrast to multigene phylogenies with a large number of accessions with respect to the relationships of downy mildews and Phytophthora species. In the current study, we sequenced the whole genome of the economically important basil pathogen Peronospora belbahrii, and performed in-depth comparative genomics and phylogenomics towards clarifying some aspects of downy mildew and Phytophthora evolution