Incomplete tricarboxylic acid cycle and proton gradient in Pandoravirus massiliensis: is it still a virus?

The discovery of Acanthamoeba polyphaga Mimivirus, the first isolated giant virus of amoeba, challenged the historical hallmarks defining a virus. Giant virion sizes are known to reach up to 2.3µm, making them visible by optical microscopy. Their large genome sizes of up to 2.5Mb can encode proteins involved in the translation apparatus. We have investigated possible energy production in Pandoravirus massiliensis. Mitochondrial membrane markers allowed for the detection of a membrane potential in purified virions and this was enhanced by a regulator of the tricarboxylic acid cycle but abolished by the use of a depolarizing agent. Bioinformatics was employed to identify enzymes involved in virion proton gradient generation and this approach revealed that 8 putative P. massiliensis proteins exhibited low sequence identities with known cellular enzymes involved in the universal tricarboxylic acid cycle. Further, all 8 viral genes were transcribed during replication. The product of one of these genes, ORF132, was cloned and expressed in Escherichia coli, and shown to function as an isocitrate dehydrogenase, a key enzyme of the tricarboxylic acid cycle. Our findings show for the first time that a membrane potential can exist in Pandoraviruses, and this may be related to tricarboxylic acid cycle. The presence of a proton gradient in P. massiliensis makes this virus a form of life for which it is legitimate to ask the question ‘what is a virus?’

Rapid MALDI-TOF MS identification of commercial truffles

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Insight into the Lifestyle of Amoeba Willaertia magna during Bioreactor Growth Using Transcriptomics and Proteomics

Willaertia magna C2c maky is a thermophilic free-living amoeba strain that showed ability to eliminate Legionella pneumophila, a pathogenic bacterium living in the aquatic environment. The amoeba industry has proposed the use of Willaertia magna as a natural biocide to control L. pneumophila proliferation in cooling towers. Here, transcriptomic and proteomic studies were carried out in order to expand knowledge on W. magna produced in a bioreactor. Illumina RNA-seq generated 217 million raw reads. A total of 8790 transcripts were identified, of which 6179 and 5341 were assigned a function through comparisons with National Center of Biotechnology Information (NCBI) reference sequence and the Clusters of Orthologous Groups of proteins (COG) databases, respectively. To corroborate these transcriptomic data, we analyzed the W. magna proteome using LC-MS/MS. A total of 3561 proteins were identified. The results of transcriptome and proteome analyses were highly congruent. Metabolism study showed that W. magna preferentially consumed carbohydrates and fatty acids to grow. Finally, an in-depth analysis has shown that W. magna produces several enzymes that are probably involved in the metabolism of secondary metabolites. Overall, our multi-omic study of W. magna opens the way to a better understanding of the genetics and biology of this amoeba

Experimental Analysis of Mimivirus Translation Initiation Factor 4a Reveals Its Importance in Viral Protein Translation during Infection of Acanthamoeba polyphaga

International audienceThe Acanthamoeba polyphaga mimivirus is the first giant virus ever described, with a 1.2-Mb genome which encodes 979 proteins, including central components of the translation apparatus. One of these proteins, R458, was predicted to initiate translation, although its specific role remains unknown. We silenced the R458 gene using small interfering RNA (siRNA) and compared levels of viral fitness and protein expression in silenced versus wild-type mimivirus. Silencing decreased the growth rate, but viral particle production at the end of the viral cycle was unaffected. A comparative proteomic approach using two-dimensional difference-in-gel electrophoresis (2D-DIGE) revealed deregulation of the expression of 32 proteins in silenced mimivirus, which were defined as up-or downregulated. Besides revealing proteins with unknown functions, silencing R458 also revealed deregulation in proteins associated with viral particle structures, transcriptional machinery, oxidative pathways, modification of proteins/lipids, and DNA topology/repair. Most of these proteins belong to genes transcribed at the end of the viral cycle. Overall, our data suggest that the R458 protein regulates the expression of mimivirus proteins and, thus, that mimivirus translational proteins may not be strictly redundant in relation to those from the amoeba host. As is the case for eukaryotic initiation factor 4a (eIF4a), the R458 protein is the prototypical member of the ATP-dependent DEAD box RNA helicase mechanism. We suggest that the R458 protein is required to unwind the secondary structures at the 5' ends of mRNAs and to bind the mRNA to the ribosome, making it possible to scan for the start codon. These data are the first experimental evidence of mimivirus translation-related genes, predicted to initiate protein biosynthesis. IMPORTANCE The presence in the genome of a mimivirus of genes coding for many translational processes, with the exception of ribosome constituents, has been the subject of debate since its discovery in 2003. In this work, we focused on the R458 mimivirus gene, predicted to initiate protein biosynthesis. After silencing was performed, we observed that it has no major effect on mimivirus multiplication but that it affects protein expression and fitness. This suggests that it is effectively used by mimivirus during its developmental cycle. Until large-scale genetic manipulation of giant viruses becomes possible, the silencing strategy used here on mimivirus translation-related factors will open the way to understanding the functions of these translational genes

Lactonase Specificity Is Key to Quorum Quenching in Pseudomonas aeruginosa

The human opportunistic pathogen Pseudomonas aeruginosa orchestrates the expression of many genes in a cell density-dependent manner by using quorum sensing (QS). Two acyl-homoserine lactones (AHLs) are involved in QS circuits and contribute to the regulation of virulence factors production, biofilm formation, and antimicrobial sensitivity. Disrupting QS, a strategy referred to as quorum quenching (QQ) can be achieved using exogenous AHL-degrading lactonases. However, the importance of enzyme specificity on quenching efficacy has been poorly investigated. Here, we used two lactonases both targeting the signal molecules N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C-12 HSL) and butyryl-homoserine lactone (C-4 HSL) albeit with different efficacies on C-4 HSL. Interestingly, both lactonases similarly decreased AHL concentrations and comparably impacted the expression of AHL-based QS genes. However, strong variations were observed in Pseudomonas Quinolone Signal (PQS) regulation depending on the lactonase used. Both lactonases were also found to decrease virulence factors production and biofilm formation in vitro, albeit with different efficiencies. Unexpectedly, only the lactonase with lower efficacy on C-4 HSL was able to inhibit P. aeruginosa pathogenicity in vivo in an amoeba infection model. Similarly, proteomic analysis revealed large variations in protein levels involved in antibiotic resistance, biofilm formation, virulence and diverse cellular mechanisms depending on the chosen lactonase. This global analysis provides evidences that QQ enzyme specificity has a significant impact on the modulation of QS-associated behavior in P. aeruginosa PA14

Proteomics of the Oomycete <i>Phytophthora parasitica</i> Strain INRA 310

The phytopathogen Phytophthora parasitica, from the Oomycetes class, known to be the tobacco black shank agent, can induce devastating diseases in various crop, plant and forest ecosystems. The genus Phytophthora has been studied at the cellular level, suggesting that different developmental steps are induced by the expression of some specific genes. However, these studies have only been carried out on certain species, such as Phytophthora infestans and Phytophthora cactorum. As for Phytophthora parasitica, which can be considered as one of the top ten oomycete pathogens due to the economic impact and effect it has on food security, even less functional analyses and transcriptomics data are available. To date, little is known about the protein expression of Phytophthora parasitica, information that is essential for achieving a better understanding of this species. In this study, we aimed to gain insight into the proteomics of the mycelium of the Phytophthora parasitica strain INRA 310 by addressing the following questions: (i) how many predicted proteins can be detected on the mycelium of P. parasitica INRA 310, and (ii) what proteins can be detected? The proteomics experiments were performed on the mycelium of the strain Phytophthora parasitica INRA310, using the nanoliquid chromatography-MS/MS technique. A total of 219 proteins were identified, including ten unknown proteins and 209 proteins involved in lipid, carbohydrate, nucleotide, energy production and other metabolic pathways. This proteomics study is, to our knowledge, the first to be performed on the mycelium of Phytophthora parasitica INRA 310. It gives a brief first insight into its in vitro-expressed proteins. This work may be the first step before further, more comprehensive studies are undertaken with the aim of better understanding the biology of this species and its pathogenicity

Human galectin-1 and galectin-3 promote Tropheryma whipplei

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Generation of Infectious Mimivirus Virions Through Inoculation of Viral DNA Within Acanthamoeba castellanii Shows Involvement of Five Proteins, Essentially Uncharacterized

International audienceOne of the most curious findings associated with the discovery of Acanthamoeba polyphaga mimivirus (APMV) was the presence of many proteins and RNAs within the virion. Although some hypotheses on their role in Acanthamoeba infection have been put forward, none have been validated. In this study, we directly transfected mimivirus DNA with or without additional proteinase K treatment to extracted DNA into Acanthamoeba castellanii. In this way, it was possible to generate infectious APMV virions, but only without extra proteinase K treatment of extracted DNA. The virus genomes before and after transfection were identical. We searched for the remaining DNA-associated proteins that were digested by proteinase K and could visualize at least five putative proteins. Matrix-assisted laser desorption/ionization time-of-flight and liquid chromatography–mass spectrometry comparison with protein databases allowed the identification of four hypothetical proteins—L442, L724, L829, and R387—and putative GMC-type oxidoreductase R135. We believe that L442 plays a major role in this protein–DNA interaction. In the future, expression in vectors and then diffraction of X-rays by protein crystals could help reveal the exact structure of this protein and its precise role

Human galectin-1 and galectin-3 promote Tropheryma whipplei infection

International audienc

Proteomics and Lipidomics Investigations to Decipher the Behavior of Willaertia magna C2c Maky According to Different Culture Modes

International audienceWillaertia magna C2c Maky is a free-living amoeba that has demonstrated its ability to inhibit the intracellular multiplication of some Legionella pneumophila strains, which are pathogenic bacteria inhabiting the aquatic environment. The Amoeba, an industry involved in the treatment of microbiological risk in the water and plant protection sectors, has developed a natural biocide based on the property of W. magna to manage the proliferation of the pathogen in cooling towers. In axenic liquid medium, amoebas are usually cultivated in adhesion on culture flask. However, we implemented a liquid culture in suspension using bioreactors in order to produce large quantities of W. magna. In order to investigate the culture condition effects on W. magna, we conducted a study based on microscopic, proteomics and lipidomics analyzes. According to the culture condition, amoeba exhibited two different phenotypes. The differential proteomics study showed that amoebas seemed to promote the lipid metabolism pathway in suspension culture, whereas we observed an upregulation of the carbohydrate pathway in adherent culture. Furthermore, we observed an over-regulation of proteins related to the cytoskeleton for W. magna cells grown in adhesion. Regarding the lipid analysis, suspension and adhesion cell growth showed comparable lipid class compositions. However, the differential lipid analysis revealed differences that confirmed cell phenotype differences observed by microscopy and predicted by proteomics. Overall, this study provides us with a better insight into the biology and molecular processes of W. magna in different culture lifestyles