Nicolas Papon

Microbial antioxidant defense enzymes

Free radicals are often described as chemical compounds characterized by unpaired electrons in their outer orbital rendering them highly reactive species. In mammalians, studies on free radicals were focused on reactive oxygen species (ROS) or reactive nitrogen species (RNS) due to their relative importance in physiological as well as in pathological processes. These cellular compounds are produced by different physiological systems such as the aerobic metabolism and play a major role in cell signaling pathways but also in the host immune defenses against pathogenic microorganisms. ROS and RNS are highly reactive species with potentially harmful effects on any cellular components (lipids, proteins and nucleic acids) when produced with a high level. To maintain ROS and RNS at a non-toxic concentration, enzymatic and non-enzymatic cellular antioxidants coordinate the balance between their production and their degradation. Superoxide dismutases, catalases, glutathione system, thioredoxin system, peroxidase systems, flavohemoglobins and nitrate or nitrite reductases represent the prominent enzymatic antioxidants used to scavenge excess of internal as well as external ROS and RNS. Bacteria, fungi and parasites also display similar enzymatic activities to escape the host oxidative defenses during the immune response against infectious processes. Here we summarize current knowledge on the enzymatic systems that allow microorganisms to fight against ROS and RNS, and shed light on the role that take some of them in microbial infections. Such microbial protective systems are considered as virulence factors, and therefore represent key targets for diagnosis of the infections or development of anti-infectious drugs.

Major Sensing Proteins in Pathogenic Fungi: The Hybrid Histidine Kinase Family.

The pioneering discovery of histidine kinases (HKs) from Escherichia coli was made in the early 1980s with the identification of the envZ gene [1] (Fig 1A). Further biochemical characterization of the corresponding protein revealed a new type of protein kinase activity, namely HK, to add to the well-known serine/threonine and tyrosine kinases. For a decade, HKs were considered to be restricted to bacteria, but in the 1990s, HKs were identified in plants [2], fungi [3], archaea [4], cyanobacteria [5], and amoebae (Fig 1A) [6]. Soon after, evidence suggested that HKs regulate essential processes in pathogenic bacteria and fungi [7]. Although some HKs appear to be present in humans, typical bacterial or fungal HK-like sensor proteins have not been reported yet in mammals [8], promoting these proteins as ideal targets for future therapeutics [9]. It is now accepted that HKs are involved in cell signaling systems referred to as His-to-Asp phosphorelays and several canonical schemes depicting transduction pathways involving HKs in bacteria, amoebae, plants, and fungi have emerged (Fig 1B). To date, HKs act as primary sensors for various environmental stimuli, and, upon activation, initiate phosphate transfer events between various proteins, leading to an adaptive response. Although these mechanistic models are largely described in bacteria and plants, limited evidence is available for amoebae and fungi.

The Identification of Phytohormone Receptor Homologs in Early Diverging Fungi Suggests a Role for Plant Sensing in Land Colonization by Fungi

ABSTRACT Histidine kinases (HKs) are among the most prominent sensing proteins studied in the kingdom Fungi. Their distribution and biological functions in early diverging fungi (EDF), however, remain elusive. We have taken advantage of recent genomic resources to elucidate whether relationships between the occurrence of specific HKs in some EDF and their respective habitat/lifestyle could be established. This led to the unexpected discovery of fungal HKs that share a high degree of similarity with receptors for plant hormones (ethylene and cytokinin). Importantly, these phytohormone receptor homologs are found not only in EDF that behave as plant root symbionts or endophytes but also in EDF species that colonize decaying plant material. We hypothesize that these particular sensing proteins promoted the interaction of EDF with plants, leading to the conquest of land by these ancestral fungi.

Class II Cytochrome P450 Reductase Governs the Biosynthesis of Alkaloids

Class II cytochrome P450 reductase in Madagascar periwinkle displays a prominent contribution toward specialized metabolism by acting as the main partner of P450s dedicated to alkaloid biosynthesis. Expansion of the biosynthesis of plant specialized metabolites notably results from the massive recruitment of cytochrome P450s that catalyze multiple types of conversion of biosynthetic intermediates. For catalysis, P450s require a two-electron transfer catalyzed by shared cytochrome P450 oxidoreductases (CPRs), making these auxiliary proteins an essential component of specialized metabolism. CPR isoforms usually group into two distinct classes with different proposed roles, namely involvement in primary and basal specialized metabolisms for class I and inducible specialized metabolism for class II. By studying the role of CPRs in the biosynthesis of monoterpene indole alkaloids, we provide compelling evidence of an operational specialization of CPR isoforms in Catharanthus roseus (Madagascar periwinkle). Global analyses of gene expression correlation combined with transcript localization in specific leaf tissues and gene-silencing experiments of both classes of CPR all point to the strict requirement of class II CPRs for monoterpene indole alkaloid biosynthesis with a minimal or null role of class I. Direct assays of interaction and reduction of P450s in vitro, however, showed that both classes of CPR performed equally well. Such high specialization of class II CPRs in planta highlights the evolutionary strategy that ensures an efficient reduction of P450s in specialized metabolism.

Prequels to Synthetic Biology: From Candidate Gene Identification and Validation to Enzyme Subcellular Localization in Plant and Yeast Cells

Natural compounds extracted from microorganisms or plants constitute an inexhaustible source of valuable molecules whose supply can be potentially challenged by limitations in biological sourcing. The recent progress in synthetic biology combined to the increasing access to extensive transcriptomics and genomics data now provide new alternatives to produce these molecules by transferring their whole biosynthetic pathway in heterologous production platforms such as yeasts or bacteria. While the generation of high titer producing strains remains per se an arduous field of investigation, elucidation of the biosynthetic pathways as well as characterization of their complex subcellular organization are essential prequels to the efficient development of such bioengineering approaches. Using examples from plants and yeasts as a framework, we describe potent methods to rationalize the study of partially characterized pathways, including the basics of computational applications to identify candidate genes in transcriptomics data and the validation of their function by an improved procedure of virus-induced gene silencing mediated by direct DNA transfer to get around possible resistance to Agrobacterium-delivery of viral vectors. To identify potential alterations of biosynthetic fluxes resulting from enzyme mislocalizations in reconstituted pathways, we also detail protocols aiming at characterizing subcellular localizations of protein in plant cells by expression of fluorescent protein fusions through biolistic-mediated transient transformation, and localization of transferred enzymes in yeast using similar fluorescence procedures. Albeit initially developed for the Madagascar periwinkle, these methods may be applied to other plant species or organisms in order to establish synthetic biology platform.

Disruption of Protein Mannosylation Affects Candida guilliermondii Cell Wall, Immune Sensing, and Virulence

The fungal cell wall contains glycoproteins that interact with the host immune system. In the prominent pathogenic yeast Candida albicans, Pmr1 acts as a Golgi-resident ion pump that provides cofactors to mannosyltransferases, regulating the synthesis of mannans attached to glycoproteins. To gain insight into a putative conservation of such a crucial process within opportunistic yeasts, we were particularly interested in studying the role of the PMR1 homolog in a low-virulent species that rarely causes candidiasis, Candida guilliermondii. We disrupted C. guilliermondii PMR1 and found that loss of Pmr1 affected cell growth and morphology, biofilm formation, susceptibility to cell wall perturbing agents, mannan levels, and the wall composition and organization. Despite the significant increment in the amount of β1,3-glucan exposed at the wall surface, this positively influenced only the ability of the mutant to stimulate IL-10 production by human monocytes, suggesting that recognition of both mannan and β1,3-glucan, is required to stimulate strong levels of pro-inflammatory cytokines. Accordingly, our results indicate C. guilliermondii sensing by monocytes was critically dependent on the recognition of N-linked mannans and β1,3-glucan, as reported in other Candida species. In addition, chemical remotion of cell wall O-linked mannans was found to positively influence the recognition of C. guilliermondii by human monocytes, suggesting that O-linked mannans mask other cell wall components from immune cells. This observation contrasts with that reported in C. albicans. Finally, mice infected with C. guilliermondii pmr1Δ null mutant cells had significantly lower fungal burdens compared to animals challenged with the parental strain. Accordingly, the null mutant showed inability to kill larvae in the Galleria mellonella infection model. This study thus demonstrates that mannans are relevant for the C. guilliermondii-host interaction, with an atypical role for O-linked mannans.

Candida guilliermondii as a potential biocatalyst for the production of long-chain α,ω-dicarboxylic acids

ObjectivesTo explore Candida guilliermondii for the production of long-chain dicarboxylic acids (DCA), we performed metabolic pathway engineering aiming to prevent DCA consumption during β-oxidation, but also to increase its production via the ω-oxidation pathway.ResultsWe identified the major β- and ω-oxidation pathway genes in C. guilliermondii and performed first steps in the strain improvement. A double pox disruption mutant was created that slowed growth with oleic acid but showed accelerated DCA degradation. Increase in DCA production was achieved by homologous overexpression of a plasmid borne cytochrome P450 monooxygenase gene.ConclusionC. guilliermondii is a promising biocatalyst for DCA production but further insight into its fatty acid metabolism is necessary.

A synthetic construct for genetic engineering of the emerging pathogenic yeast Candida auris

Candida auris has recently emerged as a global cause of severe hospital-acquired fungal infections. To enable functional genomic approaches for this prominent pathogen, we designed a synthetic construct that can be used to genetically transform the genome-sequenced strain VPCI 479/P/13 of C. auris following an efficient electroporation procedure.

Mechanical stress rapidly induces E-resveratrol and E-piceatannol biosynthesis in grape canes stored as a freshly-pruned byproduct

Grape canes represent a promising source of bioactive phytochemicals. However the stabilization of the raw material after pruning remains challenging. We recently reported the induction of stilbenoid metabolism after winter pruning including a strong accumulation of E-resveratrol and E-piceatannol during the first six weeks of storage. In the present study, the effect of mechanical wounding on freshly-pruned canes was tested to increase the induction of stilbenoid metabolism. Cutting the grape canes in short segments immediately after pruning triggered a transient expression of phenylalanine ammonia-lyase (PAL) and stilbene synthase (STS) genes, followed by a rapid accumulation of E-resveratrol and E-piceatannol. The degree of stilbenoid induction was related to the intensity of mechanical wounding. Data suggest that a global defense response is triggered involving jasmonate signaling, PR proteins and stilbenoid metabolism. Mechanical wounding of freshly-pruned canes drastically shortens the time required to reach maximal stilbenoid accumulation from 6 to 2weeks.

Comparison of a commercial real-time PCR assay, RealCycler® PJIR kit, progenie molecular, to an in-house real-time PCR assay for the diagnosis of Pneumocystis jirovecii infections

We compared the RealCycler® PJIR kit (Progenie Molecular), available in Europe, to an in-house real-time PCR assay for the diagnosis of Pneumocystis jirovecii infections. Excellent agreement was found (concordance rate, 97.4%; Cohens kappa, 0.918>0.8) showing that this commercial assay represents an alternative method for the diagnosis of P. jirovecii infections.