Mechanisms of Surface Antigenic Variation in the Human Pathogenic Fungus <i>Pneumocystis jirovecii</i>.

Microbial pathogens commonly escape the human immune system by varying surface proteins. We investigated the mechanisms used for that purpose by &lt;i&gt;Pneumocystis jirovecii&lt;/i&gt; This uncultivable fungus is an obligate pulmonary pathogen that in immunocompromised individuals causes pneumonia, a major life-threatening infection. Long-read PacBio sequencing was used to assemble a core of subtelomeres of a single &lt;i&gt;P. jirovecii&lt;/i&gt; strain from a bronchoalveolar lavage fluid specimen from a single patient. A total of 113 genes encoding surface proteins were identified, including 28 pseudogenes. These genes formed a subtelomeric gene superfamily, which included five families encoding adhesive glycosylphosphatidylinositol (GPI)-anchored glycoproteins and one family encoding excreted glycoproteins. Numerical analyses suggested that diversification of the glycoproteins relies on mosaic genes created by ectopic recombination and occurs only within each family. DNA motifs suggested that all genes are expressed independently, except those of the family encoding the most abundant surface glycoproteins, which are subject to mutually exclusive expression. PCR analyses showed that exchange of the expressed gene of the latter family occurs frequently, possibly favored by the location of the genes proximal to the telomere because this allows concomitant telomere exchange. Our observations suggest that (i) the &lt;i&gt;P. jirovecii&lt;/i&gt; cell surface is made of a complex mixture of different surface proteins, with a majority of a single isoform of the most abundant glycoprotein, (ii) genetic mosaicism within each family ensures variation of the glycoproteins, and (iii) the strategy of the fungus consists of the continuous production of new subpopulations composed of cells that are antigenically different. &lt;b&gt;IMPORTANCE&lt;/b&gt; &lt;i&gt;Pneumocystis jirovecii&lt;/i&gt; is a fungus causing severe pneumonia in immunocompromised individuals. It is the second most frequent life-threatening invasive fungal infection. We have studied the mechanisms of antigenic variation used by this pathogen to escape the human immune system, a strategy commonly used by pathogenic microorganisms. Using a new DNA sequencing technology generating long reads, we could characterize the highly repetitive gene families encoding the proteins that are present on the cellular surface of this pest. These gene families are localized in the regions close to the ends of all chromosomes, the subtelomeres. Such chromosomal localization was found to favor genetic recombinations between members of each gene family and to allow diversification of these proteins continuously over time. This pathogen seems to use a strategy of antigenic variation consisting of the continuous production of new subpopulations composed of cells that are antigenically different. Such a strategy is unique among human pathogens

Potential new drug targets and mating process of the human pathogenic fungus Pneumocystis jirovecii

Pneumocystis organisms are extracellular fungal parasites that colonize the lungs of mammals. They constitute a group of species, each displaying strict host specificity for a given mammalian species. Pneumocystis jirovecii infects specifically humans. It is an opportunistic pathogen causing severe pneumonia in immunocompromised individuals that can be fatal if not treated. 1,3 and 1,6 -β glucans are essential constituents of cell walls of most fungi. Enzymes involved in their synthesis represent ideal drug targets, especially as inhibitors of these enzymes are known. The first part of my thesis project consisted in the identification and functional characterization of these two potential drug targets in the human pathogen P. jirovecii by rescue of the null allele of the orthologous gene in Saccharomyces cerevisiae. Echinocandins are a class of antifungal drugs composed by caspofungin, micafungin and anidulafungin, that inhibits the catalytic subunit Gsc1 of the enzymatic complex ensuring the synthesis of 1,3-β glucan. Even if caspofungin efficacy was demonstrated against P. carinii and P. murina in the rodent model, its efficacy against P. jirovecii is controversial in the clinical studies presently available. Moreover, the response to caspofungin of P. jirovecii could be different than those of P. carinii and P. murina because these species have a mean genetic divergence of ca. 20% at nucleotide level. In the fungal pathogen Candida albicans, resistance to caspofungin is conferred by point mutations leading to two amino acid substitutions within the hotspot no. 1 of mutations of Gsc1. We used site-directed mutagenesis to introduce the corresponding mutations within the gsc1 gene of the three Pneumocystis species. Upon expression of Pneumocystis gsc1 genes on plasmid in the S. cerevisiae null mutant, a partial restoration of the wild type growth was observed on medium containing caspofungin in presence of one substitution, and increased more in presence of the two substitutions. Our results suggest that the Gsc1 enzyme of P. jirovecii is sensitive to caspofungin, similarly to those of P. carinii and P. murina. In the third and last part, we investigated the sexual reproduction of Pneumocystis organisms, that would be essential for the formation of asci, the particles necessary for the spread of the infection. Recent comparative genomic analysis performed by our group suggested the existence of a single mating type locus that includes both plus (P) and minus (M) genes, suggesting that Pneumocystis species are primary homothallic organisms. However, the mating type of single cells remained unknown (M or P, or M and P at the same time). The aim of the third part was to analyze the expression of the M-factor receptor (mam2) and the P-factor receptor (map3) genes, and to verify if both are present at the same time at the surface of single Pneumocystis cells. Several bronco-alveolar lavage (BAL) fluid samples from patient with P. jirovecii pneumonia were analyzed for mam2 and map3 expression. The majority of them resulted positive for both pheromone receptors, suggesting that they are concomitantly expressed during Pneumocystis infection. P. jirovecii Mam2 and Map3 pheromone receptors were afterwards observed at the cellular surface using a specific immunofluorescent staining. The majority of the presumed trophic cells we identified were positive for both pheromone receptors, suggesting that each cell is of M and P mating types at the same time, and consequently that any cell can mate with any other cell present in the population. -- Les organismes du genre Pneumocystis sont des champignons parasites extracellulaires qui peuvent coloniser les poumons des mammifères. Ils font partie d’un groupe d’espèces ayant chacune une spécificité stricte pour un mammifère donné, Pneumocystis jirovecii étant celle qui infecte spécifiquement l’homme. Cette dernière est un pathogène opportuniste, qui peut causer une pneumonie très sérieuse chez les individus dont le système immunitaire est déficient, à savoir la pneumonie à Pneumocystis ou pneumocystose (PCP). Les glucans sont des constituantes essentielles de la paroi de la cellule fongique dans une grande majorité des champignons. Les enzymes impliquées dans leur synthèse représentent des cibles thérapeutiques idéelles, spécialement parce que les inhibiteurs de ces enzymes sont connus. La première partie de mon projet de thèse a consisté en l’identification et la caractérisation fonctionnelle de deux cibles thérapeutiques potentielles du pathogène humain P. jirovecii grâce la complémentation fonctionnelle d’une souche de Saccharomyces cerevisiae avec les allèles orthologues deletés. Les échinocandines sont une classe de médicaments antifongiques qui comprend la caspofungine, la micafungine et l’anidulafungine. Ces antifongiques inhibent spécifiquement la sous unité catalytique Gsc1 du complexe enzymatique nécessaire à la synthèse du 1,3-β glucane. Même si l’efficacité de la caspofungin a été démontrée contre P. carinii et P. murina dans le modèles animaux, l’utilisation de ce médicament contre P. jirovecii est controversé dans les études clinique disponibles. En plus, la réponse de P. jirovecii à ce traitement pourrait être différente de P. carinii et P. murina, parce que ces espèces ont une divergence génétique moyenne de 20% aux niveau nucléotidique. Chez le champignon pathogène Candida albicans, des mutations spécifiques dans Gsc1 amenant à la substitution de deux acides aminées, confèrent une résistance à la caspofungine. En utilisant un système de mutagenèse dirigé, j’ai introduit les mutations correspondantes dans les gènes gsc1 de P. jirovecii, P. carinii et P. murina. J’ai ensuite exprimé ces gènes dans la souche S. cerevisiae deletée pour GSC1. Comme déjà observé précédemment, l’expression des gènes gsc1 des différents Pneumocystis permet une restauration partielle de la croissance par rapport à la souche sauvage. Cette restauration de croissance augmente en présence d’une mutation, et augmente encore plus en présence des deux mutations. Nos résultats suggèrent que l’enzyme Gsc1 de P. jirovecii serait sensible à la caspofungin à un niveau similaire que ces de P. carinii et P. murina. Dans la troisième et dernière partie de ma thèse, j’ai étudié la reproduction sexuée des organismes du genre Pneumocystis, lequel semble être indispensable pour la formation de l’asque, qui peut infecter un nouvel hôte par transmission aérienne. Les analyses de génomiques comparatives qui ont été réalisées par notre groupe, ont démontré l’existence d’un seul locus de mating type, lequel inclus les gènes plus (P) et minus (M). Cela impliquerait que Pneumocystis est un organisme homothallique primaire, et par conséquent que chaque souche serait auto-fertile. Toutefois, le mating type de chaque cellule reste inconnu (M ou P, ou M et P à la fois). Le but de cette troisième partie était d’analyser l’expression des récepteurs pour le facteur de mating M (mam2) et pour le factor P (map3), et de vérifier si un seul ou les deux récepteurs étaient présents ensemble à la surface de chaque cellule de P. jirovecii. J’ai testé l’expression de mam2 et map3 sur plusieurs lavages broncho-alvéolaire (LBA) venant de patients souffrants de pneumonie causée par P. jirovecii. La majorité des LBA était positive pour les deux récepteurs. Ces résultats suggèrent que les deux récepteurs sont exprimés de façon concomitante pendant la pneumonie. Mam2 et Map3 de P. jirovecii ont été observés à la surface des cellules trophiques en utilisant une coloration immuno-fluorescente spécifique pour chacun des récepteurs. La majorité des cellules trophiques présumées étaient positives pour les deux récepteurs. Ces résultats suggèrent que chaque cellule est des deux mating types (M et P) à la fois, et de fait, chaque cellule pourrait s’accoupler dans les poumons avec n’importe quelle autre cellule de Pneumocystis présente

Investigating nanomotion-based technology (Resistell AST) for rapid antibiotic susceptibility testing among adult patients admitted to a tertiary-care hospital with Gram-negative bacteraemia: protocol for a prospective, observational, cross-sectional, single-arm study

Introduction Effective treatment of bloodstream infections (BSIs) is relying on rapid identification of the causing pathogen and its antibiotic susceptibility. Still, most commercially available antibiotic susceptibility testing (AST) methods are based on monitoring bacterial growth, thus impacting the time to results. The Resistell AST is based on a new technology measuring the nanomotion caused by physiologically active bacterial cells and detecting the changes in nanomotion caused by the exposure to a drug.Methods and analysis This is a single-centre, prospective, cross-sectional, single-arm diagnostic accuracy study to determine the agreement of the Resistell AST on Gram-negative bacteria isolated from blood cultures among patients admitted to a tertiary-care hospital with the reference method. Up to 300 patients will be recruited. Starting with a pilot phase, enrolling 10%–20% of the subjects and limited to Escherichia coli BSI tested for ceftriaxone susceptibility, the main phase will follow, extending the study to Klebsiella pneumoniae and ciprofloxacin.Ethics and dissemination This study has received ethical approval from the Swiss Ethics Committees (swissethics, project 2020-01622). All the case report forms and clinical samples will be assigned a study code by the local investigators and stored anonymously at the reference centre (Lausanne University Hospital). The results will be broadly distributed through conference presentations and peer-reviewed publications.Trial registration number ClinicalTrials.gov Registry (NCT05002413)