Expression Pattern of the Pneumocystis jirovecii Major Surface Glycoprotein Superfamily in Patients with Pneumonia.

BACKGROUND: The human pathogen Pneumocystis jirovecii harbors 6 families of major surface glycoproteins (MSGs) encoded by a single gene superfamily. MSGs are presumably responsible for antigenic variation and adhesion to host cells. The genomic organization suggests that a single member of family I is expressed at a given time per cell, whereas members of the other families are simultaneously expressed. METHODS: We analyzed RNA sequences expressed in several clinical samples, using specific weighted profiles for sorting of reads and calling of single-nucleotide variants to estimate the diversity of the expressed genes. RESULTS: A number of different isoforms of at least 4 MSG families were expressed simultaneously, including isoforms of family I, for which confirmation was obtained in the wet laboratory. CONCLUSION: These observations suggest that every single P. jirovecii population is made of individual cells with distinct surface properties. Our results enhance our understanding of the unique antigenic variation system and cell surface structure of P. jirovecii.

Identification and Functional Ascertainment of the Pneumocystis jirovecii Potential Drug Targets Gsc1 and Kre6 Involved in Glucan Synthesis.

The most efficient drug against the human pathogenic fungus Pneumocystis jirovecii is cotrimoxazole targeting the folate biosynthesis. However, resistance toward it is emerging and adverse effects occur in some patients. Studies in rodent models suggested that echinocandins could be useful to treat Pneumocystis pneumonia. Echinocandins inhibit the catalytic subunit Gsc1 of the enzymatic complex ensuring the synthesis of 1,3-ß glucan, an essential constituent of cell walls of most fungi. Besides, inhibitors of the enzyme Kre6 involved in the synthesis of 1,6-ß glucan, another essential component of fungal walls, were recently described. We identified and functionally characterized these two potential drug targets in the human pathogen P. jirovecii by rescue of the null allele of the orthologous gene in Saccharomyces cerevisiae. The P. jirovecii proteins Gsc1 and Kre6 identified using those of the relative Pneumocystis carinii as the query sequence showed high sequence identity to the putative fungal orthologs (53-97% in conserved functional domains). The expression of their encoding genes on plasmid rescued the increased sensitivity to, respectively, caspofungin or calcofluor white of the corresponding S. cerevisiae null allele. The uniqueness and likely essentiality of these proteins suggest that they are potential good drug targets.

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).

Mechanisms of Surface Antigenic Variation in the Human Pathogenic Fungus Pneumocystis jirovecii.

Microbial pathogens commonly escape the human immune system by varying surface proteins. We investigated the mechanisms used for that purpose by Pneumocystis jirovecii 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 P. jirovecii 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 P. jirovecii 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.IMPORTANCEPneumocystis jirovecii 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.