Isolation of the Pneumocystis carinii dihydrofolate synthase gene and functional complementation in Saccharomyces cerevisiae

The Pneumocystis carinii gene encoding the enzyme dihydrofolate synthase (DHFS), which is involved in the essential biosynthesis of folates, was isolated from clones of the Pneumocystis genome project, and sequenced. The deduced P. carinii DHFS protein shares 38% and 35% identity with DHFS of Schizosaccharomyces pombe and Saccharomyces cerevisiae, respectively. P. carinii DHFS expressed from a plasmid functionally complemented a S. cerevisiae mutant with no DHFS. Comparison of available DHFSs with highly similar folylpolyglutamate synthases allowed the identification of potential signatures responsible for the specificities of these two classes of enzymes. The results open the way to experimentally analyse the structure and function of P. carinii mono-functional enzyme DHFS, to investigate a possible role of DHFS in the resistance to antifolates of P. jirovecii, the species infecting specifically humans, and to develop a new class of antifolate

Prophylaxis Failure Is Associated with a Specific Pneumocystis carinii Genotype

To investigate the possible association between Pneumocystis carinii types and various clinical and demographic parameters, we used molecular typing to analyze 93 bronchoalveolar lavage specimens from patients with P. carinii pneumonia (PCP). Multivariate regression analysis revealed an association between being infected with a specific P. carinii genotype and receiving anti-PCP prophylaxis (odds ratio, 4.4; 95% confidence interval, 1.0-18.6; P = .05), although no association with a specific drug was detecte

Association between a Specific Pneumocystis jiroveci Dihydropteroate Synthase Mutation and Failure of Pyrimethamine/Sulfadoxine Prophylaxis in Human Immunodeficiency Virus-Positive and -Negative Patients

To investigate the possible association between different prophylactic sulfa drugs and the genotype of the Pneumocystis jiroveci dihydropteroate synthase (DHPS) gene, we examined DHPS polymorphisms in clinical specimens from 158 immunosuppressed patients (38 HIV-negative and 120 HIV-positive), using polymerase chain reaction-single-strand conformation polymorphism. Fifty-seven (36.1%) of 158 patients were infected with a mutant DHPS genotype. All patients who developed P. jiroveci pneumonia (PcP) while receiving pyrimethamine/sulfadoxine (PM/SD) prophylaxis (n=14) had a strain harboring DHPS with an amino acid change at position 57 (Pro→Ser). This mutation was only present in 20 (14%) of 144 patients not receiving prophylaxis (P<.001). Hospitalization in a specific hospital was an independent risk factor for having P. jiroveci harboring the same DHPS mutation, which indirectly supports that interhuman transmission may affect the dissemination of the mutant strain

Interhuman Transmission as a Potential Key Parameter for Geographical Variation in the Prevalence of Pneumocystis jirovecii Dihydropteroate Synthase Mutations

Background. Pneumocystis jirovecii dihydropteroate synthase (DHPS) mutations are associated with failure of prophylaxis with sulfa drugs. This retrospective study sought to better understand the geographical variation in the prevalence of these mutations. Methods. DHPS polymorphisms in 394 clinical specimens from immunosuppressed patients who received a diagnosis of P. jirovecii pneumonia and who were hospitalized in 3 European cities were examined using polymerase chain reaction (PCR) single-strand conformation polymorphism. Demographic and clinical characteristics were obtained from patients' medical charts. Results. Of the 394 patients, 79 (20%) were infected with a P. jirovecii strain harboring one or both of the previously reported DHPS mutations. The prevalence of DHPS mutations was significantly higher in Lyon than in Switzerland (33.0% vs 7.5%; P<.001). The proportion of patients with no evidence of sulfa exposure who harbored a mutant P. jirovecii DHPS genotype was significantly higher in Lyon than in Switzerland (29.7% vs 3.0%; P<.001). During the study period in Lyon, in contrast to the Swiss hospitals, measures to prevent dissemination of P. jirovecii from patients with P. jirovecii pneumonia were generally not implemented, and most patients received suboptimal prophylaxis, the failure of which was strictly associated with mutated P. jirovecii. Thus, nosocomial interhuman transmission of mutated strains directly or indirectly from other individuals in whom selection of mutants occurred may explain the high proportion of mutations without sulfa exposure in Lyon. Conclusions. Interhuman transmission of P. jirovecii, rather than selection pressure by sulfa prophylaxis, may play a predominant role in the geographical variation in the prevalence in the P. jirovecii DHPS mutation

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

The \u3cem\u3eChlamydomonas\u3c/em\u3e Genome Reveals the Evolution of Key Animal and Plant Functions

Chlamydomonas reinhardtii is a unicellular green alga whose lineage diverged from land plants over 1 billion years ago. It is a model system for studying chloroplast-based photosynthesis, as well as the structure, assembly, and function of eukaryotic flagella (cilia), which were inherited from the common ancestor of plants and animals, but lost in land plants. We sequenced the ∼120-megabase nuclear genome of Chlamydomonas and performed comparative phylogenomic analyses, identifying genes encoding uncharacterized proteins that are likely associated with the function and biogenesis of chloroplasts or eukaryotic flagella. Analyses of the Chlamydomonas genome advance our understanding of the ancestral eukaryotic cell, reveal previously unknown genes associated with photosynthetic and flagellar functions, and establish links between ciliopathy and the composition and function of flagella

Risk for Pneumocystis carinii Transmission among Patients with Pneumonia: a Molecular Epidemiology Study

We report a molecular typing and epidemiologic analysis of Pneumocystis carinii pneumonia (PCP) cases diagnosed in our geographic area from 1990 to 2000. Our analysis suggests that transmission from patients with active PCP to susceptible persons caused only a few, if any, PCP cases in our setting