Genes Encoding Antigenic Surface Glycoproteins in Pneumocystis from Humans

Pneumocystis is a eukaryotic microbe that causes pneumocystosis, an AIDS-associated pneumonia. Pneumocystosis also occurs in many other mammalian species, and animal-derived organisms have been extensively utilized in Pneumocystis research. Pneumocystis from diverse hosts contain a large glycoprotein (gpA/MSG) on the surface. Antibodies elicited against gpA/MSG of Pneumocystis from humans sometimes cross-react with epitopes on proteins of similar size from Pneumocystis from other host species. Here we report the isolation and partial sequence of two presumptive gpA/MSG genes from human-derived Pneumocystis. The cloned human-derived Pneumocystis gpA/MSG genes and predicted peptides were different from those previously isolated from Pneumocystis from rats and ferrets. The genome of human-derived Pneumocystis contained multiple copies of sequences related to the two cloned gpA/MSG genes.     

Relationship Between Pneumocystis carinii Burden and the Degree of Host Immunosuppression in an Airborne Transmission Experimental Model

To quantitatively assess the risk of contamination by Pneumocystis depending on the degree of immunosuppression (ID) of the exposed rat hosts, we developed an animal model, where rats went through different doses of dexamethasone. Then, natural and aerial transmission of Pneumocystis carinii occurred during cohousing of the rats undergoing gradual ID levels (receivers) with nude rats developing pneumocystosis (seeders). Following contact between receiver and seeder rats, the P. carinii burden of receiver rats was determined by toluidine blue ortho staining and by qPCR targeting the dhfr monocopy gene of this fungus. In this rat model, the level of circulating CD4⁺ and CD8⁺ T lymphocytes remained significantly stable and different for each dose of dexamethasone tested, thus reaching the goal of a new stable and gradual ID rat model. In addition, an inverse relationship between the P. carinii burden and the level of circulating CD4⁺ or CD8⁺ T lymphocytes was evidenced. This rat model may be used to study other opportunistic pathogens or even co‐infections in a context of gradual ID.     

Molecular diagnosis of Pneumocystis pneumonia

The detection of Pneumocystis DNA in clinical specimens by using PCR assays is leading to important advances in Pneumocystis pneumonia (PcP) clinical diagnosis, therapy and epidemiology. Highly sensitive and specific PCR tools improved the clinical diagnosis of PcP allowing an accurate, early diagnosis of Pneumocystis infection, which should lead to a decreased duration from onset of symptoms to treatment, a period with recognized impact on prognosis. This aspect has marked importance in HIV-negative immunocompromised patients, who develop often PcP with lower parasite rates than AIDS patients. The specific amplification of selected polymorphous sequences of Pneumocystis jirovecii genome, especially of internal transcribed spacer regions of the nuclear rRNA operon, has led to the identification of specific parasite genotypes which might be associated with PcP severity. Moreover, multi-locus genotyping revealed to be a useful tool to explore person-to-person transmission. Furthermore, PCR was recently used for detecting P. jirovecii dihydropteroate synthase gene mutations, which are apparently associated with sulfa drug resistance. PCR assays detected Pneumocystis-DNA in bronchoalveolar lavage fluid or biopsy specimens, but also in oropharyngeal washings obtained by rinsing of the mouth. This non-invasive procedure may reach 90%-sensitivity and has been used for monitoring the response to treatment in AIDS patients and for typing Pneumocystis isolates.     

Characterizing Pneumocystis in the Lungs of Bats: Understanding Pneumocystis Evolution and the Spread of Pneumocystis Organisms in Mammal Populations

Bats belong to a wide variety of species and occupy diversified habitats, from cities to the countryside. Their different diets (i.e., nectarivore, frugivore, insectivore, hematophage) lead Chiroptera to colonize a range of ecological niches. These flying mammals exert an undisputable impact on both ecosystems and circulation of pathogens that they harbor. Pneumocystis species are recognized as major opportunistic fungal pathogens which cause life-threatening pneumonia in severely immunocompromised or weakened mammals. Pneumocystis consists of a heterogeneous group of highly adapted host-specific fungal parasites that colonize a wide range of mammalian hosts. In the present study, 216 lungs of 19 bat species, sampled from diverse biotopes in the New and Old Worlds, were examined. Each bat species may be harboring a specific Pneumocystis species. We report 32.9% of Pneumocystis carriage in wild bats (41.9% in Microchiroptera). Ecological and behavioral factors (elevation, crowding, migration) seemed to influence the Pneumocystis carriage. This study suggests that Pneumocystis-host association may yield much information on Pneumocystis transmission, phylogeny, and biology in mammals. Moreover, the link between genetic variability of Pneumocystis isolated from populations of the same bat species and their geographic area could be exploited in terms of phylogeography.     

Pneumocystis jirovecii colonization is associated with enhanced Th1 inflammatory gene expression in lungs of humans with chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease (COPD) is a complex disease, the pathogenesis of which remains incompletely understood. Colonization with Pneumocystis jirovecii may play a role in COPD pathogenesis; however, the mechanisms by which such colonization contributes to COPD are unknown. The objective of this study was to determine lung gene expression profiles associated with Pneumocystis colonization in patients with COPD to identify potential key pathways involved in disease pathogenesis. Using COPD lung tissue samples made available through the Lung Tissue Research Consortium (LTRC), Pneumocystis colonization status was determined by nested PCR. Microarray gene expression profiles were performed for each sample and the profiles of colonized and non‐colonized samples compared. Overall, 18 participants (8.5%) were Pneumocystis‐colonized. Pneumocystis colonization was associated with fold increase in expression of four closely related genes: INF‐γ and the three chemokine ligands CXCL9, CXCL10, and CXCL11. These ligands are chemoattractants for the common cognate receptor CXCR3, which is predominantly expressed on activated Th1 T‐lymphocytes. Although these ligand–receptor pairs have previously been implicated in COPD pathogenesis, few initiators of ligand expression and subsequent lymphocyte trafficking have been identified: our findings implicate Pneumocystis as a potential trigger. The finding of upregulation of these inflammatory genes in the setting of Pneumocystis colonization sheds light on infectious‐immune relationships in COPD.     

Evidence of Airborne Excretion of Pneumocystis carinii during Infection in Immunocompetent Rats. Lung Involvement and Antibody Response

To better understand the role of immunocompetent hosts in the diffusion of Pneumocystis in the environment, airborne shedding of Pneumocystis carinii in the surrounding air of experimentally infected Sprague Dawley rats was quantified by means of a real-time PCR assay, in parallel with the kinetics of P. carinii loads in lungs and specific serum antibody titres. Pneumocystis-free Sprague Dawley rats were intratracheally inoculated at day 0 (d0) and then followed for 60 days. P. carinii DNA was detected in lungs until d29 in two separate experiments and thereafter remained undetectable. A transient air excretion of Pneumocystis DNA was observed between d14 and d22 in the first experiment and between d9 and d19 in the second experiment; it was related to the peak of infection in lungs. IgM and IgG anti-P. carinii antibody increase preceded clearance of P. carinii in the lungs and cessation of airborne excretion. In rats receiving a second challenge 3 months after the first inoculation, Pneumocystis was only detected at a low level in the lungs of 2 of 3 rats at d2 post challenge and was never detected in air samples. Anti-Pneumocystis antibody determinations showed a typical secondary IgG antibody response. This study provides the first direct evidence that immunocompetent hosts can excrete Pneumocystis following a primary acquired infection. Lung infection was apparently controlled by the immune response since fungal burdens decreased to become undetectable as specific antibodies reached high titres in serum. This immune response was apparently protective against reinfection 3 months later.     

Population Structure of Rat-Derived Pneumocystis carinii in Danish Wild Rats

The rat model of Pneumocystis carinii pneumonia is frequently used to study human P. carinii infection, but there are many differences between the rat and human infections. We studied naturally acquired P. carinii in wild rats to examine the relevance of the rat model for human infection. P. carinii DNA was detected in 47 of 51 wild rats and in 10 of 12 nonimmunosuppressed laboratory rats. Evidence for three novel formae speciales of rat-derived P. carinii was found, and these were provisionally named Pneumocystis carinii f. sp. rattus-secundi, Pneumocystis carinii f. sp. rattus-tertii, and Pneumocystis carinii f. sp. rattus-quarti. Our data suggest that low-level carriage of P. carinii in wild rats and nonimmunosuppressed laboratory rats is common and that wild rats are frequently coinfected with more than one forma specialis of P. carinii. We also examined the diversity in the internally transcribed spacer (ITS) regions of the nuclear rRNA operon of Pneumocystis carinii f. sp. carinii by using samples from wild rats and laboratory rats and spore trap samples. We report a lack of variation in the ITS1 and ITS2 regions that is consistent with an evolutionary bottleneck in the P. carinii f. sp. carinii population. This study shows that human- and rat-derived P. carinii organisms are very different, not only in genetic composition but also in population structure and natural history.     

Pulmonary fungus flora in experimental pneumocystosis of cortisone-treated rats

Summary In cortisone-treated rats, in addition to the observation of heavy bacterial infections and pneumocystosis in almost all animals, numerous fungus species were isolated from the lung tissue in 75 percent of the animals.The luxurious bacterial and fungus flora in rats with artificial pneumocystosis (cortisone-treated) constitutes a serious obstacle in the attempts to growPneumocystis.This work was supported in part by Grant E 576 from the National Institute of Health.     

Polymorphism of the Thymidylate Synthase Gene of Pneumocystis carinii from Different Host Species

Pneumocystis carinii is an opportunistic agent found in the lung of various mammals which often causes severe pneumonia in immunocompromised humans, especially in AIDS patients. In the past several years significant additions have been made to the collection of knowledge we have concerning the genetic diversity of P. carinii. These additions provide new understanding of Pneumocystis transmission and the effect of possible reservoirs of Pneumocystis in the various species. In this study, a 400-bp fragment of the thymidylate synthase (TS) gene of P. carinii has been amplified by PCR from 43 parasite isolates obtained from 4 mammalian host species: rat, mouse, rabbit and human. A probe selected from the TS gene sequence of rat-derived P. carinii was hybridized with the amplified products from rat- and mouse-derived P. carinii, but not with rabbit or human P. carinii DNA. Restriction profiles were performed on amplified fragments from all isolates, and the 4 nucleotide sequences of the TS gene fragment amplifed from rat, mouse, rabbit and human P. carinii were determined. Differences were detected in the gene fragment in P. carinii isolates from the 4 host species; however no difference was revealed in P. carinii isolates within a single host species, whatever the host strain or its geographic origin. Thus, the sequence differences of the P. carinii TS gene appeared as host-species specific. A specific probe which recognized all human P. carinii isolates was defined.     

Synthetic p55 tandem DNA vaccine against Pneumocystis carinii in rats

Pneumocystis spp. are opportunistic fungal pathogens that are closely associated with severe pneumonia and pulmonary complications in patients with impaired immunity. In this study, the antigenic epitopes of the gene encoding the 55 kDa antigen fragment of Pneumocystis (p55), which may play an important role in Pneumocystis pneumonia, were analyzed. A gene containing tandem variants of the p55 antigen was synthesized and named the tandem antigen gene (TAG). TAG's potential as a DNA vaccine was assessed in immunosuppressed rats. Immunization with p55‐TAG DNA vaccine significantly reduced both the pathogen burden and lung–weight to body–weight ratios. Additionally, p55‐TAG vaccination in immunosuppressed rats elicited both cell‐mediated and humoral immunity.     

<Emphasis Type="BoldItalic">Pneumocystis Jirovecii</Emphasis> Pneumonia: Current Knowledge and Outstanding Public Health Issues

Antiretroviral therapy and cotrimoxazole prophylaxis have caused a decline in Pneumocystis jirovecii incidence in developing countries since the 1990s. Nevertheless, Pneumocystis jirovecii pneumonia (PCP) remains the primary AIDS-defining disease around the world. Much about the disease remains unknown, including its global burden, best diagnostic practices, the frequency of drug resistance, and the risks associated with Pneumocystis colonization. This review describes current knowledge about Pneumocystis infection and highlights areas where new research could benefit public health.     

Evidence for DNA Synthesis in Pneumocystis carinii Trophozoites Treated with the β‐1,3‐Glucan Synthesis Inhibitor Pneumocandin L‐693,989

ABSTRACT. Pneumocandins inhibit β‐1,3‐glucan synthesis preventing the development of Pneumocystis cysts that are absent from the lungs of treated rats. To determine whether treated trophozoites are capable of DNA replication, cytochemical analyses were performed on 4′,6‐diamidino‐2‐phenylindole (DAPI)‐ and DB181‐stained Pneumocystis carinii isolated from pneumocandin L‐693‐989‐treated rats. Fluorescence intensities of trophozoite nuclei from drug‐treated rats were greater than those of untreated controls, suggesting that DNA replication was not inhibited but that cytokinesis and perhaps karyokinesis were blocked.     

Clearance of Pneumocystis murina infection is not dependent on MyD88

To determine if myeloid differentiation factor 88 (MyD88), which is necessary for signaling by most TLRs and IL-1Rs, is necessary for control of Pneumocystis infection, MyD88-deficient and wild-type mice were infected with Pneumocystis by exposure to infected seeder mice and were followed for up to 106 days. MyD88-deficient mice showed clearance of Pneumocystis and development of anti-Pneumocystis antibody responses with kinetics similar to wild-type mice. Based on expression levels of select genes, MyD88-deficient mice developed immune responses similar to wild-type mice. Thus, MyD88 and the upstream pathways that rely on MyD88 signaling are not required for control of Pneumocystis infection.     

Pneumocystis carinii Delanoë, its Ultrastructure and Ultrastructural Affinities*

SYNOPSIS The fine structure of Pneumocystis carinii Delanoë is described in detail and is compared to the fine structure of protozoa and fungi. Pneumocystis does not have ultrastructural affinities to Protozoa but rather to fungi. Specifically, the process of intracystic body formation in the cyst is similar to the formation of ascospores inside a yeast ascus. The significance of the structural similarity between Pneumocystis and fungi is discussed. It is concluded from the ultrastructural evidence that Pneumocystis may indeed be a yeast or have a yeast-like stage in its life cycle. However, Pneumocystis is not completely similar to any fungus whose ultrastructure has been described so far. Perhaps its particular structure may be an adaptation to the parasitic way of life in mammalian lungs. It should be cultivated in order to be sure of its exact taxonomic position.     

What Do Pneumocystis Organisms Tell Us about the Phylogeography of Their Hosts? The Case of the Woodmouse Apodemus sylvaticus in Continental Europe and Western Mediterranean Islands

Pneumocystis fungi represent a highly diversified biological group with numerous species, which display a strong host-specificity suggesting a long co-speciation process. In the present study, the presence and genetic diversity of Pneumocystis organisms was investigated in 203 lung samples from woodmice (Apodemus sylvaticus) collected on western continental Europe and Mediterranean islands. The presence of Pneumocystis DNA was assessed by nested PCR at both large and small mitochondrial subunit (mtLSU and mtSSU) rRNA loci. Direct sequencing of nested PCR products demonstrated a very high variability among woodmouse-derived Pneumocystis organisms with a total number of 30 distinct combined mtLSU and mtSSU sequence types. However, the genetic divergence among these sequence types was very low (up to 3.87%) and the presence of several Pneumocystis species within Apodemus sylvaticus was considered unlikely. The analysis of the genetic structure of woodmouse-derived Pneumocystis revealed two distinct groups. The first one comprised Pneumocystis from woodmice collected in continental Spain, France and Balearic islands. The second one included Pneumocystis from woodmice collected in continental Italy, Corsica and Sicily. These two genetic groups were in accordance with the two lineages currently described within the host species Apodemus sylvaticus. Pneumocystis organisms are emerging as powerful tools for phylogeographic studies in mammals.     

Interstitial pneumonia in immunocompetent laboratory rats caused by natural infection with Pneumocystis carinii

Pneumocystis (P.) carinii is known to cause fatal pneumonia in immunocompromised rats. Cases of P. carinii interstitial pneumonia in immunocompetent rats have been shown histologically to present with perivascular lymphoid cuffs, which have previously been attributed to rat respiratory virus. This study aims to determine the prevalence and pathological characteristics of P. carinii in immunocompetent laboratory rats in experimental facilities in Japan. An epidemiological survey for this agent was performed using PCR to assess 1,981 immunocompetent rats from 594 facilities in Japan. We observed that 6 of the 1,981 rats (0.30%) from 4 out of 594 facilities (0.67%) were positive for P. carinii without infection of other known pathogens. Gross pulmonary lesions were found in 4 of the 6 affected rats. The lungs of these rats contained scattered dark red/gray foci. Histopathologically, the lungs exhibited interstitial pneumonia with lymphoid perivascular cuffs: Pneumocystis cysts were observed using Grocott’s methenamine silver stain. To our knowledge, this report is the first to reveal the prevalence of natural P. carinii infection in immunocompetent laboratory rats in Japan.     

Pneumocystis jirovecii pneumonia in developing countries

Pneumocystis pneumonia (PcP) is a serious fungal infection among immunocompromised patients. In developed countries, the epidemiology and clinical spectrum of PcP have been clearly defined and well documented. However, in most developing countries, relatively little is known about the prevalence of pneumocystosis. Several articles covering African, Asian and American countries were reviewed in the present study. PcP was identified as a frequent opportunistic infection in AIDS patients from different geographic regions. A trend to an increasing rate of PcP was apparent in developing countries from 2002 to 2010.     

The Ecology of Pneumocystis: Perspectives,Personal Recollections,and Future Research Opportunities

I am honored to receive the second Lifetime Achievement Award by International Workshops on Opportunistic Protists and to give this lecture. My research involves Pneumocystis, an opportunistic pulmonary fungus that is a major cause of pneumonia (“PcP”) in the immunocompromised host. I decided to focus on Pneumocystis ecology here because it has not attracted much interest. Pneumocystis infection is acquired by inhalation, and the cyst stage appears to be the infective form. Several fungal lung infections, such as coccidiomycosis, are not communicable, but occur by inhaling < 5 μm spores from environmental sources (buildings, parks), and can be affected by environmental factors. PcP risk factors include environmental constituents (temperature, humidity, SO₂, CO) and outdoor activities (camping). Clusters of PcP have occurred, but no environmental source has been found. Pneumocystis is communicable and outbreaks of PcP, especially in renal transplant patients, are an ongoing problem. Recent evidence suggests that most viable Pneumocystis organisms detected in the air are confined to a patient's room. Further efforts are needed to define the risk of Pneumocystis transmission in health care facilities; to develop more robust preventive measures; and to characterize the effects of climatological and air pollutant factors on Pneumocystis transmission in animal models similar to those used for respiratory viruses.     

Frequency of trichomonads as coinfecting agents in Pneumocystis pneumonia

OBJECTIVE: To investigate the incidence of the association of Trichomonas and Pneumocystis in the lung. STUDY DESIGN: Sixty-six bronchoalveolar lavage fluid (BALF) samples from immunocompromised patients with pneumocystosis were retrospectively examined microscopically. RESULTS: Trichomonads were found as coinfecting agents in 60% of BALF samples. The frequency and abundance of trichomonads was increased, up to 100%, in cases rich in Pneumocystis. CONCLUSION: The data suggest that pulmonary Trichomonas infection occurs frequently in the course of Pneumocystis pneumonia. The role of trichomonads in causing alveolar damage during Pneumocystis pneumonia is hypothetical.     

Transcription Factor Genes from Rat Pneumocystis carinii

Genes encoding the TFIID TATA-box binding protein (TBP) from two probable species of rat Pneumocystis carinii (prototype and variant) were sequenced. The two P. carinii TBP gene sequences were 91% identical to each other, and 65-77% identical to TBP genes from other species. A cDNA from one of the two P. carinii TBP genes was sequenced, which showed that four small introns resided in identical positions within the TBP genes from the prototype and variant rat P. carinii. Conservation of the 180 amino acids that constitute the conserved core of TBP was 97% between the P. carinii TBP, which were 95% and 97% identical to conserved core sequences of TBP from Saccharomyces cerevisiae and Schizosaccharomyces pombe respectively.