Pneumocystis: from a doubtful unique entity to a group of highly diversified fungal species

At the end of the 20th century the unique taxonomically enigmatic entity called Pneumocystis carinii was identified as a heterogeneous group of microscopic Fungi, constituted of multiple stenoxenic biological entities largely spread across ecosystems, closely adapted to, and coevolving in parallel with, mammal species. The discoveries and reasoning that led to the current conceptions about the taxonomy of Pneumocystis at the species level are examined here. The present review also focuses on the biological, morphological and phylogenetical features of Pneumocystis jirovecii, Pneumocystis oryctolagi, Pneumocystis murina, P. carinii and Pneumocystis wakefieldiae, the five Pneumocystis species described until now, mainly on the basis of the phylogenetic species concept. Interestingly, Pneumocystis organisms exhibit a successful adaptation enabling them to dwell and replicate in the lungs of both immunocompromised and healthy mammals, which can act as infection reservoirs. The role of healthy carriers in aerial disease transmission is nowadays recognized as a major contribution to Pneumocystis circulation, and Pneumocystis infection of nonimmunosuppressed hosts has emerged as a public health issue. More studies need to be undertaken both on the clinical consequences of the presence of Pneumocystis in healthy carriers and on the intricate Pneumocystis life cycle to better define its epidemiology, to adapt existing therapies to each clinical context and to discover new drug targets.     

Pneumocystis oryctolagi sp. nov., an uncultured fungus causing pneumonia in rabbits at weaning: review of current knowledge, and description of a new taxon on genotypic, phylogenetic and phenotypic bases

The genus Pneumocystis comprises noncultivable, highly diversified fungal pathogens dwelling in the lungs of mammals. The genus includes numerous host-species-specific species that are able to induce severe pneumonitis, especially in severely immunocompromised hosts. Pneumocystis organisms attach specifically to type-1 epithelial alveolar cells, showing a high level of subtle and efficient adaptation to the alveolar microenvironment. Pneumocystis species show little difference at the light microscopy level but DNA sequences of Pneumocystis from humans, other primates, rodents, rabbits, insectivores and other mammals present a host-species-related marked divergence. Consistently, selective infectivity could be proven by cross-infection experiments. Furthermore, phylogeny among primate Pneumocystis species was correlated with the phylogeny of their hosts. This observation suggested that cophylogeny could explain both the current distribution of pathogens in their hosts and the speciation. Thus, molecular, ultrastructural and biological differences among organisms from different mammals strengthen the view of multiple species existing within the genus Pneumocystis. The following species were subsequently described: Pneumocystis jirovecii in humans, Pneumocystis carinii and Pneumocystis wakefieldiae in rats, and Pneumocystis murina in mice. The present work focuses on Pneumocystis oryctolagi sp. nov. from Old-World rabbits. This new species has been described on the basis of both biological and phylogenetic species concepts.     

Nosocomial Pneumocystis jirovecii infections

Airborne transmission of Pneumocystis sp. from host to host has been demonstrated in rodent models and several observations suggest that interindividual transmission occurs in humans. Moreover, it is accepted that the Pneumocystis organisms infecting each mammalian species are host specific and that the hypothesis of an animal reservoir for Pneumocystis jirovecii (P. jirovecii), the human-specific Pneumocystis species, can be excluded. An exosaprophytic form of the fungus cannot be strictly ruled out. However, these data point toward the potential for the specific host to serve as its own reservoir and for Pneumocystis infection in humans as an anthroponosis with humans as a reservoir for P. jirovecii. This review highlights the main data on host-to-host transmission of Pneumocystis in rodent models and in humans by the airborne route and provides a rationale for considering the occurrence of nosocomial infections and measures for their prevention     

Molecular phylogeny of Pneumocystis based on 5.8S rRNA gene and the internal transcribed spacers of rRNA gene sequences

To clarify the phylogenetic relationships and species status of Pneumocystis, the 5.8S rRNA gene and the internal transcribed spacers (ITS, 1 and 2) of Pneumocystis rRNA derived from rat, gerbil and human were amplified, cloned and sequenced. The genetic distance matrix of six Pneumocystis species compared with other fungi like Taphrina and Saccharomyces indicated that the Pneumocystis genus contained multiple species including Pneumocystis from gerbil. The phylogenetic tree also showed that Pneumocystis from human and monkey formed one group and four rodent Pneumocystis formed another group. Among the four members, Pneumocystis wakefieldiae was most closely related to Pneumocystis murina and Pneumocystis carinii, and was least related to gerbil Pneumocystis.     

Phylogenetic relationships among Pneumocystis from Asian macaques inferred from mitochondrial rRNA sequences

The presence of Pneumocystis organisms was detected by nested-PCR at mitochondrial large subunit (mtLSU) rRNA gene in 23 respiratory samples from Asian macaques representing two species: Macaca mulatta and M. fascicularis. A very high level of sequence heterogeneity was detected with 18 original sequence types. Two genetic groups of Pneumocystis could be distinguished from the samples. Within each group, the extent of genetic divergence was low (2.5+/-1.4% in group 1 and 2.3+/-1.7% in group 2). Genetic divergences were systematically higher when macaque-derived sequence types were compared with Pneumocystis mtLSU sequences from other primate species (from 5.3+/-2.7% to 19.3+/-3.0%). The two macaque-derived groups may be considered as distinct Pneumocystis species. Surprisingly, these Pneumocystis species were recovered from both M. mulatta and M. fascicularis suggesting that host-species restriction may not systematically occur in the genus Pneumocystis. Alternatively, these observations question about the species concept in macaques.     

Ultrastructural observations on the attachment of Pneumocystis carinii in vitro.

Pneumocystis carinii trophozoites grow in vivo in close contact with host cells. The attachment of Pneumocystis to the lung cells seems to be a critical step in the parasite's development. Up to now, the contact of Pneumocystis with mammalian tissue culture cells was shown using light and scanning electron microscopy. The methods are not sufficient to observed in detail the parasite-feeder cell area of contact. In this work, the attachment of Pneumocystis trophozoites to feeder cells was examined in serial sections using transmission electron microscopy. When the contact of a trophozoite with a feeder cell took place, the development of filopodia penetrating deeply into invaginations of the feeder cell plasma membrane was observed. Then, the apical tips of filopodia become bulged anchoring the parasite to the feeder cell. The behaviour of Pneumocystis in feeder cell cultures is compared to that of the parasite in other in vitro or in vivo experimental models.     

The characteristics of the course of Leishmania and Pneumocystis opportunistic infections in immunosuppressed laboratory animals

Specific features of the course of Leishmania and Pneumocystis infections were studied on experimental models. Laboratory animals were subjected to medicinal suppression with Tricort-40. Leishmania infection in naturally susceptible animals was considerably aggravated (in comparison with the controls) in the presence of even short-term immunosuppression. Pneumocystis infection developed in experimental animals under the effect of double suppression. The process of the reactivation of Pneumocystis infection was influenced by Leishmania infection, rapidly developing in the presence of prolonged immunodeficiency. The results thus obtained make it possible to regard Leishmania infection in animals with reactivated Pneumocystis infection as co-infection having the properties of a biological, immunosuppressant. Such situation may be extrapolated on HIV/visceral leishmaniasis co-infection.     

Phylogenetic systematics and evolution of primate-derived Pneumocystis based on mitochondrial or nuclear DNA sequence comparison

Previous studies have demonstrated that the agent of Pneumocystis pneumonia (PcP), Pneumocystis carinii, is actually a complex of eukaryotic organisms, and cophylogeny could explain the distribution of the hosts and parasites. In the present work, we tested the hypothesis of cophylogeny between the primate-derived Pneumocystis group and their hosts. Specific strains isolated from 20 primate species, including humans, were used to produce a phylogeny of the parasites. Aligned sequences corresponding to DNA sequences of three genes (DHPS, mtSSU-rRNA, and mtLSU-rRNA) were separately analyzed and then combined in a single data set. The resulting parasite phylogeny was compared with different controversial phylogenies for the hosts. This comparison demonstrated that, depending upon which topology is accepted for the hosts, at least 61% and perhaps 77% of the homologous nodes of the respective cladograms of the hosts and parasites may be interpreted as resulting from codivergence events. This finding and the high specificity of these parasites suggests that cophylogeny may be considered the dominant pattern of evolution for Pneumocystis organisms, representing a new example of parallel evolution between primates and their specific parasites. Because the phylogeny of Pneumocystis followed very closely the differentiation of their hosts at the species level, the study of the parasites could provide valuable information on the phylogeny of their hosts. We used this information to explore controversial hypotheses of the phylogeny of the Platyrrhini by comparison with the phylogeny of their specific Pneumocystis parasites. If these organisms were closely associated as lung parasites with primates through the ages, the hypothesis of the Pneumocystis spp. being new pathogenic agents could be refuted. However, these organisms are opportunistic symbionts, becoming pathogenic whenever the immunological defences of their hosts decline. This study also provides support for the hypothesis that the different Pneumocystis species are genetically independent organisms, helping to clarify their taxonomic status.     

Serial propagation of Pneumocystis carinii in cell line cultures.

Pneumocystis carinii was propagated on three cell lines routinely cultured in many laboratories; the method is practical and convenient. Organisms produced were found to be reactive to Pneumocystis antisera. Studies of antigenic relationships, life cycles, and diagnostic methods will be made easier by these cultures.     

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.     

Pneumocystis: immune recognition and evasion

Pulmonary infection caused by the opportunistic fungal organism Pneumocystis continues to be a leading AIDS defining illness. The initiation of highly active antiretroviral therapy (HAART) in the HIV-infected population has led to a significant reduction in the incidence of Pneumocystis pneumonia (PCP), although recent trends suggest the incidence has plateaued rather than decreased. Host defense against Pneumocystis involves a delicate, concerted balance between the inflammatory response and immune-mediated clearance. Innate cellular immunity is a cornerstone in this response as it provides the initial recognition event that precipitates an immune response, ultimately leading to clearance of the organism from the host. This review will focus on carbohydrate moieties found in the Pneumocystis cell wall and the immune events that occur following their recognition.     

Comparison of pulsed field gel electrophoresis karyotypes of Pneumocystis carinii derived from rat lung, cell culture, and ferret lung

Pulsed field gel electrophoretic karyotypes of Pneumocystis carinii derived from three sources were compared: immunosuppressed virus-free rats transtracheally inoculated with Pneumocystis-infected rat lung; WI-38 cell/Cytodex bead cell cultures inoculated with the same material; and immunosuppressed ferrets which reactivated latent Pneumocystis pneumonia. Karyotypes of DNA from Pneumocystis trophozoites or cysts from rat lung, and trophozoites from cell culture were identical. In contrast, ferret Pneumocystis DNA karyotypes were distinctly different. Rat Pneumocystis gene probes reacted with Southern- transferred rat Pneumocystis DNA but not with ferret Pneumocystis DNA. We concluded that neither the source nor life stage of rat Pneumocystis carinii influenced genomic karyotype, and that rat and ferret Pneumocystis are genetically diverse.     

Influence of Climatic Factors on Pneumocys Carriage within a Socially Organized Group of Immunocompetent Macaques (Macaca fascicularis)

ABSTRACT. As monkeys-derived Pneumocystis is closely related to P. jirovecii , simian populations should be considered as valuable models for the understanding of the epidemiology of human pneumocystosis. In the present study, the impact of environmental factors on the carriage of Pneumocystis was evaluated in socially organized group of immunocoinpetent macaques (Macuca fascicularis). The tribe, maintained in partial release al the Primatology Center of Strasbourg in France, comprised 29 animals at the end of the study. From December 2000 to November 2002, deep nasal swab samples were collected monthly from each animal under general anaesthesia. The presence of Pneurmocystis DNA was assessed by nested PCR of mtLSU rRNA gene. No case of pneumocystosis was reported during the study. Pneumocystis DNA was detected in 166 out 01 481 swab samples examined (34.5%). The number of macaques with detectable Pneumocystis DNA was highly variable from one month to another. However, Pneumocystis carriage was clearly correlated to the mean precipitation rates.     

Transmission of Pneumocystis infection in humans

Is Pneumocystis pneumonia (PcP) a transmissible fungal disease? Does nosocomial PcP occur? Is there Pneumocystis transmission in the community? These questions, which could not be tackled before the 2000s, may at present be approached using either noninvasive detection methods or experimental transmission models. Represented by a unique entity (P.?carinii) for almost one century, the Pneumocystis genus was shown to contain several species, being P.?jirovecii the sole species identified in humans hitherto. Molecular methods combined with cross infection experiments revealed strong host specificity that precludes Pneumocystis inter-species transmission. In contrast, respiratory transmission between mammals of a same species is usually highly active, even between immunocompetent hosts. Other transmission ways could also exist. New data show that human being is the unique P.?jirovecii reservoir; it would constitute the sole infection source in both hospital and community.     

Pneumocystis PCINT1, a molecule with integrin-like features that mediates organism adhesion to fibronectin

Pneumocystis species cause severe pneumonia during chronic immunosuppression, especially in patients with AIDS or malignancy. Adhesion of Pneumocystis to extracellular matrix proteins, particularly fibronectin, associated with alveolar epithelial cell surfaces, triggers organism proliferative pathways. Herein, we report the characterization of a novel Pneumocystis molecule with considerable structural features of an integrin-like extracellular matrix adhesion receptor. A PCINT1115 bp probe was initially identified from partial sequence present within the Pneumocystis genome project database. A full-length 3018 bp cDNA was subsequently obtained with extensive homology to the C-terminal region of Candida albicans INT1 (31% blastx), a gene originally described as encoding an integrin-like molecule implicated in adhesion, growth, and virulence. Sequence analysis of PCINT1 indicated that the Pneumocystis molecule contained both a putative internal RGD motif and four Metal Ion-Dependent Attachment Sites (MIDAS) motifs required for coordination of divalent cations, as well as a specific tyrosine residue found in the cytoplasmic tails of some integrin receptors and C. albicans INT1. Northern, Western and immunofluorescence studies demonstrated that the trophic forms of Pneumocystis, known to be the life cycle forms that tightly adhere to lung epithelium, expressed the molecule to a substantially greater degree than cystic forms. Heterologous expression of PCINT1 in yeast followed by application to human fibronectin-coated surfaces demonstrated these yeast display PCINT1 on their surfaces and subsequently gain the ability to bind fibronectin in a cation dependent fashion. Taken together, these results indicate that Pneumocystis expresses a novel integrin-like PCINT1 molecule sufficient to mediate interactions with extracellular matrix fibronectin, an integral component of host-cell organism interactions during this infection.     

肺孢子菌肺炎辅助诊断方法研究进展

肺孢子菌肺炎是AIDS、器官移植受者、抗肿瘤放、化疗等各类继发或原发性免疫机能低下人群最常见的机会感染性疾病。通过显微镜检发现肺孢子菌是诊断肺孢子菌肺炎的金标准。但是,由于肺孢子菌主要寄生于肺泡腔内,目前临床采用的病原学检测方法或受到创伤性取材方法的限制或受病原体检出率极低的困惑。而目前盛行的基因检测方法因其操作过程的复杂及昂贵的费用难以适应临床应用。探索和建立敏感、特异的、可以从非创伤性标本中诊断肺孢子菌肺炎的快速诊断方法为临床之急需。学者们对肺孢子菌的主要表面糖蛋白及葡聚糖等菌体组分及其抗体等的检测方法进行了不断的探索,取得了一定进展。我们就该领域的研究进展及其在肺孢子菌肺炎辅助诊断方面的意义进行了综述。     

In vitro systems in pneumocystis research

Most groups involved in Pneumocystis research need large quantities of well preserved, viable Pneumocystis organisms free of host cell contamination. Biological, biochemical, immunological, genetic or other studies on Pneumocystis usually involve the separation of Pneumocystis from lung tissue as well as elimination of host cell debris from parasite extracts. In other investigations, such as transmission, infectivity, life cycle, biochemical, in vitro culture or drug-screening studies, viable and infectious Pneumocystis organisms are urgently required. However, there is no generally accepted methodology for obtaining Pneumocystis from experimental hosts or from human clinical samples; methods are still far from reaching standardization, as discussed here by the members of the European Concerted Action (ECA) on Pneumocystis carinii, which is co-ordinated by Eduardo Dei-Cas and Jean-Charles Cailliez.