Cécile-Marie Aliouat-Denis

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.

Growth and Airborne Transmission of Cell-Sorted Life Cycle Stages of Pneumocystis carinii

Pneumocystis organisms are airborne opportunistic pathogens that cannot be continuously grown in culture. Consequently, the follow-up of Pneumocystis stage-to-stage differentiation, the sequence of their multiplication processes as well as formal identification of the transmitted form have remained elusive. The successful high-speed cell sorting of trophic and cystic forms is paving the way for the elucidation of the complex Pneumocystis life cycle. The growth of each sorted Pneumocystis stage population was followed up independently both in nude rats and in vitro. In addition, by setting up a novel nude rat model, we attempted to delineate which cystic and/or trophic forms can be naturally aerially transmitted from host to host. The results showed that in axenic culture, cystic forms can differentiate into trophic forms, whereas trophic forms are unable to evolve into cystic forms. In contrast, nude rats inoculated with pure trophic forms are able to produce cystic forms and vice versa. Transmission experiments indicated that 12 h of contact between seeder and recipient nude rats was sufficient for cystic forms to be aerially transmitted. In conclusion, trophic- to cystic-form transition is a key step in the proliferation of Pneumocystis microfungi because the cystic forms (but not the trophic forms) can be transmitted by aerial route from host to host.

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

ABSTRACT 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.

Ploidy of cell-sorted trophic and cystic forms of Pneumocystis carinii.

Once regarded as an AIDS-defining illness, Pneumocystis pneumonia (PcP) is nowadays prevailing in immunocompromised HIV-negative individuals such as patients receiving immunosuppressive therapies or affected by primary immunodeficiency. Moreover, Pneumocystis clinical spectrum is broadening to non-severely-immunocompromised subjects who could be colonized by the fungus while remaining asymptomatic for PcP, thus being able to transmit the infection by airborne route to susceptible hosts. Although the taxonomical position of the Pneumocystis genus has been clarified, several aspects of its life cycle remain elusive such as its mode of proliferation within the alveolus or its ploidy level. As no long-term culture model exists to grow Pneumocystis organisms in vitro, an option was to use a model of immunosuppressed rat infected with Pneumocystis carinii and sort life cycle stage fractions using a high-through-put cytometer. Subsequently, ploidy levels of the P. carinii trophic and cystic form fractions were measured by flow cytometry. In the cystic form, eight contents of DNA were measured thus strengthening the fact that each mature cyst contains eight haploid spores. Following release, each spore evolves into a trophic form. The majority of the trophic form fraction was haploid in our study. Some less abundant trophic forms displayed two contents of DNA indicating that they could undergo (i) mating/fusion leading to a diploid status or (ii) asexual mitotic division or (iii) both. Even less abundant trophic forms with four contents of DNA were suggestive of mitotic divisions occurring following mating in diploid trophic forms. Of interest, was the presence of trophic forms with three contents of DNA, an unusual finding that could be related to asymmetrical mitotic divisions occurring in other fungal species to create genetic diversity at lower energetic expenses than mating. Overall, ploidy data of P. carinii life cycle stages shed new light on the complexity of its modes of proliferation.

Pneumocystis diversity as a phylogeographic tool

Parasites are increasingly used to complement the evolutionary and ecological adaptation history of their hosts. Pneumocystis pathogenic fungi, which are transmitted from host-to-host via an airborne route, have been shown to constitute genuine host markers of evolution. These parasites can also provide valuable information about their host ecology. Here, we suggest that parasites can be used as phylogeographic markers to understand the geographical distribution of intra-specific host genetic variants. To test our hypothesis, we characterised Pneumocystis isolates from wild bats living in different areas. Bats comprise a wide variety of species; some of them are able to migrate. Thus, bat chorology and migration behaviour can be approached using Pneumocystis as phylogeographic markers. In the present work, we find that the genetic polymorphisms of bat-derived Pneumocystis are structured by host chorology. Therefore, Pneumocystis intra-specific genetic diversity may constitute a useful and relevant phylogeographic tool.

High-Speed Cell Sorting of Infectious Trophic and Cystic Forms of Pneumocystis carinii

ABSTRACT. The separation of Pneumocystis carinii life‐cycle stages while preserving infectivity is a hitherto unresolved challenge. We describe an original, reproducible, and efficient method for separating trophic from cystic forms of P. carinii using a high‐speed cell sorter. The large amounts of highly purified (99.6±0.3%) infectious trophic and cystic forms can now be used to elucidate the poorly understood P. carinii life cycle.

Transcriptomic Insights into the Oxidative Response of Stress-Exposed Aspergillus fumigatus

NADPH-oxidase mediated production of Reactive Oxygen Species (ROS) by alveolar macrophages and neutrophils is a critical mechanism for immune defence against Aspergillus fumigatus. Fungal oxidative stress response includes enzymatic response by superoxide dismutases (SOD), catalases, and enzymes from the thioredoxin and glutathione systems, which are regulated by the transcription factor Yap1. Secondary metabolites are also involved in defense against ROS. Some of the secondary metabolite clusters are controlled by the transcriptional regulator LaeA. The redundancy of antioxidant systems, and the variable impact of SOD or catalase gene deletions on in vitro oxidative stress sensitivity and in vivo virulence suggest a complex regulation of oxidative stress response in A. fumigatus, making high-throughput approaches, such as microarray or next generation sequencing (NGS), highly relevant to study their respective role. These approaches have been widely applied to A fumigatus, in order to characterize its metabolic response to different stresses mimicking in vivo conditions (such as antifungals, or neutrophils), or to transcription factor deletion (including LaeA). In some studies, oxidative stress response process and antioxidant enzymes have been identified as key metabolic pathways. However, oxidative stress response has not been analyzed systematically and a further data analysis could be helpful to clarify the role of A. fumigatus antioxidant systems and, potentially, to identify new drug targets. In this review, we synthesized available A. fumigatus microarrays and NGS data, focusing on the role of antioxidant systems. We analyzed the different methodologies that were used for transcriptomic analysis, and we compared biological processes and antioxidant system modulations in A. fumigatus exposed to stress.

A review of methods for nematode identification

Nematodes are non-segmented roundworms found in soil, aquatic environment, plants, or animals. Either useful or pathogenic, they greatly influence environmental equilibrium, human and animal health, as well as plant production. Knowledge on their taxonomy and biology are key issues to answer the different challenges associated to these organisms. Nowadays, most of the nematode taxonomy remains unknown or unclear. Several approaches are available for parasite identification, from the traditional morphology-based techniques to the sophisticated high-throughput sequencing technologies. All these techniques have advantages or drawbacks depending on the sample origin and the number of nematodes to be processed. This review proposes an overview of all newly available methods available to identify known and/or unknown nematodes with a specific focus on emerging high-throughput molecular techniques.

An Hcp100 gene fragment reveals Histoplasma capsulatum presence in lungs of Tadarida brasiliensis migratory bats

Histoplasma capsulatum was sampled in lungs from 87 migratory Tadarida brasiliensis bats captured in Mexico (n=66) and Argentina (n=21). The fungus was screened by nested-PCR using a sensitive and specific Hcp100 gene fragment. This molecular marker was detected in 81·6% [95% confidence interval (CI) 73·4-89·7] of all bats, representing 71 amplified bat lung DNA samples. Data showed a T. brasiliensis infection rate of 78·8% (95% CI 68·9-88·7) in bats captured in Mexico and of 90·4% (95% CI 75·2-100) in those captured in Argentina. Similarity with the H. capsulatum sequence of a reference strain (G-217B) was observed in 71 Hcp100 sequences, which supports the fungal findings. Based on the neighbour-joining and maximum parsimony Hcp100 sequence analyses, a high level of similarity was found in most Mexican and all Argentinean bat lung samples. Despite the fact that 81·6% of the infections were molecularly evidenced, only three H. capsulatum isolates were cultured from all samples tested, suggesting a low fungal burden in lung tissues that did not favour fungal isolation. This study also highlighted the importance of using different tools for the understanding of histoplasmosis epidemiology, since it supports the presence of H. capsulatum in T. brasiliensis migratory bats from Mexico and Argentina, thus contributing new evidence to the knowledge of the environmental distribution of this fungus in the Americas.

In Memoriam: Eduardo Dei-Cas (1945–2014)

Eduardo Dei-Cas, one of the leading parasitologists studying the opportunistic fungus Pneumocystis, died in Tournai, Belgium on January 12, 2014, after a 2-yr fight against a terrible illness. Born in Montevideo (Uruguay), on October 1, 1945, he studied both Biology (Facultad de Humanidades y Ciencias) and Medicine (Facultad de Medicina) at the Universidad de la Rep ublica, Montevideo. On May 12, 1966, he married Norah Giraldi, Professor of Hispanic languages (University of Lille 3) and who was decorated for services to education. They had four daughters, Paula (Montevideo, 1969), Beatriz (Montevideo, 1971), Mathilde (Tourcoing, 1978), and Chlo e (Tourcoing, 1980). The medical training of Eduardo Dei-Cas started with Prof. J.E. Mackinnon, a prestigious mycologist, and Prof. J.J. Osimani, a main contributor to understanding toxoplasmosis in Uruguay. Both professors successively headed the Parasitology-Mycology Department at the Faculty of Medicine, Universidad de la Rep ublica between 1960 and 1974. From them, Eduardo Dei-Cas learned medical mycology diagnosis and the fascinating biology of Spirometra cestodes. In 1969, Eduardo Dei-Cas also became a collaborator of Prof. F. Ma~ n e-Garz on, a renowned specialist of South-American trematodes, who shared with him his passion for the zoology of invertebrates and taught him about rigors in scientific research. In this laboratory, Eduardo DeiCas studied three organisms of the Rio de la Plata estuary: (i) the anatomy of nemertine organisms which are free-living aceolomates, (ii) the adaptation of the polychaete annelid Nereis succinea and the crab Chasmagnathus granulata to salinity changes. He graduated in Biology in 1973 and was granted the degree of Doctor of Medicine in 1975. At that time, he also registered at the school of fine arts to take evening classes as he had a passion for music and plastic arts (Fig. 1). Eduardo Dei-Cas had always been keen to pass on his knowledge and at the age of 23, he started to teach natural history and biology to undergraduate students (1968–1975). During that period, he also taught parasitology, zoological taxonomy, and invertebrate zoology at the Facultad de Medicina and the Facultad de Humanidades y Ciencias of Montevideo. To pursue his graduate studies, Eduardo Dei-Cas applied to undertake a Ph.D. in Parasitology abroad. He received a grant from the French government (1975–1977) and soon was welcomed to study parasitology with Pr. J. Biguet (Facult e de M edecine, Universit e Droit et Sant e, Lille, France) and Pr. S. Deblock (Facult e de Pharmacie, Universit e Droit et Sant e, Lille, France). He was awarded his Ph.D. in 1978 under the direction of Pr. M. Durchon and Pr. N. Dhainaut (Universit e des Sciences et Techniques, Lille, France). The subject of his thesis was the nervous system and parenchyma of the flatworm Schistosoma mansoni adult form. The same year, he was given official permission to practice medicine in France. In 1976, he joined the INSERM research unit 42 (Biochimie et Biologie Parasitaires et Fongiques, Villeneuve d’Ascq, France) headed by Pr. A. Vernes (later by Pr. D. Camus) where he worked on malaria. This experience allowed him to witness the in vitro cultures of Plasmodium falciparum (thanks to William Trager, who was the first to succeed in culturing P. falciparum in 1979) and to study the mechanisms of action of antimalarial drugs, Eduardo Dei-Cas (Institut Pasteur de Lille, 2009).