M I N I R E V I E W

Pneumocystis: fromadoubtful unique entity toagroupof highlydiversi¢ed fungal speciesMagali Chabe1,2, Cecile-Marie Aliouat-Denis1,2, Laurence Delhaes1,3, El Moukhtar Aliouat1,2,Eric Viscogliosi1 & Eduardo Dei-Cas1,2,3

1Biology and Diversity of Emergent Eukaryotic Pathogens (BDEEP)–Center for Infection and Immunity of Lille, Pasteur Institute of Lille, Inserm U1019,

CNRS UMR 8204, University Lille-Nord-de-France, Lille, France; 2Department of Parasitology–Mycology, Faculty of Biological and Pharmaceutical

Sciences, University of Lille-Nord-de-France, Lille, France; and 3Department of Parasitology–Mycology, Faculty of Medicine, University of Lille-Nord-

de-France, Biology-Pathology Centre, University Hospital Center, Lille, France

Correspondence: Magali Chabe,

Department of Parasitology–Mycology,

Faculty of Biological and Pharmaceutical

Sciences, University of Lille-Nord-de-France,

3, rue du Professeur Laguesse BP83, 59006

Lille, France. Tel.: 133 3 2096 4010; fax: 133

3 2087 7276; e-mail: magali.chabe@univ-

lille2.fr

Received 11 May 2010; revised 10 October

2010; accepted 12 October 2010.

Final version published online 26 November

2010.

DOI:10.1111/j.1567-1364.2010.00698.x

Editor: Teun Boekhout

Keywords

Pneumocystis spp.; phylogenetic species

concept; life cycle; epidemiology.

Abstract

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 epidemio-

logy, to adapt existing therapies to each clinical context and to discover new

drug targets.

Introduction

The present review examines the discoveries and reasoning

that led to the current conceptions about the taxonomy of

Pneumocystis at the species level. Considered for a long time

an enigmatic pulmonary unicellular parasite of unknown

biological significance in man and other mammals, it is now

well established that the ascomycetous Pneumocystis genus

contains numerous host species-specific species widely spread

across ecosystems. Interestingly, advances in this field have

had a beneficial impact on the understanding of the natural

history of Pneumocystis infection. On the basis of the identi-

fication of several species in the genus and the demonstration

that each Pneumocystis species can only infect its own specific

host (Aliouat et al., 1993b, 1994; Furuta et al., 1993; Gigliotti

et al., 1993; Atzori et al., 1999; Durand-Joly et al., 2002), new

patterns of Pneumocystis pneumonia (PcP) epidemiology are

emerging. These new concepts, associated with the fact that

Pneumocystis organisms are actively transmitted by the air-

borne route (Walzer et al., 1977; Hughes, 1982; Soulez et al.,

1991; Dumoulin et al., 2000; Chabe et al., 2004), and perhaps

also vertically (Cere et al., 1997; Demanche et al., 2003;

Sanchez et al., 2007; Montes-Cano et al., 2009), provide new

insights to identify reservoirs and infection sources, and

therefore to conceive rational, efficient and cost-effective

prevention measures against PcP (Vargas et al., 2000; Miller

et al., 2001; Rabodonirina, 2001; Durand-Joly et al., 2003;

Calderon et al., 2004).

FEMS Yeast Res 11 (2011) 2–17c� 2010 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

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The Pneumocystis genus: from Protoctiststo Fungi

Carlos Chagas (1879–1934) first discovered Pneumocystis

cystic forms in 1909 after he had been charged with the task

of malaria control during the construction of the central

railroad in Minas Gerais state, Brazil (Delaporte, 1999).

Chagas observed such forms in the lungs of guinea pigs

inoculated with the blood of two children with trypanoso-

miasis. He misidentified them as a new schizogonic state of

Trypanosoma cruzi and proposed a new trypanosome genus

named Schizotrypanum (Chagas, 1909). Chagas observed

the same cystic forms in the lungs of a human case of acute

American trypanosomiasis (Chagas, 1911; Delaporte, 1999).

In 1910, at the Pasteur Institute of Sao Paulo, Antonio

Carini (1872–1950) found similar cysts in the lungs of

Rattus norvegicus infected by Trypanosoma lewisi (Carini,

1910). He sent the slides to Alphonse Laveran, whose two

fellows, husband and wife Delanoe, observed similar pul-

monary cysts in trypanosome-free Parisian rats (R. norvegi-

cus). After inoculating Trypanosoma-free rats with the cysts

and checking that they did not lead to blood-circulating

trypanosomes, Delanoe & Delanoe concluded that pulmon-

ary cystic forms reported by Chagas and Carini indeed

constituted a new microorganism unrelated to trypano-

somes (Delanoe & Delanoe, 1912; Calderon-Sandubete

et al., 2002). Pneumocystis carinii was named and described

as a new biological entity (Delanoe & Delanoe, 1912). The

choice of the Latin name stems from the tropism of the

organism for the lungs (Pneumo-), its round shape

(-cystis) and the name of Dr Antonio Carini, who provided

the tissue samples (Delanoe & Delanoe, 1912). Chagas then

retracted his first description and ascertained the French

couple’s position by supportive data (Chagas, 1913).

At this time, P. carinii was hypothetically linked with

coccidian protozoa (Aragao, 1913; Chagas, 1913). The

controversy about the taxonomic classification of Pneumo-

cystis, especially its fungal vs. protozoan nature, began and

continued to gain momentum until the end of the 1980s.

However, since 1988 many pieces of evidence have been

brought together that indicate that Pneumocystis belongs in

the group of Fungi. For example, phylogenetic analyses of

Pneumocystis based on rRNA gene (Edman et al., 1988,

1989a; Stringer et al., 1989) as well as mitochondrial gene

sequences (Pixley et al., 1991; Sinclair et al., 1991; Wakefield

et al., 1992) showed significant homology with fungal DNA

sequences. Recently, analyses of P. carinii expressed sequence

tags (EST) and cDNA sequences in the framework of the

Pneumocystis genome project (PGP) initiative (http://pgp.

cchmc.org) confirmed the overwhelming homology of

Pneumocystis to Fungi (Cushion et al., 2007). Furthermore,

Pneumocystis genes encoding thymidylate synthase (TS) and

dihydrofolate reductase (DHFR) were found to be located

on different chromosomes, indicating that TS and DHFR

enzymatic functions reside in two distinct polypeptide

chains, unlike all protozoa studied to date (Krungkrai et al.,

1990; Anderson, 2005; Sienkiewicz et al., 2008). Finally, a

gene encoding a protein that showed marked structural

similarity with the elongation factor 3 (EF-3) of Saccha-

romyces cerevisiae and Candida albicans, was characterized

in Pneumocystis (Ypma-Wong et al., 1992). Whereas EF-1

and EF-2 are conserved among Fungi and higher eukaryotes,

EF-3 is unique to Fungi.

Today, the assignment of the Pneumocystis genus to the

group of Fungi needs no further proof, but the identity of

the closest extant relative to the Pneumocystis genus has been

the subject of debate. In fact, the Pneumocystis genus was

placed in the fungal phylum Ascomycota, subphylum Taph-

rinomycotina (Eriksson & Winka, 1997), Order Pneumocys-

tidales (Eriksson, 1994), Class Pneumocystidomycetes (sensu

Eriksson & Winka, 1997), Family Pneumocystidaceae (Eriks-

son, 1994), Genus Pneumocystis (Delanoe & Delanoe, 1912).

The Taphrinomycotina (formerly Archiascomycota) taxon

was initially created based on the rRNA gene phylogeny and

comprises highly diverse members such as the fission yeast

Schizosaccharomyces pombe, the plant pathogen Taphrina

deformans, the anamorphic yeast-like Saitoella complicata,

Protomyces sp. and Neolecta irregularis, the only member

bearing a fruiting body structure (Nishida & Sugiyama,

1993, 1994). The question whether Taphrinomycotina is a

monophyletic (James et al., 2006; Liu et al., 2006; Spatafora

et al., 2006; Sugiyama et al., 2006) or a paraphyletic

(Eriksson, 1999; Baldauf et al., 2000; Landvik et al., 2001;

Diezmann et al., 2004) group has long been debated. Indeed,

the inferred phylogenetic analyses testing this issue differ in

the sampling of genes and fungal species as well as in the

chosen method of comparison. However, a recent phyloge-

nomic analysis, combining both nuclear and mitochondrial

gene sequences, revealed the monophyly of Taphrinomyco-

tina, which was placed as a sister group of Saccharomycoti-

na1Pezizomycotina (Liu et al., 2009). This confirmed the

assignment of the Pneumocystis genus to the group of

Taphrinomycotina, with S. pombe as the closest extant

relative species (Liu et al., 2009).

Pneumocystis organisms: what are they?

Little notice was paid to Pneumocystis organisms until they

were linked to the epidemics of interstitial plasma cell

pneumonia that plagued malnourished infants and small

children living in European orphanages as a consequence of

World War II (see Calderon-Sandubete et al., 2002 for

review). As a primary cause of mortality among AIDS

patients in the 1980s, Pneumocystis and pneumocystosis

have became a focus of research attention.

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3Pneumocystis species

Lack of efficient culture systems to propagate Pneumocys-

tis organisms in vitro has hindered the understanding of the

basic biology of Pneumocystis. In addition, as culture meth-

ods to isolate current Pneumocystis jirovecii from clinical

samples are unavailable, most Pneumocystis cell biology

studies have been developed using parasites of animal

origin. Most data about Pneumocystis cell structure resulted

from research on Pneumocystis oryctolagi in the 1990s,

thanks to significant improvements in transmission electron

microscopy (TEM)-fixation methods (Goheen et al., 1989;

Palluault et al., 1992a, b), ultrastructural cytochemistry

(Yoshikawa & Yoshida, 1987; Yoshikawa et al., 1988; Pal-

luault et al., 1990, 1992a, b; Kwon et al., 1998), and compu-

ter-aided 3D reconstructions (Palluault et al., 1991a, b).

These studies revealed specific ultrastructural features of

P. oryctolagi (Dei-Cas et al., 2006), but most morphological

data on this species can be extended to other Pneumocystis

species (Dei-Cas et al., 2004) and are briefly summarized

below.

All known Pneumocystis life-cycle stages are found in the

lungs of infected hosts, although parasites may spread

exceptionally to other organs. The usually accepted life cycle

(Fig. 1) of Pneumocystis species involves an ameboid, thin-

walled, mononuclear vegetative or trophic form which

becomes a thick-walled cystic stage (ascus), in which a

multiple nuclear division leads to the formation of eight

ascospores. These forms are able to leave the cyst, presum-

ably by a pore-like zone located at the thickest part of the

cyst cell wall (Itatani, 1994), to attach specifically to type-I

epithelial alveolar cells and to evolve towards the cystic stage.

The transition from trophic forms to mature cyst or ascus

occurs in three consecutive sporocytic stages (early, inter-

mediate and late sporocytes) (Yoshida, 1989; Dei-Cas et al.,

2004).

Ascospores, which result from invaginations of the spor-

ocyte cell membrane, present a single mitochondrion, a

well-developed rough or smooth endoplasmic reticulum

and an electron-dense one-layered cell wall with a clearly

visible outer membrane (Barton & Campbell, 1967; Vavra &

Kucera, 1970; Haque et al., 1987; Palluault et al., 1992a, b).

Pneumocystis trophic forms and early sporocytes also have a

thin, monolayered, electron-dense cell wall with an outer

membrane (Palluault et al., 1992a). Mononuclear trophic

forms are irregular in size (4–8 mm long) and shape and

present numerous filopodia, filiform structures markedly

abundant and tree-like in mouse-derived Pneumocystis

(Dei-Cas et al., 1991). A single mitochondrion with budding

zones occupies a considerable part of the cell volume

(Palluault et al., 1991a, b). Early sporocytes are usually

rounded, mononuclear cells with a cell wall similar to that

Cyst

Late sporocyte

n

Intermediatesporocyte

n

Earlysporocyte

R!

2n

n + n

Trophicforms

Conjugation• ste2• ste3• mapk• ste11/ste20

Synaptonemalcomplexe

Meiosis:• mei2• ran1

Mitosis:• cdc2• cdc13• cdc25• cdc42

Spindle polebody

Fig. 1. A hypothetical life-cycle of Pneumocystis species. Parasites are represented as observed in the lung using TEM. Pleomorphic, thin-walled

mononuclear trophic forms are shown attached to type-I epithelial alveolar cells (at the top). Trophic form (2n) evolves into early sporocyte in which a

synaptonemal complex is indicated. Meiotic nuclear division (R!) leads to thick-walled sporocytic and cystic stages, in which multiple nuclear divisions in

intermediate and late sporocytes lead to the formation of eight haploid spores or ascospores (n). These forms are able to leave the cyst, to attach

specifically to type-I epithelial alveolar cells and, likely, to develop conjugation as illustrated at the top (left) (n1n), where spindle pole bodies are clearly

visible. Main molecular factors involved in conjugation, meiosis or mitosis identified in Pneumocystis carinii have been listed in the figure (see

homologies, functions and references in Table 1). Conjugation and synaptonemal complexes have been drawn according to Yoshida et al. (1984) and

Itatani (1996), respectively.

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4 M. Chabe et al.

of trophic forms. Transition from early to intermediate

sporocyte is denoted (1) by an increase of the cell wall

thickness (from 40 to 100 nm), resulting mainly from the

synthesis of a b-(1,3) glucan-rich, electron-lucent middle

layer, and (2) by the occurrence of multiple nuclear divi-

sions. In the late sporocytes, the resulting eight nuclei are

visible and a plasma membrane invagination progressively

demarcates the ascospores. The mature cyst or ascus is about

4–6mm in diameter, round and thick-walled. This life stage

also presents an outer membrane (De Stefano et al., 1990;

Palluault et al., 1992a). It contains a maximum of eight

spores. Mature cysts were detected in the bronchial lumen,

suggesting that they could gain the external environment to

be transmitted to other hosts by the airborne route (Dei-

Cas, 2000). However, the infectious forms of Pneumocystis

species have not been identified (Chabe et al., 2009).

In the parasite cell wall there is a highly polymorphic

116–120-kDa mannose-rich glycoprotein (major surface

glycoprotein) that constitutes the major surface antigen of

Pneumocystis (Kovacs et al., 1993). Cholesterol constitutes

about 78% of the total sterols in Pneumocystis (Kaneshiro,

1998) and is largely present in the parasite plasma mem-

brane. Pneumocystis organisms, like rust fungi, do not

contain ergosterol (Kaneshiro, 1998).

Pneumocystis fungi: an ascomycetous lifecycle

Pneumocystis species are ascomycetous pathogens appar-

ently able to sexually reproduce (mating, meiotic division,

production of ascospores) within their hosts, a property

more frequent in pathogenic fungi of plants than animals

(Dei-Cas & Vernes, 1986; Sexton & Howlett, 2006).

On the basis of detailed ultrastructural investigations

(Matsumoto & Yoshida, 1984; Yoshida, 1989; Peters et al.,

2001; Dei-Cas et al., 2004), cell sorting (Aliouat-Denis et al.,

2009; Martinez et al., 2009) and other approaches (Yamada

et al., 1986; Wyder et al., 1994, 1998; Cornillot et al., 2002),

crucial steps in the Pneumocystis life cycle are being clarified

(Fig. 1). At least in the species P. carinii, meiosis occurs in

early sporocytes, as revealed by the detection of synaptone-

mal complexes in the nucleus of these life-cycle stages

(Matsumoto & Yoshida, 1984; Peters et al., 2001). The

nucleus of the ascospore should be haploid, and a process

of nuclear fusion may therefore occur (Itatani, 1996; Smu-

lian et al., 2001) to produce diplophasic forms able to

resume the process of cyst generation. Although haploidy

has been predicted for most stages based on karyotype and

quantitative fluorescence analyses (Yamada et al., 1986;

Wyder et al., 1994, 1998; Cushion, 1998; Stringer & Cushion,

1998), more recent data indicate that haploidy and diploidy

coexist in Pneumocystis populations (Cornillot et al., 2002;

Dei-Cas et al., 2004; Aliouat-Denis et al., 2009). Thus,

replication of Pneumocystis organisms results at least

from the generation of haploid ascospores in the cyst, and

in vitro observations have suggested that trophic forms

could result exclusively from cyst development (Aliouat

et al., 1999).

In Fungi, the mating process is usually initiated after

mutual secretion of pheromones by fungal cells of opposite

mating types. Pheromone secretion is also stimulated by

environmental stress such as nutrition deprivation (Li et al.,

2007). Pheromones recognize a heterotrimeric G-coupled

transmembrane receptor located at the cell surface of the

opposite mating type. In turn, a mitogen-activated protein

kinase (Mapkp) signal transduction cascade is activated (Li

et al., 2007). Once activated, Mapkp controls many cell

effectors that halt the mitotic cell cycle, initiate transcription

of genes involved in mating, and eventually allow the fusion

of both cells (Harigaya & Yamamoto, 2007).

Many genetic factors involved in mating, meiosis and

mitosis regulation (Table 1, Fig. 1) have been identified in

P. carinii, thanks, at least in part, to the PGP (http://pgp.

cchmc.org/) (Cushion, 2004; Cushion et al., 2007). Also,

using degenerate PCR, library screening, heterologous

expression, yeast complementation, computer modeling,

immunoprecipitation and biochemical characterization,

key Pneumocystis genes or proteins potentially involved in

mating, meiosis or mitosis have been identified (Table 1, Fig.

1). The topic was reviewed recently by Aliouat-Denis et al.

(2009). Thomas et al. (1998b) identified a gene encoding a

Mapkp in P. carinii that is homologous to other fungal

Mapkps. Thus, heterologous expression of P. carinii mapk

was shown to restore pheromone signaling in S. cerevisiae

fus3/kss1 double mutants (Vohra et al., 2003a). In addition,

P. carinii Mapkp was reported to phosphorylate the P. carinii

homologue of S. pombe ste11 (ste12 in S. cerevisiae), a gene

which encodes a transcriptional factor needed for the

pheromone-induced expression of genes required for mat-

ing (Vohra et al., 2003b).

Once conjugation has occurred, activation of ste11 indir-

ectly turns on mei2, which plays pivotal roles in both the

induction and the progression of meiosis (Harigaya &

Yamamoto, 2007). Pneumocystis carinii mei2, a homologue

of S. pombe mei2, has recently been identified using the PGP

database (Burgess et al., 2008). The same group also

identified another kinase gene, P. carinii ran1, as an S. pombe

ran1 homologue. Ran1p is known to directly phosphorylate

and inhibit the activity of Mei2p. Both P. carinii genes

exhibited functional activity in meiotic control when ex-

pressed in S. pombe.

Pneumocystis carinii ste3, an a-factor pheromone receptor

homologue, was identified from an EST database that was

created as part of the PGP (Smulian et al., 2001). This

G-protein-coupled receptor was later reported to be exclu-

sively expressed in a subpopulation of trophic forms (Vohra

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5Pneumocystis species

et al., 2004), a finding that is consistent with the expression

pattern of pheromone receptors in other fungi. So far, no

ligand has been identified for this receptor. Genes encoding

functional elements of the pheromone response signal

transduction cascade, such as ste12 and ste20 orthologues,

were found to be clustered around the Pneumocystis ste3

gene (Smulian et al., 2001). Pneumocystis carinii ste20 was

shown to be expressed following adherence of the fungus to

extracellular matrix components or lung epithelial cells

(Kottom et al., 2003). Heterologous expression of P. carinii

ste20 conferred pseudohyphal growth and also revealed the

gene to be functional in mating signaling pathways in S.

cerevisiae mutant strains (Kottom et al., 2003). Additional

orthologues of fungal genes associated with either the

mating/sexual mode of replication or stress/nutritional

deprivation were identified in the P. carinii EST database,

pointing to these conditions as triggers of Pneumocystis

mating (Cushion et al., 2007). A b-1,3-endoglucanase gene,

potentially involved in the regulation of cell wall biosynthe-

sis, was recently characterized in P. carinii (Villegas et al.,

2010). Lastly, many cell division-cycle genes have been

identified and characterized in Pneumocystis species (Tho-

mas et al., 1998a; Kaiser et al., 1999; Kottom et al., 2000;

Gustafson et al., 2001; Chabe et al., 2004; Arcenas et al.,

2006; Cushion et al., 2007; Krajicek et al., 2010).

Pneumocystis genus: from one tomultiple species

A notion, which lasted for almost a century after the

description of Pneumocystis in rats, guinea pigs, humans

and other mammals, was that Pneumocystis pneumonia

(PcP) has a zoonotic pattern. This misleading belief was

consistent with the erroneous concept of a unique Pneumo-

cystis species (i.e. P. carinii Delanoe & Delanoe, 1912).

Frenkel (1976) was the first to consider rat- and human-

derived Pneumocystis as distinct species on the basis of

apparent host species specificity, antigenic and ultrastruc-

tural differences. He proposed to name the human Pneumo-

cystis organisms ‘P. jiroveci’ (Frenkel, 1976). But it was only

in the early 1990s that the idea really emerged of a

Pneumocystis genus containing numerous highly divergent

genuine taxonomic entities. Indeed, molecular techniques

have revealed the existence of great genomic diversity

among isolates of Pneumocystis from different mammalian

species (see Aliouat-Denis et al., 2008 for review). These

Table 1. Pneumocystis life cycle: main Pneumocystis genes/proteins associated with mating, meiosis or mitosis

Gene Protein Homologue to� Function References

ste2w – Saccharomyces cerevisiae a-mating factor pheromone

receptor

Smulian et al. (2001); Cushion (2004)

ste3 Ste3p Coprinopsis cinerea a-mating factor pheromone

receptor

Smulian et al. (2001); Vohra et al. (2004)

mapk Mapkp Fusarium solani,

Schizosaccharomyces pombe SPK1,

Saccharomyces cerevisiae FUS3

Mitogen-activated protein kinase

(fungal differentiation and

proliferation)

Thomas et al. (1998b); Vohra et al.

(2003a)

ste11/ste20z Ste11p/Ste20p Schizosaccharomyces pombe

Ste11p, Cryptococcus neoformans

Ste20p

Ste11/Ste20p are involved in

pheromone response signal

transduction cascade

Smulian et al. (2001); Vohra et al.

(2003a); Kottom et al. (2003)

mei2 Mei2p Schizosaccharomyces pombe Induction and progression of

meiosis

Burgess et al. (2008)

ran1 Ran1p Pneumocystis carinii Temperature-dependent meiosis

inhibition activity

Burgess et al. (2008); Burgess et al.

(2009)

cdc2 Cdc2p Schizosaccharomyces pombe Cell-division-cycle serine–threonine

kinase (mitosis inductor)

Thomas et al. (1998a)‰

cdc13 Cdc13p Pneumocystis carinii Cdc13p binds to Cdc2 (needed for

Cdc2p kinase activity)

Kottom et al. (2000)

con7w – Magnaporthe grisea Likely involved in fungal

sporogenesis

Cushion et al. (2007)

cdc25 Cdc25p Schizosaccharomyces pombe Cell-division-cycle mitotic inducer

phosphatase

Gustafson et al. (2001)

cdc42 Cdc42p Schizophyllum commune Interacting with Ste20. Regulatory

GTPase function.

Krajicek et al. (2010)

�Only the greatest or most significant fungal homologies are mentioned.wGenes that were shown to be transcribed in Pneumocystis carinii.zAlso homologue to Saccharomyces cerevisiae Ste12p.‰cdc2 has also been described in Pneumocystis murina (Chabe et al., 2004) and in Pneumocystis jirovecii (Kaiser et al., 1999; Arcenas et al., 2006).

FEMS Yeast Res 11 (2011) 2–17c� 2010 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

6 M. Chabe et al.

observations were strengthened by the demonstration of a

narrow Pneumocystis host specificity revealed by cross-

infection experiments (Aliouat et al., 1993b, 1994; Gigliotti

et al., 1993).

In 1994, at the 3rd International Workshops on Oppor-

tunistic Protists (IWOP-3, Cleveland), the use of a tempor-

ary trinomial nomenclature (i.e. ‘formae speciales’) to name

Pneumocystis organisms from different mammals, was

unanimously accepted (Anonymous, 1994). Indeed, the

nomenclature of special forms, which is governed by the

requirements of the International Code of Botanical

Nomenclature (ICBN), is used to distinguish, within a

fungal parasite species, the special forms (formae speciales)

that are exclusively characterized by their adaptation to

different hosts (ICBN, chapter 1, article no. 4, note no. 3).

For instance, the human Pneumocystis strain was named

P. carinii f.sp. hominis, the mouse strain P. carinii f.sp. muris,

the rabbit strain P. carinii f.sp. oryctolagi, etc. Since 1994,

sequence comparisons, phylogenetic analyses, population

genetics and even ultrastructural studies revealed that diver-

gence existing among the best-characterized Pneumocystis

special forms reached the level of divergence found among

fungal species (Dei-Cas et al., 1994, 2006; Keely et al., 1994,

2003, 2004; Banerji et al., 1995; Mazars et al., 1995, 1997;

Wakefield, 1998; Frenkel, 1999; Stringer et al., 2001; Cushion

et al., 2004; Redhead et al., 2006). In accordance with this,

Frenkel (1999) described again human-derived Pneumocystis

at the species level, naming it P. jirovecii, but this time in the

framework of ICBN. He took advantage of this work to

characterize rat-derived Pneumocystis according to ICBN

rules, retaining the name P. carinii (Frenkel, 1999) proposed

by Delanoe & Delanoe (1912). Afterwards, a discussion

among the Pneumocystis investigators at the IWOP-7 (Cin-

cinnati, 2001) ended with a general agreement to pursue the

process of describing and naming the Pneumocystis species

(Cushion & Beck, 2001). To date, three other Pneumocystis

species have been formerly described: Pneumocystis wake-

fieldiae Cushion et al. (2004), in R. norvegicus, Pneumocystis

murina Keely et al. (2004), in laboratory mouse (Mus

musculus), and P. oryctolagi Dei-Cas et al. (2006), in Old

World rabbits (Oryctolagus cuniculus) (Table 2).

Which species concept for Pneumocystis?

Fungal taxonomy issues are often complex, and Pneumocys-

tis taxonomy does not depart from this rule. The reasons for

this complexity include the very nature and odd character-

istics of some fungi, such as the inability to grow in vitro, but

also the heterogeneity of the species concepts. Indeed, at

least 20 defined species concepts exist, leading to many ways

of conceiving a species (Mayden, 1997; Hey, 2001). Fungal

taxonomy issues, especially criteria and strategies to describe

new species, have been comprehensively clarified (Taylor

et al., 2000). Taylor highlighted the distinction made by

Mayden between theoretical species concepts, such as the

evolutionary species concept (ESC), and operational ones,

such as the morphological species concept (MSC), the

biological species concept (BSC), and the phylogenetic

species concept (PSC) (Mayden, 1997; Taylor et al., 2000).

MSC, BSC and PSC specify criteria to recognize species and

are thus in common use, whereas the ESC cannot be directly

applied (Mayden, 1997). Therefore, Taylor proposed the

term ‘species recognition’ for the operational species con-

cepts, i.e. morphological species recognition (MSR), biolo-

gical species recognition (BSR) and phylogenetic species

recognition (PSR).

MSR is the dominant method and has permitted the

diagnosis of nearly 70 000 fungal species, which serve as a

base for future comparisons (Taylor et al., 2000). At first

sight, MSR is of little use for describing new species in the

Pneumocystis genus because Pneumocystis species show little

difference at the light microscopic level. However, host

species-related divergence, at least among some Pneumocys-

tis species, was found using TEM (Dei-Cas et al., 1994, 2004,

2006; Nielsen et al., 1998). Indeed, ultrastructural differ-

ences allowed rabbit-derived P. oryctolagi to be distinguished

from rodent-derived Pneumocystis species (Dei-Cas et al.,

1994; Nielsen et al., 1998). Pneumocystis murina organisms

show thinner and more numerous filopodia than P. orycto-

lagi (Dei-Cas et al., 2006) and the density of membrane-

limited cytoplasmic granules in Pneumocystis is different

among rats, mice and rabbits (Nielsen et al., 1998). In

addition to these morphological differences, phenotypic

divergence based on other features was reported to exist

among Pneumocystis species from diverse mammals. These

phenotypic differences, such as cystic-to-trophic form ratio,

location in the pulmonary alveolus, in vivo doubling time

and pathology, are detailed in Table 2. Furthermore, cross-

infection experiments, performed by several groups, in

SCID mice or Nude rats with inocula of parasites isolated

from rats, mice, monkeys, ferrets, rabbits or immuno-

suppressed patients, revealed that Pneumocystis-free labora-

tory animals were refractory to infection elicited by Pneu-

mocystis organisms derived from a heterologous host species

(Aliouat et al., 1993b, 1994; Furuta et al., 1993; Gigliotti

et al., 1993; Atzori et al., 1999; Durand-Joly et al., 2002).

Thus, Pneumocystis species show an outstanding selective

infectivity, demonstrating narrow host-species specificity.

Further differences were reported in the Pneumocystis in

vitro behavior (Aliouat et al., 1993a). For instance, rat-

derived Pneumocystis seems to have a higher capacity for

attaching to target cells in vitro than mouse-derived patho-

gens. Moreover, in vitro attachment of rat Pneumocystis

seems to be more sensitive to pentamidine or cytochalasin-

B than attachment of mouse-derived organisms (Aliouat

et al., 1993a).

FEMS Yeast Res 11 (2011) 2–17 c� 2010 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

7Pneumocystis species

Table 2. Genotypic and phenotypic signatures differentiating Pneumocystis species (modified from Aliouat-Denis et al., 2008)

Features Pneumocystis carinii

Pneumocystis

wakefieldiae Pneumocystis murina

Pneumocystis

oryctolagi Pneumocystis jirovecii

Specific host Rat (Rattus norvegicus) Rat (Rattus norvegicus) Mouse (Mus musculus)

Rabbit (Oryctolagus

cuniculus) Man (Homo sapiens)Genes�

mtLSUrRNA U20169 (Cushion,

1998)

U20173 (Cushion,

1998)

AF257179 (S. Ruan

et al., unpublished

data)

S42915 (Wakefield

et al., 1992)

S42926 (Wakefield

et al., 1992)

mtSSUrRNA Hunter & Wakefield

(1996)

Hunter & Wakefield

(1996)

Hunter & Wakefield

(1996)

Hunter & Wakefield

(1996)

nucrRNA M86760 (Edman et al.,

1988)

L27658 (Ortiz-Rivera

et al., 1995; Cushion

et al., 1993)

AY532651 (Keely

et al., 2004)

DQ010098 (L. Li et al.,

unpublished data)

Liu et al. (1992)

ITS Ortiz-Rivera et al.

(1995)

L27658 (Ortiz-Rivera

et al., 1995)

AY532651 (Keely

et al., 2004)

DQ010098 (L. Li et al.,

unpublished data)

Ortiz-Rivera et al.

(1995); Lu et al. (1994)

TS M25415 (Edman et al.,

1989b)

Keely et al. (1994) Mazars et al. (1995) Mazars et al. (1995) Mazars et al. (1995)

HSP70 U80967 (Stedman

et al., 1998)

U80969 (Stedman

et al., 1998)

AY353254 (C.R.

Icenhour et al.,

unpublished data)

DQ435616 (Dei-Cas

et al., 2006)

U80970 (Stedman

et al., 1998)

Electrophoretic

karyotypes (number

and size range of

bands)

12–16 bands

(308–680 kb) Cushion

et al. (2004)

14 bands

(308–660 kb) Cushion

et al. (2004)

17 bands

(309–634 kb) Keely

et al. (2004)

14 bands

(300–700 kb) Cho

et al. (1999)

13 bands

(370–810 kb) Stringer

& Cushion (1998)

Size of

Pneumocystis

genome

8.2 Mb (Keely et al.,

2004)

7.7 Mb (Keely et al.,

2004)

8.2 Mb (Keely et al.,

2004)

7.7 Mb (Stringer &

Cushion, 1998)

Main phenotypic signatures (from Dei-Cas et al., 2006 unless otherwise indicated)

Organisms in lung

dry smears (TBO or

Giemsa stains)

Closely clustered Closely clustered Clustered Detached from each

other

Closely clustered

Cystic-to-trophic

form ratio

0.02–0.05 ? 0.02–0.05 0.10–0.15 ?

Size of cystic forms 4.03–4.42mm

(Laakkonen & Sukura,

1997)

5–8mm (Cushion

et al., 2004)

5–8mm (Keely et al.,

2004)

4–6 mm (Dei-Cas et al.,

2006)

3.5–5mm (Frenkel,

1976)

Filopodia thickness

and richness

Smaller than those of

rabbit

? Thinner and more

numerous than those

of rabbit, human and

macaque

Thicker and less

abundant than those

of mice

Thicker and less

abundant than those

of mice

Location Filling alveolar lumen Filling alveolar lumen Filling alveolar lumen Lining alveolar

epithelium

Filling alveolar lumen

(AIDS) or lining

alveolar epithelium

(epidemic or infantile

PcP)

In vivo doubling

time

4.5 days (in corticoid-

treated rats) (Aliouat

et al., 1999)

? 10.5 days (SCID mice)

(Aliouat et al., 1999)

1.7 days (untreated

rabbits) (Aliouat et al.,

1999)

?

Intra-alveolar

eosinophilic

honeycomb material

Present ? Present Rare Present

Fibrosis Frequent ? Frequent Rare Frequent

�Gene sequences available from at least two different Pneumocystis species. Accession numbers of the Pneumocystis DNA sequences and/or references

are given when they are available.

mtLSUrRNA, large subunit of mitochondrial rRNA; mtSSUrRNA, small subunit of mitochondrial rRNA; nucrRNA: rRNA from nuclear genome; ITS,

internal transcribed spacer from nuclear rRNA gene locus; TS, thymidylate synthase; HSP70, heat shock protein 70; ?, unknown.

FEMS Yeast Res 11 (2011) 2–17c� 2010 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

8 M. Chabe et al.

Concepts of biological species (‘groups of actually or

potentially interbreeding natural populations, which are

reproductively isolated from other such groups’; Mayr,

1963) play a prominent role in describing new fungal species

(Taylor et al., 2000). In Fungi, BSR has been used to identify

groups of mating compatible individuals, thus equated

with species. To some degree, BSC could be applied to

identify new Pneumocystis species. Indeed, sexual forms of

Pneumocystis have not been identified and sustained in vitro

cultures are not yet feasible, thus preventing laboratory

mating experiments from being set up. To answer the

question of whether gene flow occurs between Pneumocystis

subpopulations, population genetic approaches were uti-

lized (Mazars et al., 1997). Actually, multilocus enzyme

electrophoresis (MEE) was used to analyze five Pneumocystis

enzyme systems: malate dehydrogenase (MDH), glucose

phosphate isomerase (GPI), leucine aminopeptidase

(LAP), malic enzyme (ME) and 6-phosphogluconate

dehydrogenase (6PGDH) in Pneumocystis isolates from

rabbits, mice and rats (22 weaning rabbits, 30 corticoste-

roid-treated rats and 17 corticosteroid-treated mice) (Ma-

zars et al., 1997). Linkage disequilibrium analysis of allele

segregation and recombination of genotypes occurring

at these five loci clearly showed that Pneumocystis strains

from different host species constitute genetically distinct

populations, isolated from each other for a very long time

and therefore representing truly diverse lineages (Mazars

et al., 1997).

Today, the PSC (evolutionary lineages with a unique

combination of DNA orthologue sequences) is of growing

importance in fungal taxonomy (Taylor et al., 2000). For

Taylor, PSR comes closer than MSR and BSR to recognizing

species consistent with the ESC because, once progeny

evolutionary species have formed from an ancestor, changes

in gene sequences occur and can be recognized before

changes have occurred in mating behavior or morphology

(Taylor et al., 2000). Moreover, the genealogical concor-

dance phylogenetic species recognition method (Taylor

et al., 2000), which assesses the relationships among several

phylogenetic trees constructed on the basis of gene

sequences, increases the reliability of PSC-based species deter-

mination, making it possible to detect the occurrence of

genetic exchange (i.e. a concordance of gene trees is indica-

tive of genetic isolation) (Taylor et al., 2000). Beyond doubt,

PSC is the main species concept for use in the description of

new Pneumocystis species. Thanks to the abundance of

Pneumocystis gene sequence data, the genealogical concor-

dance condition, required to describe new fungal species

(Taylor et al., 2000), has been fulfilled by all Pneumocystis

species described until now, and all have emerged as mono-

phyletic clades on the basis of several genes (Fig. 2; Dei-Cas

et al., 2006). In agreement with this, the topological con-

cordance among seven Pneumocystis gene trees suggests that

the Pneumocystis organisms found in different host species

do not mate (Keely & Stringer, 2005). Genetic variation at

rRNA-encoding and DHFR loci between Pneumocystis spe-

cies were also used to estimate the times of Pneumocystis

speciation (Keely et al., 2003, 2004). According to these

estimations, P. carinii and P. jirovecii diverged early from

each other, about 100 million years ago (Keely et al., 2003).

Although this kind of estimation has to be viewed with

caution, the chronology matches well the divergence time of

primates from rodents (Nei et al., 2001). The same approach

applied to Pneumocystis from rodent subfamily Murinae

(P. carinii and P. murina) suggested they diverged from each

other 30–40 million years ago (Keely et al., 2004), which is

Fig. 2. Maximum-likelihood phylogeny of 12

Pneumocystis taxa, including the five described

Pneumocystis species, inferred from

mitochondrial large subunit ribosomal RNA

(mtLSUrRNA) and mitochondrial small-subunit

rRNA (mtSSUrRNA) concatenated gene

sequences, carried out using MRBAYES v3.1.2

(Ronquist & Huelsenbeck, 2003). Bayesian

posterior probabilities, calculated using a

Markov chain Monte Carlo sampling approach

(Green, 1995) implemented in MRBAYES v3.1.2,

are given as percentages near the individual

nodes. Nodes with values of o 50% are not

shown. Scale bar: 0.1 substitutions (corrected)

per base pair (from Aliouat-Denis et al., 2008).

FEMS Yeast Res 11 (2011) 2–17 c� 2010 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

9Pneumocystis species

consistent with the evolutionary time frame of their hosts

(Nei et al., 2001).

To date, the five accepted Pneumocystis species (P. carinii

Frenkel, 1999; P. jirovecii Frenkel, 1999; P. wakefieldiae

Cushion et al., 2004; P. murina Keely et al., 2004; P. oryctolagi

Dei-Cas et al., 2006) have all been described in the frame-

work of ICBN, on the basis of the morphological and/or

biological (Mayr, 1963) and phylogenetic (Taylor et al.,

2000) species concepts (Dei-Cas et al., 2006) examined

above.

Practical and theoretical implications ofnew notions on Pneumocystis taxonomy

The discovery of the genetic diversity of Pneumocystis genus

was likely the most important achievement in Pneumocystis

research for the last 20 years. Research on this topic revealed

that the genus contains numerous species with diverse

biological properties, each one being stenoxenous, i.e.

strictly infectious to one mammal species. Relatively large

molecular studies have been developed to explore the

presence and the circulation of Pneumocystis species in

ecosystems. Thus, Pneumocystis isolates from domestic,

synanthropic or wild mammals could be identified on the

basis of new species-specific DNA sequences that hitherto

confirmed the narrow host range of Pneumocystis species

(see Aliouat-Denis et al., 2008 for review). The results of

these researches have radically changed our conceptions

about Pneumocystis development, infection sources and

reservoir.

It was considered until recently that Pneumocystis organ-

isms were able to multiply exclusively in deeply immunode-

pressed hosts. Recent research has drastically changed

this view. Sensitive molecular methods allowed the detection

of Pneumocystis organisms in respiratory samples from

healthy or hospitalized subjects without severe immuno-

depression (Calderon et al., 1996, 2004; Dei-Cas, 2000;

Peterson & Cushion, 2005). In addition, experiments

focused on the behavior of Pneumocystis organisms in

immunocompetent hosts, demonstrated that Pneumocystis

cells are able to multiply in their lungs (Chabe et al., 2004).

It was also long believed that infection could be con-

tracted by the airborne route from animal or hypothetical

environmental sources, where the unique species of the

genus (P. carinii) able to cause PcP in both humans and

animals developed saprophytic growth. We know now

that the Pneumocystis genus includes numerous species,

each one restricted to one mammal species. Therefore,

animals cannot constitute a source of Pneumocystis infection

in humans (Gigliotti et al., 1993; Durand-Joly et al., 2002).

The idea of a zoonotic pattern to Pneumocystis infection,

although still retained by some authors (Stewart et al., 2005;

Youn, 2009), is no longer valid, as only human beings

could represent a potential infection source for other human

beings.

Which humans play the role of Pneumocystis infection

source? It might be considered that only hosts with PcP

transmit the infection. However, it was shown that immu-

nocompetent hosts with subclinical Pneumocystis infection

can transmit the infection by the airborne route to both

susceptible and healthy hosts (Chabe et al., 2004). Several

pieces of evidence suggest that the real impact of Pneumo-

cystis infection in humans or other mammals is beyond PcP.

For instance, seroconversion revealed Pneumocystis primary

infection in 4 90% of healthy children worldwide (re-

viewed in Aliouat-Denis et al., 2008), a condition that could

be associated with benign respiratory symptoms (Stagno

et al., 1980; Totet et al., 2004; Larsen et al., 2007). Immuno-

competent adults are often found to be Pneumocystis carriers

( = subjects without PcP but harboring very low Pneumocys-

tis rates), and Pneumocystis carriage was shown to be more

frequent in subjects with chronic respiratory conditions, e.g.

chronic obstructive pulmonary disease (COPD) (Calderon

et al., 1996, 2007), which is a major cause of disability and

the fourth leading cause of death in the world (Tan & Ng,

2008). Pneumocystis infection of immunocompetent hosts is

emerging as a relevant issue for human as well as animal

health (Rajagopalan-Levasseur et al., 1998; Cavallini-

Sanches et al., 2007) and, interestingly, typical PcP seems to

be a rare event in the natural history of Pneumocystis

infection.

What about the environment as a potential Pneumocystis

infection source? It cannot be formally excluded that (1)

Pneumocystis growth could be supported by unknown

inanimate, abiotic or vegetal substrates, or (2) some Pneu-

mocystis life-cycle stages could retain their infectious power

for some time after being released into the environment

from infected hosts. Hypotheses (1) and (2) were strength-

ened by the detection of Pneumocystis DNA in air- and

water-filtrate samples (Wakefield, 1996; Casanova-Cardiel &

Leibowitz, 1997). Also, Pneumocystis DNA was successfully

amplified from the air at conventional animal facilities

where Pneumocystis-infected laboratory animals were

housed (Olsson et al., 1996; Latouche et al., 1997) and from

hospital wards housing PcP patients (Bartlett et al., 1994;

Olsson et al., 1998). Against hypothesis (1) is that no

evidence of Pneumocystis environmental growth has been

reported so far. However, some Pneumocystis forms could

retain their viability after being in the air, as P. jirovecii

mRNA could be amplified from hospital air samples

(Latouche et al., 2001; Maher et al., 2001). In agreement with

this, there is some evidence of the existence of a Pneumocystis

‘dormant’ life-cycle stage (Kaneshiro & Maiorano, 1996; Chin

et al., 1999). Alternatively, the fact that Pneumocystis DNA or

even mRNA can be detected in the environment of Pneumo-

cystis-infected animals or humans (Bartlett et al., 1997;

FEMS Yeast Res 11 (2011) 2–17c� 2010 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

10 M. Chabe et al.

Olsson et al., 1998; Sing et al., 1999; Latouche et al., 2001;

Maher et al., 2001) suggests that Pneumocystis cells are

released with the exhaled air from infected hosts. This

observation supports the existence of an active host-to-host

transmission rather than the existence of hypothetical Pneu-

mocystis life stages able either to grow out of the hosts or to

remain viable in the environment for extended periods.

As Pneumocystis organisms are able to replicate in the

lungs of immunocompetent hosts, healthy members of

mammal populations, as well as severely infected susceptible

hosts, could play a major role as a Pneumocystis reservoir

(Dei-Cas, 2000; Chabe et al., 2004; Cushion et al., 2007;

Aliouat-Denis et al., 2008). The following features of Pneu-

mocystis organisms support this hypothesis: (1) a long

genetic isolation and coevolution with their host species

entailing Pneumocystis speciation and narrow host-species

specificity (Banerji et al., 1995; Mazars et al., 1995, 1997;

Demanche et al., 2001; Guillot et al., 2001; Hugot et al.,

2003; Keely et al., 2003, 2004; Dei-Cas et al., 2006; Aliouat-

Denis et al., 2008); (2) a highly compatible relationship with

mammalian host physiology (Dei-Cas et al., 1991, 2004;

Settnes & Nielsen, 1991; Aliouat et al., 1993a; Beck et al.,

1998; Cushion et al., 2007); and (3) a low pathogenic power

in healthy hosts (Chabe et al., 2004). In addition, Pneumo-

cystis colonized hosts are usually much more numerous than

hosts with PcP (Aliouat-Denis et al., 2008).

On the whole, molecular taxonomy research has played a

crucial role in the shift from an old conceptual Pneumocystis

infection framework to a new one. Main issues involve

taxonomy, host range and terminology. In the old concep-

tual framework, Pneumocystis organisms were considered

undefined or even enigmatic protists belonging to a unique

euryxenic taxonomic entity (P. carinii) transmissible by the

airborne route between mammals of different species. In this

old framework, terminology used to name Pneumocystis life-

cycle stages was taken from protistology (trophozoites,

precysts, cysts, intracystic bodies). In the new conceptual

framework, the Pneumocystis genus is a highly diversified

group of parasitic microfungi that contains numerous

stenoxenic species closely adapted to, and coevolving with,

mammal species. Accordingly, in this new framework, the

terminology used to name Pneumocystis life-cycle stages was

taken from mycology (trophic forms, sporocytes, ascus,

ascospores).

Conclusion

Hitherto, the identification of Pneumocystis species

(P. jirovecii excepted) was performed on parasites harvested

from immunosuppressed laboratory rodents or nonimmuno-

compromised laboratory, domestic, meat or wild Old World

rabbits. We need to explore Pneumocystis species diversity in

natural ecosystems to avoid bias derived from conventional

breeding. In fact, besides the potential impact of breeding

conditions on the Pneumocystis populations (e.g. housing in

close host-to-host proximity and isolation from the natural

environment), it could also be speculated that laboratory

animals come from a limited number of standard colonies

infected originally by a small number of Pneumocystis

strains. Furthermore, the characterization of Pneumocystis

species living in the lungs of wild animals remains a crucial

way forward to understand how these pathogens dissemi-

nate across ecosystems. Thanks to long adaptation of

Pneumocystis to its mammalian host, the topology of

Pneumocystis phylogenetic trees could shed light on the

mammalian host evolution history, helping to solve taxo-

nomic uncertainties. Although MSR and BSR have signifi-

cantly contributed to new species descriptions in the

Pneumocystis genus, the PSR seems to be a promising tool

to describe new Pneumocystis species, which are usually

represented in wild mammals by low parasite rates (Mazars

et al., 1997; Laakkonen, 1998; Palmer et al., 2000; Aliouat-

Denis et al., 2008; Chabe et al., 2010).

As a result of advancing knowledge about the natural

history of Pneumocystis infection, the perception of its

clinical impact on public health is evolving. The role of

healthy carriers in airborne disease transmission is nowa-

days recognized as a major contribution to Pneumocystis

circulation (Chabe et al., 2004). Recent data indicate that the

transplacental route is another human-to-human mode of

transmission (Montes-Cano et al., 2009). Finally, the low

parasite burden usually found in Pneumocystis carriers could

be the cause of benign respiratory symptoms in healthy

small children (Stagno et al., 1980; Totet et al., 2004; Larsen

et al., 2007) or worsening of the symptoms in COPD

patients (Calderon et al., 2007). More studies need to be

undertaken both on the clinical consequences of the pre-

sence of Pneumocystis in chronic pulmonary contexts and on

the intricate Pneumocystis life cycle to better define preven-

tion measures, adapt existing therapies to each clinical

context and discover new drug targets.

Acknowledgements

This work was supported by the ANR-ERA-NET ‘Pneumo-

cystis’ PathoGenoMics Program (ANR-06-PATHO-009-01),

by ANR ‘Biodiversite’ Program (CERoPath network, ANR-

07-BDIV-012) and by the French Ministry of Research

(EA3609-University Lille-Nord-de-France & Lille Pasteur

Institute).

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17Pneumocystis species