P astor b onus y sus reformas

1.2.2.1. Many “asexual” human fungal pathogens retain their sexual machinery

Interestingly, many important human fungal pathogens appear to be asexual in nature, such as C. albicans, C. glabrata, A. fumigatus, C. immitis, and C. posadasii (Heitman, 2006). In addition, it seems that not only do many important human fungal pathogens tend to have clonal population structures, but strains that are more virulent or more successful within these species also are more clonal than the remainder of the species, for instance, C. neoformans var. grubii strains (Lengeler et al., 2000; Simwami et al., 2011) or GPG strains in C. albicans (Schmid et al., 2008). An explanation for these observations could be that loss of sex or severe restriction of its frequency is positively correlated with success in the human host. This led to the hypothesis that sexual reproduction would have destroyed complex combinations of genes which arose on rare occasions and allowed a small number of fungi to adapt to survival in the human, and that loss of sex is a necessary

prerequisite for a fungus to become a human pathogen, explaining why human pathogenic fungi are predominantly asexual (Whelan, 1987).

However, recent studies revealed that many “asexual” human pathogenic fungi actually retain the machinery to undergo sexual reproduction and population studies also show some evidence of recombination from these organisms. Also while mating has not been directly observed in nature, it has been observed in the laboratory under particular conditions for some of these fungi. It seems likely that while these human fungal pathogens are capable of sexual reproduction, mating occurs either rarely (or using alternative ways) or never in their natural environment (Nielsen and Heitman, 2007).

For example, a sexual cycle was observed over 30 years ago for C. neoformans in the laboratory (Kwon-Chung, 1975; Kwon-Chung, 1976), but never in nature. C. neoformans seems to prevent or limit its sexual reproduction in nature by having a nearly unisexual population, i.e. ∝ mating type strains predominate in both environmental and clinical isolates. In var. neoformans, a mating type strains account for less than 2% of the population. In var. grubii, the predominant pathogenic variety in this species, only 17 a mating type strains have been identified from >2000 isolates worldwide and 15 of them were clinical isolates from sub- Saharan Africa; thus mating could be geographically restricted as well (Litvintseva et al., 2003). C. neoformans can undergo a homothallic sexual reproduction (∝-∝ mating) in the laboratory, which involved only strains of one mating type (Lin et al., 2005) . It is also reported that there is some evidence for same-sex mating of C. gattii in nature (Fraser et al., 2005).

Another important fungal pathogen of humans, A. fumigatus has also been considered as an asexual organism for a long time. However, recent genome analysis has not only identified two different mating type loci but also genes involving pheromones, receptors, and components of the pheromone-signaling pathway (Paoletti et al., 2005). Although sexual reproduction was not discovered in nature, mating was observed in this fungus in the laboratory by O’Gorman et al. (2009). They discovered an extant sexual cycle in A. fumigatus by mating strains of opposite mating types. However, mating in the laboratory required half of a year of incubation of the mating compatible strains from a naturally occurring recombinant population in the dark on specialized medium (Heitman, 2011 ; O'Gorman et al., 2009).

In C. albicans, sexual forms have never been observed in clinical isolates. It has been discovered that strains can be induced to undergo mating (Hull et al., 2000; Magee and Magee, 2000) and undergo a parasexual cycle, without meiotic recombination, in the laboratory (Bennett and Johnson, 2003). To mate, strains however first need to become homozygous at the mating type locus to generate a/a or ∝/∝ strains that are mating compatible and then need to undergo white/opaque switching to become mating competent (the details of mating in C. albicans will be described in section 1.3.). This would make mating very rare in C. albicans’ natural environment, as most of the clinical isolates (90-97%) are a/α heterozygous strains and not able to mate with each other. Other than this, an alternative mating route, unisexual mating (a-a mating) has also been discovered recently in C. albicans (Alby et al., 2009), which is very similar to unisexual mating discovered previously in C. neoformans. It was found that alpha-pheromone produced by MTLα cells

promoted low frequency same-sex mating between wild-type MTLa cells.

Genome sequencing reveals that all of the other common human fungal pathogens including H. capsulatum, P. carinii, A. flavus, C. immitis, and C. posadasii have mating-type loci, although a sexual cycle has not yet been identified in these species (Nielsen et al., 2007).

1.2.2.2. Population genetic studies reveal some evidence of recombination in many human fungal pathogens

Mating in the laboratory shows that the potential for recombination remains but may not accurately predict the impact in nature. For example, Trypanosoma cruzi, the agent of Chagas disease has long been known to undergo sexual recombination in the laboratory (Gaunt et al., 2003), but its natural populations are highly clonal, indicating that recombination does not contribute to its population structure (Gauthier and Tibayrenc, 2005; Tibayrenc et al., 1981; Tibayrenc et al., 1986). This implies that recombination either does not happen in its natural environment or that the recombination-generated lineages do not (usually) survive.

The biological significance of mating (out-breeding) can only be determined by population genetic methods which assess if a species contains recombinant1 lineages. The principle of these methods is as follows: A clonal population structure means the mutations occur independently, are confined to individual lineages and

are not exchanged between lineages. Thus only a limited number of combinations of mutations should be detected. On the other hand, a recombinant structure means that mutations can be exchanged between lineages and most possible combinations of mutations can be observed (Schmid et al., 2004; Tibayrenc, 1999; Xu and Mitchell, 2002).

Population genetic studies of many important “asexual” human fungal pathogens revealed that although their population structures are largely clonal, some evidence of recombination was observed in these organisms (Nielsen and Heitman, 2007). For example, in C. neoformans, population genetic studies in the early years examining strains throughout the world suggested only a very low level of recombination (Brandt et al., 1995; Xu and Mitchell, 2003; Xu et al., 2000). However, newer population genetics studies examining strains from HIV/AIDS patients in southern California strongly support the presence of ongoing, or very recent, recombination in C. gattii (Edmond et al., 2011). Other population studies also showed evidence of recombination in C. neoformans and var. grubii (Campbell et al., 2005; Litvintseva et al., 2003). In C. albicans, population genetic studies revealed some degree of genetic recombination (as will be discussed in more detail in section 1.3.6). For the Aspergillus species, genetic variation was detected within populations of both “asexual” pathogenic species A. fumigatus and A. flavus, although mating has never been detected in nature (Bertout et al., 2001; Paoletti et al., 2005; Pildain et al., 2004; Varga and Toth, 2003).

These population genetic studies indicate that the “asexual” human fungal pathogens may have some form of genetic exchange between individuals. However,

the recombination levels in these organisms are normally very low, probably due to the lack of a classical sexual cycle. Therefore, based on this evidence, an alternative hypothesis of reproductive biology of these organisms proposed by Neilson et al., (2007) is that: “the human pathogenic fungi have retained the ability to generate either clonal or recombining population structures in response to either constant or changing environments by preserving their ability to undergo sexual (or parasexual) reproduction but limiting the conditions under which sexual reproduction occurs in unique ways”. However, indications of a low level of recombination in a population genetics analysis may not necessarily be caused by ongoing rare sex. This evidence could also be explained as an “echo” of past sex (Schmid et al., 2004), which will be mentioned in detail below.