Chapter 1
WORKING WITH CANDIDA PARAPSILOSIS:
A GENERAL INTRODUCTION
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C. parapsilosis belongs to the genus Candida, which comprises more than 150 related species that grow mainly as oval-shaped unicellular cell, called blastoconidia. Candida genus is recognized as the most common human fungal pathogen despite the fact that, among all the species belonging to this genus, less than 30 are associated with colonization and human diseases. The most commonly isolated species include C. albicans, C. glabrata, C. parapsilosis and C. tropicalis [1]. C. parapsilosis grows as yeast with a round or elongated shape but, under inducing condition, filamentous structures, called pseudohyphae, can also be observed. The presence of different morphological forms is a characteristic shared by all the Candida species and correlates with environmental adaptation and pathogenicity. Most of the species are detectable as yeast or pseudohyphae, both characterized by asymmetric budding. Only two species, C. albicans and C. dubliniensis, are also able to form true hyphae. In pseudohyphae the cytokynesis is not followed by cell separation and branched chain of cells, with constriction at the bud neck, are observed. In contrast, hyphae have no constriction at the site of separation, they are slender, parallel sided with individual and uninucleate compartments (Figure 1-1) [2].
Figure 1-1. Fungal morphologies. The most common cellular morphologies of human fungal pathogens are visualized in C. albicans by differential interference contrast (DIC) microscopy (top). Schematic representation of each morphology is illustrated in the bottom. Bar = 10 µm. Obtained from reference [2]. .
C. parapsilosis is one of the few Candida species able to grow at 37°C, an essential trait of an invasive human pathogen, even if the optimal growth temperature ranges between 28-30°C. The morphology of C. parapsilosis colony is regular, smooth and the color tends to be white-creamy. However, environmental conditions (temperature, pH, nutrient composition of the medium) may led to a switch in the colony phenotype which often correlates with a dimorphic transition in cell morphology (single cell yeast, pseudohypha) [3].
The first isolate of C. parapsilosis, the reference strain ATCC 22019, was collected from a stool specimen in Puerto Rico. The strain was first named in 1928 as Monilia parapsilosis and later reclassified in the Candida genus in 1932 [4]. More recently, DNA typing methods helped scientist to outline the taxonomic relationship within the genus Candida, while the old taxonomy, which mainly relied on physical structures related to sexual reproduction, has being reviewed.
Most of the medically relevant species, C. parapsilosis included, are found to be associated in a phylogenetic subgroup, characterized by the translation of the CTG codon as serine instead of leucine (Figure 1-2) [5].
Figure 1-2. Schematic representation illustrating the phylogeny of Candida within the CTG clade. The majority of Candida species responsible for human mycosis is grouped in the CTG clade. However, the clinically relevant pathogen C. glabrata, belongs to the “whole genome duplication” (WGD) clade, as well as C. krusei [6].
Over the past decades, according to DNA typing methods, strains belonging to C. parapsilosis were revealed genotypically heterogeneous and subdivided in 3 different groups: I, II and III [7]. However, in 2005, cumulative evidences from the analysis of several molecular markers, supported by new typing techniques, such as MLST (Multilocus Sequence Typing), led to the classification of these groups in 3 distinct
species: C. parapsilosis sensu strictu (formerly group I); C. orthopsilosis (II) and C. metapsilosis (III) [8]. Among the “psilosis” group isolates, C. parapsilosis is the most frequent species, followed by C. orthopsilosis and C. metapsilosis [9]. Conventional biochemical tests for the identification of fungi, commonly used in the clinical routine (such as API, Vitek or chromogenic agar plates), fail to discriminate the different “psilosis” species. Several DNA-based approaches have been successfully applied to identify members of the C. parapsilosis species complex, including restriction profiles of secondary alcohol dehydrogenase gene fragments [8], pyrosequencing [10], amplification fragment length polymorphism [11, 12], a microarray based system [13], ITS sequencing [8], even though these techniques are often time consuming and require skilled personnel. Recently, the high-throughput MALDI-TOF MS (Matrix-assisted laser desorption/ionization Mass Spectrometry) technique has emerged as a powerful and rapid tool for the discrimination of these cryptic species from C. parapsilosis [14].
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The genomic sequence of Candida parapsilosis reference strain CDC317 was published in 2009 [15]. The genome size reaches 13 Kb and is organized in 7 chromosome pairs. As annotated in one of the most updated database on Candida species, the Candida Genome Database (www.Candidagenome.org), the genome actually includes 5,810 uncharacterized open reading frames (ORFs) (99.54% of the genomic sequence), and only 27 verified ORFs (0.56%).
Evidences collected in early studies of C. parapsilosis genome, including electrophoretic karyotyping, flow cytometry, gene disruption experiments [16, 17, 18], confirmed that C. parapsilosis is a natural diploid organism. The analysis of the genomic content indicated a low level of heterozygosis between chromosomes (25-70 fold lower than the other Candida species) [19], with a single nucleotide polymorphism (SNP) frequency of 1 per 15,553 bp [15]. It is important to point out that the current knowledge about genomic variability in C. parapsilosis is restricted to the genomic sequence of a single strain. In this respect, it has been postulated that C. parapsilosis may have undergone a recent population bottleneck during speciation [20]. Moreover, the clonal expansion correlated with the absence of a documented sexual reproduction in this species. In fact, C. parapsilosis was originally thought to be the anamorph of Lodderomyces elongisporus [21], since a parasexual cycle was not observed in any C. parapsilosis isolates. The ability to mate in diploid Candida species is associated with the mating-type locus (MTL, with two allelic forms: MTLa and MTLα ). Mating takes place in homozygous strain and
it is regulated by 2 genes, located in the locus (a1 and a2 in MTLa; α1 and α2 in MTLα). In a heterozygous diploid a/α cell, the a1/α2 heterodimeric complex represses expression of mating genes. [22]. To date, MTLa idiomorphs were identified in several C. parapsilosis tested strains, while the MTLa1 gene was recognized as a pseudogene, containing four termination codons and an intron, which is not spliced out [23]. In conclusion, the mating process seems unlikely to occur in C. parapsilosis, which does not seem to take advantage from one of the main source of genetic variability.
Nevertheless, the continuous development of sequencing tools facilitated the analysis of C. parapsilosis genome by whole genome sequencing, which has become faster and cheaper. In a recent paper, Pryszcz and coworkers presented the entire genomic sequence analysis of 3 additional C. parapsilosis strains, questioning the “old” hypothesis of a clonal expansion and documenting the first evidence of genomic recombination in this species. The average SNPs frequency reported in this work for the 3 strains was calculated to be 1 SNP every 2,376 bp. The high genomic variability also included variation in gene copy number and rearrangements, such as deletion and gene fusion [19]. The authors hypothesized that recombination events between different strain could occur and explain the presence of high variable genomic regions. These “revolutionary” findings demonstrate the necessity to increase the number of sequenced genomes, from both clinical and environmental isolates, to expand upon our partial knowledge of the genomic traits of C. parapsilosis.
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C. parapsilosis is an ubiquitous microorganism, commonly isolated from a wide variety of natural environments, including soil, fresh and sea water, plants and insects [24]. Medically relevant Candida species are often described as opportunistic pathogens of the human body. In most of the cases, yeast coexist with the host on mucosal surfaces in a commensal state, establishing a silent warfare with the microbial flora and with the host defenses [25]. Any element affecting this balance, such as a weakness either in the host defense or in the competing microbial flora, can promote the fungus overgrowth and the disease onset. The term “defense” not only includes innate and adaptive immune response, but also intact anatomical barriers [1]. In this regard, it is not surprising to find out that the majority of the patients who develop disseminated candidiasis due to C. parapsilosis are not severely immunocompromised, but in most cases alterations of different factors concur to increase host susceptibility toward the pathogen [26].
Among the Candida genus, the isolation of C. parapsilosis from physical surfaces in the hospital environment can be considered a unique characteristic of this species. Statistically, the main risk factors associated with a disseminated candidiasis are iatrogenic and/or nosocomial factors and the majority of the infections are due to exogenous acquisition [27]. In clinical environment, C. parapsilosis is able to cause invasive infection in patients without prior evidence of colonization via horizontal transmission [28]. Common sources of infection in the clinical settings are the hands of healthcare workers and contaminated medical devices, such as catheters [29]. C. parapsilosis is mainly isolated in premature newborns, hospitalized in neonatal intensive care units (NICU), elderly patients receiving prolonged medical attendance, intensive care units (ICU), catheterized patients, AIDS or cancer affected individuals. Other risk factors reported in surveillance studies include transplant receipt, long antibiotics treatment, heavy burns [29, 30, 31, 32, 33]. Moreover, the eradication of the infection can be particularly difficult in the presence of intravenous hyperalimentation, since the presence of high lipid and glucose concentrations are associated with C. parapsilosis biofilm formation [34, 35]. The ability to produce biofilm by the infecting microorganism is not only associated with potential outbreaks, but also with a higher mortality rate of patients [36].
C. parapsilosis is considered less pathogenic than C. albicans but, due to the wide range of infections caused and the increasing isolation frequency, it has gained the role of emerging yeast pathogen [29]. In fact, this species is involved in superficial, mucosal and systemic infections. Both superficial and mucosal candidiasis are more common than invasive infections, although the latter receive far more attention due to the significative mortality rate.
Superficial infections are mainly onychomicosis even if cases of folliculitis and infections of middle and external ear are also documented [37, 38].
Mucosal infections due to C. parapsilosis include vaginitis [39, 40] and oral candidiasis, especially observed in HIV-infected individuals [41]. As illustrated in Table 1-1, C. parapsilosis is one of the most common Candida species responsible of candidemia (bloodstream infection, BSI), with isolation percentages often influenced by geographical areas [42]. C. parapsilosis is actually the third, second, cause of BSI or invasive candidiasis (IC), which include BSI and other deep seated infections, depending on the studies and it has become one of the main causative agent in several location in Europe, Asia and South America (Table 1-1, 1-2).
Table 1-1. Distribution of Candida species from bloodstream infection by region, from ARTEMIS global surveillance program, 2004-2007a,b
a: Data obtained from [43]. b: Table adapted from [42].
The worldwide IC isolation frequency of C. parapsilosis has slightly increased in the past years (Table 1-2), but in some local cases a higher percentage has also been reported. C. parapsilosis has been isolated with high frequency in patients affected by hematologic malignancies or involved in hematopoietic stem cell transplantation (HSCT), reaching 24% among all the Candida species analyzed (together with C. albicans) [44]. Another study documented a surprising increase in C. parapsilosis BSI cases (+400%) in correlation with caspofungin usage [45]. An increasing frequency has also been reported in Atlanta, and Baltimore, between 1992-1993 and 2008-2009 [46].
Table 1-2. Distribution of Candida species from patients with IC: ARTEMIS Global Surveillance Program, 2001 to 2007 a,b
% of total by year (no. tested)
Species (21,804) 2001 (24,680) 2002 (33,106) 2003 (33,406) 2004 (28,412) 2005 (29,167) 2006 (31,078) 2007 C. albicans 65.4 61.4 62.3 62.8 65.9 65.1 64.0 C. glabrata 11.1 10.7 12.1 11.7 11.2 11.7 12.0 C. tropicalis 7.5 7.4 7.6 7.5 7.6 8.0 8.3 C. parapsilosis 6.9 6.6 7.3 6.7 5.6 5.9 5.4 C. krusei 2.5 2.6 2.7 2.3 2.4 2.5 2.6 C. guillermondi 0.7 1.0 0.8 0.7 0.7 0.5 0.5 C. lusitanie 0.6 0.5 0.6 0.6 0.6 0.7 0.7 C. kefyr 0.3 0.4 0.5 0.5 0.6 0.5 0.6 Candida sp.NOSc 4.0 9.4 6.1 7.2 5.4 5.1 5.9 a: Data from [47].
b: Table adapted from [42].
% of total by region (n) Species North America (2,116) Latin America (1,348) Europe (2,151) Asia-Pacific (1,064) Total (7,191) C. albicans 51.8 46.0 58.5 49.1 52.7 C. glabrata 20.3 6.8 14.8 12.1 14.2 C. parapsilosis 14.4 18.5 9.8 13.8 13.9 C. tropicalis 8.5 18.5 9.8 13.8 11.8 C. krusei 1.9 4.5 4.7 2.5 3.3 C. lusitaniae 1.6 0.5 1.4 0.9 1.2 C. guillermondii 0.4 3.3 0.6 1.2 1.1 C. keyfr 0.5 0.7 1.1 1.1 0.8 C. famata 0.2 0.6 0.2 0.7 0.4 C. pelliculosa 0.1 0.3 0.1 0.4 0.2
Data obtained from the global surveillance programs clearly illustrate that although the 5 most common species causing IC are the same worldwide, the distribution among the different clinical wards may differ. C. parapsilosis is most common in NICU and in surgical units (Table 1-3).
Table 1-3. Species distribution of Candida BSI isolates by clinical ward.
Species
% Isolates by species and clinical service (n)a
GME D (1,339) HEM E (197) SCT
(58) HIV (41) NICU (26) (166) SOT (351) ST SURG (662) (2,019) Total
C. albicans 46.3 27.4 22.4 43.9 62.2 39.2 47.6 47.9 45.6 C. glabrata 26.6 25.9 32.8 29.3 0.0 38.6 26.8 24.0 26.0 C. parapsilosis 15.7 11.7 15.5 9.8 26.9 12.0 12.8 17.7 15.7 C. tropicalis 7.5 17.3 8.6 7.3 0.0 6.0 7.4 7.3 8.1 C. krusei 1.9 13.7 15.5 4.9 0.0 1.8 2.6 1.4 2.5 Otherb 2.0 4.0 5.2 4.8 3.9 2.4 2.8 1.7 2.1 a:Data from [42].
b: Other includes C. lusitanie, C. guillermondii, C. dubliniensis and other unknown Candida spp.
GMED, general medicine; HEME, hematologic malignancy; SCT, stem cell transplant; HIV, human immunodeficiency virus/AIDS; SOT, solid–organ transplant; ST, solid tumor; SURG, surgical (non transplant).
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The knowledge about pathogenesis and virulence in C. parapsilosis is still poor if compared to the information collected for C. albicans. However, in the last years, the increasing interest about the traits that enable C. parapsilosis to cause infection went hand to hand with the increment in the prevalence of this yeast in nosocomial infections. The research of putative virulence factors in C. parapsilosis is essentially based on gene ontology studies, starting from known virulence factors in C. albicans. These virulence factors, include: adherence to biotic surfaces, such as epithelial or endothelial cells, and abiotic materials, such as prostetic devices and catheters; production and secretion of extracellular hydrolytic enzymes, such as acid proteinases, phospolypases and lypases; hemolytic activity; biofilm formation; phenotypic switching [24].
1.4.1
Adherence
The ability to adhere is considered an essential trait that enable microorganisms to colonize and eventually infect the host. The stable presence of a microbe in the host, especially on skin or mucosal surfaces, is often related to the strong interactions between molecules that play the role of receptors and ligands. Such interactions can contribute to the unsuccessful clearance and promote the stable colonization of the surfaces. The interaction with biotic or abiotic surfaces takes place through aspecific mechanisms, such as electrostatic or hydrophobic forces [48], but the main protagonists are specific elements, localized on the cell wall surface. The concept of adhesion not only includes the interaction of the yeast cell with the host, but can also refers to flocculation (the ability of yeast cell to adhere to other yeast cells) and coaggregation (adhesion to other microbes). Adhesion is also considered a necessary condition for biofilm formation.
In fungi, proteins in the cell wall (CWPs) mediate the primary interactions with the host cell and with the environment. Even if the basic structure of the cell wall is rather conserved, each Candida species and even each isolate belonging to the same species is characterized by an unique cell wall proteome. This surely reflects the result of adaptation to different environments and the microevolution response to different selective pressure. The cell wall “bricks” are represented by mannoproteins, β-glucans and chitin. The major structural support is provided by branched β-1,3-glucan polymers (40%), covalently linked to β-1,6-glucans (20%) and chitin (2%). The CWPs are located at the top of this intricate basic structure and provide a mannosylated coat, able to mask the structural glucans to the host immune system. The coat is composed by mannoproteins (35-40%), proteic structures stabilized by O-linked glycans and highly N-glycosilated at the N terminal globular domain [49, 50] (Figure 1-3).
Figure 1-3. Structure of Candida cell wall. On the left, freeze-substitution electron micrograph showing the outer fibrillar mannoprotein layer and the inner skeletal layer, composed by polysaccharides, chitin and β-1,3-glucan. On the right, a schematic representation of the cell wall components. Figure taken from reference [51].
Most of CPWs are glycosilphospatidylinositol (GPI)- modified proteins, covalently linked via β-1,6-glucans to the β-1,3-glucan-chitin skeleton [52]. Different proteins belonging to this specific CWPs class have a documented role in adhesion and are therefore identified with the name of adhesins. The function of most of CWPs was unknown since the last decade and the main reason was related to the difficult procedures required for their extraction and purification. New techniques are now available to collect and analyze the cell wall proteome, mainly based on the efficient digestion of the covalent linkage that tightly binds the protein to the cell wall skeleton [53]. Moreover, the availability of genomic sequences has given the opportunity to extend the study on computer-based algorithm predictions, gene knockout approaches, ontology analysis. In C. albicans case, the use of prediction software led to the identification of 108 putative GPI-anchored proteins [53] but only few of them have been detected by cell wall proteomics [53]. A wide study performed by Butler and co-workers on the genomic sequence of six Candida
species gave interesting information about predicted GPI-anchored proteins in C.
parapsilosis (Table 1-4) [15]. After sequence comparison, these GPI-anchored
glycoproteins were classified in 23 families, most of them composed of only 2-3 members [15, 54]. The most numerous families identified in C. albicans and C. parapsilosis are the Als and the Hyr/Iff families (Table 1-3). Nowadays, ALS (Agglutinin-like sequence) gene family is one of the best studied adhesin family in C. albicans.
Table 1-4. Summary of predicted GPI protein families in C. albicans and C. parapsilosis Family C. albicans C. parapsilosis
Chitinase 2 (2) 4 (2) Dfg-like 2 2 Ecm33-like 2 (1) 1 (1) Exoglucanase 2 (1) 1 (2) Pga37/57 2 0 Pga24/59/62 3+1 3 Yapsins 2 (1) 11 Crh-like 2 (1) 2 (1) Sod-like 3 2 Pga15/41/42 3 0 8 conserved Cys 5 2 (1)
Family C. albicans C. parapsilosis
Hwp-like 3 2 Pga30-like 3 2 Plb-like 3 4 Rbt-like 4 (1) 4 (3) Gas-like 4 (1) 2 (3) Als-like 8 5 Hyr/Iff-like 10 (2) 7 (11) Pga52-like 1 (1) 1 (1) Kre1-like 2 2 Muc1-like / / Flo9-like / /
Predicted number of GPI and non-GPI (in brackets) proteins, +1 refers to pseudogene. Data obtained from reference [15] supplementary material.
1.4.1.1 ALS family
Past studies about adhesion, aggregation and biofilm formation in C. albicans often displayed common protagonists directly or indirectly involved in the regulation of analyzed phenotype. These elements were further identified as Als proteins. C. albicans genome shows 8 different ALS genes, named ALS1-ALS7 and ALS9, while in C. parapsilosis only 5 putative ALS genes were annotated (Table 1-4). These GPI-anchored adhesins are conserved structure, observed in other Candida species. C. glabrata, another important human pathogens, exhibits a wide set of adhesins and among them the big family of Epa proteins can be functionally and structurally compared to Als proteins.
Structure of GPI-anchored adhesins
The structure of yeast adhesins is overall conserved. A small signal peptide is located at the N-terminus, followed by a Ig-like region, organized in a globular structure. A region rich in Thr connects the Ig-like region to a domain characterized by tandem repeats. A long stalk, rich in Ser and Thr is located at the C-terminus and it is linked to β-1,6-glucans via a remnant of the GPI-anchor (Figure 1-4).
Figure 1-4. Schematic representation of the Als structure. Adapted from reference [56].
The signal sequence encodes for a 20-30 amino acids sequence, required for the correct modification and secretion of the protein and guides the transit of the protein into the endoplasmic reticulum (ER). In the ER the GPI anchor replaces the C-terminal hydrophobic domain (responsible of the attachment to the ER membrane during the transit), which is cleaved away [57, 58]. The GPI-anchor is further processed and the
mature adhesin is translocated from the plasma membrane and covalently linked into the cell wall [50].
The Ig-like domain shares similarity with bacterial adhesins and invasins of the immunoglobulin superfamily. The region consists of 3 tandem β-sheet domains, homologous to the Ig- -agglutinin in S. cerevisiae (which explains the ALS
name). The N-terminal is thought to be primarily involved in the recognition of ligands [58]. Different studies were focused on the analysis and structural characterization of this single important part of the Als protein, revealing the specific binding of different Als N-terminals to a wide range of substrate, including fibronectin, laminin, casein, BSA, type IV collagen, cadherins, and ferritin [60, 61, 62]. This region appears to be the most conserved among the ALS domains and shows the highest level of similarity, with 41– 84% identities, among all the ALS sequences in C. albicans [61].
The threonine-rich region is highly conserved too [62] and in C. albicans Als5p it has been associated with aggregation and with the formation of amyloid fibers [63, 64], an interesting trait that could be involved in the adherence and the interaction between yeast cells (e.g. flocculation).
Tandem repeat region (TRR) is composed by a series of tandemly repeated 36-amino acid units, heavily N and O-glycosilated. The number of repeats may vary among Als proteins, strains, and even a single strain may present two different allelic form of the same Alsp [65]. The precise amino acid sequence in the TRR may vary as well. It has also been observed that the number of tandem repeats, related to the stalk length, affects the adhesive properties of the protein, regulating the localization of the N terminus (more or less protruding towards the extracellular environment) [66]. TRR seems also directly involved in binding, especially in cell-cell aggregation (as already discussed for the Thr-rich region) [67].
The C-terminal region of Als proteins is the least conserved element and it is mainly constituted by serine and threonine [59]. The high percentage of O-glycosilation forces the extended conformation of this domain, which appears like a stalk, embedded into the cell wall. The length of the stalk, together with TRR contribution, is responsible for the projection of the ligand-binding region towards the environment. The C-terminus possess a sequence that specifies for the addition of GPI anchor [56].
The role of Als in adhesion
Als-mediated adhesion can be considered the result of the binding activity of the different protein domains: the specific binding of the Ig-like region; the weak, non-specific hydrophobic interaction within TRR; the amyloid-mediated interaction of the
Thr-region, which promotes the formation of Alsp cluster in the cell wall and the aggregation. The role of Alsp in C. albicans has been investigated since early 21th century. The main strategy used is based on the analysis of mutant strains but transcriptional profiling performed in different environmental conditions and in different infection models, binding test to different substrates and heterologous expression in S. cerevisiae are also included. The analysis of C. albicans knock out strains revealed the different ligand-specificity already hypothesized for the Als family. ALS1 has been shown to be involved in adhesion to HUVECs (Human Umbilical Vein Endothelial Cells) but its presence was dispensable for BECs (Buccal Epithelial Cells) adhesion [65]. Among all the Als proteins, the absence of Als3p had the greater effect. A strong reduction in the ability to adhere to and damage both endothelial and epithelial cells was observed [68], with a significant reduction in the cytokine production [69]. In addition, biofilm formation was also impaired by ALS3 deletion [70]. The use of specific antibodies recognizing the N-terminal of Als3, can block adherence to BECs and endothelial cells [71]. Together with Als1p, Als3p exhibits the highest level of adherence to the broadest array of substrates, and for this reason, they have been selected as potential targets for a vaccine development. Promising results have been achieved in murine disseminated candidiasis [72]. However, even if the role of C. albicans Als3p has been demonstrated in several in vitro models, als3Δ strain showed a virulence comparable to the wild type in a murine model of disseminated candidiasis [73].
In some cases, the deletion of ALS genes resulted in an increasing adhesion, such as in als5Δ/als5Δ, als6Δ/als6Δ and als7Δ/als7Δ [74]. The analysis by gene deletion of a single gene, belonging to a gene family, may be complicated by presumed redundancy of function and the depletion of one member of the family may led to a modification in the transcriptional patterns to compensate the absence of a specific gene. In this respect, the heterologous expression of an Alsp in a model that does not naturally adhere to substrates or human tissue, such as S. cerevisiae, may help to better understand the function of the single protein. Such system has been used to investigate the substrate specificity of Als1 and Als5 to different peptides [75]. However, this system presents the risk to alter the properties of the protein, since the cellular context is completely different from the original.
Studies on ALS expression were also published. The transcription of the entire gene family in vitro and during in vivo infection has been detected, but some genes demonstrated low level of transcripts. In vitro, ALS1, ALS2 and ALS3 showed the most dynamic range of expression and they were expressed in different growth conditions, with an increased expression during germ tube formation (ALS1) and hyphal growth
(ALS3) [76]. A weak expression was documented for ALS4, ALS6 and ALS7 [73]. To assess the specific role of each CaALS during infection, the ALS expression in human clinical specimens and in models of vaginal candidiasis was monitored by RT-PCR. Even in this case, ALS1, ALS2, ALS3 together with ALS9 were the most abundant transcripts. An increasing in the basal level of ALS4 expression during vaginal infection has been documented in later phases but the highest induction was observed in oral infection models, pointing to a potential role of Als4p in the colonization/infection of the oral cavity [77, 78]. Until now, only few studies reported information about the regulatory system that controls the transcription of the ALS family but Efg1 and Tup1, involved in the morphogenesis regulation in C. albicans, seem to be the best candidates to the role of central regulators. Other transcriptional regulators that control morphogenesis, such as Bcr1 (mainly associated with biofilm formation) contribute to the control network [79]. Only few information about C. parapsilosis ALS are documented in literature. Studies on biofilm characterization demonstrated that only 2 out of the 5 CpALS displayed minor increase in the transcription level during biofilm formation [80], and that CpBCR1 played no obvious role in the regulation of CpALS gene [81]. These findings suggest the importance to clarify the ALS role in C. parapsilosis and highlight the differences in orthologous genes regulation in related but different species.
1.4.2
Biofilm formation
Biofilms are defined as surface-associated microbial communities, surrounded by an extracellular matrix. Yeast biofilms are characterized by the presence of cells with different morphologies, including budding yeast, pseudohypae and hyphae, embedded in an extracellular matrix composed of carbohydrates, proteins, phosphorus, glucose, hexosamines and other molecularly undefined elements [82]. This matrix is thought to contribute to the structural organization of the biofilm: it acts as protective barrier, defending the yeast from phagocytic cells and toxic substances. In late stages, a colorless matrix of exopolysaccharide, composed by glucose, mannose, rhamnose and N-acetylglucosamine, can be observed [83]. Biofilm sessile cells are physiologically distinct from planktonic cells: they exhibit a significant increased resistance to antimicrobial agents and, in some cases, a metabolic quiescence is also observed (persister cells) [84]. Biofilm are formed on most biological and non biological substrates: infections involving biofilm formation are commonly found in the oral cavity (soft tissues, teeth, dental implants); in the middle ear; the gastrointestinal tract; the urogenital tract; indwelling catheters for hemodialysis and for chronic administration of chemotherapeutic agents;
prosthesis; ventilator tubing [85]. Biofilm production can be considered an important factor, which can influence the development of candidemia. A study on patients with C. parapsilosis systemic infections revealed that 70% of the patients infected with a biofilm-producing isolate deceased, while only 28% of the patients infected by biofilm-negative isolates shared the same outcome [86].
Biofilm formation begins with adherence to the substrate and proliferation of the yeast; cells start to differentiate in hyphae (or pseudohyphae, in C. parapsilosis case) and to secrete the extracellular matrix. Mature biofilm is composed by a basal layer of attached cells, surrounded by filamentous cells and extracellular matrix. In a final step, non adherent budding cells are released from the biofilm into the environment and dispersed [85].
The ability to form biofilm can be considered isolate-specific, rather than species-related. C. albicans and C. parapsilosis differs in the nature of the biofilm formed. C. parapsilosis forms smaller and less complex structures (Figure 1-5) and the amount of extracellular matrix is lower if compared with C. albicans production [87].
Figure 1-5. C. parapsilosis biofilm morphology. 1) Scanning electron microscopy (SEM) image of C.
parapsilosis biofilm after 48 h incubation on a catheter surface (1500x) [88]. 2) Schematic representation of
the different steps of in vitro biofilm formation: A) yeast cells adhere to a substrate, B) proliferation and the extracellular matrix production begins; C) extracellular matrix envelops the biofilm, which is now composed by yeast cells and pseudohyphal/hypal cells. D) In the final step, yeast cell are dispersed to further colonize the surrounding environment [85].
The genetic regulatory mechanism that guides biofilm production in C. parapsilosis is still poorly understood. The role of CpBCR1 is still unclear: even thought in some cases it acted as a major regulator [81], as already documented for C. albicans, other studies observed a marginal role for this gene, which was not required for initial adhesion and formation of mature biofilm in biofilm producing strains [89].
Since adherence of yeast cells to solid substrates and to other cells (aggregation) is vital for biofilm-related formation, Als proteins have been considered excellent candidates for biofilm adhesins. In C. albicans, several ALS gene are overexpressed during biofilm
formation [77], and the deletion of ALS3 showed that the protein is important for biofilm formation in vitro but is dispensable for the in vivo production. The deletion of both CaALS1 and CaALS3 resulted in a highly defective in vitro biofilm. In general, several studies indicated that there may be a large overlap in the functions of the Als proteins during biofilm production [90].
1.4.3
Phenotyipic switching
As already mentioned in paragraph 1.1, C. parapsilosis is able to grow in two different morphological forms: the ovoid-shaped budding yeast and elongated ellipsoid, with constrictions at the septa (pseudohypha). The main distinction between yeast and pseudohypae is that pseudohyphae spend more time in G2 phase of the cell cycle than yeast cells [91] and, during this time, cell continue to elongate. During filamentous growth, daughter buds appear larger and longer than the buds observed during yeast form proliferation. The daughter pseudohyphal cells tend to reach the mother cell size and often the following cell cycle start synchronously. As observed in hyphal growth, pseudohyphal formation is induced by many environmental stimuli including growth temperature of 37°C, the presence of serum [92] or specific amino acids [93]. These aspects can be related to the environment found by a pathogen within the host. The hyphal form has been clearly correlated to an increased virulence, with higher expression of virulence factors, an improved ability to adhere to and invade tissues [2]. Pseudohyphal growth shares some of this features, even if the advantages gained are less significative in comparison to the hyphal morphology. In this respect, the absence of a true hyphal form in C. parapsilosis is thought to be related to the lower virulence, compared to C. albicans. C. parapsilosis pseudohyphae acquire cell invading ability and certain isolates, characterized by a significative filamentous growth, generate more biofilm and are more invasive into agar than strains predominantly growing in the yeast form [94]. It has also been hypothesized that pseudohyphae, as well as C. albicans hyphae, could express a higher level of resistance against phagocytosis, compared to the yeast form. Moreover, the production of filamentous forms was observed with a higher frequency in clinical isolated strains and it often correlates to a higher percentage of phagocyte killing. [95; 96; 97; 98]. Morphological switching is also induced in presence of endothelial and epithelial cells [99; 100].
1.4.4
Secreted enzymes
Extracellular secreted enzymes are considered potential virulence factors in most microbial pathogens and the study of their role during infection is often associated with the discovery of new targets for the infection control. Well-known and characterized secreted enzymes, considered important virulence determinants in C. albicans, have been also identified in C. parapsilosis. Once again the presence of conserved gene families among the Candida pathogenic species can be related to a key aspect connected to the yeast survival within the host environment. In fact, the secretion of hydrolytic enzymes by the yeast cell not only allows the digestion of molecules for the nutrient acquisition, but it also may affect the host cell membranes or tissues, with a direct damage that promotes adhesion and tissue invasion. The damage can also involve cells of the host immune system and thus contribute to the pathogen resistance against microbial clearance from the host [98]. The most studied secreted enzymes in C. albicans and in C. parapsilosis belong to three enzymatic classes: aspartic proteinases; phospholipases; lipases. Recently, the characterization of other enzymatic activities has also been investigated, such as esterase and hemolysin activities [101].
Secreted aspartic proteinases (Saps)
Pathogenic Candida species usually possess a SAP gene family, even if the number of genes encoded by each family may differ. In C. albicans, 10 Sap proteins have been identified (Sap1-Sap10), two of which (Sap9 and Sap10) are predicted to be GPI anchored. In C. parapsilosis, the number of characterized SAP genes is much lower: the family is actually composed of 3 members, named SAPP1-3, and only 2 of the encoded proteins (Sapp 1 and Sapp2) have been biochemically analyzed [102; 103]. The phylogenetic analysis of C. parapsilosis genome revealed further 11 sequences potentially encoding secreted aspartic proteinases [15], but no information about the presence of any transcript or protein has been provided yet. The production of Sapp1p is induced in presence of an exogenous protein as a sole nitrogen source, as in the case of the Sap2 enzyme from C. albicans; Sapp1p also displays a higher catalytic activity than Sapp2p [104]. Saps are also able to recognize a wide broad of substrates, including host defense and structural proteins such as IgG heavy chains, C3 protein, collagen and fibronectin [105] In Candida spp, SAP genes encode for pre-pro-enzymes processed during the transit into the endoplasmic reticulum and the Golgi apparatus. The mature proteins are transported via the secretory pathway to the cell surface. In C. parapsilosis, it has been shown that Sapp1p is temporarily retained in the cell wall prior to secretion into the
extracellular space. During this period, the proteinase is already active and the catalytic site is exposed to the environment, allowing the cleavage of substrates. For C. parapsilosis, no typical GPI-specific sequence has been identified in the available SAPP genes and no C. albicans Sap9-10 equivalents were detected [15]. Saps production varies among C. parapsilosis isolates and an association between the proteinase production and the site of isolation has also been unveiled: skin and mucosal isolates exhibit higher in vitro activity than bloodstream isolates [29], confirming the hypothesis of a key role of proteinases in invasion and colonization of host tissues. However, even if proteinases appear to be important facilitators of the tissue invasion, their role is not essential since the use of specific inhibitors do not avoid yeast penetration [106]. Their activity is pH and temperature dependent; each Sap has its own optimal condition of activity, with different pH and temperature values enabling the yeast to exhibit proteolitic activity in different host districts [107]. Moreover, the SAP gene expression is also modulated according to the infected tissue [108].
Phospholipases
Phospholipases (PL) are a heterogeneous group of enzymes that share the ability to hydrolyze one or more ester linkage in glycerophospholipids. Each enzyme has the ability to cleave a specific ester bond of the targeted phospholipid and, according to this feature, phospholipases are classified in 4 groups: A, B, C and D [109]. Among Candida spp, once again C. albicans exhibit the highest level of enzymes production [110]. The function of phospholypases during infection is still not well understood, although it has been hypothesized a role in the disruption of host membranes [110]. Several experimental systems were used to clarify the role of phospholitic activity in the infection process and confirmed the involvement of PL in C. albicans virulence: murine infection models, adhesion to epithelial cells, host cell penetration, invasion of a reconstituted human epithelium [29]. Cloning, disruption, and virulence evaluation of genes encoding extracellular phospholipases have been completed only for C. albicans. CaPLB1, CaPLB2 and CaPLD have been successfully deleted to assess the role of each PL in C. albicans virulence. The role of PLB was analyzed through in vivo mice infection models: in a hematogenous-dissemination mice model, an increasing survival percentage was observed in mice infected with the mutant strain, also confirmed by a significative lower tissue fungal burden. An oral-intragastric infant-mouse model indicated that the absence of PL activity limited the candidal transmigration across the gastrointestinal tract and the subsequent dissemination to target organs [110]. Nevertheless, the knowledge regarding C. parapsilosis phospholipases is still poor. Most of the data
collected for C. parapsilosis PL refer to the presence/absence of phospholipase activity in collected isolates, suggesting that this enzymatic activity is a strain-dependent trait [110; 111; 112]. An exhaustive protein characterization of C. parapsilosis PL is still awaiting to be completed.
Lipases
Lipases catalyze both the hydrolysis and the synthesis of triacylglycerols. Lipases are considered virulence factors in a broad range of bacteria and fungi, including Candida spp. [29]. Their activity ranges from lipid digestion for nutrient acquisition, to adhesion to host cells and tissues, or interactions with immune cells [29]. Only two LIP gene (CpLIP1 and CpLIP2), as against the 10 identified in C. albicans, have been identified and analyzed in C. parapsilosis. Investigation, performed by gene knock-out strategy, provided evidence for a role of lipases in phatogenesis: mutants exhibited an impaired biofilm production; they were more efficiently phagocyted and killed by macrophage-like cells; they were less virulent in infection of reconstituted human oral epithelium and during murine intraperitoneal challenges [99; 113]. Moreover, the inability of Lip null mutants to grow on lipid-rich media appears particularly interesting since C. parapsilosis is frequently associated with patients receiving lipid-rich parenteral nutrition (expecially low birthweight newborns). In this context, a lipase inhibiting strategy could be developed to improve the chance to avoid yeast colonization in hospitalized patients receiving lipid emulsions [29].
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Most of the knowledge acquired on the role and function of genes relied on the genetic manipulation of microorganisms. The possibility to transfer exogenous, in vitro modified DNA into microbial cells significantly contributed to the understanding of various biological phenomena at the molecular level. Direct transformation of yeast species has been firstly reported in Saccharomyces cerevisiae, in late 1960s [114]. Since then, different techniques were developed to improve the chance of a successful genetic transformation in yeast [115]. Certainly, one of the most captivating approach to study the gene function is to inactivate the gene by targeted mutagenesis. The optimization of gene disruption tools were achieved in C. albicans, considered a model system for human pathogenic fungi in clinical research. The construction of mutants in C. albicans, as wellas in C. parapsilosis, is complicated by its diploid nature, which implies two round of transformation.
At first, genetic engineering in C. albicans was largely based on the use of auxotrophic strains and episomal plasmids. Allelic replacement was then introduced for the study of knock out mutants and the URA3 blaster approach soon became the gold standard for the generation of null mutants. This system relies on the repeated use of a URA3 marker for the selection of uridine-prototrophic transformants from ura3-negative host strains [116; 117]. The transformed cassette contains C. albicans URA3 gene, flanked by direct repeats of Salmonella typhimurium hisG, and two homology regions, specific for the target gene. The cassette is able to disrupt the chromosomal copy of the gene by homologous recombination and a subsequent intrachromosomal recombination between the hisG repeats allows the recycle of the URA3 marker from the allele. Ura-revertants can be selected on the basis of their resistance to 5-fluoroorotic acid (5-FOA) [116; 117]. Nevertheless, problems associated with the use of URA3 gene in genetic selections have been well documented, and altered expression of Ura3 have a marked effect on growth and filamentation [118]. Moreover, the expression level of an ectopically inserted URA3 gene depends on the integration locus [119] and the uridine auxotrophy causes a reduction in C. albicans virulence [120], leading to a difficult interpretation of the mutant phenotypes. First approaches in C. parapsilosis mutagenesis followed the same path with the identification of selectable markers (CpGAL1 and CpURA3) complementing the corresponding auxotrophic mutation; the subsequent construction of plasmid vectors carrying C. parapsilosis autonomously replicating sequences (AARS) and the selective marker, complementing the gal1/ura3 mutation in the host strain [121]. Attempts to improve the efficiency of the URA-based deletion system led to the inclusion of a site specific recombinase, such as Flp and Cre, into the deletion cassette [122; 123]. However, the necessity to find a dominant selection marker to simplify the selection of transformants (avoiding all the potential problems related to the use of auxotrophic markers) was overcome with the construction of a gene deletion cassette containing the MPAR marker that confers resistance to mycophenolic acid (MPA) [124]. This new
disruption tool offered different advantages and was considered an improvement of the URA3 system. The MPAR cassette was provided with a C. albicans adapted FLP gene
(already tested in the URA-flipper method), placed under the control of the inducible SAP2 promoter. The cassette was flanked by repeats of the minimal FLP recombination target sequence (FRT) and by the homology regions amplified from the target gene. The selection of transformants was assessed in presence of MPA, followed by activation of FLP transcription in a SAP2-inducing medium. The MPAR flipping cassette could then
be used again for a second round of transformation and the generation of a null mutant. This system has also been successfully used for the direct transformation of C. parapsilosis [18]. Unfortunately, this method required up to 7 days for the selection of resistant mutants [125]. In addition, the MPAR marker was obtained from the mutation
of the target enzyme of MPA, encoded by the gene IMH3. It has been observed that the mutated IMH3 in the cassette could mediate itself a homologous recombination with the wild type IMH3 in the genome of C. albicans. In this case, the acquired resistance to MPA did not indicate the interruption of the target gene and the advantage in the selection of the transformants gained by the selective pressure was lost [124]. Thanks to Reuss and coworkers, the promising disruption system, based on the FLP site specific recombination, was refined by replacing the MPAR marker with a new dominant
selection marker, CaSAT1, conferring resistance to nourseothricin. [125]. As already described for the other system, the homologous recombination in the target locus is ensured by the presence of homologous flanking sequences on both sides of the cassette and two rounds of integration/excision generate homozygous mutants that differ from the wild-type parent strain only by the absence of the target gene. The construction of a reconstituted strain by reintegration of an intact gene copy for complementation of mutant phenotypes could be achieved in the same way. FLP-recombinase is put under the control of an inducible promoter (such as MAL2p or SAP2p) (Figure 1-4) and FLP-mediated excision of the SAT1 flipper cassette can be achieved by simply growing the transformants for a few hours in a medium containing the inducing agent without selective pressure.
Figure 1-4. Structure of the SAT1 flipper cassette. Schematic representation illustrating the main features of the SAT1 flipper cassette. Elements are not drawn to scale. The exact coordinates of each component are indicated in the panel. The figure has been modified from Reuss et al. [125].
The SAT1 flipper method was applied with success for the generation of knock out strains also in C. parapsilosis. In most cases, no modification of the C. albicans-derived sequences (CaMAL2p, CaACT1p, CaACT1t, CaSAT1, CaFLP and CaURA3t) was required and the genes CpLIP1-2 andCpFAS2 were successfully deleted by simply adding the required flanking regions of homology [113; 126]; on the other hand, the replacement of CaMAL2 and CaACT1 promoters with the equivalent sequences from C. parapsilosis was required to optimize the deletion of CpBCR1 [81]. The development of new and efficient tools for the genetic manipulation, along with the improving protocols for high-efficency transformation, will provide a great enhancement in the quality of molecular studies performed on Candida basic biology, as well as on its virulence mechanisms.
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The development of new and more efficient diagnostic tools and therapeutic approaches for Candida infections is strictly correlated with the current knowledge of pathogenic mechanisms. To this extent, experimental infection models have the advantage to allow disease development to be monitored from the initial phases; in addition the complex host-fungus interactions can be investigated in their multifactorial expressions [127]. The majority of Candida virulence studies have been assessed in rodent infection models. Different type of mucosal infections, such as the oral and vaginal ones, have been developed in rats and mice. Denture-associated fungal biofilm was also studied in a rodent acrylic denture model [128]. However, the establishment of a mucosal infection generally requires treatment of the host with immunosuppressive agents, oestrogen, or antibiotics [129, 130]. An alternative option consists in the use of germ free-animals or nude mice [131]. Mouse/rat models are often used to compare the virulence of different Candida species, C. parapsilosis included [132, 133]; to elucidate the effects of in vivo drug treatments of infected host [134; 135]; to assess differences between mutant and wild type strains [136; 137].
Invasive infection models can be considered the most popular methods to investigate the in vivo virulence. The two major models of Candida invasive infection which have been developed in mouse consist in a gastrointestinal (GI) colonization with subsequent dissemination and a intravenous (IV) challenge model [127]. In the GI model, the infection is localized in stomach, caecum and small intestine, reflecting some of the traits
of a human invasive infection. Candida colonization is monitored by the count of yeast colony forming unit (CFU) retrieved from the mice feces and, after dissemination, the organ burden can be evaluated by culturing liver, kidney and spleen homogenates. This model was used to demonstrate the lower pathogenic potential of C. parapsilosis compared to C. albicans and C. tropicalis [138] and, as well as in mucosal infection models, mutant strains were also tested for their ability to cause disease [127]. IV challenge model reproduces an invasive human infection occurring via catheter colonization/infection. Yeast cells were injected into the lateral tail vein, and the dissemination directly starts from the bloodstream. Disease progresses only in the kidneys and brain of the mouse. The disease progression eventually leads to mouse death due to sepsis. As in the GI model, infection is monitored through the annotation of survival rate or by quantifying the fungal burden in selected organs [127]. In C. parapsilosis IV infection, fungi are usually cleared from the host [139], and only a high inoculum potentially allows the disease development [140].
Although their diffusion in the research, mouse infection models are often criticized for their limits. Most of the Candida species associated with human diseases, C. albicans and C. parapsilosis included, are not natural mouse commensal or pathogens [141] and an alteration of the animal immune system or a drug treatment altering the natural bacterial flora are required to start the infection. Moreover, even if the immune system of mice and men are similar, fundamental differences could affect the results obtained [127]. Other important aspects to be considered are the costs, the necessity of specialized personnel and the availability of appropriate structures to manage animals. To this end, researchers have begun to test alternative infection models, mainly in invertebrate hosts, including Caenorhabditis elegans, Drosophila melanogaster and the wax moth Galleria mellonella. Among them, Galleria mellonella provides important advantages such as the opportunity to maintain the larvae at 37°C, equivalent to the temperature of mammalian hosts, and the easy to interpret mortality monitoring. In this model fungi are directly injected into larvae via proleg and survival can be monitored over a short time period. The model is cheap and large numbers of larvae can be infected and monitored for each experiment, increasing the statistical power of the assay. The results obtained during C. albicans infection roughly reproduce those found in the murine model [142]. Even if G. mellonella larvae do not possess an adaptive immune system, the innate response, crucial during the infections due to Candida [143], is conserved and G. mellonella hemocytes act as phagocytic cells [97]. This model has been successfully optimized to determine the virulence of genetically modified C. albicans strains [144;
145] and to evaluate the effect of antifungal drugs against Candida species [146; 147]. Species which are not able to cause mortality, even in immunocompromised mice, such as C. parapsilosis, revealed a substantial virulence potential in G. mellonella infection model with a significant mortality of infected larvae [97; 98]. In this model, the infection monitoring does not only include the daily annotation of dead larvae but hemocytes density (correlated to survival post Candida infection [148] or phagocytosis assay could also be investigated [97]. This alternative infection model to assess Candida virulence and study the pathogenicity shows a promising potential. However, standardized protocols for propagation and maintenance of larvae, as well as experimental guidelines still need to be established to allow the comparison of the results obtained in different laboratories. Moreover, investigations on the molecular mechanism at the basis of the immune system of G. mellonella are also required to better optimize the infection model and interpret the observed results.
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The present project was aimed at extending the current knowledge on the opportunistic yeast Candida parapsilosis. Pathogenetic mechanisms contributing to the development of symptomatic mycoses by this species are only partially characterized. It is known, however, that the adhesion ability is an essential factor involved in the first stages of the infection. Therefore, the identification of genes encoding adhesion molecules is a key factor for a better understanding of the infection process. C. albicans possesses different cell wall glycoproteins, anchored to the wall structure via GPI residues. Among them, Als protein family is one of the most characterized and its involvement in adhesion to biotic and abiotic surfaces has been well documented. In silico analysis of the genomic sequence of C. parapsilosis indicated the existence of 5 potential homologues of CaALS genes. This study was firstly focused on the identification of ALS genes in the reference strain ATCC 22019 and in a collection of C. parapsilosis clinic isolates, followed by a qualitative analysis of ALS genes transcription in different strains of C. parapsilosis by RT-PCR. In silico homology studies was performed to identify the ortholog of CaAls3p, which is considered one of the most important Alsp, among the 5 CpAlsp. The function of the selected gene, named CpALS3, was investigated by targeted gene deletion, performed with the SAT1-flipper cassette system. The effect of the deletion of one of both the CpALS3 copies was observed in two independent lineages of mutant strains, each including a ALS3/als3Δ and a als3Δ/als3Δ strains. The characterization of the mutants included on some phenotypic traits that could be affected by the depletion of the adhesin
such as growth in the presence of cell wall perturbing agents, adhesion to human buccal epithelial cells, cytotoxicity, virulence and pathogenicity.
The present PhD project was carried out at the Department of Biology of the University of Pisa, Via San Zeno 35-39, under the supervision of Dr. Arianna Tavanti.
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![Figure 1-4. Schematic representation of the Als structure. Adapted from reference [56]](https://thumb-eu.123doks.com/thumbv2/123dokorg/7957568.120444/10.892.363.575.638.938/figure-schematic-representation-als-structure-adapted-reference.webp)
