1 National Institute of Public Health-National Institute of Hygiene, Warsaw, Poland
2 Faculty of Chemistry, Warsaw University of Technology, Warsaw, Poland
- Corresponding author: M. Staniszewska, Independent Laboratory of Streptomyces and Fungi Imperfecti, National Institute of Public Health-National Institute of Hygiene, Warsaw, Poland; e-mail: firstname.lastname@example.org
Submitted 16 August 2014, revised 25 January 2015, accepted 13 April 2015
Secreted aspartic proteases (Saps) are considered as key virulence factors of Candida albicans. Hopefully our outlook will widen the knowledge of SAP7’s role in C. albicans pathogenesis. The goal of our study was to investigate SAP7 expression during C. albicans adhesion to intestinal human cells. Another objective was to study the role of SAP8-10 and transcriptional regulators: EFG1 and CPH1, using the mutants: Δsap, Δefg1, Δcph1 during growth on Caco-2 monolayer. SAP7 expression was analyzed using real time RT-PCR; relative quantification was normalized against ACT1 in cells after growth on Caco-2. Adherence assay of C. albicans to Caco-2 was performed in a 24-well-plate. The results proved that SAP7 can play a role during the initial adaptation of C. albicans to intestinal tract and decreases over time. Up-regulation of SAP7 occured in the absence of SAP8 and SAP10 (genetic alternations dependence). SAP7 can be regulated by the morphogensis’ regulators during C. albicans growth on epithelium. Adhesion of the mutants was indistinguishable from SC5314. The lack of neither SAP8-10 nor EFG1/CPH1 influences the adhesive behaviour of C. albicans. Deletion of SAP8-10 resulted in no filamentation defects. The results help better understand the role of SAP7 during adhesion and morphogenesis in C. albicans.
Key words: Candida albicans, adhesion, SAP7 gene expression, secreted aspartyl proteinase Sap7-10, true hyphae
The incidence and severity of candidiasis in immunocompromised or otherwise debilitated hosts, coupled with diagnostic difficulties and the high cost of treatment, have persuaded several investigators to focus on Candida albicans which is by far the most prevalent etiological agent of candidiasis. The most characteristic features of C. albicans are its extraordinary range of virulence factors, particularly adherence and spread on and/or through epithelial and endothelial tissues by inducing the filamentous growth and hyphal-associated aspartic proteases (Saps). Adherence to host tissues and morphological versatility are thought to be important in C. albicans virulence (Bertini et al., 2013; Braga-Silva and Santos, 2011; Naglik et al., 2011). C. albicans contains the hypha-associated genes: EFG1 and CPH1 that mediate adhesion of C. albicans to mucosal surfaces (Moazeni et al., 2012; Lo et al., 1997). Efg1 and Cph1 play a major role in promoting filamentous growth and regulate the expression of several genes with a crucial function in the invasion of host cells or in biofilm formation (Moazeni et al., 2012; Staniszewska et al., 2013). As adhesion and morphogenesis are crucial for the latter, it is fundamental to study the role of EFG1 and CPH1 associated with morphogenesis and adhesion during C. albicans epithelial cells colonization. The secreted aspartic protease (Sap) family encompasses at least ten members (Saps1-10) containing a signal peptide, which are secreted, except for Sap9 and Sap10 that remain bound to the cell wall. Individual SAP genes are expressed at various stages of the infection process (Correia et al., 2010; Dalle et al., 2010; Jackson et al., 2007; Martin et al., 2011; Pietrella et al., 2013). Saps are characterized by a broad-spectrum proteolytic activity and virulence properties. Sap1-3 are active at acidic pH, whereas Sap4-6 are more active at neutral to slightly alkaline pH and are associated with hyphal growth (Aoki et al., 2011; Cadicamo et al., 2013; Naglik et al., 2011). Saps directly attack and degrade host proteins that are involvedin both innate as well as adaptive immunity (for example, complement, E‑cadherin, histatins, antibodies), and development of inflammations (Aoki et al., 2011; Cassone and Cauda, 2012; Mayer et al., 2013; Pietrella et al., 2013). Although the combined role of Sap1-6 in virulence has now come into question, and the Sap9 and Sap10 isoenzymes’ role in the cell surface integrity, cell separation, and adhesion, has been described (Albrecht et al., 2006; Schild et al., 2011), almost nothing is known of Sap7 and Sap8 competence (Taylor et al., 2005). Therefore, we presented our outlook in the hope that it will lead to widening knowledge of the role of SAP7-10 in C. albicans pathogenesis.
The goal of the present study was to investigate theaspartic protease SAP7 gene expression during C. albicans adhesion to intestinal human cells. A further objective was to study the role of SAP8-10 and ofthe transcriptional regulators: EFG1 and CPH1, using the mutants: Δsap, Δefg1, Δcph1 during growth onthe Caco-2 monolayer. We tested the ability of the Δsap8-10 mutants to form hyphae and to adhere to intestinal cells comparing them with the mutants: Δcph1 and efg1 (attenuated in morphogenesis).
Materials and Methods
Strains and Media. C. albicans strains used in the current study are listed in Table I (Lo et al., 1997; Fonzi and Irwin, 1993; Gillum et al., 1984; Liu et al., 1994;Puri et al., 2012; Schild et al., 2011; Staniszewska, 2009).C. albicans (strain no 82) was isolated from blood samples from a patient treated for Ependymoma anaplasticum (Staniszewska, 2009; Staniszewska et al., 2014a; 2014b). The clinical isolate had been previously identified according to colony colour on CHROMagar Candida medium and evaluated following API 20C AUX carbohydrate assimilation patterns (Staniszewska, 2009; Staniszewska et al., 2012; Staniszewska et al., 2014a;2014b). Additionally, the identification procedure was confirmed with genetic methods using rDNA sequencing as previously described (Staniszewska, 2009; Staniszewska et al., 2012; Staniszewska et al., 2014b). All the strains used in the present study were stored on ceramic beads in Microbank tube (Prolab Diagnostics, Richmond Hill, ON, Canada) at –70°C. Prior to the respective examinations, routine culturing of strainsfor growth was conducted at 30°C for 18 h in YEPD (Ness et al., 2010).
Cultivation and Infection of Caco-2 Cell Line (ATCC HTB27, LGC, Poland). Following the supplier’s guidelines, monolayers of the colon adenocarcinoma derived cell line were cultured in the Eagle’s Minimum Essential Medium (EMEM) containing 10% (v/v) FCS, 1mM pyruvic acid, without antibiotics or antifungal agents and maintained in a humidified incubator at 37°C in 5% (v/v) CO2. For the experiment 1.2 × 105 of Caco-2 cells (/ml EMEM) were seeded into 24-well-plates (Corning, USA) and cultured up to 18 h. Next, after 18 h post seeding the Caco-2 monolayers were inoculated with 105 log phase yeast cells (/ml EMEM) of C. albicans wild type and mutants. After 18 h of incubation the Caco-2 was lysed by adding sterile water resulting in recovery of C. albicans cells.
Candida albicans strains used in this study
|Strain||Parental strain||Relevant characteristics or genotype||Reference|
|SC5314||none||Prototrophic wild-type strain||(Gillum et al., 1994)|
|CAI4||SC5314||ura3Δ::imm434/ura3Δ::imm434||(Fonzi and Irwin, 1993)|
|CAF2-1||SC5314||ura3Δ::imm434/URA3||(Fonzi and Irwin, 1993)|
|no. 82||none||Prototrophic wild-type strain||(Staniszewska, 2009)|
|sap8Δ, sap9Δ, sap10Δ and sap9/10Δ mutants**|
|Δsap8||Δsap8::hisG/ Δsap8::hisG-URA3-hisG||(Puri et al., 2012)|
|Δsap9||CAI4||CAI4, sap9Δ::hisG/sap9Δ::hisG + pCIp10 (integration)||(Schild et al., 2011)|
|Δsap10||CAI4||CAI4, sap10Δ::hisG/sap10Δ::hisG + pCIp10 (integration)||(Schild et al., 2011)|
|Δsap9/10||CAI4||CAI4, sap10Δ::hisG/sap10Δ::hisG sap9Δ::his/sap9Δ::hisG + pCIp10 (integration)||(Schild et al., 2011)|
|efg1Δ and cph1Δ mutants**|
|Δcph1||CAI4||ura3::1imm434/ura3::1imm434 cph1::hisG/cph1::hisG-URA3-hisG||(Liu et al., 1994)|
|Δcph1 (CPH1)||CAI4||ura3::1imm434/ura3::1imm434 cph1::hisG/cph1::hisG(CPH1)||(Lo et al., 1997)|
|Δefg1||CAI4||ura3::1imm434/ura3::1imm434 efg1::hisG/efg1::hisG-URA3-hisG||(Lo et al., 1997)|
|Δefg1 (EFG1)||CAI4||ura3::1 imm434/ura3::1 imm434 efg1::hisG/efg1::hisG (EFG1)||(Lo et al., 1997)|
|Δcph1Δefg1 (EFG1)||CAI4||ura3::1 imm434/ura3::1 imm434 cph1::hisG/cph1::hisG efg1::hisG/efg1::hisG (EFG1)||(Lo et al., 1997)|
* apart from indicated features all strains are identical to their parental strain; ** full genotype
RNA Isolation, cDNA preparation and quantification. Total RNA from C. albicans cells was extracted as described by Amberg et al. (2005). Prior to further examinations C. albicans total RNA was stored at –20°C. RNA was reverse transcribed into first-strand cDNA using the Enhanced Avian HS RT-PCR kit (Sigma-Aldrich, USA) according to the manufacturer’s instructions. Briefly, total reaction volume (10 μl) contained 1 μl of total RNA, and 1 μl of oligo (dT)23 (3.5 μM), and 1 μl of dNTP mix (500 μM each dNTP), and 7 μl of water (nuclease-free) was prepared. Incubation step was carried out at 50°C for 10 min. Then subsequently, the remaining components: 1 μl of Enhanced avian AMV-RT (1 U/μl), 1 μl of 10 × buffer for AMV-RT, 8 μl of water (nuclease-free) were added to obtain 20 μl of final volume. The RT reaction was carried out at 50°C for 50 min. cDNA was quantified using the QuantiTect Taq-Man probe RT-PCR kit (Qiagen, Germany) according to the manufacturer’s instructions. Appropriate Taq-Man primer and probe sets for SAP7 (SAP7-1 5’-ATGGACACAGTGTGAAATATGAAGTG-3’; SAP7-2 5’-TCAGTGGAGGATGGACCATTAGA-3’) and ACT1 (ACT-1 5’-GACAATTTCTCTTT CAGCACTAGTAGTGA-3’; ACT-2 5’-GCTGGTAGAGACTTGACCAACCA-3’) were designed as described previously by Naglik et al. (2008). For the real time RT-PCR analysis each reaction mixture contained 1 × RT-PCR buffer, 4 mM MgCl2, 250 nM forward and reverse primer, 200 nM Taq-Man probe, HotStarTaq DNA polymerase, and template RNA (Naglik et al., 2008). To validate our normalization we determined differences in the SAP7 expression levels between C. albicans cells grown for 3 h and 18 h respectively on the Caco-2 cell line. Moreover, for reliable normalization of the SAP7 gene expression data in C. albicans cells grown for 3 h and 18 h respectively we used the housekeeping gene ACT1 as a reference gene. The real time RT-PCR reactions were performed as described previously by Naglik et al. (2008): at 95°C for 15 min, followed by 45 cycles of 15 s at 94°C and 1 min at 60°C with the LightCycler® 96 (Roche Diagnostics GmbH, Germany). The CT values were provided from real time RT-PCR instrumentation and were imported into a spreadsheet Microsoft Excel 2010. The relative quantification was calculated using Eq. (Livak and Schmittgen, 2001), where
ΔCT = Avg. SAP7 CT – Avg. ACT1 CT;
ΔΔCT = ΔCT – ΔCT parental strain = 2–ΔΔCT.
Assay of adherence to human line Caco-2 epithelial cells. Adherence of C. albicans to the Caco-2 cell line (ATCC HTB-37TM) was performed as described previously Hashash et al. (2011). Briefly, the Caco-2 cell line was cultivated in the EMEM containing 10% (v/v) FCS at 37°C at 5% (v/v) CO2. After trypsinisation, with the use of 0.25% trypsin (Biomed-Lublin, Lublin, Poland) 1.2 × 105 Caco-2 cells (/ml EMEM) were incubated for at least 16 h on a 24-well-plate (Costar, Corning, NY, USA) to generate a confluent layer. Subsequently, the blastoconidia were grown overnight in the YEPD medium at 30°C. Then, 104 blastoconidial cells(/ml saline) were added to each well of the epithelial cells to be afterwards incubated for 90 min (adhesion phase). Next, the non-adherent cells were removed by standard rinsing, and the wells were overlaid with Sabouraud dextrose agar and allowed to solidify. After 18 h growth at 37°C, the number of adherent cells was determined by colony counting and compared with the controls on the Sabouraud dextrose agar plates. Adherence was expressed as a percentage of the total number of cells added (control cells).
Microscopy. The morphological transition from yeast-to-hyphal cells was performed by using LEXT 3D Measuring LASER Microscope OLS4000 (Olympus, USA) and Scanning Electron Tabletop Microscope TM 100 (Hitachi, Japan). Briefly, Caco-2 cells grown on 12 mm glass coverslips were inoculated for 21 days post seeding with 104 log phase yeast cells (/ml saline) of strains tested. After 90-min incubation at 37°C,the cells were washed three times with PBS to remove non-adherent yeast and then fixed in 2% glutaraldehyde for 10 min.
Statistical analysis. Each experiment was performed in triplicate on three separate occasions. The percentage of cell adhesion and the SAP7 expression were formulated as a mean ± standard deviation. Statistical differences were evaluated through comparison with the Wilcoxon test, P values ≤ 0.05 were considered significant. Based on recently published data (Naglik et al., 2008; Staniszewska et al., 2014a), the latter method was used to calculate statistical differences between relative gene expression and cell adhesion.
The SAP7 gene expression during adhesion to the Caco-2 Monolayer. The level of SAP7 transcript normalized to the transcription of ACT1 is presented in Table II. The expression of the SAP7 gene in the cells of the parental strain CAF2-1 and Δsap8 decreased 1.7- and 1.3-fold respectively after 18-h growth on Caco-2. Conversely, in the cells of Δsap8, SAP7 mRNA increased 3.6- and 4.8-fold after 3-h and 18-h growth on the intestinal cells respectively compared to the parental strain CAF2-1. Moreover, the expression of SAP7 decreased 2.3- and 1.1-fold in the cells of Δsap9, and Δsap9-10 respectively after 18-h growth on Caco-2. On the other hand, the SAP7 mRNA slightly increased 1.1-fold in the null Δsap10 mutant. In the cells of the parental strain CAI4, SAP7 decreased 2.5-fold after 18-h growth on Caco-2. After 3-h growth on Caco-2, SAP7 was up-regulated in Δsap9, Δsap10, and Δsap9-10 (1.3-, 1.5- and 1.3-fold as compared respectively to the parental strain CAI4). After 18-h growth, SAP7 was up-regulated in Δsap9, Δsap10, and Δsap9-10 (1.4-, 3.9-, and 3.1-fold as compared respectively to the parental strain CAI4). We showed that after 18-h growth, Δcph1 cells showed SAP7 decreased 2.3-fold, while Δefg1 displayed a slight up-regulation of this gene (1.2-fold). The expression of SAP7 on the line Caco-2 at 3 h post-inoculation was higher than in 18-h grown cells. The obtained results proved that SAP7 can play a role during the initial adaptation of C. albicans to the intestinal tract and that it decreases over time.
Analysis of the SAP7 gene relative expression compared to the ACT1 reference gene in C. albicans cells. The cells were grown on Caco-2 cell line at 37°C
C(t)- mean for three independent experiment ± SD; 1strain SC5314 – calibrator in 2-∆ΔC(t)
Determination of C. albicans adhesion to Caco-2 monolayer. To study whether the genetic alternations found in SAP and EFG1 and CPH1 genes could have an impact on in vitro virulence, we investigated the adherence of the C. albicans null mutants in a model of epithelial cells, and compared them with that of the wild type strains: SC5314 and no 82 as well as the rescued strains. As depicted in Table III, the adherence ability varied within each species with the values from 0.533 ± 0.321 to 11.3 ± 10.324 for the wild type strains: SC5314 and no 82, and from 0.68 ± 0.593 to 11.8 ± 7.532 for the mutants. A significant trend toward an affected adhesion of morphogenesis mutants was noted by comparison with the wild type isolate no 82 (P ≤ 0.05). Our results demonstrated that lack of Cph1 protein influence significant reduction in their adhesive ability (P ≤ 0.05) compared to the wild type strain no 82. With Δcph1 adhesion was significantly lower (16.6-fold) than with Δefg1 (2.1-fold) compared to the wild type strain no 82. In opposition, the SC5314 and Δcph1 strains showed almost similar adhesion, whereas Δefg1 revealed 9.85-fold increase in adhesive mode compared to the parental strain (SC5314). In this particular case of Δcph1, reintroduction of the one copy of CPH1 restored adhesion. Thus the presence or absence of CPH1 affected the adhesiveness behaviour of C. albicans cells.
The adhesion properties depended on the morphogenesis mutants (compared to the wild type strain 82) while for the mutants Δsap9 and Δsap10 almost the same level of adhesion was observed (P ≥ 0.05). Moreover, the mutant Δsap8 adhered less efficiently to epithelial cells (6.2-fold reduction) compared to strain no 82. In the case of the Δsap8 mutant, adhesion of the latter strain was reduced to statistically significant levels (P ≤ 0.05). Moreover, when strain SC5314 was analyzed, there were no detectable differences in its percentage of adhesion in comparison to the mutants (P ≥ 0.05, Table III). In this regard, all the tested C. albicans strains were able to adhere to the epithelial cell line to a different degree.
Adherence of C. albicans morphologies in an in vitro model of intestinal candidiasis (monolayer of Caco-2 cell line ATCC). Data are expressed as the mean ± SD of three independent experiments
106 cells (ml saline)-1
|Percentage of cells adhesion|
Significant reduction of adhesive properties (P≤0.05) in bold (compared to the wild type strain no. 82)
C. albicans morphology on the intestinal Caco-2 monolayer. Electron microscope studies revealed a, clearly reduced capacity of hyphal growth of the mutant Δefg1 in comparison with the wild type strain SC5314 on enterocyte monolayer (Fig. 1). As shown microscope micrographs, following an 90-min attachment phase of yeast cells, we observed that all fungal strains had switched to hyphal growth form. TheΔefg1 mutant latter strain showed morphologies tended to be slightly distorted compared to those of the wild type strain. Microscopically, no differences in the Δsap8-10 mutants’ morphogenesis potential during adhesion to Caco-2 monolayer were observed after 90 min, indicating epithelial adherence and possibly cell proliferation.
As far it is known (Cadicamo et al., 2013; Correiaet al., 2010; Dalle et al., 2010; Jackson et al., 2007;Martin et al., 2011; Naglik et al., 2003; 2008; Taylor et al., 2005), the increased expression of specific Saps at various stages of the infection plays a special role in tissue invasion. As the role of SAP7 in C. albicans virulence had remained unknown, we showed in our study, that the level of the SAP7 expression correlates with the importance of this gene for the early stage of the Caco-2 intestinal tissues invasion. It is worth noting, that differences in the SAP7 expression between C. albicans cells colonizing Caco-2 depend on genetic alternations. These results showed clearly that the up-regulation of SAP7 occurs in the absence of SAP8 and SAP10. The compensation for the absence of Sap10 activity by Sap7 demonstrated their similar function. As showed Bocheńska et al. (2013) these two Saps out of 10 isoenzymes are unable to release bactericidal peptides from human Hb. As Sap10 participated in the processing of cell wall proteins (Schild et al., 2011), the function of Sap7 was speculated and needed experimental verification. Our results perfectly agree with the previous study concerning Sap7 activity at neutral pH. We demonstrated (Staniszewska et al., 2014a) that SAP7 may help the fungus to cause systemic infections. We also showed that morphogenesis factors can be regulators of SAP7 at early stage of epithelial infection. As desribed Pierce and Kumamoto (2012), Efg1 is a transcritional regulator of a large number of genes and many differences in the gene expression are suspected to occur in response to changes in the EFG1 expression. Moreover, according to the latter authors, in the human gastrointestinal (GI) tract, wild type cells and cells with low Efg1 activity are expected to express factors that allow them to interact with host epithelial cells. We proposed that the SAP7 activity provides a mechanism which allows the colonizing of human cells. As showed Nobile et al. (2012) C. albicans is one of the very few fungal species that can efficiently form biofilms in healthy mammalian cells. Following Nobile et al. (2012), we suggested that SAP7 is involved in the biofilm network in C. albicans as a ‘young’ gene engaged in an early organisation of biofilm (adhesion to the intestinal monolayer). On the other hand, C. albicans hydrolytic enzymes might be regulated differently during various experiments and what is more, these genes’ expression is strain-specific. Lermann and Morschhäuser (2008) showed that Saps are not required for the invasion of reconstituted human epithelia (RHE) by C. albicans. Therefore, environmental conditions have an important impact on the SAP expression pattern in C. albicans. Moreover, the proteinase encoded by the SAP7 gene, if translated, may be associated with C. albicans early infection and is quite different from laboratory culture conditions (Cadicamo et al., 2013; Taylor et al., 2005).
C. albicans possesses a remarkable capacity to adhere to tissues (Dalle et al., 2010; Yan et al., 2013). Moreover, adhesion is influenced by enhanced expression and production of Saps (Albrecht et al., 2006; Braga-Silva and Santos, 2011; Dalle et al., 2010; Martin et al., 2011; Seabra et al., 2013). In our in vitro model of epithelial infection, Δsap8-10 and morphogenesis mutants displayed adherence to Caco-2 monolayer, where hyphae germinated. We characterized the morphology of the two morphogenesis mutants and four Δsap compared to the wild type by CLSM, using the Caco-2 cell line. The strains proliferated, underwent morphogenesis and caused epithelial cells lysis. Although deletion of CPH1 reduces hyphal growth on solid medium (Tsai et al., 2013), we showed that it still forms hyphae during adhesion to a polarized monolayer of Caco-2 epithelial cells (Fig. 1). In stark contrast, Δefg1 was slightly attenuated in developing filaments (Fig. 1). While, the extent of adhesion was strain-depended, a general trend was observed among all the strains tested, confirming a slightly reduced adhesion ability of the morphogenesis mutants, with Δsap9 and Δsap10 strongly adhering to the epithelial cells, followed by the wild type strain no 82 (Table III). Although, Cph1 appears to contribute to adhesion (Δcph1 was reduced in this capacity, Table III), other factors, particularly agglutinin-like sequence (ALS) are the major contributor to epithelial cells adhesion (Tsai et al., 2013). As discussed Brand (2012), deletion of one member of SAPs effected on the compensation of genes encoding surface proteins that are involved in adhesion i.e., ALS3, HWP1.
In our study, deletion of SAP9 resulted in only slightly altered adhesion to Caco-2 compared to the wild type strains (Table III). To be more precise, Δsap9 was more effective in adhesion than its parental strain SC5314. Strikingly, our data showed that adhesion activity of Δsap10 was reduced in higher level compared to SC5314. We also found that strains were not adhesion activity correlated, pointing to a strain-dependent phenomenon rather than a significant association between Saps and adhesion. It was demonstrated that the ability of the mutants: Δsap, Δefg1, and Δcph1 to adhere to epithelial cells is not altered significantly compared with the wild types. Thus we suggested that a lack of morphogenesis factors as well as Δsap8-10 cannot be important for adhesion to the intestinal layer. Furthermore, deletion of the SAP8-10 genes resulted in no filamentation defects; however these have not been reported to date. Overall, this analysis indicates that unlike SC5314, the significant differences in adhesion of Δsap, Δefg1 and Δcph1 during the growth on Caco-2 relative to the clinical strain no 82 are not meaningful for this process if the strains genetic backgrounds are In vitro SAP7 expression in Candida albicans too different. On the other hand, as reviewed by Naglik et al. (2011), Sap9 and Sap10 may indirectly contribute to adhesion by targeting covalently linked fungal cell wall proteins such as Cht2, Ywp1, Als2, Rhd3, Rbt5, Ecm33 and Pga4 and glucan cross-linking protein Pir1. Furthermore, the findings of Schild et al. (2011) on the in vivo expression profile of Saps1-6 supports a role of Sap1-3 in the adherence process to epithelial cells and Sap4 to Sap6 in assisting C. albicans cells to evade phagocytosis (Bertini et al., 2013; Braga-Silva andSantos, 2011; Han et al., 2011; Naglik et al., 2011).
To our knowledge, ours is the first study of the role of SAP7 in the early stage of the colorectal carcinoma Caco-2 invasion In order to benefit from these results in therapy (development of proper anti-fungal compounds or potentially effective combination vaccines including Sap7) additional in vivo experiments should be conducted.
The work was supported by the grant No DEC-2011/03/D/NZ7/06198 from National Science Center, Kraków, Poland. The authors would like to thank sincerely Prof. B. Hube for the strains CAF2-1, CAI4, Δsap9, Δsap10, Δsap9-10, Δsap8. We are extremely grateful to Professor Hsiu-Jung Lo from the National Health Research Institute in Zhunan (Taiwan) for providing us with the following strains: Can16, YLO323, HLC52, HLC74 and HLC84. We are very grateful to the Laboratory of Miniaturized Chemical System (Warsaw University of Technology) for access to confocal and electron microscopes.
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