Iron Limitation Shows No Significant Qualitative Changes on the Cell Wall Proteome of Candida

Qualitative changes lead to a deeper investigation of the cell wall proteome of Candida albicans under iron limited conditions Jens M. Wartenberg Bachelor Thesis

Swammerdam Institute for Life Sciences, University of Amsterdam, Nieuwe Achtergracht 166, 1018 WV, Amsterdam, The Netherlands

Summary Candida albicans is an opportunistic pathogenic fungus which is carried by 80% of the human population without causing infection. The fungus is able to become virulent at certain environmental cues and cause infection which may result in death. Immunocompromised patients are at higer risk of developing candidias. Several antifungals have been artificially developed, but overuse led to resistance and thus new drug-targets need to be identified. Iron is an important nutrient for proliferation and growth in almost every microorganism, but due to its toxic nature it is not freely accessible in the host. The human body packages iron in proteins for transport and storage and so Candida needed to develop several uptake systems. Proteins on the cell wall are known to be important in iron acquisition. By making use of mass spectrometry techniques we have looked at changes in the cell wall proteome of Candida albicans under iron limiting conditions at pH 7.4 and pH4. Transcriptional studies indicate the upregulation of several proteins, like the iron acquisition protein Rbt5, but we could not verify that many changes on qualitative protein level.

Introduction vaccines without causing severe side- effects. Fungal research is becoming A number of virulence factors have been increasingly important. Although the established in C. albicans which are current health system provides great responsible for its opportunistic care of patients, immunocompromised behaviour. It is capable of switching humans remain at high risk of from a unicelullar budding-yeast developing fungal infections and this is phenotype to an invasive multicellular still the leading cause of mortalities filamentous form called hyphae and among HIV-1 affected people. Candida there is also an intermediate form albicans is an opportunistic fungus named pseudo-hyphae. This switching which lives and thrives in 80% of the is reversible and is induced by population without causing harmful environmental cues. The cell wall effects. A lot of research has been done contains a large repertoire of adhesion on this organism and several antifungals proteins which are involved in the have been developed, but overuse of formation of stable attachments to the these has led to resistant strains and so host-tissue. The fungus also secretes the quest for new drug-targets proteases which presumably are continues. Cell-wall proteins of C. involved in nutrient supply, degradation albicans are one of the new targets to of immunoglobins and degradation of develop fungistatic and fungicidal host-barriers during invasion.

1 Candida is able to grow in different to directly take up siderophores which habitats throughout the human body. are small molecules capable of binding Three different forms of infection can be ferric iron. Surprisingly it is not able to distinguished: cutaneous, mucosal and produce siderophores itself but uses systemic. Mucosal infections can be those of other micro-organisms. Another subdivided in oropharyngeal candidiasis way of acquiring iron without reducing is (OPC), esophageal candidiasis (EPC) the direct uptake of haeme with Rbt5 a and vulvovaginal candidiasis (VVC). GPI-anchored protein capable of binding OPC and EPC are the main mucosal and transporting haeme out of infections in HIV patients. This research haemoglobin across the cell membrane. project will mainly focus on mucosal Iron uptake systems are similar among infections. most micro-organisms. So there is not For Candida to grow at different sites of only great difficulty in obtaining iron, but the human body it needs to adapt to the as well great competition for it. environment it is surrounded by. Not In this study we are investigating the only is it able to withstand different pH effects of iron limitation on the cell wall levels, but oxygen availability can range proteome of Candida albicans. The cell from a pO2 of around 100 mm Hg to walls and cell wall proteins (CWP) of almost anaerobic conditions. Iron is an fungi have several functions like water essential nutrient for growth and retention, but also functions important proliferation for almost all micro for virulence like adhesion, cell organisms. It is used in many enzymes aggregation, biofilm formation and as a cofactor due to its favourable protection against oxygen radicals. Cell reducing potential. Ferric iron is the wall proteins are also involved in the predominant form of iron, because uptake of iron. Here we show the effects under standard conditions ferrous iron of iron limitation on the cell wall will auto-oxidize to ferric iron. At pH 7 proteome. We are looking at the first ferric iron forms a complex with water in stages of a mucosal infection in the which protons are lost. This results in an human body which include epithelial amorphous gel and so virtually no free adhesion and superficial penetration of iron is available. The human body the mucosal surface in which hyphae solves this by packing and transporting are involved. Mass spectrometry iron in proteins like lactoferrin, ferritin techniques are used to identify the and transferritin. The difficulty in qualitative changes in the CWPs as a obtaining iron leads to a limited number consequence of the iron restriction of uptake mechanisms in micro- compared to untreated control cells. organisms. Iron acquisition can be Also the effects of low iron levels on the divided in two major groups: reductase ergosterol synthesis are analysed. dependent and reductase indepent. The solubility of ferrous iron at ~pH 7 is Materials and methods much higher than that of ferric iron. A logical way of iron uptake would be to Strains and growth media reduce FeIII to FeII. C. albicans has two The C. albicans strain used for the reducing uptake systems: high affinity experiments is SC5314, a clinical and low affinity. The latter directly takes isolate. Overnight cultures (ONC) were up ferrous iron by the FTR1 protein made by inoculating Candida (stored at which is a transmembrane permease. 4 C) in 20 mL YPD medium(1% Bacto- The high affinity system first reduces yeast extract, 2% Bacto peptone, 2% iron to reoxidize it again by ferroxidase. Glucose) for ~24 hours at 30 C and The reason why and how this system constant shaking (200 rpm). Cells for works is not known yet. Candida is able cell wall isolation were grown on plates

2 for 18 hours at 37 C. (3% agar, 5 mM dissolved in 23 L of 0.1% TFA for mass Mucin, 1.7% Yeast Nitrogen Base spectrometry analysis. For protein without aminoacids and ammonium identification a tandem MS system was sulfate, 75 mM Mopso (pH 7.4) or 55 used including a nano-LC column. The mM Tartaric acid (pH 4), 0.09% eluted peptides are electrosprayed in a Glucose). Iron restricted plates were Micromass quadrupole time-of-flight prepared by adding 100 M of the iron mass spectrometer. Ions were selected chelator bathophenanthroline di- as described by Sosinska et al [2008]. sulphonic acid(BPS) at pH 7.4 and 150 M BPS at pH 4. Ergosterol level determination Cells were washed off plates with MQ- water and 2 mL were suspended in 6 Cell wall isolation mL Methanol. 6 mL of cold Petroleum- Invasive cells were harvested by Benzeen (PE) were added to the solubilizing the plates with 6 M sample and vortexed for 1 minute at guanidine Thiocyanate, non-invasive high speed. The samples were cells by washing off with Mili Q water. centrifuged for 2 minutes at 3000 rpm. Cells were harvested out of the plates The upper phase containing ergosterol with 6 M Guanidine Thiocyanate or was transferred to a new tube and dryed washed of with Milli-Q water. A standard under a constant flow of gaseous cell wall isolation protocol (De Groot et nitrogen. The dryed sample was al. 2004) was used in which several dissolved in 60 L ethanol to load on the adaptations were made. Due to HPLC. As a standard for quantitation 1, problems with hyphae floating the washing with Tris buffer was reduced to 10 and 100 M ergosterol were used. one time and replaced by multiple The different samples were normalized washing steps with MQ-water. Breaking by dry weight. of cell walls by bead-beating was increased and boiling with SDS- Dry weight/growth determination Cells were harvested from soft agarose extraction buffer containing - plates and washed with Milli-Q water. mercaptoethanol (8 L per 100 mg of Samples are loaded in pre-weighted cell wall) was increased to four times to eppendorfs and centrifuged for 1 minute get rid off all cytoplasmic proteins. at 13000 rpm. The washing was Isolated cell walls were freeze-dryed repeated and the samples were dried at and stored at – 20 C. 60 C. Sample preparation for MS Results A standard protocol was used described by Sosinska et al [2008]. No changes Iron Limitation leads to a reduction in were made. biomass C. albicans is capable of growing under MS identification of cell wall proteins Iron limited conditions to a certain After the tryptic digest the cell walls extent. Here we wanted to identify a were centrifuged for 1 minute at 13000 BPS concentration which leads to a rpm. The supernatant containing the 30% biomass reduction, because this tryptic peptides was transferred to a new indicates a clear effect of the treatment, tube and a standard protein zip-tip but the cells remain viable. In Figure 1A protocol was performed using a C18 the effect of various BPS concentrations 100 L zip-tip. Zip-tipped samples were on the biomass is shown. 100 M BPS, stored at – 18 C or 2 L were which causes a 30% reduction in

3 A 100 uM BPS leads to a 30% growth reduction in Candida B 150 uM of BPS leads to a 30% growth reduction in albicans at pH 7.4 Candida albicans at pH 4

120 120 100 100

) 80 % % ( (

t h h t 60 g w

i 60 o e r w G y 40 r 40 D

0 0 50μm BPS 100μm BPS 150μm BPS pH 7.4 125μm BPS 150μm BPS pH 4

Fig 1A. Growth diagram of Candida albicans with different concentrations of BPS at pH 7.4. Growth is represented in percentages with pH 7.4 as the reference. 100 μM of BPS leads to a ~30% growth reduction which is the concentration used for further experiments. 1B Growth diagram of Candida albicans with different concentrations of BPS at pH 4. Growth is represented in percentages with pH 4 as the reference. 150 μM of BPS leads to a ~30% growth reduction which is the con-centration used for further experiments.

biomass, is used for further experiments effect of iron limitation on ergosterol at pH 7.4. Figure 1B shows the same levels. 100 M BPS shows a 40% experiment at pH 4, where 150 M BPS reduction of ergosterol in comparison leads to a 30% reduction in biomass with control cells at pH 7.4. As a positive and is used for further experiments at control, cells were treated with low pH. fluconazole, a widely used fungistatic which inhibits 14α-demethylase, an Supplementation of iron can abolish the enzyme of the ergosterol synthesis BPS induced biomass reduction pathway. 0.5 g/mL fluconazole led to a In order to establish the fact that growth 90% reduction of ergosterol levels. reduction is attributed by the chelating of iron and not by a toxic side effect of Effects of iron limitation on the cell wall BPS or the chelating of other essential proteome ions, iron was supplemented in the Peptide mixtures of each sample are Iron limited conditions lead to a 40% decrease in ergosterol media to restore growth. Figure 2A. and loaded in an LC-MS-MS Q-tofconcentrations mass- in Candida albicans

2B. show growth restoration of C. spectrometer and the120 derived peak list is albicans cells when supplemented with analysed by MASCOT. Table 1. and 2. A 100 different concentrations of Ferrous Iron show the number of peptides found for ]

) 80 % (

at pH 7.4 and pH 4. each identified proteinl at both pH 7.4 o r

e 60 t s

and pH 4 - a list o of all proteins and g r E Iron limitation leads to reduced functions is found in [ supplementary40 data ergosterol levels in C. albicans table S1. The number20 of peptides is not

Iron is essential in the ergosterol quantitative and only gives0 an indication synthesis pathway, since many of its whether the protein is presentpH 7.4 or not.Fluconazole BPS enzymes contain iron, which is needed Table 1. shows thatFigure Ssr1 3. and Ergosterol Sap9 levels are in two differently treated for their function. Figure 3. shows the found in normal treatedCandida cells albicans at pH cultures. 7.4, Ergosterol levels were determined with HPLC in iron limited conditions at pH 7.4 Iron limitation leads to a 40% reduction of ergosterol. Fluconazole Iron supplementation restores BPS caused growth inhibiton at pH 7.4 Iron supplementation (0.5restores μg/mL) BPS is caused shown growthas a positive controle. 160 B inhibiton at pH 4 160 140 140 120 120 h h 100 t t 100 w w o o r 80

r 80 g

0 0 pH 7,4 100 μM BPS 100 μM BPS + 100 μM BPS + 100 μM BPS + 100 μM BPS + pH 4 150 μM BPS 150 μM BPS + 100 150 μM BPS + 300 100 μM FeII 300 μM FeII 500 μM FeII 1000 μM FeII μM FeII μM FeII

Fig 2A. Iron supplementation graph of Candida albicans at pH 7.4. Growth is represented in percentages with pH 7.4 as the reference culture. Different concentrations of ferrous iron are supplemented in plates containing 100 μM of BPS. Higher concentrations of Iron restore growth and promote it. Fig 2B. Iron supplementation graph of Candida albicans at pH 4. Growth is represented in percentages with pH 4 as the reference culture. Different concentrations of ferrous iron are supplemented in plates containing 100 μM of BPS. Higher concentrations of Iron restore growth and promotes it. 4 but not in the iron limited and the other Iron limited conditions lead to a 40% decrease in ergosterol wy around for Als 5. At pH 4 Rbt5 as concentrations in Candida albicans well as Sod5 are found in the iron 120 depleted cells, but not in the normal 100 ] reference. The significance of these ) 80 % (

l o r e

findings is debatable and will be t 60 s o g r

discussed further. E [ 40

20 Proteins pH7.4 pH7.4 BPS 0 Als1 2 1 3 2 2 pH 7.4 Fluconazole BPS Als2 Als3 5 3 7 4 9 Figure 3. Ergosterol levels in two differently treated Als4 1 3 1 1 1 Candida albicans cultures. Ergosterol levels were Als5 2 determined with HPLC in iron limited conditions at pH 7.4 Iron limitation leads to a 40% reduction of ergosterol. Fluconazole Cht2 4 3 4 2 3 (0.5 μg/mL) is shown as a positive controle. Crh11 4 1 6 2 3 Ecm33 3 5 6 2 6 Mp65 3 1 Pga4 2 2 1 Discussion/conclusion Phr1 1 2 2 1 2 Phr2 Pir1 2 2 1 In this study we show the effects of iron Rbt5 1 3 3 3 depletion on growth, ergosterol levels Rhd3 2 9 7 1 5 and the cell wall proteome of Candida Sap9 2 1 albicans. Iron is an essential nutrient Sod4 2 2 3 3 Sod5 7 4 7 6 5 and growth is markedly affected when Ssr1 2 2 2 iron is restricted using the iron chelator Utr2 3 2 2 2 BPS (Fig. 2A/B). At pH 4 a higher Ywp1 1 1 1 1 1 Table 1. Number of peptides found for each protein identified concentration of BPS is needed to by MASCOT in normally treated cells and iron depleted cells reduce growth which is probably due to at pH 7.4 Normal pH 7.4 cells were done three times and Iron limited cells were done twice the fact that there is more free iron available than at neutral pH (Kosman, 2003). Reduction of growth by BPS is Proteins pH 4 pH 4+BPS caused by its iron chelating capabilities Als1 2 only, because when supplemented with Als2 iron, normal growth is restored and even Als3 promoted (Fig. 3A/B). As stated iron is Als4 2 1 1 1 Als5 an inducer of growth and proliferation, Cht2 5 4 5 3 thus the overshoot of growth at higher Crh11 6 3 8 2 concentrations is due to this fact. Ecm33 5 5 4 3 Mp65 3 2 2 1 Iron is used in many enzymatic Pga4 4 1 1 1 reactions because of its reducing Phr1 ? ? nature. Ergosterol is a critical Phr2 4 ? 6 ? component of the fungal cell membrane Pir1 4 1 1 1 Rbt5 2 2 and the synthesis is a multistep process Rhd3 6 8 6 3 in which iron is important. Depletion of Sap9 iron should therefore have an impact on Sod4 1 4 2 Sod5 1 2 ergosterol levels. Figure 4. shows that Ssr1 2 2 2 2 iron limitation leads to a 40% reduction Utr2 3 1 2 1 of the ergosterol concentration in the Ywp1 1 1 1 1 cell membrane of Candida. Ergosterol Table 2. Number of peptides found for each protein identified levels of cells treated with fluconazole – by MASCOT in normally treated cells and iron depleted cells at pH 4. The question marks are peptides which MASCOT wasn’t a widely used fungistatic - are shown as able to distinguish based on the peptide sequence, but at pH 4 a positive control. only PHr2 is upregulated.

5 The cell wall proteome is dynamic and findings have to be supported by further adjusted to the environment the fungus experiments. is surrounded by. The protocol used leads to about 25 identified proteins by Future prospects MASCOT in a clean sample, but normally fewer are identified at one In this research a 30% reduction of condition. This can be attributed to growth is chosen by treatment with BPS. overlapping peptide peaks and loss of The effect of iron limitation on the cell proteins during the washing and boiling wall proteome could be more at higher steps. Table 1. and 2. show the number concentrations. It might be interesting to of peptides found for each identified repeat the experiments at a BPS protein. At pH 7.4 several differences concentration which leads to a 50% can be seen. Ssr1 and Sap9 are found reduction of growth. only in the control cells. Ssr1 is a β- Although qualitatively there is no real glucan associated protein important for significant change in the cell wall cell wall structure and Sap9 is a proteome of Candida albicans under secreted aspartyl proteinase involved in iron limitation, quantitatively it might. adhesion. Although not seen in the iron The next logical step is to design an deprived samples the number of experiment by which one can quantify peptides found for the normal samples the proteins of the cell wall by making are not that many and Ssr1 should be use of mass spectrometry based present, but maybe at much lower techniques. Significant down or up- amounts in the iron limited samples as regulated cell wall proteins could then well. The significance of these findings be used as new potential drug-targets. is therefore not clear. Als5 is identified Not only iron is essential for growth and once in the two iron limited samples, but proliferation, but oxygen availability is again the number of peptides is only also a key component in many reactions two. To be able to say something about for example the ergosterol synthesis this difference the experiment might be pathway. Sosinska et al [2008] have repeated for the iron deprived cells. looked at the effect of hypoxic For pH 4 there are two differences conditions under vagina-simulative recorded: Sod5 which has a protective conditions and shown – using role in oxidative stress and Rbt5 which immunoblot assays - an up-regulation of is involved in iron acquisition. Iron is Pir1 and Hwp1, but also Pga10 and cytotoxic and especially auto-oxidation Rbt5 which are involved in iron of ferrous iron results in the formation of acquisition. Addition of the iron chelator superoxide radicals. At pH 4 more free ferrozine led to even higher up- ferrous iron is available and so Sod5 regulation of the latter. This suggest a should probably be seen in both normal related to low oxygen and iron levels. and iron limited samples. The fact that Using the techniques we applied for iron Rbt5 is seen only in the affected restriction we could take a look at samples gives an indication that Rbt5 is qualitative as well as quantitative up-regulated upon iron limited changes of the cell wall proteome conditions. This indication should be depending on oxygen availability. verified by repeating the experiment and Not all proteins on the cell wall are in the future take a quantitative look at digested by trypsine or give peptides of the cell wall proteome. which MASCOT is able to use. These Although there are some qualitative proteins can be identified using changes on the cell wall proteome of immunoblot assays. Sosinka et al. Candida albicans under iron restricted (2008) have identified several cell wall conditions, the significance of these proteins using these assays at

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8 Supplementary data.

Proteins Nomenclature Function and features Induction Als1 Agglutinin-Like Sequence 1 Adhesion, role in virulence Als2 Agglutinin-Like Sequence 2 Adhesion, biofilm formation Low iron Als3 Agglutinin-Like Sequence 3 Epithelial adhesion, Hyphae endothelial invasiveness, Iron assimilation Als4 Agglutinin-Like Sequence 4 Adhesion, germ-tube Down-regulated upon induction vaginal contact Als5 Agglutinin-Like Sequence 5 Cell-cell adhesion Cht2 Chitinase 2 Chitinase Yeast cells Crh 11 Congo Red Hypersensitive 11 predicted glycosyl hydrolase domain Ecm33 ExtraCellular Mutant 1 Cell wall architecture fluconazole Mp65 MannoProtein of 65 kDa possible role in cell-wall heat glucan metabolism Pga4 Cell wall organization Oral candidiasis Phr1 PH Responsive 1 Glycosidase High pH Phr2 PH Responsive 2 Glycosidase Low pH, High Iron, Fluconazole Pir1 Proteins with Internal Repeats 1 Structural constituent of cell Hyphal repressed, wall fluconazole Rbt5 Repressed By TUP1 5 hemoglobin utilization High pH Rhd3 Repressed during Hyphae - Decrease in hyphae, Development 3 regulated by iron Sap9 Secreted Aspartyl Proteinase Adhesion, cell surface fluconazole integrity Sod4 Superoxide dismutase 4 Superoxide dismutase Sod5 Superoxide dismutase 5 protective role against Hyphal growth, osmotic and oxidative stress oxidative stress Ssr1 Cell wall structure antifungals Utr2 Cell-Surface Factor Putative glycosidase, cell cell wall regeneration wall, adhesion Ywp1 Yeast-form Wall Protein dispersal in host growth phase Table S1. Descriptions of all proteins identified by MASCOT in a clean sample when using our cell wall isolation protocol