Role of SEF1 Gene in Iron Homeostasis in The Human Pathogen C. albicans
August 14th, 2011
Abstract
Candida albicans is a eukaryotic commensal organism and is part of the human microbiota. However, it is also an opportunistic pathogen and has the ability to cause systemic diseases – which can range from minor subcutaneous infections to life-threatening systemic candidiasis, in individuals who have weak immune systems. Iron and copper are essential nutrients for microorganisms mainly due to their use as cofactors in many processes, with high affinity iron uptake being one of them. Iron uptake has also been found to be crucial for disease establishment of C. albicans within the host.This study aimed to give further evidence about the SEF1 gene being involved in the iron acquisition process and more specifically, whether or not it is a positive regulator of iron acquisition. Results from this study suggest that SEF1 is involved in the iron acquisition pathway and is most likely involved in the regulation of the ferric reductases.
The sef1?? mutant obtained was confirmed to have both the copies of the SEF1 gene knocked out using a Polymerase Chain Reaction (PCR) approach. Then this mutant strain was tested in different conditions (by changing the concentration of iron and copper) and it was clear with the wild type strain (SC5314) that the SEF1 gene was involved with the iron acquisition pathway. Northern blots were also attempted to quantify the level of SEF1 transcript in mac1?? and sfu1?? strains grown in different conditions (as above) which would give information on regulation of the SEF1 gene. Finally cloning and transformation was attempted to produce a complemented mac1?? strain, in order to gain further information on the role of the MAC1 gene, particularly with respect to high affinity iron uptake in C. albicans.
Introduction
Candida albicans and Virulence
Fungal pathogens are common bloodstream infection causers and Candida albicans is ranked fourth (Wisplinghoff et al., 2004) among these species. Candidal infections – such as thrush (Fig 1a) and candidiasis (Fig 1b) are very common in humans and a diploid fungus called Candida albicans plays the biggest part in causing these vicious infections. This species’ significance in healthcare could be understood further with statistics from the USA, where the mortality rate reaches 50% in low weight premature infants with candidiasis (Kalyoussef, 2010); with C. albicans also being the leading cause of Sepsis in critical care patients and half of them die because of it. Candidiasis symptoms include inflammation of the skin and/or mucosal membranes, thrush (or vaginitis) – which 75% of women will get at least once in their lifetime, and something has to be done pretty quick about it as the frequency seems to show an increase all the time.
C. albicans’ genome size is approximately 15.6 Mbp and its diploid number is 16. A reductional division to an eight chromosome haploid has never been observed (Robinson, 2008). It seems to be involved in a parasexual cycle and the number of chromosomes increases to thirty two during conjugation when two different mating strains unite, which then reduces back to the diploid number with chromosomal loss. What makes this microorganism more relevant to us humans is that C. albicans is not found in the environment such as the soil, but only exists in humans as part of the human microbiota. C. albicans is also a chemoorganotrophic fungus and is an innocent commensal of the gut, mouth and the gastro-intestinal tract; but this is the case only in a healthy host.
C. albicans is very effective at causing diseases within the host because it has the ability to penetrate the vascular system, colonise mucosa and lyse erythrocytes. These abilities can be seen more commendably when the immune system is weakened with excessive usage of immunosuppressant drugs, undergoing surgery, severe radiation exposure etc. where the list can go on (Tortorano et al., 2004), which all paves the way for infection by the microorganism. C. albicans is also called an adaptable pathogen as it can easily switch to its harmful form and cause severe life-threatening bloodstream infections such as Candidaemia. Being a pathogen is not usual in the fungi kingdom, as only 200 of the 1.5 million fungi are pathogenic. AIDS and cancer patients are the most affected by C. albicans, as approximately 90% of AIDS patients have suffered candidiasis at some stage after being diagnosed with this disease. This supports the idea that the microorganism turns pathogenic when in a host with the immune system in a compromised state. A study carried out by the USA National Nosocomial Infection Surveillance System in 1993 showed that C. albicans was the most abundant pathogen with frequency of 59.7% in hospital environments (Beck-Sague and Jarvis, 1993).
While C. albicans is already is one of the most common fungal pathogens and what is leading to even more worry is C. albicans’ ability to develop resistance to anti-mycotic drugs such as fluconazole and itroconazole (triazoles). These drugs are commonly used in treatment of candidiasis, and day by day the effect is lessening and the mortality rates are increasing in immune-compromised patients. Another downside feature of candidiasis is recurrence, thus the importance of finding drugs which cures and/or prevents candidal diseases is reaching its peak.
C. albicans exists in three morphological forms; two of them being unicellular which are the pseudohyphae form as well as the normal yeast form (Fig. 2); and the final form is the hyphae (Fig 3) form which is multicellular (Sudbery et al., 2004). It switches between these forms using complex pathways which involves many regulatory interactions that respond to different environmental conditions and signals differently (Dhillon et al., 2003); how it does these intricate manoeuvres is poorly understood. Furthermore its ability to switch from a commensal form to the detrimental pathogenic form seems to involve genetic modifications but yet again, it is not very well known how, when and why these changes happen. What is known however is that numerous factors contribute to the outcome, when switching from one form to the other. These factors include resistance to anti-fungal drugs such as amphotericin B, adherence to surfaces via adhesins and amount of copper and iron available within the host. C. albicans’ has the ability to change its morphologic form with ease, and the morphological form has an effect on its capability to invade the host’s cells; and these transitions are necessary for virulence (Rocha et al., 2001). For example the unicellular form is most useful for spreading in to the host’s blood stream and the hyphae forms – whether pseudo or multicellular; is most suitable for tissue and penetration of the cells due to the presence of the hyphal tip (Whiteway et al., 2004). Due to these abilities C. albicans has been in the spotlight amongst researchers for many years. What is encouraging though is the amount of information known – especially about the mechanisms used for copper and iron uptake about another important yeast, Saccharomyces cerevisiae; which is a non-pathogen, used in the baking industry; but despite this difference shows significant amount of homology with C. albicans
Iron and Copper – Role in Pathogenicity?
For C. albicans,iron and copper are required for processes such as growth, respiration – and in the case of pathogens, infection of the host, because they are used as a cofactor in most biochemical reactions and serve as regulatory signals for the expression of virulence determinants. In respiratory reactions iron and copper are required as co-factors for enzymes such as cytochrome c oxidase which is involved in the electron transfer process. Ferrous iron transport complex proteins and superoxide dismutase, proteins which are important for virulence, require copper to function (Hwang et al., 2002). Iron and copper are present plentifully in humans with regards to what the microorganisms needs, but are not in the form C. albicans can use directly. They are present as insoluble complexes and C. albicans has acquired mechanisms which can convert these into useable form. In healthy hosts, the defence mechanisms and the blood plasma proteins such as transferrin, haemoglobin and lactoferrin mop up most of the iron with high affinity, keeping the concentrations below what is required for pathogenic microorganisms such as C. albicans, reducing its chance of survival and/or activating the mechanisms that would be harmful to the host (Marvin et al., 2004); this is why C. albicans has acquired several mechanisms for the acquisition and retention of iron, in an iron-restrictive environment.
During an infection, the human body further reduces accessible iron by triggering the hypoferraemic response which results in the iron transported into the cells and being tightly bound to an iron storage protein called ferritin, which holds around 30% of the total body iron (Fleming & Wood, 1995). Thus the mechanism to take iron from ferritin is very important for C. albicans survival even after disease has been established. To obtain iron from the proteins such as transferrin and ferritin as well as from the blood plasma (environmental iron), a reductive pathway has been developed by the microorganism. For the acquisition of iron from a range of siderophores which have been produced by other organisms, a siderophore uptake system has been established (Sritharan, 2006; Almeida et al., 2009). Ismael et al have also reported that C. albicans can also secrete its own siderophores (Ismael et al., 1985), but not much study has been carried out on this ability of C. albicansever since. Finally, for the acquirement of iron from haemoglobin and possibly from other haem-proteins – which is coupled to nearly 70% of the total body iron (Evans et al., 1999), a haemoglobin uptake and degradation system has been advanced (Fig. 4). However these mechanisms are generally activated during iron-restricted conditions and there are other mechanisms which are used when iron concentrations are high relative to the organisms’ needs; but not much is known about how the latter systems are regulated.
Another problem facing C. albicans is that excessive amounts of the two metals within the cell leads to the formation of free radicals due to the Fenton reaction which can be toxic to the cell, thus the concentrations of the two metals within the cell requires careful monitoring. Microorganisms usually synthesize iron detoxification proteins or have mechanisms which control and regulate the storage and consumption of iron (Andrews et al., 2003). Another significance of in vivo levels of iron within the host (for virulence) is that, low in vivo iron levels are recognised as an “entry” into a host and this is used as a signal to switch on the virulence factors and cause disease (Xin et al., 2010). This is strong evidence again for linkage between iron and pathogenicity.
Comparison of C. albicans and S. cerevisiae
The current understanding of the mechanisms used by S. cerevisiae during iron and copper uptake is well established (Fig. 5); and due to the similarities between the two yeasts S. cerevisiae and C. albicans, the mechanisms already studied on S. cerevisiae can also be used to illuminate the dark patches in knowledge about C. albicans’ iron and copper uptake machinery. Since the completion of the C. albicans genome sequence in 1998 by the Stanford Genome Technology Centre, identification of the high affinity iron uptake mechanisms of
C. albicans was achieved in rapid fashion, by complementing S. cerevisiae mutants with C. albicans genes. Work done in our lab has also contributed to finding similarities between the two species.
Morrissey and her coworkers reported that C. albicans had cell surface associated cupric and ferric reductases which were regulated in a very similar manner to the ones in S. cerevisiae (Morissey et al., 1996). Another finding of our lab was the activation of copper transporter gene CaCTR1, under low copper conditions, was carried out by the transcription factor CaMac1p which is what occurs in S. cerevisiae (Woodacre et al., 2008). There are significant differences however, especially from a clinical point of view, as S. cerevisiae and C. albicans are non-pathogens and (opportunistic) pathogens respectively. The presence of homologous proteins that play different biological roles in the two species also resulted in a lot of deviation in the iron and copper homeostasis between the two organisms. Our lab has also contributed to this side of the coin also by reporting that the MAC1 gene is transcribed in response to copper in C. albicans, whereas ScMAC1 is constitutively transcribed in S. cerevisiae (Woodacre et al., 2008). Moreover 19% of C. albicans’ genes do not share homology with any other organism let alone S. cerevisiae which makes this microorganism even more unique.
Two genes that have been identified to have a role in iron transfer, CaFTR1 and CaFTR2 in C. albicans, which share a high level of homology with the iron permease gene ScFTR1 in S. cerevisiae, were able to rescue S. cerevisiae ftr1?? mutants, which gives strong evidence that the high affinity iron uptake mechanisms are similar in the two organisms. In another study, ftr1?? mutants in iron-replete conditions were not able to establish candidiasis in mice (Ramanan et al., 2000; Almeida et al., 2008) which shows the prominence of high affinity iron uptake in disease establishment of C. albicans.
AFT1 and AFT2 positively regulates iron uptake in S. cerevisiae and SFU1 does the opposite in C. albicans. Homologues of AFT1 and AFT2 are also present in C. albicans but are not functional, thus the question is, what regulates this pathway in this organism? Could it be SEF1? A new regulatory branch was added by Homann et al in 2009 (Fig 6) to the iron acquisition model which involves the novel gene SEF1, which is a transcription factor thought to be positively regulating iron acquisition. What is interesting is the fact that a similar role for SEF1 was predicted in C. albicans (Lan et al., 2004); and our aim was to confirm this role of the SEF1 gene in C. albicans and identify the genes which interact with it and are regulated by it.
Iron and Copper Homeostasis in C. albicans
Copper and iron are available in the form of cupric and ferric states within the host but these must be reduced by ferric reductases to allow copper and iron transporters such as CaCTR1 and CaFTR1 respectively to take up the minerals (Woodacre et al., 2008). Furthermore work done in our lab and reported by Marvin et al in 2004 show that in the ctr1?? mutant, high affinity iron uptake was decreased by up to 96% compared to the wild type. This lead to the conclusion that iron and copper homeostasis are interlinked (Fig 7) because deficiencies in copper uptake systems lead to disfunction of the high affinity iron uptake mechanisms (Ramanan and Wang, 2000; Marvin et al., 2004). This proved that copper was compulsory for high affinity iron uptake (Knight et al., 2002).
It has been also demonstrated that the MAC1 gene in S. cerevisiae regulates iron and copper uptake genes, in response to copper levels (Fig 6). Another finding by Knight et al was that oxidase activity – which is copper dependent, is required for high affinity uptake of iron in C. albicans (Knight et al., 2002). Thus, even though it is known that the two mechanisms are interlinked, the relationship between the two metals should be further analysed to give more clues about the role the two metals play in C. albicans’ establishment
of disease within host (Marvin et al., 2003).
Project Aims and Objectives
In this project, the aim was to further understand how C. albicans acquires iron and copper from the host environment and how the uptake of these essential metals is regulated in this opportunistic pathogen. The findings from this project, in the long run, could help in developing ways of interfering with this cycle and prevent establishment and spread of disease in humans by C. albicans. The SEF1 gene can be part of the missing links in knowledge of pathogenicity of this species.
The objectives of this project were:
Identify SEF1′s (and MAC1) role in Fe related gene metabolism
Determine how SEF1 interacts with other regulatory genes eg MAC1
Experimental Strategy and Background Information
In 2009 the putative transcription factor SEF1 had been indicated by Homann et al as a possible positive regulator involved in the regulation of C. albicans iron acquisition; because there was evidence that the gene was linked to iron homeostasis as the sef1?? strain showed growth changes in the presence of bathophenanthroline disulfonate (BPS), an iron and copper chelator. When compared to the wild type, the sef1?? strain showed reduction in growth when incubated on Yeast extract peptone dextrose (YPB) with either alkaline pH or with BPS (Homann et al., 2009). Therefore, in order to find the role of SEF1, we worked with a SEF1 double knock-out strain and further investigated its role in iron and/or copper homeostasis. To test this possible role of SEF1, a sef1?? mutant was obtained and we first started by confirming the phenotypes reported by Homann and his coworkers in 2009. The sef1?? mutant was grown along with the wild type strain (SC5314) and the ctr1?? strain in low and high iron and copper media to identify new phenotypes associated with the two metals. The CTR1 gene was previously studied in our lab and the ctr1?? was known to show growth defects in low iron media (Woodacre et al., 2007). These initial experiments helped us to develop a hypothesis about the role(s) of SEF1 in iron homeostasis in C. albicans.
A lot of work has also been done in our lab on the MAC1 gene – which was shown to regulate the CaCTR1 and CaFRE7 (copper transporter and ferric/cupric reductase respectively) genes (Woodacre, W. et al., 2008); and since iron and copper homeostasis are interlinked, further analysis of this gene would allow us to better understand where SEF1 stands in the regulation of these mechanisms. Thus in this project, as an important control, we tried to produce a complemented mac1?? mutant by re-introducing the MAC1 gene in to the mac1??. To construct a complemented mac1?? strain, cloning was carried out to transform the MAC1 gene into the genome of the mac1?? mutant to produce an organism with just one copy of the MAC1 gene. To do this, external primers (to the MAC1 gene) were designed, which would bind to the DNA flanking the gene and will be polymerised using Polymerase chain reaction (PCR). The resulting fragment from the PCR reaction will be transformed into the C. albicans genome with the use of vectors. This will enable us to further study the role of MAC1 and confirm that the changes in phenotypes only occur due to the absence/presence of the MAC1 gene.
We first begun by confirming that the sef1?? mutant obtained from Homann’s lab did not contain the SEF1 gene. They knocked out both alleles of SEF1 in C. albicans (strain SC5314) using homologous recombination, replacing the SEF1 alleles with either one of HIS1 and LEU2 genes. Our confirmation was done by designing internal primers (with both forward and the reverse primers hybridising within the gene) for the SEF1 gene and another gene (which we chose to be the MAC1 gene) as a control; then extracted chromosomal DNA of the wild type and the SEF1 mutant to perform PCR with the primers designed. Final step was to electrophorese the PCR products on an agarose gel to check for the existence of the SEF1 and the MAC1 genes in the wild type and the SEF1 mutant.
To confirm the phenotypes of the SEF1 mutant reported by Homann et al, replicate experiments were designed and performed with appropriate amounts of Copper and/or iron included (and not included) in Yeast extract peptone extract agar (YPA) plates. For this the sef1?? mutant and the wild type (SC5314 strain) were incubated on YPA plates with and without BPS (150µM); also by the addition of copper accordingly and whether changes in growth and morphology were observed and checked whether the same results were obtained.
To analyse phenotypic effects of deleting SEF1, the mutant strain was compared to the wild type in different conditions to see how its growth and/or iron/copper uptake was affected when incubated in growth media with and without copper and iron with the appropriate use of BPS – paying special attention to changes occurring due to the presence and absence of iron. For this, YPA plates and Yeast nitrogen base (YNB) plates with variable amounts of copper and iron were produced, the latter not having any iron present in it, in contrast to the YPA plate which has everything required for yeast to grow. With the appropriate addition of BPS, the concentration of these two metals were changed to see how the sef1?? reacted to changes in concentration as well as in the absence and presence of copper and iron.
To investigate SEF1s role in the regulation of already identified iron proteins, northern blots were carried out. SEF1 probes were designed with the use of internal primers and the presence and quantification of mRNA was carried out in mac1??, sfu1?? and the wild type with the use of northern blotting protocols. The results from this experiment would enable us to investigate SEF1s position with the other regulators such as RIM101, SFU1 and CBF in the iron regulatory system. This will give us clues about the regulation of the SEF1 gene in response to high and low copper/iron levels.
Materials & Methods
Growth Conditions
C. albicans (SC5314): C. albicans were grown in YPB made with yeast extract (1%), glucose (2%), Bactopeptone (2%) and 50mM uridine; and incubated at 30ºC on shakers running at 200rpm. The YPA plates contained additional Bactoagar (2%).
E.coli: E. coli were grown in Luria Bertani (LB) medium made with Bacto-tryptone (1%), yeast extract (0.5%) and sodium chloride (0.5%) at pH 7.2 (adjusted with NaOH); and incubated at 37ºC overnight on shakers running at 200rpm.
Material Recipe
AE Buffer: 50mM Sodium acetate at pH 5.3 and 10mM EDTA
Breaking Buffer: Triton X (2%), SDS (1%), 10mM TrisCl (pH 8), 100mM NaCl and 1mM EDTA (pH 
Loading Buffer: Ficoll 400 (15%), Bromophenol blue (0.06%), Xylene cynanol FF (0.06%) and EDTA (30%)
TE: 1mM EDTA (pH and 10mM Tris Cl (pH
Yeast Peptone Dextrose Broth (YPB): 1% yeast extract, 2% Bactopeptone, 2% glucose and 50mM uridine
Yeast Peptone Dextrose Agar (YPA): Same as YPB with 2% Bactoagar
Yeast Nitrogen Base (YNB): Biotin, 2 ?M, Calcium pantothenate, 400 ?M, Folic acid, 2 ?M, Niacin, 400 ?M. p-Aminobenzoic acid, 200 ?M, Pyridoxine HCl, 400 ?M, Riboflavin, 200 ?M, Thiamine HCl, 400 ?M, Inositol, 2 mM, Boric acid, 500 ?M, Copper sulfate, 40 ?M, Potassium iodide, 100 ?M, Ferric chloride, 200 ?M, Manganese sulfate, 400 ?M, Sodium molybdate, 200 ?M, Zinc sulfate, 400 ?M, Potassium phosphate monobasic, 1 M, Magnesium sulfate, 0.5 M, Sodium chloride, 0.1 M, Calcium chloride, 0.1 M
BPS: Bathophenanthrolinedisulphonic acid: Iron chelator – mops up iron to reduce availability (working stock: 10mM)
CuCl2: Copper chloride – source of copper (working stock: 500mM)
FeSo4: Iron Sulfate – source of iron (working stock: 500mM)
MD media (taken from Woodacre et al., 2007): A mixture of Salt and Trace medium (10%), Vitamin solution (0.1%), glucose (2%), CaCl2 (7mM), Tri-sodium citrate (20mM) and uridine (50mM).
Cloning and Transformation
Extraction of Plasmids: E.Z.N.A. Plasmid Miniprep Kit I Spin Protocol (from E.Z.N.A.) was used to extract the PGEM-HIS1 plasmid (see Fig. from DH5? E. coli cells.
Ligation of MAC1 gene into plasmid: PCR (using 10µl of distilled water, 20µl premix D, 2µl of each primer and 2µl of Phusion DNA Polymerase) was carried out using the CaMAC1-899 and CaMAC1+1443 (Table 1) (Marvin et al., 2004) to amplify a 2362bp fragment from template DNA purified from the C. albicans WT strain. The PCR product was then digested with SalI to produce sticky ends. The plasmids were also digested with SalI and the two were ligated (using 3µl plasmid and PCR product, 1µl dATP, 1µl T4 ligase buffer, 2µl T4 ligase and 3µl distilled water) and incubated overnight at 16ºC.
Electroporation: E. coli (TOPO10) were inoculated in LB and incubated for 2-3 hours until exponential was observed (OD600 ? 0.5). The cells were then chilled on ice for 10 minutes before centrifuging them at 4000rpm for 10 minutes at 4ºC. The supernatant was removed and the pellets were washed three times in 1 volume ice cold distilled water. The cells were centrifuged again at 4000rpm for 5 minutes (4ºC) and supernatant was removed. The pellet was then washed first in ½ volume of ice cold 10% glycerol, then with 1/20 volume. The pellet was then resuspended in 1/200 volume of ice cold 10% glycerol and stored at -80ºC.
The DNA to be transformed was dialysed using nitrocellulose sheets and incubated on ice along with 50µl of electrocompetent cells for 30 minutes. Electroporation of transformation samples were carried at 25µF-1000?-1.5V and 1000µl of LB was added and was incubated at 37ºC for 6 minutes. Then the cells were centrifuged for 1 minute at 13000rpm (room temperature) and the supernatant was removed. The pellet was resuspended in 1000µl of LB and 200µl of each transformant was streaked onto Luria agar plates containing ampicillin (100mM) and incubated at 37ºC.
Nucleic Acid Preparation & Engineering
Polymerase Chain Reaction: A typical PCR reaction contained Bio X-Act Long DNA polymerase (0.5units/µl ? 2µl), 2 x Premix D buffer (20µl), reverse and forward primers (2µl each; see Table 1), template DNA (50-100ng) and distilled deionized water (to make up total volume: 40µl). The PCR involved 30 cycles of a denaturing step at 94ºC for 2 minutes, a primer annealing stage at 60ºC (depending on primer Tm) for 30 seconds and an extension stage at 68ºC for 1 minute per 1kb of PCR product.
DNA digestion using Restriction enzymes: All restriction enzymes (RE) were purchased from New England Biolabs Ltd. and all digestions were carried out using the buffers provided and by following the manufacturer’s instructions. 10 units of RE were used for every 5µg of DNA; and time allowed for digestion was typically 3 hours
Gel Electrophoresis: DNA fragments were separated using agarose (Seakem LE agarose) gels dissolved in 1 x TAE (Tris acetate electrophoresis) buffer with 25mM of ethidium bromide. Loading buffer was also added to DNA samples (at 1:5 ratio). Electrophoresis was performed at 10V (per centimetre of gel) and the gels were visualised using a UV transiluminator.
DNA Ligation: To prevent plasmids re-annealing after digestion, the phosphate groups were removed using Antarctic Phosphate purchased from New England Biolabs Ltd. by following the manufacturer’s protocol and using the buffers supplied. The ligation reactions typically had a molar ratio of 3:1 of insert:vector, 400 units of T4 DNA ligase and 1 x T4 DNA ligase reaction buffer. The solution was then incubated at 16ºC overnight. The plasmids, insert DNA and distilled water were incubated at 65 for 5 minutes then chilled on ice for 5 minutes, before adding them to the reaction.
Genomic DNA Preparation of C. albicans: The SC5314 strain was grown in 10ml of YPB overnight and centrifuged at 3000rpm for 5 minutes. After removal of supernatant, the cell were resuspended in 500µl distilled water and pipetted to a 1.5ml screw cap microcentrifuge tube. These cells were also centrifuged at 13000rpm for 1 minute; again the supernatant was removed. The pellet was then disrupted using the vortex machine (until recognisable) and was resuspended in 200µl of breaking buffer, equal volume (200µl) of acid washed glass beads and 200µl of phenol:chloroform:isoamylalcohol in 25:24:1 ratio. The solution was then mixed using the vortex machine again (for 5 mins) and 200 µl TE was added to the mix. This solution was then centrifuged at 13000rpm for 5 minutes and the supernatant was transferred to a new tube containing 500µl chloroform:amylalcohol in 24:1 ratio. This tube was then centrifuged at 13000rpm for 5 minutes and the supernatant was transferred to a microcentrifuge tube. Precipitation of DNA was done by addition of 1ml of 100% ethanol and incubated at -20ºC for 3 hours. The DNA was then centrifuged at 13000rpm for 20 minutes and the supernatant was removed. The pellet was resuspended in 0.4ml of TE (pH and RNAse A (25M) was added (to degrade contaminating RNA). Final precipitation of DNA was done by the addition of 40µl of 3M sodium acetate (pH 5.2) and 1ml of 100% ethanol and incubated at -20ºC (for 3 hours). The DNA was centrifuged at 13000rpm and the supernatant was removed; and the remaining pellet was air dried and then resuspended in 100µl of deionized water.
Extraction of (total) RNA from C. albicans: C. albicans inoculated in 10ml of YPB were incubated for 6 hours on shakers (200rpm) at 30ºC. The culture was then centrifuged for 5 minutes at 4000rpm and the supernatant was removed. The pellet was then washed with (sterile) distilled water twice and resuspended in 1ml of distilled water. Using OD600 measurements, a culture containing (approx.) 3×104 cells per ml were inoculated and incubated in MD media containing high levels of iron and copper (on shakers at 30ºC) overnight. The cultures were then centrifuged, washed and resuspended in 1ml of distilled water (as above). This culture was then used to inoculate MD media with different supplements (see Table 2) until OD600 measurements showed results which indicated 2×106 per ml cell density. These cultures were then incubated at 30ºC on shakers until the culture reached exponential growth phase (cell density ? 1×107) – which is approximately 5 hours; they were then centrifuged at 4000rpm for 5 minutes and the supernatant of each culture were removed. The pellets were then resuspended in 400µl RNAse free AE buffer and 80µl SDS and vortexed for 30 seconds. 480µl phenol (with AE buffer) was also added and again vortexed for good mix. The tubes were then incubated at 65ºC for 4 minutes before cooling in dry ice for 3 minutes; repeating this process three times before finishing with incubation at 65ºC for 4 minutes. The samples were then centrifuged for 5 minutes at 13000rpm and the supernatant was transferred to clean tube containing 500µl of phenol:chloroform:isoamylalcohol at ratio 25:24:1. These tubes were then vortexed and centrifuged for 10 minutes at 13000rpm (at 4ºC) twice. The resulting supernatant was then transferred to a different tube and precipitated by the addition of 40µl 3M sodium acetate and 2 volumes of ethanol (100%); leaving the samples for incubation, overnight at -80ºC. The samples were then centrifuged for 25 minutes at 13000rpm (at 4ºC) and washed with 500µl of ethanol (80%). The samples were centrifuged again and the pellet was air dried and resuspended in 50µl of DEPC treated water.
Northern Blotting: After performing denaturing agarose gel electrophoresis the RNA was immediately transferred to nylon membrane by northern blotting. A glass plate covered with 3MM paper soaked in 10 x SSC (with both edges of the paper in contact with the 10 x SSC solution of the plastic tray) on top was rested on a plastic tray filled with 10 x SSC. The gel was washed with distilled water and was placed on top of the 3MM paper; then a nylon filter (same size of gel and soaked in 3 x SSC) was then placed over the gel followed by two pieces of 3MM paper (also soaked in 3 x SSC), then by a stack of paper towels, then by a glass plate and finally by any weight which would increase the pressure on the gel. The blot was left to develop (& transfer) overnight. After completion, the nylon filter was then dried and the RNA were fixed using 0.7J cm-2 of energy in a UV crosslinker (by Amersham Biosciences).
Radioactive labelling of Probes: The blot was prehybridised in Church Gilberts buffer overnight at 65?C. The probes (see Table 3) were then diluted with dH2O to final amount of 30ng in volume of 18µl. This solution was left to boil in water bath for 5 minutes before cooling in ice for 5 minutes. The probes were then transferred to a clean tube with 5µl oligolabelling buffer, 1µl BSA and 1µl klenow fragment of DNA Pol 1. Using 2.5µl of 32P, the probes were incubated at 37?C for 2 hours. During this time 1600µl of elution buffer was added to a Sephadex column. When incubation is over, the elute was discarded from the columns; the labelled probes and 400µl elution buffer was added to the columns. The elute was then collected and checked for radioactivity (should give low readings). Then 400µl elution buffer was added and again collected in screw cap tube; again checked for radioactivity (should be high). After confirmation of high radioactivity, boil tube on heating block for 10 minutes before cooling on ice for 10 minutes. Finally add to hybridisation tube and hybridise overnight at 65?C.
Hybridisation of Probes for Northern Blots: Wash the blot with 3 x SSC with 0.1% SDS (final volume of 500ml) at 65?C in hybe oven and check for radioactivity. Then remove blot using forceps and dry on 3MM paper. Transfer blot to second 3MM, tape corners and wrap in cling film. Finally put in cassette with intensifier and film and expose in -80?C freezer. After 2-3 weeks remove cassette and defrost. Dip film in developing solution for 2 minutes, then neutralising solution for 30 seconds, then in fixing solution for 3 minutes and finally rinse in water.
Plate Assays
The WT and the sef1?? mutants were streaked on YPA plates and incubated at 30ºC overnight. These were then subcultured into 10ml of YPB and incubated at 30ºC overnight on shakers (200rpm). The strains were diluted using water to an OD600 of 0.160 (1x). In addition, dilutions of 1:10 and 1:100 were also made. These were then pipetted into a 96 well flat plate and with the use of a 48-pin bolt replicator, these were placed on YNB (or YPA) plates with different supplements. Finally the strains were incubated at 30ºC until a phenotype was observed (up to 1 week)
Results
Confirmation of sef1?? mutants
he aim of the project was to work on the SEF1 gene and understand its functions and where it sits in the regulation of iron uptake of C. albicans. To do this, sef1?? mutants were obtained from Homann’s lab (Homann et al., 2009) and tested in different environments such as in high and/or restricted iron and/or copper levels. Initial work was carried out to confirm that the sef1?? mutants obtained were indeed sef1??. For this objective, internal primers (hybridising within the gene) were designed (see Table 1) both for the SEF1 gene and another gene MAC1 – used as a control. These were used to check for the existence of these genes using PCR.
What should be observed is that the wild type has both the bands corresponding to the MAC1 and the SEF1 genes, while the SEF1 mutant has only one band corresponding to the MAC1 gene. This is what the results showed also (Fig 9), proving that the DNA extraction was successful (DNA was intact) and the SEF1 gene was not present in the sef1?? mutant. The marker also confirmed that the fragment sizes corresponded to the expected size of the SEF1 and MAC1 gene fragments (448bp and 279bp respectively).
Analysis of Phenotypic Effect of Deleting SEF1
After confirmation of the sef1??, the mutant was tested in different conditions to see how it reacts. This would give evidence about which pathways sef1 plays a part in and how it is regulated. For this the iron chelator BPS was used to restrict iron and copper levels as well as producing a combination of different conditions such as low iron, high copper and vice versa. Another condition produced was high copper and high iron. The sef1?? and WT was grown in YPB and then compared to see how removing the SEF1 has affected growth levels. Measurements were taken every hour until cultures reached exponential phase (to ensure growth was happening); and a final measurement was taken 24 hours later, which is the significant one as it showed growth potential of the strains.
In low Fe and low Cu conditions, the WT grew better than the sef1?? mutant indicating that SEF1 is involved in iron and/or copper homeostasis (Fig 10 a). Then testing the strains in high iron and low copper conditions showed that the growth defect was corrected and there was no difference between the WT and the sef1?? (Fig 10 b). When in high Cu and low Fe conditions, there was no significant difference between the WT and the sef1?? (Fig 10 c), indicating that SEF1 is not involved in copper homeostasis, but (specifically) in iron regulation. Finally when the strains were grown in High Cu and High Fe, the mutant grew better (Fig 10 d), giving strong evidence that SEF1 is involved in the iron acquisition process.
Furthermore, to confirm (what we found with the growth curves) the effect, the removal of SEF1 has on growth (and morphology) of C. albicans compared to the WT, plate assays were carried out. For this the sef1?? and WT were streaked on YNB plates with different supplements and incubated until a phenotype was observed (usually after 1 week). The photos were then taken using CCD cameras.
Plate assays also show that in low Fe and low Cu conditions, the WT grew better than the mutant. Then testing the strains in high iron and low copper conditions showed that the growth defect was corrected and the mutant grew better than the WT. When in high Cu and low Fe conditions, there was no significant difference between the WT and the sef1??. Finally when the strains were grown in High Cu and High Fe, the mutant grew better, confirming what was already said about SEF1 being involved in the iron acquisition process of C. albicans (Fig. 11 a-c, e). When BPS was not added, the excessive amount of Cu and/or Fe seemed to be toxic to the organism and reduced growth (Fig. 11 d,f,g), thus could not conclude with these plate assays.
Investigating the SEF1 gene’s Role in the Regulation of (known) C. albicans Fe Homeostasis related Proteins
To study the level of SEF1 transcript in different conditions in a variety of strains including the mac1?? and the sfu1??, northern blot analysis was carried out. The significance of the MAC1 gene was that it is the main Cu responsive gene regulator, whereas the SFU1 gene is the main (negative) regulator of iron acquisition. The results from this experiment would give an insight into the function of SEF1 and its interactions with other iron homeostasis related genes. mac1?? and the sfu1?? strains together with the wild type (SC5314), were grown in high copper and high iron, high copper and low iron, high iron and low copper, and finally, in low copper and low iron conditions. Attempts were then made to quantitatively analyse the mRNA levels in these cultures using radioactive probes of SEF1 (see Table 3).
In the blots, extremely faint bands were visible (data not shown) and due to the limited amount of time, the experiment could not be repeated; thus we could not make any firm conclusions from this experiment.
Complementing the mac1?? mutant
A complemented mac1?? could not be produced as transformation was unsuccessful because the MAC1 gene could not be ligated in to the pGEM-HIS1 plasmid (data not shown), thus no experiments were carried out on the MAC1 gene.
The MAC1 gene was amplified using the CaMAC1-899 and CaMAC1+1443 primers (see Table 1) and the PCR product was electrophoresed on an agarose gel. The fragment was confirmed to be the MAC1 gene using the HindIII marker. The gene and the plasmid were then digested using SalI; and with the use of T4 ligase, the two linear DNA fragments were ligated. The resulting plasmids were transformed into the (electro-competent) E.coli Topo10 cells for amplification; and the resulting cells were streaked on Luria plates with ampicillin. Since the plasmid contained an ampicillin resistance gene, only E.coli with the plasmids could survive. The colonies which did survive were tested for the presence of plasmids with the MAC1 gene using restriction digestion. However the results showed that none of the plasmids had the MAC1 gene; and because of limited time, attention was turned to the SEF1 gene and its roles in iron homeostasis.
Discussion & Future Work
Candida albicans is a commensal organism and is only present as part of the human microbiota. Besides being a commensal, C. albicans is also an opportunistic pathogen which gives the microorganism its clinical relevance. C. albicans has the ability to cause systemic infections such as candidiasis in immunocompromised hosts and it is rapidly gaining resistance against anti-fungal drugs which is becoming a major concern all over the world, not just in third world countries. Predictions made by the Division of Bacterial and Mycotic Diseases, which is a division of the US Center for Disease Control, states that 75% of women will get at least one of the candida caused diseases over their lifetime, which is an alarming statistic; and these figures are pushing scientists in trying to find solutions about how to go on about curing, and (better) preventing them.
It is known that various factors contribute to the switching between the commensal and the pathogenic forms such as morphology, adherence to surfaces and the host’s immune reactions; however it is now clear that copper and iron levels also play significant roles in pathogenicity in C. albicans (Ramanan et al., 2000; Marvin et al., 2004); thus better understanding of these processes could have therapeutic significance to humans and lead to saving people’s lives, especially in third world countries.
C. albicans has adapted to the iron-restricted environment of humans, thus has acquired iron acquisition proteins which are particularly responsive to low iron conditions. Nevertheless much remains unknown about iron regulation of C. albicans and the low affinity iron uptake mechanisms, particularly in vivo. The SFU1 gene has been identified as a regulator of many iron-related genes in C. albicans but this finding has still left many questions unanswered. Lan and his colleagues – and later re-iterated by Homann and his colleagues (Homann et al., 2009), indicated that SEF1 was a potential positive regulator of iron acquisition in C. albicans (Lan et al., 2004), as the sef1?? mutant strain had shown expression changes in the presence of BPS, an iron and copper chelator. In the same study, SEF1 was also found to be up-regulated in sfu1?? mutants. These findings indicated that the role of SEF1 needed to be discovered fully as it appeared to be a potentially significant gene in the iron acquisition process.
The aim of the project was to prove the hypothesis that SEF1 was involved in the regulation of iron uptake in C. albicans (as a positive regulator). For this we have designed experiments where the sef1?? mutant was tested in different environments and compared with the wild type strain to see whether this hypothesis was consistent with expectations. Then northern blots were carried out to study how the level of SEF1 transcript was changed in different conditions in different strains, expected to give important information about the role of the gene and its interactions.
We started off by confirming that the sef1?? strains we received from Homann’s lab were indeed a sef1?? and not even a single copy of the gene was present in the genome (Homann et al., 2009). This confirmation was then followed by testing the sef1?? mutant in different conditions by varying the copper and iron concentrations and comparing them with the wild type (SC5314) strain. The results showed that in low copper and low iron levels, the mutant did not grow as well as the WT which gave strong indications that the SEF1 gene was involved in either of copper or iron homeostasis. With the addition of iron however, the growth defect of the sef1?? mutant with respect to the WT, seemed to have been amended and the sef1?? mutant grew better than the WT suggesting that SEF1 is in truth involved in regulating iron acquisition. To check whether copper would have the same effect, the strains were incubated in low iron but high copper medium, but the results showed no significant difference between the growth levels of the WT and the sef1?? mutant indicating that SEF1 is not involved in copper homeostasis. Finally growing the strains in both high copper and iron conditions proved that it was the addition of iron that caused the increase in growth in the sef1?? mutant, as results showed that the mutant grew better than the WT. The experiments were also repeated with plate assays and the same results were observed. Additionally plate assays also showed that high levels of copper and/or iron can be toxic to the organism shown by the lack of growth in the plates without BPS.
With the use of survival plate assays, our colleagues within the lab found that the sef1?? mutant could tolerate high levels of Cu (relative to the WT strain), whereas the wild type could not survive in the same medium, indicating that copper in its 2+ state was not being reduced to the Cu1+ state – the soluble form in which it can be taken up by the cell. The ferric reductases are responsible for this process; and with the combination of this finding with results of our experiments we hypothesize that SEF1 could be playing a part in regulating the reduction of Fe3+ to Fe2+ (Fig. 12), as the same ferric reductases which reduce Cu2+ to Cu1+ also reduce Fe3+ to Fe2+. This prediction however, is still in its early stages and requires further research.
If time and funding had permitted, short term plans would be to repeat the northern blots again and simultaneously carry out a RT-PCR analysis – which is more sensitive, to verify the results. Results from the northern blots (and qPCR) would give indications about which genes SEF1 interacts with and how SEF1 regulates them (or is regulated by them).
Constructing a SEF1 protein expression strain would be a useful study to carry out. The experimental strategy would be to insert the SEF1 gene into the genome of electro-competent E. coli cells with the help of plasmids. Information about the size and identity of the SEF1 protein could then be obtained using Western blotting (or other methods such as affinity chromatography and mass spectrometry). Construction of a SEF1 protein expression strain would be a perfect fitting to confirm the genes role at a molecular level as information about binding sites, subunits of the protein (if present) etc. can be acquired.
Future work would involve producing a complemented SEF1 strain and analysing this new strain in the same environments the sef1?? mutant was tested to confirm that the changes in phenotype in the sef1?? mutant occurred only due to the presence/absence of the SEF1 gene. The results obtained from these experiments will give definitive evidence about the roles of the SEF1 gene in iron uptake and/or regulation.
Other objectives would be finding SEF1s role in the regulation of already identified C. albicans iron proteins such as FRE7p, FET3p and FTR1p, which are a ferric reductase, iron transporter and iron permease respectively. Furthermore SEF1s position in the iron regulatory system will be investigated and seeing where it stands with the other identified iron regulators such as CBF, RIM101 and SFU1 would be very useful for coming up with new models.
Long term objectives of the project are identifying differences between the three morphological forms of C. albicans, especially in the iron/copper uptake systems and discovering whether there is a difference in gene expression. In addition, further analysis would be carried out on already identified iron homeostasis genes. Double mutants can also be constructed to deduce whether SEF1 is upstream/downstream of known regulators. Searching for novel protein/genes and interactions in copper and iron homeostasis in C. albicans would be the long term aims of this project.
As cloning had not worked, no experiments could be carried out about the MAC1 gene in this project. Analysing the molecular mechanisms of this important gene would be part of the short term aims of this project had there been more time, as the MAC1 gene is an important player in iron homeostasis in C. albicans.
The findings of this project could help better differentiate between the iron and the copper homeostasis systems of C. albicans and S. cerevisiae. Finding these differences could answer why C. albicans responds more precisely to environmental changes (especially in vivo levels of copper and iron) and gaining an advantage over the human immune system when causing disease.
Toxicity of free cellular ions is a major problem for micro-organisms, and for pathogens careful monitoring of these is even more significant as iron and copper are both restricted in vivo except during inflammation, when copper levels are increased. It is known that the low level of iron of the host is used as a signal to express virulence determinants by many pathogens. Better comprehension of copper and iron homeostasis of C. albicans and other pathogens is certain to increase understanding of different signals and pathways these organisms use to establish diseases in humans; and hopefully lead to better treatment and prevention.
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