Candidal infections - like Thrush (Fig 3) and Candidiasis are very common in humans and a diploid fungus called Candida albicans plays the biggest part in 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. Candidiasis symptoms include inflammation of the skin and/or mucosal membranes, thrush and vaginitis.
C. albicans' diploid number is 16, and a reductional division to an eight chromosome haploid has not been observed (Robinson R, 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. C. albicans is also a chemo-organotrophic fungus which is an "innocent" commensal of the gut, mouth and the gastro-intestinal tract; but this is the case only in a healthy host. The immune system is weakened with usage of immunosuppressant drugs, surgery, radiation and the list can go on (Tortorano et al., 2004) which paves the way for infection by pathogens. C. albicans is an adaptable pathogen and can easily switch to its harmful form and cause severe life-threatening bloodstream infections like Candidaemia. This explains why AIDS and Cancer patients are most affected, due to their immune system being in a compromised state, which this microorganism craves for.
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C. albicans is already is the most common fungal pathogen and what is leading to even more worry is C. albicans' ability to develop resistance to antimycotic drugs like fluconazole. 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; thus the importance of finding drugs which cures and/or prevents candidal diseases is reaching its peak.
Figure 1 C. albicans grown in YPB: Unicellular budding yeast form Figure 2 C. albicans grown in YPB + %20Serum: Multicellular hyphae and the pseudohyphae form in red box
Fig. 3 Oral Thrush (taken from thrushpictures.com)
C. albicans exists in three morphological forms; two of them being unicellular which are the pseudohyphae form as well as the normal (Fig. 1); and the final form being multicellular hyphae (Fig 2) (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 not very well known how, when and why these changes happen. What is known however is that numerous factors contribute to the decision taken, when switching from one form to the other. These factors include resistance to anti-fungal drugs such as amphotericin B, adherence to surfaces and amount of copper and iron available within the host. There is also evidence that C. albicans' ability to change its morphologic form, has an effect on its capability to invade the host's cells and these transitions are necessary (Rocha R et al., 2001) for virulence. 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 M et al., 2004). Due to these abilities C. albicans has been in the spotlight amongst researchers. What is encouraging though is the amount of information known - especially about the mechanisms used for copper and iron uptake; about another clinically important yeast, Saccharomyces cerevisiae; which shows significant amount of homology with C. albicans.
Iron, Copper and Pathogenicity
For C. albicans, iron and copper are required for processes like growth, respiration - and in the case of pathogens, infection of the host, because they are used as a cofactor in most biochemical reactions. In respiratory reactions iron and copper are required as co-factor for enzymes like cytochrome oxidase to prevent oxidative stress. Ferrous iron transport complex proteins and superoxide dismutase, proteins which are important for virulence require copper (Hwang C et al., 2002).
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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 mechanisms which can convert these into useable form. In healthy hosts, the defence mechanisms and/or 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 like 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. To obtain iron from the proteins transferrin and ferritin as well as from the blood plasma (environmental iron), a "reductive pathway" has been developed. For the acquisition of iron from a range of siderophores which have been produced by other organisms a "siderophore uptake system" has been established (Almeida R et al., 2009). Finally but not least, for acquirement of iron from haemoglobin and possibly from other haem-proteins, a "haemoglobin uptake and degradation" system has been advanced (Fig 4). However these 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.
Fig 4 How C. albicans exploits iron within the host with different mechanisms. (taken from Almeida et al., 2009)
Another problem facing C. albicans is that excessive amounts of the two metals leads to the formation of free radicals which can be toxic to the cell, thus the concentrations of the two in vivo requires careful monitoring. (write more here)
The concentration of iron has another significance for virulence, as low in vivo iron level is recognised as an "entry" to a host and used as a signal to start the virulence factors and cause disease. This is strong evidence again for linkage between iron and pathogenicity. (Write more here)
C. albicans and S. cerevisiae (write a bit more)
The current understanding of the mechanisms used by S. cerevisiae during iron and copper uptake is well established; and due to the similarities between the two yeasts, this information can also be used to illuminate the dark patches in knowledge about C. albicans' iron and copper uptake machinery. There are significant differences however - especially from the clinical point of view; as S. cerevisiae and C. albicans are non-pathogen and an opportunistic pathogen respectively.
Two genes that have been identified to have a role in iron transfer, CaFTR1 and CaFTR2 in C. albicans - which shares a high level of homology with the iron permease gene ScFTR1 in S. cerevisiae; were able to rescue S. cerevisiae ftr1âˆ† mutants which gives strong evidence that the high affinity iron uptake mechanisms are similar in the two (Tang J, 2008). In another study, CaFTR1 double mutants in iron-replete conditions were not able to establish candidiasis in mice (Ramanan N et al., 2000) which shows the prominence of high affinity iron uptake in disease establishment of C. albicans.
Fig 5 A model of the differences between S. cerevisiae and C. albicans in iron homeostasis regulation (taken from Homann et al., 2009)
Iron and Copper (write more)
It is now known that iron and copper homeostasis are interlinked 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 suggests that copper is compulsory for high affinity iron uptake (Knight et al., 2002) and the relationship between the two metabolisms should be further analysed to give clues about the role the two play in C. albicans' establishment of disease within host (Marvin et al., 2003).
Project Aims and Objectives
In this project, the aim is 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. Through this project we hope to develop ways of interfering with this cycle and prevent establishment and spread of disease in humans.
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In 2009 the putative transcription factor Sef1p has been indicated by Homann et al as a possible positive regulator involved in the regulation of C. albicans iron acquisition; because there is evidence that this gene is linked to iron as the SEF1âˆ†âˆ† showed expression changes in the presence of an iron and copper chelator, bathophenanthroline disulfonate (BPS); and compared to wild type it showed reduction in growth when incubated on Yeast extract peptone dextrose (YPED) with either alkaline pH or with BPS (Homann et al., 2009). Therefore we will be working with a SEF1 double knock-out mutant strain and investigating its role in iron and/or copper homeostasis further. To test this hypothesis, a SEF1âˆ†âˆ† mutant will be produced and first confirm the phenotypes reported by Homann et al in 2009. Then a complemented SEF1 strain will be constructed and this new strain will be analysed in the same environments the SEF1âˆ†âˆ† mutant was tested to confirm that the changes in phenotype in the SEF1âˆ†âˆ† mutant occurred only due to the presence/absence of the SEF1 gene. The results obtained from these experiments will give strong evidence about the roles of the SEF1 gene in iron uptake and/or regulation. Then a SEF1 protein expression strain will be constructed to confirm the genes role at a molecular level. If time permits, SEF1s role in the regulation of already identified C. albicans iron proteins such as FRE7, FET3 and FTR1 which are a ferric reductase, an iron transporter and an iron permease respectively. Furthermore SEF1s position in the iron regulatory system will investigated and will be tried to deduce where it stands with the other identified iron regulators such as CBF, RIM101 and SFU1.
We will firstly begin by confirming that the SEF1âˆ†âˆ† mutant produced is definitely a SEF1âˆ†âˆ† mutant. This will be done by designing internal primers for the SEF1 gene and another gene (which we chose to be the MAC1 gene) as a control; then extraction of chromosomal DNA of the wild type and the SEF1 mutant will be carried out to perform Polymerase Chain Reaction (PCR) with the primers designed. Final step would be 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. 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.
To confirm the phenotypes of the SEF1 mutant reported by Homann et al, replicate experiments will be designed and performed with appropriate amounts of Copper and/or iron included (and not included) in Yeast extract peptone extract agar (YPA) plates.
To construct a complemented SEF1 strain, the SEF1 gene will be introduced to the SEF1âˆ†âˆ† mutant to a locus somewhere else in the genome with respect to its actual locus. To do this, external primers will be designed - which spans the whole gene; and the resulting fragment from the PCR reaction will be transformed into the C. albicans with the use of vectors (check if true).
To analyse phenotypic effects of deleting SEF1, the mutant strain will be compared with the wild type in different conditions; to see how is growth and/or iron/copper uptake affected when incubated in growth media with and without copper and iron with appropriate use of BPS. Also if time permits, finding the answer to whether there is a difference between the three morphological forms in iron/copper uptake could be interesting.
To construct a SEF1 protein expression strain, electro competent cells will be produced. (write more)
To investigate SEF1s role in the regulation of already identified iron proteins, northern blots will be carried out. SEF1 probes will be designed with the use of internal primers and the presence and quantification of mRNA will be carried out in MAC1âˆ†âˆ†, SFU1âˆ†âˆ† and the Wild type with the use of northern blotting. Further analysis could be carried out on already identified iron homeostasis genes if time permits. The results from this experiment will enable us to investigate SEF1s position with the other regulators like RIM101, SFU1 and CBF in the iron regulatory system. Double mutants will be constructed to deduce whether SEF1 is upstream/downstream of these regulators.