Saccharomyces cerevisiae survival against heat stress entails a communication between CCT and cell wall integrity pathway.

The chaperonin TRiC/CCT is cytosolic cylindrical complex of 16 subunits encoded by eight essential genes CCT1-8. It contributes to folding 10% of cellular polypeptides in yeast. The strain carrying substitution point mutation G412E in the equatorial domain of Cct7p resulted in the improper folding of substrates. In this study, the Cct7p mutant exhibited sensitivity to non-optimal growth temperatures and cell wall stressors. Heat shock is known to disrupt cell wall and protein stability in budding yeast. Mitogen-activated protein kinase-mediated cell wall integrity pathway gets activated to compensate the perturbed cell wall. Overexpression of the PKC1 and SLT2 genes of MAPK signaling pathway in mutant rescued the growth and cell division defects. Additionally, the genes of the CWI pathway such as SED1, GFA1, PIR1, and RIM21 are down-regulated. The Cct7p mutant strain (G412E) is unable to withstand the heat stress due to the underlying defects in protein folding and cell wall maintenance. Taken together, our results strongly indicate the interaction between CCT and cell wall integrity pathway.

CSU52, a novel regulator functions as a repressor of L-sorbose utilization in Candida albicans.

BACKGROUND AND OBJECTIVES: Monosomy of chromosome 5 associated with utilization of non-canonical sugar L-sorbose is one of the well-studied aneuploidies in Candida albicans. Stress-induced ploidy changes are crucial determinants for pathogenicity and genetic diversity in C. albicans. The five scattered regulatory regions (A, B, C, 135, and 139) comprising of two functionally redundant pathways (SUR1 and SUR2) were found to be responsible for the growth on L-sorbose. So far, three genes such as CSU51, CSU53 and CSU57 have been identified in region A, region 135 and region C, respectively. In this study we have verified the role of region B in this regulatory pathway. MATERIALS AND METHODS: We employed a combinatorial gene deletion approach to verify the role of region B followed by co-over expression studies and qRT-PCR to identify the regulatory role of this region. RESULTS: We confirmed the role of region B in the regulation of SOU1 gene expression. The qRT-PCR results showed that regulation occurs at transcriptional level along with other two regions in SUR1 pathway. A previously uncharacterized open reading frame in region B has been implicated in this regulation and designated as CSU52. Integrating multiple copies of CSU52 in the genome at tandem, suppresses the growth of recipient strain on L-sorbose, establishing it as a repressor of SOU1 gene. CONCLUSION: This finding completes the identification of regulators in SUR1 pathway. This result paves the way to study the underlying molecular mechanisms of SOU1 gene regulation that in-turn helps to understand stress induced aneuploidy.

Sorbitol and PKC1 overexpression alleviate temperature sensitivity in chaperonin mutants of Saccharomyces cerevisiae.

CCT (Chaperonin containing TCP-1) is a constitutively expressed eukaryotic chaperonin complex involved in the proper folding of proteins like actin and tubulin. Temperature sensitive mutants of CCT complex have been employed in various genetic screens, acting as models to study human CCT, the defects of which are implicated in disease conditions like neurodegeneration. Mutants of CCT complex are sensitive to cell wall stress agents. In this study, we have tested the effects of sorbitol and protein kinase C overexpression on the temperature sensitivity of cct mutants. We report that both the factors alleviated temperature sensitivity of cct mutants, indicating the possible role of CCT in maintaining cell wall integrity in S. cerevisiae.

Systematic truncations of chromosome 4 and their responses to antifungals in Candida albicans.

BACKGROUND: Candida albicans is an opportunistic human fungal pathogen responsible for superficial and systemic life-threatening infections. Treating these infections is challenging as many clinical isolates show increased drug resistance to antifungals. Chromosome (Chr) 4 monosomy was implicated in a fluconazole-resistant mutant. However, exposure to fluconazole adversely affects Candida cells and can generate numerous mutations. Hence, the present study aimed to truncate Chr4 and challenge the generated Candida strains to antifungals and evaluate their role in drug response. RESULTS: Herein, Chr4 was truncated in C. albicans using the telomere-mediated chromosomal truncation method. The resulting eight Candida strains carrying one truncated homolog of Chr4 were tested for response to multiple antifungals. The minimal inhibitory concentration (MIC) for these strains was determined against three classes of antifungals. The MIC values against fluconazole, amphotericin B, and caspofungin were closer to that of the wild type strain. Microdilution assay against fluconazole showed that the mutants and wild type strains had similar sensitivity to fluconazole. The disc diffusion assay against five azoles and two polyenes revealed that the zones of inhibition for all the eight strains were similar to those of the wild type. Thus, none of the generated strains showed any significant resistance to the tested antifungals. However, spot assay exhibited a reasonably high tolerance of a few generated strains with increasing concentrations of fluconazole. CONCLUSION: This analysis suggested that Chr4 aneuploidy might not underlie drug resistance but rather drug tolerance in Candida albicans.

Chaperonin point mutation enhances cadmium endurance in Saccharomyces cerevisiae.

OBJECTIVE: To study the effect of the mutation in conserved G412E in Cct7p subunit of CCT complex on its cellular fate. RESULTS: TriC/CCT is a dynamic multimeric protein that assists in protein folding in an energy-dependent manner. A point mutation in the ATP binding pocket in the equatorial domain of the Cct7p subunit delays the doubling time. The cell size was twice the wild type, and the formation of protein aggregates suggests disturbed folding of the proteins. Upon growing in stressful conditions of arsenous acid and cadmium chloride, the mutant was lethal in As3+ but grew well in Cd2+ with 10.5 µg cadmium uptake mg-1 compared to the wild type. The increased expression of vacuole transporters YCF1 and BPT1 by ten-fold and two-fold in mutant indicates the metal transportation to the vacuole. CONCLUSION: CCT complex was vulnerable to the mutation in G412E in the Cct7p subunit of protein folding molecular machinery. Interestingly, already stressed cells provided robustness against oxidative stress and cadmium sequestration in the vacuole.

The interactome of CCT complex - A computational analysis.

The eukaryotic chaperonin, CCT (Chaperonin Containing TCP1 or TriC-TCP-1 Ring Complex) has been subjected to physical and genetic analyses in S. cerevisiae which can be extrapolated to human CCT (hCCT), owing to its structural and functional similarities with yeast CCT (yCCT). Studies on hCCT and its interactome acquire an additional dimension, as it has been implicated in several disease conditions like neurodegeneration and cancer. We attempt to study its stress response role in general, which will be reflected in the aspects of human diseases and yeast physiology, through computational analysis of the interactome. Towards consolidating and analysing the interactome data, we prepared and compared the unique CCT-interacting protein lists for S. cerevisiae and H. sapiens, performed GO term classification and enrichment studies which provide information on the diversity in CCT interactome, in terms of protein classes in the data set. Enrichment with disease-associated proteins and pathways highlight the medical importance of CCT. Different analyses converge, suggesting the significance of WD-repeat proteins, protein kinases and cytoskeletal proteins in the interactome. The prevalence of proteasomal subunits and ribosomal proteins suggest a possible cross-talk between protein-synthesis, folding and degradation machinery. A network of chaperones and chaperonins that function in combination can also be envisaged from the CCT interactome-Hsp70 interactome analysis.

Defects in Protein Folding Machinery Affect Cell Wall Integrity and Reduce Ethanol Tolerance in S. cerevisiae.

The chaperonin complex CCT/TRiC (chaperonin containing TCP-1/TCP-1 ring complex) participates in the folding of many crucial proteins including actin and tubulin in eukaryotes. Mutations in genes encoding its subunits can affect protein folding and in turn, the physiology of the organism. Stress response in Saccharomyces cerevisiae is important in fermentation reactions and operates through overexpression and underexpression of genes, thus altering the protein profile. Defective protein folding machinery can disturb this process. In this study, the response of cct mutants to stress conditions in general and ethanol in specific was investigated. CCT1 mutants showed decreased resistance to different conditions tested including osmotic stress, metal ions, surfactants, reducing and oxidising agents. Cct1-3 mutant with the mutation in the conserved ATP-binding region showed irreversible defects than other mutants. These mutants were found to have inherent cell wall defects and showed decreased ethanol tolerance. This study reveals that cell wall defects and ethanol sensitivity are linked. Genetic and proteomic analyses showed that the yeast genes RPS6A (ribosomal protein), SCL1 (proteasomal subunit) and TDH3 (glyceraldehyde-3-phosphate dehydrogenase) on overexpression, improved the growth of cct1-3 mutant on ethanol. We propose the breakdown of common stress response pathways caused by mutations in CCT complex and the resulting scarcity of functional stress-responsive proteins, affecting the cell's defence against different stress agents in cct mutants. Defective cytoskeleton and perturbed cell wall integrity reduce the ethanol tolerance in the mutants which are rescued by the extragenic suppressors.

Significance of αThr-349 in the catalytic sites of Escherichia coli ATP synthase.

This paper describes the role of α-subunit VISIT-DG sequence residue αThr-349 in the catalytic sites of Escherichia coli F1Fo ATP synthase. X-ray structures show the highly conserved αThr-349 in the proximity (2.68 Å) of the conserved phosphate binding residue ßR182 in the phosphate binding subdomain. αT349A, -D, -Q, and -R mutations caused 90-100-fold losses of oxidative phosphorylation and reduced ATPase activity of F1Fo in membranes. Double mutation αT349R/ßR182A was able to partially compensate for the absence of known phosphate binding residue ßR182. Azide, fluoroaluminate, and fluoroscandium caused insignificant inhibition of αT349A, -D, and -Q mutants, slight inhibition of the αT349R mutant, partial inhibition of the αT349R/ßR182A double mutant, and complete inhibition of the wild type. Whereas NBD-Cl (7-chloro-4-nitrobenzo-2-oxa-1,3-diazole) inhibited wild-type ATPase and its αT349A, -D, -R, and -Q mutants essentially completely, ßR182A ATPase and double mutant αT349A/ßR182A were inhibited partially. Inhibition characteristics supported the conclusion that NBD-Cl reacts in ßE (empty) catalytic sites, as shown previously by X-ray structure analysis. Phosphate protected against NBD-Cl inhibition in the wild type, αT349R, and double mutant αT349R/ßR182A but not in αT349A, αT349D, or αT349Q. The results demonstrate that αThr-349 is a supplementary residue involved in phosphate binding and transition state stabilization in ATP synthase catalytic sites through its interaction with ßR182.

Candida infections and their prevention.

Infections caused by Candida species have been increased dramatically worldwide due to the increase in immunocompromised patients. For the prevention and cure of candidiasis, several strategies have been adopted at clinical level. Candida infected patients are commonly treated with a variety of antifungal drugs such as fluconazole, amphotericin B, nystatin, and flucytosine. Moreover, early detection and speciation of the fungal agents will play a crucial role for administering appropriate drugs for antifungal therapy. Many modern technologies like MALDI-TOF-MS, real-time PCR, and DNA microarray are being applied for accurate and fast detection of the strains. However, during prolonged use of these drugs, many fungal pathogens become resistant and antifungal therapy suffers. In this regard, combination of two or more antifungal drugs is thought to be an alternative to counter the rising drug resistance. Also, many inhibitors of efflux pumps have been designed and tested in different models to effectively treat candidiasis. However, most of the synthetic drugs have side effects and biomedicines like antibodies and polysaccharide-peptide conjugates could be better alternatives and safe options to prevent and cure the diseases. Furthermore, availability of genome sequences of Candida albicans and other non-albicans strains has made it feasible to analyze the genes for their roles in adherence, penetration, and establishment of diseases. Understanding the biology of Candida species by applying different modern and advanced technology will definitely help us in preventing and curing the diseases caused by fungal pathogens.

Candida albicans: A Model Organism for Studying Fungal Pathogens.

Candida albicans is an opportunistic human fungal pathogen that causes candidiasis. As healthcare has been improved worldwide, the number of immunocompromised patients has been increased to a greater extent and they are highly susceptible to various pathogenic microbes and C. albicans has been prominent among the fungal pathogens. The complete genome sequence of this pathogen is now available and has been extremely useful for the identification of repertoire of genes present in this pathogen. The major challenge is now to assign the functions to these genes of which 13% are specific to C. albicans. Due to its close relationship with yeast Saccharomyces cerevisiae, an edge over other fungal pathogens because most of the technologies can be directly transferred to C. albicans from S. cerevisiae and it is amenable to mutation, gene disruption, and transformation. The last two decades have witnessed enormous amount of research activities on this pathogen that leads to the understanding of host-parasite interaction, infections, and disease propagation. Clearly, C. albicans has emerged as a model organism for studying fungal pathogens along with other two fungi Aspergillus fumigatus and Cryptococcus neoformans. Understanding its complete life style of C. albicans will undoubtedly be useful for developing potential antifungal drugs and tackling Candida infections. This will also shed light on the functioning of other fungal pathogens.

Functional Subunits of Eukaryotic Chaperonin CCT/TRiC in Protein Folding.

Molecular chaperones are a class of proteins responsible for proper folding of a large number of polypeptides in both prokaryotic and eukaryotic cells. Newly synthesized polypeptides are prone to nonspecific interactions, and many of them make toxic aggregates in absence of chaperones. The eukaryotic chaperonin CCT is a large, multisubunit, cylindrical structure having two identical rings stacked back to back. Each ring is composed of eight different but similar subunits and each subunit has three distinct domains. CCT assists folding of actin, tubulin, and numerous other cellular proteins in an ATP-dependent manner. The catalytic cooperativity of ATP binding/hydrolysis in CCT occurs in a sequential manner different from concerted cooperativity as shown for GroEL. Unlike GroEL, CCT does not have GroES-like cofactor, rather it has a built-in lid structure responsible for closing the central cavity. The CCT complex recognizes its substrates through diverse mechanisms involving hydrophobic or electrostatic interactions. Upstream factors like Hsp70 and Hsp90 also work in a concerted manner to transfer the substrate to CCT. Moreover, prefoldin, phosducin-like proteins, and Bag3 protein interact with CCT and modulate its function for the fine-tuning of protein folding process. Any misregulation of protein folding process leads to the formation of misfolded proteins or toxic aggregates which are linked to multiple pathological disorders.

Dietary bioflavonoids inhibit Escherichia coli ATP synthase in a differential manner.

The aim of this study was to determine if the dietary benefits of bioflavonoids are linked to the inhibition of ATP synthase. We studied the inhibitory effect of 17 bioflavonoid compounds on purified F1 or membrane bound F1Fo E. coli ATP synthase. We found that the extent of inhibition by bioflavonoid compounds was variable. Morin, silymarin, baicalein, silibinin, rimantadin, amantidin, or, epicatechin resulted in complete inhibition. The most potent inhibitors on molar scale were morin (IC50 approximately 0.07 mM)>silymarin (IC50 approximately 0.11 mM)>baicalein (IC50 approximately 0.29 mM)>silibinin (IC50 approximately 0.34 mM)>rimantadin (IC50 approximately 2.0 mM)>amantidin (IC50 approximately 2.5 mM)>epicatechin (IC50 approximately 4.0 mM). Inhibition by hesperidin, chrysin, kaempferol, diosmin, apigenin, genistein, or rutin was partial in the range of 40-60% and inhibition by galangin, daidzein, or luteolin was insignificant. The main skeleton, size, shape, geometry, and position of functional groups on inhibitors played important role in the effective inhibition of ATP synthase. In all cases inhibition was found fully reversible and identical in both F1Fo membrane preparations and isolated purified F1. ATPase and growth assays suggested that the bioflavonoid compounds used in this study inhibited F1-ATPase as well as ATP synthesis nearly equally, which signifies a link between the beneficial effects of dietary bioflavonoids and their inhibitory action on ATP synthase.

Human fungal pathogen Candida albicans in the postgenomic era: an overview.

Candida albicans is an opportunistic human fungal pathogen and is responsible for candidiasis. Owing to the improvement in healthcare, the number of immunocompromised patients in hospitals has increased worldwide and these individuals are susceptible to infections caused by many pathogenic microbes, among which C. albicans is one of the major players. Currently, the complete genome sequence of this pathogen is available and the size of this was estimated to be of 16 Mb. Annotation of C. albicans genome revealed that there are 6114 open reading frames (ORFs), of which 774 are specific to C. albicans. This poses a challenge as well as an opportunity to the Candida community to understand the functions of the unknown genes, especially those specific to C. albicans. Efforts have been made by the Candida community to systematically delete the ORFs and assign the functions. This will, in turn, help in understanding the biology of C. albicans and its interactions with animals as well as humans, and better drugs can be developed to treat Candida infections. In this article, we review updates on the Candida biology in the context of the availability of the genome sequence, its functional analysis and anti-Candida therapy. Finally, in the light of present trends in Candida research and current challenges, various opportunities are identified and suggestions are made.

Overexpressed ribosomal proteins suppress defective chaperonins in Saccharomyces cerevisiae.

The chaperonin Cct complex of the yeast Saccharomyces cerevisiae is composed of eight different subunits encoded by eight essential genes, CCT1-CCT8. This Cct complex is responsible for the folding of a number of proteins including actin and tubulin. We have isolated and characterized 22 multicopy suppressors of the temperature-sensitive allele, cct4-1, which encodes an altered protein with a G345D replacement that diminishes ATP hydrolysis. Fourteen of the suppressors encode ribosomal proteins, four have roles in ribosome biogenesis, two have phosphatase activities, one is involved in protein synthesis and one of the suppressors corresponded to Cct4p. Some of the suppressors also acted on certain cct1, cct2, cct3 and cct6 mutations. We suggest that certain overexpressed ribosomal and other proteins can act as weak chaperones, phenotypically alleviating the partial defects of mutationally altered Cct subunits.

Chromosome instability and unusual features of some widely used strains of Candida albicans.

Electrophoretic karyotyping of the Candida albicans revealed a different migration pattern of ChR in three different stocks of the sequencing strain SC5314. In one stock, the high instability of ChR size prevented the migration of ChR as a compact band; ChR appeared, instead, as a smear. In some stocks, ChR and/or Ch1 ploidy diminished, suggesting mixed populations of disomic and monosomic cells. Similarly, some stocks of widely used derivatives CAI4 and BWP17 contained smearing of ChR. In addition, the most manipulated strain in the lineage of SC5314, the last derivative, BWP17, acquired an increase in the size of Ch7b and revealed an unusual property. BWP17 did not tolerate a well-established procedure of telomere-mediated fragmentation of a chromosome; the remaining intact homologue always duplicated. We suggest that some stocks of SC5314 are unstable and that BWP17 may not be appropriate for general studies. Instead of BWP17 or CAI4, we recommend using for general research CAF4-2, which is a relatively stable Ura- derivative, and which has been successfully used for more than a decade in our laboratory.

5-fluoro-orotic acid induces chromosome alterations in genetically manipulated strains of Candida albicans.

We previously reported the occurrence of chromosome alterations in a Candida albicans prototrophic strain 3153A treated with 5-fluoro-orotic acid (5-FOA). In this study we investigated the mutagenic properties of 5-FOA with two derivatives of C. albicans strain CAF4-2 (ura3/ura3), each containing an ectopic copy of URA3 gene (ura3/ ura3 URA3) on a different chromosome. As expected, after the ura3/ura3 URA3 constructs were applied to 5-FOA containing solid medium, the "pop-outs" that lost URA3 appeared. However most of the "pop-outs" acquired various chromosome alterations. Thus constructs exposed to 5-FOA should be examined for chromosome alterations or the use of 5-FOA should be avoided.

Loss and gain of chromosome 5 controls growth of Candida albicans on sorbose due to dispersed redundant negative regulators.

A reversible decrease or increase of Candida albicans chromosome copy number was found to be a prevalent means of survival of this opportunistic pathogen, under conditions that kill cells or inhibit their propagation. The utilization of a secondary carbon source, l-sorbose, by reversible loss of chromosome 5, serves as a model system. We have determined that an approximately 209-kbp portion of the right arm of chromosome 5 contains at least five spatially separated, functionally redundant regions that control utilization of l-sorbose. The regions bear no structural similarity among themselves, and four of them contain sequences that bear no similarity with any known sequence. We identified a regulatory gene in region A that encodes a helix-loop-helix protein. Most important, the multiple redundant regulators scattered along chromosome 5 explain, in a simple, elegant way, why the loss of the entire homologue is usually required for growth on sorbose. Thus, an entire chromosome acts as a single regulatory unit, a feature not previously considered. Our finding appears to be a paradigm for the control of other phenotypes in C. albicans that also depend on chromosome loss, thus implying that C. albicans genes are not distributed randomly among different chromosomes.

Physiological effects of unassembled chaperonin Cct subunits in the yeast Saccharomyces cerevisiae.

Eukaryotic chaperonins, the Cct complexes, are assembled into two rings, each of which is composed of a stoichiometric array of eight different subunits, which are denoted Cct1p-Cct8p. Overexpression of a single CCT gene in Saccharomyces cerevisiae causes an increase of the corresponding Cct subunit, but not of the Cct complex. Nevertheless, overexpression of certain Cct subunits, especially CCT6, suppresses a wide range of abnormal phenotypes, including those caused by the diverse types of conditional mutations tor2-21, lst8-2 and rsp5-9 and those caused by the concomitant overexpression of Sit4p and Sap155p. The examination of 73 altered forms of Cct6p revealed that the cct6-24 mutation, containing GDGTT --> AAAAA replacements of the conserved ATP-binding motif, was unable to suppress any of these traits, although the cct6-24 allele was completely functional for growth. These results provide evidence for functional differences among Cct subunits and for physiological properties of unassembled subunits. We suggest that the suppression is due to the competition of specific Cct subunits for activities that normally modify various cellular components. Furthermore, we also suggest that the Cct subunits can act as suppressors only in certain states, such as when associated with ATP.

Determination of gaps by contig alignment with telomere-mediated chromosomal fragmentation in Candida albicans.

We have adopted a method of telomere-mediated chromosome fragmentation in order to demonstrate the alignment of contigs and determination of gaps. We established the order and orientation of four contigs of Candida albicans chromosome 5 and determined the sizes of three gaps between these contigs. We confirmed this proposed alignment of contigs, as well as gap sizes, by sequencing one gap and analyzing three mega deletions of approximately 41 kbp, 58 kbp, and 77 kbp, which covered two other gaps. These gaps could be also conveniently sequenced, which is an important step in establishing a complete sequence. The combined length of contigs and gaps covered approximately 422 kbp, which is one third of chromosome 5. Telomere-mediated chromosome fragmentation, used here for the first time to align the contigs of C. albicans and determine the gaps, proved to be a reliable method. The method could be helpful in sequencing projects of other diploid organisms, in particular those in which centromeres have not been identified. In addition, our approach can be used to assign any contig to a chromosome, or to induce the loss of a specific chromosome.

Role of the 14-3-3 protein in carbon metabolism of the pathogenic yeast Candida albicans.

We previously demonstrated that the pathogenic yeast Candida albicans effectively adapts to utilize L-sorbose (Sou+) by a novel mechanism based on the loss of one copy of chromosome 5, probably due to the reduction of copy number of a negative regulator located on this chromosome. We report here another negative regulator of L-sorbose utilization, an orthologue of the Saccharomyces cerevisiae BMH1 gene, which encodes the evolutionarily conserved protein 14-3-3. This essential gene is located on chromosome 1, does not have paralogues, and is supposedly a component of the regulatory network. Experiments involving disruption of one allele of BMH1 and overexpression of BMH1 revealed that BMH1 represses the transcription of SOU1, which is responsible for the utilization of L-sorbose. Although the exact mechanism of the interaction between BMH1 and SOU1 is not known, it is clear that the control is based on the ratio of gene copy number, and that BMH1 does not control the loss of chromosome 5, the major mechanism producing Sou+ mutants. We propose that function of BMH1 as a negative regulator of SOU1 contributes to a general cellular homeostasis. This is a first report on the role of the C. albicans essential gene BMH1 as a negative regulator of the utilization of secondary carbon source in yeast, which further substantiates the involvement of 14-3-3 proteins in diverse functions.