Respiratory-deficient Strains Research Articles

Listeria monocytogenes requires cellular respiration for NAD+ regeneration and pathogenesis.

Cellular respiration is essential for multiple bacterial pathogens and a validated antibiotic target. In addition to driving oxidative phosphorylation, bacterial respiration has a variety of ancillary functions that obscure its contribution to pathogenesis. We find here that the intracellular pathogen Listeria monocytogenes encodes two respiratory pathways which are partially functionally redundant and indispensable for pathogenesis. Loss of respiration decreased NAD+ regeneration, but this could be specifically reversed by heterologous expression of a water-forming NADH oxidase (NOX). NOX expression fully rescued intracellular growth defects and increased L. monocytogenes loads >1000-fold in a mouse infection model. Consistent with NAD+ regeneration maintaining L. monocytogenes viability and enabling immune evasion, a respiration-deficient strain exhibited elevated bacteriolysis within the host cytosol and NOX expression rescued this phenotype. These studies show that NAD+ regeneration represents a major role of L. monocytogenes respiration and highlight the nuanced relationship between bacterial metabolism, physiology, and pathogenesis.

Cytoduction and Plasmiduction in Yeast.

Cytoduction, and a related technique referred to as plasmiduction, have facilitated substantial advancements in the field of yeast prion biology by providing a streamlined method of transferring prions from one yeast strain to another. Prions are cytoplasmic elements consisting of aggregated misfolded proteins, and as such, they exhibit non-Mendelian patterns of inheritance. While prion transfer through mating and sporulation, or through protein transformation, is possible, these approaches yield non-isogenic strains or are technically complex, respectively. Cytoduction is a mating-based technique that takes advantage of a kar1 mutation with impaired nuclear fusion (karyogamy). It is a straightforward method for introducing a prion to any yeast strain (referred to as the recipient) by mating it with a donor strain containing the prion of interest. The only absolute requirement is that one of these two strains (donor or recipient) must carry the kar1-1 mutation to limit nuclear fusion. The resulting cytoductant contains the original nucleus of the recipient strain, but a cytoplasm reflecting a mix of all elements from the donor and the recipient. Modifications to the basic cytoduction strategy provide several options for successful cytoduction, including when working with slow growing or respiratory deficient strains. A significant advantage of the plasmiduction protocol presented is the ability to transfer a plasmid encoding a fluorescently tagged version of the prion protein, which allows for the direct verification of the prion state through visual protein aggregates. Graphic abstract: Transfer of Yeast Cytoplasmic Elements such as Prions using Cytoduction.

ВТОРИННИЙ РІСТ У ДИХАЛЬНО-ДЕФІЦИТНОМУ ШТАМІ ДРІЖДЖІВ SACCHAROMYCES CEREVISIAE ВНАСЛІДОК ДЕЛЕЦІЇ ГЕНА YKU70

It is known that significant causes of malignant tumors are destabilization of the nuclear genome and mitochondrial dysfunction. Adaptive regrowth in yeast colonies (the appearance of cell subpopulations more adapted to unfavorable conditions under conditions of the death of the original culture) is used as a model of the initial stages of carcinogenesis. To study the features of the formation of adaptive regrowth, a reparationdefective and respiratory-deficient yeast strain of Saccharomyces cerevisiae was created. The thermosensitive mutation in the yku70 gene was used as an inducer of nuclear genome instability (at 37 оC it causes cell cycle arrest due to a reduction of the length of telomeric regions of chromosomes). Damage to the mitochondrial DNA of the ∆yku70 strain led to its respiratory deficiency (petite mutation). The isolated petite mutant ∆yku70 strain was cultured at optimal 28 оC and restrictive 37 оC temperatures, the state of the cell suspension was evaluated by light and fluorescence microscopy, to determine the viability of cells was used the analysis of microcolonies growth. Isolation of adaptive regrowth clones and analysis of their properties by the method of serial dilutions were conducted. To assess the genome stability of selected clones of adaptive regrowth, PCR analysis of the microsatellite sequences YOR267C, SC8132X, SCPTSY7 was conducted. When culturing the petite mutant of the strain ∆yku70 at a restrictive temperature of 37 оC for 7 days, the formation of viable subpopulations was detected, which can overcome the arrest of the cell cycle in the G2 / M phase. Further analysis of the isolated clones of adaptive regrowth showed that they differ in cell survival at restrictive temperature, resistance to UV radiation and the ability to form adaptive regrowth on colonies. In the analysis of microsatellite repeats in adaptive regrowth clones, no manifestations of instability of the studied sequences were detected.

Flavin-based metabolic cycles are integral features of growth and division in single yeast cells

The yeast metabolic cycle (YMC) is a fascinating example of biological organization, in which cells constrain the function of specific genetic, protein and metabolic networks to precise temporal windows as they grow and divide. However, understanding the intracellular origins of the YMC remains a challenging goal, as measuring the oxygen oscillations traditionally associated with it requires the use of synchronized cultures growing in nutrient-limited chemostat environments. To address these limitations, we used custom-built microfluidic devices and time-lapse fluorescence microscopy to search for metabolic cycling in the form of endogenous flavin fluorescence in unsynchronized single yeast cells. We uncovered robust and pervasive metabolic cycles that were synchronized with the cell division cycle (CDC) and oscillated across four different nutrient conditions. We then studied the response of these metabolic cycles to chemical and genetic perturbations, showing that their phase synchronization with the CDC can be altered through treatment with rapamycin, and that metabolic cycles continue even in respiratory deficient strains. These results provide a foundation for future studies of the physiological importance of metabolic cycles in processes such as CDC control, metabolic regulation and cell aging.

Recombinant Diploid Saccharomyces cerevisiae Strain Development for Rapid Glucose and Xylose Co-Fermentation

Cost-effective production of cellulosic ethanol requires robust microorganisms for rapid co-fermentation of glucose and xylose. This study aims to develop a recombinant diploid xylose-fermenting Saccharomyces cerevisiae strain for efficient conversion of lignocellulosic biomass sugars to ethanol. Episomal plasmids harboring codon-optimized Piromyces sp. E2 xylose isomerase (PirXylA) and Orpinomyces sp. ukk1 xylose (OrpXylA) genes were constructed and transformed into S. cerevisiae. The strain harboring plasmids with tandem PirXylA was favorable for xylose utilization when xylose was used as the sole carbon source, while the strain harboring plasmids with tandem OrpXylA was beneficial for glucose and xylose cofermentation. PirXylA and OrpXylA genes were also individually integrated into the genome of yeast strains in multiple copies. Such integration was beneficial for xylose alcoholic fermentation. The respiration-deficient strain carrying episomal or integrated OrpXylA genes exhibited the best performance for glucose and xylose co-fermentation. This was partly attributed to the high expression levels and activities of xylose isomerase. Mating a respiration-efficient strain carrying the integrated PirXylA gene with a respiration-deficient strain harboring integrated OrpXylA generated a diploid recombinant xylose-fermenting yeast strain STXQ with enhanced cell growth and xylose fermentation. Co-fermentation of 162 g L−1 glucose and 95 g L−1 xylose generated 120.6 g L−1 ethanol in 23 h, with sugar conversion higher than 99%, ethanol yield of 0.47 g g−1, and ethanol productivity of 5.26 g L−1·h−1.

Bacterial xylose isomerases from the mammal gut Bacteroidetes cluster function in Saccharomyces cerevisiae for effective xylose fermentation.

BackgroundXylose isomerase (XI) catalyzes the conversion of xylose to xylulose, which is the key step for anaerobic ethanolic fermentation of xylose. Very few bacterial XIs can function actively in Saccharomyces cerevisiae. Here, we illustrate a group of XIs that would function for xylose fermentation in S. cerevisiae through phylogenetic analysis, recombinant yeast strain construction, and xylose fermentation.ResultsPhylogenetic analysis of deposited XI sequences showed that XI evolutionary relationship was highly consistent with the bacterial taxonomic orders and quite a few functional XIs in S. cerevisiae were clustered with XIs from mammal gut Bacteroidetes group. An XI from Bacteroides valgutus in this cluster was actively expressed in S. cerevisiae with an activity comparable to the fungal XI from Piromyces sp. Two XI genes were isolated from the environmental metagenome and they were clustered with XIs from environmental Bacteroidetes group. These two XIs could not be expressed in yeast with activity. With the XI from B. valgutus expressed in S. cerevisiae, background yeast strains were optimized by pentose metabolizing pathway enhancement and adaptive evolution in xylose medium. Afterwards, more XIs from the mammal gut Bacteroidetes group, including those from B. vulgatus, Tannerella sp. 6_1_58FAA_CT1, Paraprevotella xylaniphila and Alistipes sp. HGB5, were individually transformed into S. cerevisiae. The known functional XI from Orpinomyces sp. ukk1, a mammal gut fungus, was used as the control. All the resulting recombinant yeast strains were able to ferment xylose. The respiration-deficient strains harboring B. vulgatus and Alistipes sp. HGB5 XI genes respectively obtained specific xylose consumption rate of 0.662 and 0.704 g xylose gcdw−1 h−1, and ethanol specific productivity of 0.277 and 0.283 g ethanol gcdw−1 h−1, much comparable to those obtained by the control strain carrying Orpinomyces sp. ukk1 XI gene.ConclusionsThis study demonstrated that XIs clustered in the mammal gut Bacteroidetes group were able to be expressed functionally in S. cerevisiae and background strain anaerobic adaptive evolution in xylose medium is essential for the screening of functional XIs. The methods outlined in this paper are instructive for the identification of novel XIs that are functional in S. cerevisiae.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0253-1) contains supplementary material, which is available to authorized users.

Evaluation of the tolerance of acetic acid and 2-furaldehyde on the growth of Pichia stipitis and its respiratory deficient

The use of lignocellulosic residues for ethanol production is limited by toxic compounds in fermenting yeasts present in diluted acid hydrolysates like acetic acid and 2-furaldehyde. The respiratory deficient phenotype gives the cell the ability to resist several toxic compounds. So the aim of this work was to evaluate the tolerance to toxic compounds present in lignocellulosic hydrolysates like acetic acid and 2-furaldehyde in Pichia stipitis and its respiratory deficient strains. The respiratory deficient phenotype was induced by exposure to chemical agents such as acriflavine, acrylamide and rhodamine; 23 strains were obtained. The selection criterion was based on increasing specific ethanol yield (gethanolg(-1)biomass) with acetic acid and furaldehyde tolerance. The screening showed that P. stipitis NRRL Y-7124 ACL 2-1RD (lacking cytochrome c), obtained using acrylamide, presented the highest specific ethanol production rate (1.82gg(-1)h(-1)). Meanwhile, the ACF8-3RD strain showed the highest acetic acid tolerance (7.80gL(-1)) and the RHO2-3RD strain was able to tolerate up to 1.5gL(-1) 2-furaldehyde with a growth and ethanol production inhibition of 23 and 22%, respectively. The use of respiratory deficient yeast phenotype is a strategy for ethanol production improvement in a medium with toxic compounds such as hydrolysed sugarcane bagasse amongst others.

Characteristics of the high malic acid production mechanism in Saccharomyces cerevisiae sake yeast strain No. 28

We characterized a high malic acid production mechanism in sake yeast strain No. 28. No considerable differences in the activity of the enzymes that were involved in malic acid synthesis were observed between strain No. 28 and its parent strain, K1001. However, compared with strain K1001, which actively took up rhodamine 123 during staining, the cells of strain No. 28 were only lightly stained, even when cultured in high glucose concentrations. In addition, malic acid production by the respiratory-deficient strain of K1001 was 2.5-fold higher than that of the wild-type K1001 and wild-type No. 28. The findings of this study demonstrated that the high malic acid production by strain No. 28 is attributed to the suppression of mitochondrial activity.

Improvement of xylose fermentation in respiratory-deficient xylose-fermenting Saccharomyces cerevisiae

Effective conversion of xylose in lignocelluloses is expected to reduce the production cost of second-generation biofuels significantly. The factors affecting xylose fermentation in Saccharomyces cerevisiae that express xylose reductase–xylitol dehydrogenase (XR–XDH) are studied. Although overproduction of non-oxidative pentose phosphate pathway significantly increased the aerobic-specific growth rate on xylose and slightly improved conversion of xylose to ethanol under oxygen-limited conditions, the elimination of respiration by deleting cytochrome C oxidase subunit IV gene impeded aerobic growth on xylose. However, the adaptive evolution of the respiratory-deficient strain with an NADP +-preferring XDH mutant in xylose media dramatically improved its xylose-fermenting ability. The specific growth rate, ethanol yield, and xylitol yield of the evolved strain on xylose were 0.06 h −1, 0.39 g g −1, and 0.13 g g −1 consumed xylose, respectively. Similar to anaerobic fermentation, the evolved strain exhibited accumulated ethanol rather than recycled it under aerobic conditions.

Energetic limits to metabolic flexibility: responses of Saccharomyces cerevisiae to glucose–galactose transitions

Glucose is the favoured carbon source for Saccharomyces cerevisiae, and the Leloir pathway for galactose utilization is only induced in the presence of galactose during glucose-derepressed conditions. The goal of this study was to investigate the dynamics of glucose-galactose transitions. To this end, well-controlled, glucose-limited chemostat cultures were switched to galactose-excess conditions. Surprisingly, galactose was not consumed upon a switch to galactose excess under anaerobic conditions. However, the transcripts of the Leloir pathway were highly increased upon galactose excess under both aerobic and anaerobic conditions. Protein and enzyme-activity assays showed that impaired galactose consumption under anaerobiosis coincided with the absence of the Leloir-pathway proteins. Further results showed that absence of protein synthesis was not caused by glucose-mediated translation inhibition. Analysis of adenosine nucleotide pools revealed a fast decrease of the energy charge after the switch from glucose to galactose under anaerobic conditions. Similar results were obtained when glucose-galactose transitions were analysed under aerobic conditions with a respiratory-deficient strain. It is concluded that under fermentative conditions, the energy charge was too low to allow synthesis of the Leloir proteins. Hence, this study conclusively shows that the intracellular energy status is an important factor in the metabolic flexibility of S. cerevisiae upon changes in its environment.

Respiratory Deficiency Enhances the Sensitivity of the Pathogenic Fungus Candida to Photodynamic Treatment

Mucosal infections caused by the pathogenic fungus Candida are a significant infectious disease problem and are often difficult to eradicate because of the high frequency of resistance to conventional antifungal agents. Photodynamic treatment (PDT) offers an attractive therapeutic alternative. Previous studies demonstrated that filamentous forms and biofilms of Candida albicans were sensitive to PDT using Photofrin as a photosensitizer. However, early stationary phase yeast forms of C. albicans and Candida glabrata were not adversely affected by treatment. We report that the cationic porphyrin photosensitizer meso-tetra (N-methyl-4-pyridyl) porphine tetra tosylate (TMP-1363) is effective in PDT against yeast forms of C. albicans and C. glabrata. Respiratory-deficient (RD) strains of C. albicans and C. glabrata display a pleiotropic resistance pattern, including resistance to members of the azole family of antifungals, the salivary antimicrobial peptides histatins and other types of toxic stresses. In contrast to this pattern, RD mutants of both C. albicans and C. glabrata were significantly more sensitive to PDT compared to parental strains. These data suggest that intact mitochondrial function may provide a basal level of anti-oxidant defense against PDT-induced phototoxicity in Candida, and reveals pathways of resistance to oxidative stress that can potentially be targeted to increase the efficacy of PDT against this pathogenic fungus.

Membrane potential governs lateral segregation of plasma membrane proteins and lipids in yeast

The plasma membrane potential is mainly considered as the driving force for ion and nutrient translocation. Using the yeast Saccharomyces cerevisiae as a model organism, we have discovered a novel role of the membrane potential in the organization of the plasma membrane. Within the yeast plasma membrane, two non-overlapping sub-compartments can be visualized. The first one, represented by a network-like structure, is occupied by the proton ATPase, Pma1, and the second one, forming 300-nm patches, houses a number of proton symporters (Can1, Fur4, Tat2 and HUP1) and Sur7, a component of the recently described eisosomes. Evidence is presented that sterols, the main lipid constituent of the plasma membrane, also accumulate within the patchy compartment. It is documented that this compartmentation is highly dependent on the energization of the membrane. Plasma membrane depolarization causes reversible dispersion of the H(+)-symporters, not however of the Sur7 protein. Mitochondrial mutants, affected in plasma membrane energization, show a significantly lower degree of membrane protein segregation. In accordance with these observations, depolarized membranes also considerably change their physical properties (detergent sensitivity).

High-efficiency biolistic transformation of Chlamydomonas mitochondria can be used to insert mutations in complex I genes

Mitochondrial transformation of Chlamydomonas reinhardtii has been optimized by using a particle-gun device and cloned mitochondrial DNA or PCR fragments. A respiratory-deficient strain lacking a 1.2-kb mitochondrial DNA region including the left telomere and part of the cob gene could be rescued as well as a double-frameshift mutant in the mitochondrial cox1 and nd1 genes. High transformation efficiency has been achieved (100-250 transformants per microgram of DNA), the best results being obtained with linearized plasmid DNA. Molecular analysis of the transformants suggests that the right telomere sequence can be copied to reconstruct the left telomere by recombination. In addition, both nondeleterious and deleterious mutations could be introduced. Myxothiazol-resistant transformants have been created by introducing a nucleotide substitution into the cob gene. Similarly, an in-frame deletion of 23 codons has been created in the nd4 mitochondrial gene of both the deleted and frameshift recipient strains. These 23 codons are believed to encode the first transmembrane segment of the ND4 protein. This Deltand4 mutation causes a misassembly of complex I, with the accumulation of a subcomplex that is 250-kDa smaller than the wild-type complex I. The availability of efficient mitochondrial transformation in Chlamydomonas provides an invaluable tool for the study of mitochondrial biogenesis and, more specifically, for site-directed mutagenesis of mitochondrially encoded subunits of complex I, of special interest because the yeast Saccharomyces cerevisiae, whose mitochondrial genome can be manipulated virtually at will, is lacking complex I.

Shared molecular characteristics of successfully transformed mitochondrial genomes in Chlamydomonas reinhardtii

Three types of respiratory deficient mitochondrial strains have been reported in Chlamydomonas reinhardtii: a deficiency due to (i) two base substitutions causing an amino acid change in the apocytochrome b (COB) gene (i.e., strain named dum-15), (ii) one base deletion in the COXI gene (dum-19), or (iii) a large deletion extending from the left terminus of the genome to somewhere in the COB gene (dum-1, -14, and -16). We found that these respiratory deficient strains of C. reinhardtii can be divided into two groups: strains that are constantly transformable and those could not be transformed in our experiments. All transformable mitochondrial strains were limited to the type that has a large deletion in the left arm of the genome. For these mitochondria, transformation was successful not only with purified intact mitochondrial genomes but also with DNA-constructs containing the compensating regions. In comparison, mitochondria of all the non-transformable strains have both of their genome termini intact, leading us to speculate that mitochondria lacking their left genome terminus have unstable genomes and might have a higher potential for recombination. Analysis of mitochondrial gene organization in the resulting respiratory active transformants was performed by DNA sequencing and restriction enzyme digestion. Such analysis showed that homologous recombination occurred at various regions between the mitochondrial genome and the artificial DNA-constructs. Further analysis by Southern hybridization showed that the wild-type genome rapidly replaces the respiratory deficient monomer and dimer mitochondrial genomes, while the E. coli vector region of the artificial DNA-construct likely does not remain in the mitochondria.

Assembly factors of F1Fo‐ATP synthase across genomes

Work with respiration-deficient strains of Saccharomyces cerevisiae has provided evidence that assembly of the mitochondrial ATP synthase is dependent on proteins that serve substrate-specific, chaperone-type functions: Atp10p, Atp11p, Atp12p, Atp22p, and Fmc1p. Atp11p and Atp12p mediate the formation of the F1 moiety via interaction with subunits F1-beta and F1-alpha, respectively. The role of Fmc1p is less clear. Atp10p and Atp22p are essential for the formation of the F(O) part, during which Atp10p assists in the incorporation of the F(O)-a subunit. Here we present a comprehensive analysis of ATP synthase assembly factors from all available genomes. The mechanism of the F1 assembly is preserved in all eukaryotic lineages that are capable of ATP synthesis via oxidative phosphorylation and requires Atp11p and Atp12p. Conversely, composition of the F(O) part as well as its assembly is more versatile. We found two distinct subtypes of the F(O)-a subunit, one of which seems to be dependent on the action of Atp10p while the other does not. Restricted occurrence of Fmc1p and Atp22p suggests the existence of lineage-specific assembly factors. Our phylogenetic data served as a source for comparative sequence analysis, which identified evolutionarily conserved residues, putative functional domains and their basic structural features for Atp10p, Atp11p, and Atp12p orthologs. These results provide the basis for detailed molecular analysis of the ATP synthase-specific chaperones.

Transcription of TIM9 , a new factor required for the petite-positive phenotype of Saccharomyces cerevisiae , is defective in spt7 mutants

TIM9 has been identified as an additional novel gene required for the petite-positive phenotype in Saccharomyces cerevisiae. tim9-1 was obtained through a screen for respiratory-deficient strains that are unable to survive in the absence of mitochondrial DNA. A point mutation found in the tim9-1 coding region converts codon 71 from Gly to Arg. Examination of genes encoding other Tim components indicated that the temperature-conditional alleles of essential genes for the viability of S. cerevisiae, TIM9, TIM10 and TIM12, are required for petite survival, while deletion of TIM8 and TIM13 has no notable effect on petite cell viability. Northern hybridization results suggested that the Spt7 transcription factor is strictly involved in transcription of TIM9 and that the synergistic lethality of tim9-1/spt7Delta dual mutations is due to the deficiency of TIM9 transcription together with defective function of the tim9-1 protein.

H(2)O(2) generation in Saccharomyces cerevisiae respiratory pet mutants: effect of cytochrome c.

Impaired electron transport chain function has been related to increases in reactive oxygen species (ROS) generation. Here we analyzed different pet mutants of Saccharomyces cerevisiae in order to determine the relative contribution of respiratory chain components in ROS generation and removal. We found that the maintenance of respiration strongly prevented mitochondrial H(2)O(2) release and increased cellular H(2)O(2) removal. Among all respiratory-deficient strains analyzed, cells lacking cytochrome c (cyc3 point mutants) presented the highest level of H(2)O(2) synthesis, indicating that the absence of functional cytochrome c in mitochondria leads to oxidative stress. This finding was supported by the presence of high levels of catalase and peroxidase activity despite the lack of respiration. Furthermore, the addition of exogenous cytochrome c to isolated yeast mitoplasts significantly reduced H(2)O(2) detection in a manner enhanced by cytochrome c reduction and the presence of a functional respiratory chain. Together, our results indicate that the maintenance of electron transport by cytochrome c prevents ROS generation by the respiratory chain.

Analysis of respiratory mutants reveals new aspects of the control of glycogen accumulation by the cyclin-dependent protein kinase Pho85p

The PHO85 gene of Saccharomyces cerevisiae encodes a cyclin-dependent protein kinase that can interact with 10 different cyclins (Pcls). In conjunction with Pcl8p and Pcl10p, Pho85p phosphorylates and regulates glycogen synthase. Respiratory-deficient strains, such as coq3 mutants, have reduced glycogen stores and contain hyperphosphorylated and inactive glycogen synthase. We show here that pho85 coq3 mutants have dephosphorylated and active glycogen synthase yet do not maintain glycogen reserves. In contrast, deletion of PCL8 and PCL10 in the coq3 mutant background partially restores glycogen accumulation. This suggested the existence of inputs from Pho85p into glycogen storage, independent of Pcl8p and Pcl10p, and acting antagonistically.

Replication-dependent and selection-induced mutations in respiration-competent and respiration-deficient strains of Saccharomyces cerevisiae.

Adaptive or selection-induced mutations are defined as mutations that occur in non-dividing cells as a response to prolonged non-lethal selective pressure such as starvation for an essential amino acid. In the absence of DNA replication, the processing of endogenous DNA lesions by repair enzymes probably acts as a source of mutations. We are studying selection-induced reversions of frameshift alleles in the eukaryote Saccharomyces cerevisiae. Here we show that respiration-deficient strains, totally devoid of mitochondrial DNA, yield selection-induced mutants at slightly elevated frequencies compared to isonucleic respiration-competent strains. Therefore factors of mitochondrial origin such as reactive oxygen species or hypothetical recombinogenic DNA fragments are unlikely to be mediators of selection-induced nuclear frameshift mutation in yeast. Furthermore we compared sequence spectra of reversions of the +1 hom3-10 frameshift allele and found a strong preference for -1 deletions in mononucleotide repeats in selection-induced and replication-dependent revertants, indicating slippage errors during DNA repair synthesis as well as during DNA replication. Remarkably, a higher degree of variation in the site of the reverting frameshift and accompanying base substitutions was found among selection-induced revertants.

Adhesion of Candida albicans mutant strains to host tissue

Adhesion of Candida albicans to host cells is believed to represent a fungal virulence factor and a significant step in the development of candidiasis. As C. albicans strains may differ in their in vitro adhesion ability we initiated a study to investigate whether mutant strains differ in this respect from their parent wild-type. We assessed the in vitro adhesion of C. albicans CBS562 and two mutants obtained by mutagenesis with N′-nitrosoguanidine: a histidine auxotroph, SAG5, derived from CBS562, and a respiratory-deficient strain (a petite mutant), SAR1, derived from SAG5. The adhesion was tested in vitro using two target cell systems: (1) exfoliated human buccal epithelial cells (BEC); and (2) human keratinocyte tissue line cells (HaCaT cells). Adhesion to BEC was evaluated microscopically and that to HaCaT cells by a direct ELISA technique. The results indicated a 54% reduction in adhesion to BEC for SAG5 and 30% for SAR1 as compared to the wild-type, and a 25% reduction in adhesion to HaCaT cells for SAG5 and 20% for SAR1. To verify whether the prototrophy restores the adhesion ability, we complemented the his-negative auxotroph by transforming the strain with the HIS4 gene. Then we assayed the adhesion to BEC of the complemented his-negative mutant in comparison to that of the wild-type, the his-negative mutant (SAG5) and the plasmid-cured transformant. The adhesion values of the complemented his-negative strain were similar to those of the wild-type, whereas the values of the plasmid-cured strain were similar to those of SAG5.