Bacillus subtilis stress-associated mutagenesis and developmental DNA repair.

SUMMARYThe metabolic conditions that prevail during bacterial growth have evolved with the faithful operation of repair systems that recognize and eliminate DNA lesions caused by intracellular and exogenous agents. This idea is supported by the low rate of spontaneous mutations (10-9) that occur in replicating cells, maintaining genome integrity. In contrast, when growth and/or replication cease, bacteria frequently process DNA lesions in an error-prone manner. DNA repairs provide cells with the tools needed for maintaining homeostasis during stressful conditions and depend on the developmental context in which repair events occur. Thus, different physiological scenarios can be anticipated. In nutritionally stressed bacteria, different components of the base excision repair pathway may process damaged DNA in an error-prone approach, promoting genetic variability. Interestingly, suppressing the mismatch repair machinery and activating specific DNA glycosylases promote stationary-phase mutations. Current evidence also suggests that in resting cells, coupling repair processes to actively transcribed genes may promote multiple genetic transactions that are advantageous for stressed cells. DNA repair during sporulation is of interest as a model to understand how transcriptional processes influence the formation of mutations in conditions where replication is halted. Current reports indicate that transcriptional coupling repair-dependent and -independent processes operate in differentiating cells to process spontaneous and induced DNA damage and that error-prone synthesis of DNA is involved in these events. These and other noncanonical ways of DNA repair that contribute to mutagenesis, survival, and evolution are reviewed in this manuscript.

Fungal Glycosidases in Sporothrix Species and Candida albicans.

Glycoside hydrolases (GHs) are enzymes that participate in many biological processes of fungi and other organisms by hydrolyzing glycosidic linkages in glycosides. They play fundamental roles in the degradation of carbohydrates and the assembly of glycoproteins and are important subjects of studies in molecular biology and biochemistry. Based on amino acid sequence similarities and 3-dimensional structures in the carbohydrate-active enzyme (CAZy), they have been classified in 171 families. Members of some of these families also exhibit the activity of trans-glycosydase or glycosyl transferase (GT), i.e., they create a new glycosidic bond in a substrate instead of breaking it. Fungal glycosidases are important for virulence by aiding tissue adhesion and colonization, nutrition, immune evasion, biofilm formation, toxin release, and antibiotic resistance. Here, we review fungal glycosidases with a particular emphasis on Sporothrix species and C. albicans, two well-recognized human pathogens. Covered issues include a brief account of Sporothrix, sporotrichosis, the different types of glycosidases, their substrates, and mechanism of action, recent advances in their identification and characterization, their potential biotechnological applications, and the limitations and challenges of their study given the rather poor available information.

New Insights on the Origin of Life: The Role of Silico-Carbonates of Ba (II) to Preserve DNA against Highly Intense UV Radiation.

Understanding the origin of life on our planet has generated diverse theories. Currently, the theory is that life has a single origin; however, its starting point has not been defined. As evidenced, it is indispensable to unify the different theories to reach a single theory that would also allow linking the different areas of knowledge to finally understand the mechanism by which life originated on Earth. In this regard, aiming at contributing to the unification of the diverse theories on the origin of life, in this work, the hypothesis based on the condition that silica-carbonates of alkaline earth metals, called biomorphs, are the ones that could unify all the proposed theories on the origin of life is proposed. Aimed at evaluating if this hypothesis is viable, this work assessed whether biomorphs are able to protect the DNA from continuous UV radiation under two conditions that emulate the habitats that could have co-existed in the Precambrian and, after the radiation, evaluated the time during which DNA remained inside the biomorphs. Our results showed that biomorphs can protect the DNA for months after continuous UV exposure. It was also determined that biomorphs protect the DNA from external factors in different habitats, like normal atmospheric conditions and in aqueous environments. The obtained data allowed me to infer that biomorphs may be the gap that unifies the diverse proposed theories on the origin of life in our Planet.

Cell compensatory responses of fungi to damage of the cell wall induced by Calcofluor White and Congo Red with emphasis on Sporothrix schenckii and Sporothrix globosa. A review.

The cell wall (CW) of fungi exhibits a complex structure and a characteristic chemical composition consisting almost entirely of interacting crystalline and amorphous polysaccharides. These are synthesized by a number of sugar polymerases and depolymerases encoded by a high proportion of the fungal genome (for instance, 20% in Saccharomyces cerevisiae). These enzymes act in an exquisitely coordinated process to assemble the tridimensional and the functional structure of the wall. Apart from playing a critical role in morphogenesis, cell protection, viability and pathogenesis, the CW represents a potential target for antifungals as most of its constituents do not exist in humans. Chitin, ß-glucans and cellulose are the most frequent crystalline polymers found in the fungal CW. The hexosamine biosynthesis pathway (HBP) is critical for CW elaboration. Also known as the Leloir pathway, this pathway ends with the formation of UDP-N-GlcNAc after four enzymatic steps that start with fructose-6-phosphate and L-glutamine in a short deviation of glycolysis. This activated aminosugar is used for the synthesis of a large variety of biomacromolecules in a vast number of organisms including bacteria, fungi, insects, crustaceans and mammalian cells. The first reaction of the HBP is catalyzed by GlcN-6-P synthase (L-glutamine:D-fructose-6-phosphate amidotransferase; EC 2.6.1.16), a critical enzyme that has been considered as a potential target for antifungals. The enzyme regulates the amount of cell UDP-N-GlcNAc and in eukaryotes is feedback inhibited by the activated aminosugar and other factors. The native and recombinant forms of GlcN-6-P synthase has been purified and characterized from both prokaryotic and eukaryotic organisms and demonstrated its critical role in CW remodeling and morphogenesis after exposure of some fungi to agents that stress the cell surface by interacting with wall polymers. This review deals with some of the cell compensatory responses of fungi to wall damage induced by Congo Red and Calcofluor White.

Protection of the DNA from Selected Species of Five Kingdoms in Nature by Ba(II), Sr(II), and Ca(II) Silica-Carbonates: Implications about Biogenicity and Evolving from Prebiotic Chemistry to Biological Chemistry.

The origin of life on Earth is associated with the Precambrian era, in which the existence of a large diversity of microbial fossils has been demonstrated. Notwithstanding, despite existing evidence of the emergence of life many unsolved questions remain. The first question could be as follows: Which was the inorganic structure that allowed isolation and conservation of the first biomolecules in the existing reduced conditions of the primigenial era? Minerals have been postulated as the ones in charge of protecting theses biomolecules against the external environment. There are calcium, barium, or strontium silica-carbonates, called biomorphs, which we propose as being one of the first inorganic structures in which biomolecules were protected from the external medium. Biomorphs are structures with different biological morphologies that are not formed by cells, but by nanocrystals; some of their morphologies resemble the microfossils found in Precambrian cherts. Even though biomorphs are unknown structures in the geological registry, their similarity with some biological forms, including some Apex fossils, could suggest them as the first "inorganic scaffold" where the first biomolecules became concentrated, conserved, aligned, and duplicated to give rise to the pioneering cell. However, it has not been documented whether biomorphs could have been the primary structures that conserved biomolecules in the Precambrian era. To attain a better understanding on whether biomorphs could have been the inorganic scaffold that existed in the primigenial Earth, the aim of this contribution is to synthesize calcium, barium, and strontium biomorphs in the presence of genomic DNA from organisms of the five kingdoms in conditions emulating the atmosphere of the Precambrian era and that CO2 concentration in conditions emulating current atmospheric conditions. Our results showed, for the first time, the formation of the kerogen signal, which is a marker of biogenicity in fossils, in the biomorphs grown in the presence of DNA. We also found the DNA to be internalized into the structure of biomorphs.

A GDPase/UDPase bifunctional enzyme from Candida albicans: purification and biochemical characterization.

The most frequently isolated human fungal pathogen is Candida albicans which is responsible for about 50% of all Candida infections. In healthy individuals, this organism resides as a part of the normal microbiota in equilibrium with the host. However, under certain conditions, particularly in immunocompromised patients, this opportunistic pathogen adheres to host cells causing serious systemic infections. Thus, much effort has been dedicated to the study of its physiology with emphasis on factors associated to pathogenicity. A representative analysis deals with the mechanisms of glycoprotein assembly as many cell surface antigens and other macromolecules that modulate the immune system fall within this chemical category. In this regard, studies of the terminal protein glycosylation stage which occurs in Golgi vesicles has led to the identification of nucleotidases that convert glycosyltransferase-generated dinucleotides into the corresponding mononucleotides, thus playing a double function: their activity prevent inhibition of further glycosyl transfer by the accumulation of dinucleotides and the resulting mononucleotides are exchanged by specific membrane transporters for equimolecular amounts of sugar donors from the cytosol. Here, using a simple protocol for protein separation we isolated a bifunctional nucleotidase from C. albicans active on GDP and UDP that was characterized in terms of its molecular mass, response to bivalent ions and other factors, substrate specificity and affinity. Results are discussed in terms of the similarities and differences of this nucleotidase with similar counterparts from other organisms thus contributing to the knowledge of a bifunctional diphosphatase not described before in C. albicans.

Proteomic Analysis of Sporothrix schenckii Exposed to Oxidative Stress Induced by Hydrogen Peroxide.

Sporothrix schenckii modulates the expression of its cell wall proteins (CWPs) in response to reactive oxygen species (ROS) generated by the phagocytic cells of the human host, which allows it to evade and escape the immune system. In this study, we performed a comparative proteomic analysis of the CW of S. schenckii after exposure and nonexposure to H2O2. Several CWPs involved in CW remodeling and fungal pathogenesis that modulated their expression in response to this oxidizing agent were identified, as were a number of antioxidant enzymes and atypical CWPs, called moonlighting proteins, such as the Hsp70-5, lipase 1 (Lip1), enolase (Eno), and pyruvate kinase (Pk). Moreover, RT-qPCR assays demonstrated that the transcription of genes HSP70-5, LIP1, ENO, and PK is regulated in response to the oxidant. The results indicated that S. schenckii differentially expressed CWPs to confer protection against ROS upon this fungus. Furthermore, among these proteins, antioxidant enzymes and interestingly, moonlighting-like CWPs play a role in protecting the fungus from oxidative stress (OS), allowing it to infect human host cells.

Enzyme activity and expression of catalases in response to oxidative stress in Sporothrix schenckii.

Sporothrix schenckii is a dimorphic fungus, pathogenic to humans and animals, which is usually infective in the yeast form. Reactive oxygen species (ROS) play an important role in the host's defense, damaging the pathogen's DNA, proteins, and lipids. To prevent oxidative damage, the ROS are detoxified by pathogen-derived antioxidant enzymes such as catalases (CATs). In this work, we analyzed the activity and expression level of three S. schenckii genes, designated as CAT1, CAT2, and CAT3, that putatively encoded for three isoforms of monofunctional CAT with a predicted molecular weight of 57.6, 56.2, and 81.4 kDa, respectively. Our results demonstrate that oxidative stress induced by exogenous H2O2 leads to an altered lipid peroxidation, modifying CAT activity and the expression levels of the CAT genes, being CAT1 and CAT3 the genes with the highest expression in response to the oxidizing agent. These results show that CAT isoforms in S. schenckii can be regulated in response to oxidative stress and might help to control ROS homeostasis in the fungus-host interaction.

Responses of Sporothrix globosa to the cell wall perturbing agents Congo Red and Calcofluor White.

It is well documented that disturbance of cell surface by some agents triggers compensatory responses aimed to maintain the cell wall integrity in fungi and other organisms. Here, the thermodimorphic fungus Sporothrix globosa, a member of the pathogenic clade of the Sporothrix complex, was propagated in yeast-peptone-dextrose medium under conditions to obtain the mycelium (pH 4.5, 27-28 °C) or the yeast (pH 7.8, 32-34 °C) morphotypes in the absence and presence of the wall-interacting dyes Congo Red (CR) and Calcofluor White (CFW) either alone or in combination. After different periods of time, growth, cell morphology and activity of glucosamine-6-phosphate synthase (GlcN-6-P synthase), an ubiquitous enzyme that plays a crucial role in cell wall biogenesis, were determined. CR and to a lower extent CFW affected growth and morphology of both fungal morphotypes and significantly increased enzyme activity. Notoriously, CR or CR in combination with CFW induced the transient conversion of yeasts into conidia-forming filamentous cells even under culture conditions adjusted for yeast development, most likely as a strategy to evade the noxious effect of the dye. After sometime, hypha returned to yeast cells. An hypothetical model to explain the effect of CR on morphology and enzyme activity based on the possible role of membrane-spanning proteins known as mechanosensors is proposed. Results are discussed in terms of the fungal responses to cell wall damage.

A comparative proteomic analysis of Candida species in response to the oxidizing agent cumene hydroperoxide.

Candida genus comprises several species that can be found in the oral cavity and the gastrointestinal and genitourinary tracts of healthy individuals. Under certain conditions, however, they behave as opportunistic pathogens that colonize these tissues, most frequently when the immune system is compromised by a disease or under certain medical treatments. To colonize the human host, these organisms require to express cell wall proteins (CWP) that allowed them to adhere and adapt to the reactive oxygen (ROS) and nitrogen (RNS) species produced in the macrophage during the respiratory burst. The aim of this study was to determine how four Candida species respond to the oxidative stress imposed by cumene hydroperoxide (CHP). To this purpose, C. albicans, C. glabrata, C. krusei and C. parapsilosis were exposed to this oxidant which is known to generate ROS in the membrane phospholipids. Accordingly, both mock and CHP-exposed cells were used to extract and analyze CWP and also to measure catalase activity and the levels of protein carbonylation. Results indicated that all four species express different CWP to neutralize ROS. Most relevant among these proteins were the glycolytic enzymes enolase and glyceraldehyde-3-phosphate dehydrogenase, known as moonlight proteins because in addition to participate in glycolysis they play an important role in the cell response to ROS. In addition, a thiol-specific antioxidant enzyme (Tsa) was also found to counteract ROS.

Transcriptional coupling and repair of 8-OxoG activate a RecA-dependent checkpoint that controls the onset of sporulation in Bacillus subtilis.

During sporulation Bacillus subtilis Mfd couples transcription to nucleotide excision repair (NER) to eliminate DNA distorting lesions. Here, we report a significant decline in sporulation following Mfd disruption, which was manifested in the absence of external DNA-damage suggesting that spontaneous lesions activate the function of Mfd for an efficient sporogenesis. Accordingly, a dramatic decline in sporulation efficiency took place in a B. subtilis strain lacking Mfd and the repair/prevention guanine oxidized (GO) system (hereafter, the ∆GO system), composed by YtkD, MutM and MutY. Furthermore, the simultaneous absence of Mfd and the GO system, (i) sensitized sporulating cells to H2O2, and (ii) elicited spontaneous and oxygen radical-induced rifampin-resistance (Rifr) mutagenesis. Epifluorescence (EF), confocal and transmission electron (TEM) microscopy analyses, showed a decreased ability of ∆GO ∆mfd strain to sporulate and to develop the typical morphologies of sporulating cells. Remarkably, disruption of sda, sirA and disA partially, restored the sporulation efficiency of the strain deficient for Mfd and the ∆GO system; complete restoration occurred in the RecA- background. Overall, our results unveil a novel Mfd mechanism of transcription-coupled-repair (TCR) elicited by 8-OxoG which converges in the activation of a RecA-dependent checkpoint event that control the onset of sporulation in B. subtilis.

Synthesis of Crystalline Silica-Carbonate Biomorphs of Ba(II) under the Presence of RNA and Positively and Negatively Charged ITO Electrodes: Obtainment of Graphite via Bioreduction of CO2 and Its Implications to the Chemical Origin of Life on Primitive Earth.

Since Earth was formed, in the Precambrian era up until our present days, electric current has participated in the morphology and chemical composition of organic and inorganic structures. Attempting to elucidate the mechanism by which electric current participated in the creation of the first cell in the Precambrian era is an intriguing and of a permanent subject of interest to be studied. One way of emulating the formation of structures similar to those that might have existed in the Precambrian era in the presence of a biomolecule and an electric current source is to use as a model, the silica-carbonate of alkaline earth metal compounds known as biomorphs. The objective of this work was to assess the influence exerted by an electric current (negatively or positively charged indium tin oxide electrodes) on the formation of biomorphs in the presence of RNA. The compounds obtained under both electric charges were visualized through scanning electron microscopy (SEM), and their chemical composition was analyzed through Raman spectroscopy. The biomorphs obtained under a positive electric current correspond to aragonite-type BaCO3(I) and calcite-type BaCO3(II). Whereas, under a negative current, carbon graphite and aragonite-type BaCO3(I) were obtained. To the best of our knowledge, this is the first evidence showing that the presence of RNA and the electric current is fundamental in the rearrangement of atoms, suggesting that organic and inorganic compounds have coexisted since the primitive era.

Proteomic analysis of Sporothrix schenckii cell wall reveals proteins involved in oxidative stress response induced by menadione.

Sporotrichosis is an emergent subcutaneous mycosis that is a threat to both humans and other animals. Sporotrichosis is acquired by the traumatic implantation of species of the Sporothrix genus. Added to the detoxification systems, pathogenic fungi possess different mechanisms that allow them to survive within the phagocytic cells of their human host during the oxidative burst. These mechanisms greatly depend from the cell wall (CW) since phagocytic cells recognize pathogens through specific receptors associated to the structure. To date, there are no studies addressing the modulation of the expression of S. schenckii CW proteins (CWP) in response to reactive oxygen species (ROS). Therefore, in this work, a proteomic analysis of the CW of S. schenckii in response to the oxidative agent menadione (O2•-) was performed. Proteins that modulate their expression were identified which can be related to the fungal survival mechanisms within the phagocyte. Among the up-regulated CWP in response to the oxidative agent, 13 proteins that could be involved in the mechanisms of oxidative stress response in S. schenckii were identified. The proteins identified were thioredoxin1 (Trx1), superoxide dismutase (Sod), GPI-anchored cell wall protein, ß-1,3-endoglucanase EglC, glycoside hydrolase (Gh), chitinase, CFEM domain protein, glycosidase crf1, covalently-linked cell wall protein (Ccw), 30 kDa heat shock protein (Hsp30), lipase, trehalase (Treh), fructose-bisphosphate aldolase (Fba1) and citrate synthase (Cs). The identification of CWP that modulates their expression in response to superoxide ion (O2•-) in S. schenckii is a useful approach to understand how the fungus defends itself against ROS, in order to evade the phagocytic cells from the host and cause the infection.

Biosynthesis of chemical compounds by Candida albicans and Candida glabrata.

BACKGROUND: In the last three decades the species of Candida have been of great interest due to the high mortality rates that they cause in immunocompromised and hospitalized patients. These species are opportunistic pathogens and they have inhabited other environments long before colonizing human cells. Among these environments we find wastewater from mines, and water from aquifers and soils that contain high concentrations of precious metals as well as toxic and base metals. AIMS: The aim of this study was to assess whether Candida albicans and Candida glabrata are able to maintain homeostasis in the presence of zinc, copper, cobalt or silver. METHODS: To achieve the objective, each of the Candida species was exposed to every single metal individually in a salt solution. Subsequently the treated cells were lysed to evaluate the compounds formed by means of Scanning Electron Microscopy-Energy Dispersive X-ray spectroscopy (SEM-EDS). RESULTS: When analyzing the compounds that both C. albicans and C. glabrata formed in the presence of each of the metals, we found that they had synthesized silver sulfide (Ag2S), cobalt sulfate (CoSO4), zinc phosphate (Zn3(PO4)2), or copper oxide (CuO). CONCLUSIONS: Our results indicate that both C. albicans and C. glabrata have enzymatic and non-enzymatic mechanisms that allow them to achieve homeostasis in a different specific manner for each of the single metals to which they were exposed. To our knowledge, this is the first work reporting that C. albicans and C. glabrata can reduce different metals, with the subsequent formation of sulfides, sulfates, phosphates and oxides. This ability, developed over time by these Candida species, is probably a kind of biochemical mechanism in order to survive and colonize many different environments, from water or soil to humans. For this reason, C. albicans and C. glabrata make up an excellent model of study, both from a medical and biotechnical point of view.

Congo Red affects the growth, morphology and activity of glucosamine-6-phosphate synthase in the human pathogenic fungus Sporothrix schenckii.

Sporothrix schenckii is the etiological agent of sporotrichosis, a mycosis of humans and other mammals. Little is known about the responses of this thermodimorphic pathogen to perturbations in the cell wall (CW) by different stress conditions. Here we describe the effect of Congo Red (CR) on the fungal growth, morphogenesis and activity of glucosamine-6-phosphate (GlcN-6-P) synthase. Under conditions of yeast development, 15 µM CR abolished conidia (CN) germination, but when yeast cells were first obtained in the absence of the dye and then post-incubated in its presence, yeasts rapidly differentiated into mycelial cells. On the other hand, under conditions of mycelium development, 150 µM CR did not affect CN germination, but filamentous cells underwent structural changes characterized by a distorted CW contour, the loss of polarity and the formation of red-pigmented, hyphal globose structures. Under these conditions, CR also induced a significant and transient increase in the activity of GlcN-6-P synthase, an essential enzyme in CW biogenesis.

Moonlighting proteins induce protection in a mouse model against Candida species.

In recent years, C. albicans and C. glabrata have been identified as the main cause of candidemia and invasive candidiasis in hospitalized and immunocompromised patients. In order to colonize the human host, these fungi express several virulence factors such as the response to oxidative stress and the formation of biofilms. In the expression of these virulence factors, the cell wall of C. albicans and C. glabrata is of fundamental importance. As the outermost structure of the yeast, the cell wall is the first to come in contact with the reactive oxygen species (ROS) generated during the respiratory outbreak, and in the formation of biofilms, it is the first to adhere to organs or medical devices implanted in the human host. In both processes, several cell wall proteins (CWP) are required, since they promote attachment to human cells or abiotic surfaces, as well as to detoxify ROS. In our working group we have identified moonlighting CWP in response to oxidative stress as well as in the formation of biofilms. Having identified moonlighting CWP in Candida species in response to two virulence factors indicates that these proteins may possibly be immunodominant. The aim of the present work was to evaluate whether proteins of this type such as fructose-bisphosphate aldolase (Fba1), phosphoglycerate kinase (Pgk) and pyruvate kinase (Pk), could confer protection in a mouse model against C. albicans and C. glabrata. For this, recombinant proteins His6-Fba1, His6-Pgk and His6-Pk were constructed and used to immunize several groups of mice. The immunized mice were infected with C. albicans or C. glabrata, and subsequently the liver, spleen and kidney were extracted and the number of CFU was determined. Our results showed that Pk confers immunity to mice against C. albicans, while Fba1 to C. glabrata. This data allows us to conclude that the moonlighting CWP, Fba1 and Pk confer in vivo protection in a specific way against each species of Candida. This makes them promising candidates for developing specific vaccines against these pathogens.

Evaluation of cell wall damage by dimethyl sulfoxide in Candida species.

Studies dealing with the response of microorganisms to oxidative stress require the dissolution of oxidant agents in an appropriate solvent. A commonly used medium is dimethyl sulfoxide, which has been considered as an innocuous polar solvent. However, we have observed significant differences between control, untreated cells and those receiving increasing amounts of the oxidant and hence increasing amounts of DMSO, to the maximum allowed of 1%. Here we show that, while this solvent does not influence yeast cell viability, it does affect expression of cell wall proteins as well as catalase activity. Therefore, its use in future studies of oxidative stress as an innocuous solvent should be reconsidered.

Identification of proteins involved in the adhesionof Candida species to different medical devices.

Adhesion is the first step for Candida species to form biofilms on medical devices implanted in the human host. Both the physicochemical nature of the biomaterial and cell wall proteins (CWP) of the pathogen play a determinant role in the process. While it is true that some CWP have been identified in vitro, little is known about the CWP of pathogenic species of Candida involved in adhesion. On this background, we considered it important to investigate the potential role of CWP of C. albicans, C. glabrata, C. krusei and C. parapsilosis in adhesion to different medical devices. Our results indicate that the four species strongly adher to polyvinyl chloride (PVC) devices, followed by polyurethane and finally by silicone. It was interesting to identify fructose-bisphosphate aldolase (Fba1) and enolase 1 (Eno1) as the CWP involved in adhesion of C. albicans, C. glabrata and C. krusei to PVC devices whereas phosphoglycerate kinase (Pgk) and Eno1 allow C. parapsilosis to adher to silicone-made implants. Results presented here suggest that these CWP participate in the initial event of adhesion and are probably followed by other proteins that covalently bind to the biomaterial thus providing conditions for biofilm formation and eventually the onset of infection.

Moonlight-like proteins of the cell wall protect sessile cells of Candida from oxidative stress.

Biofilms of Candida species are associated with high morbidity and hospital mortality. Candida forms biofilms by adhering to human host epithelium through cell wall proteins (CWP) and simultaneously neutralizing the reactive oxygen species (ROS) produced during the respiratory burst by phagocytic cells. The purpose of this paper is to identify the CWP of Candida albicans, Candida glabrata, Candida krusei and Candida parapsilosis expressed after exposure to different concentrations of H2O2 using a proteomic approach. CWP obtained from sessile cells, both treated and untreated with the oxidizing agent, were resolved by one and two-dimensional (2D-PAGE) gels and identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Some of these proteins were identified and found to correspond to moonlighting CWP such as: (i) glycolytic enzymes, (ii) heat shock, (iii) OSR proteins, (iv) general metabolic enzymes and (v) highly conserved proteins, which are up- or down-regulated in the presence or absence of ROS. We also found that the expression of these CWP is different for each Candida species. Moreover, RT-PCR assays allowed us to demonstrate that transcription of the gene coding for Eno1, one of the moonlight-like CWP identified in response to the oxidant agent, is differentially regulated. To our knowledge this is the first demonstration that, in response to oxidative stress, each species of Candida, differentially regulates the expression of moonlighting CWP, which may protect the organism from the ROS generated during phagocytosis. Presumptively, these proteins allow the pathogen to adhere and form a biofilm, and eventually cause invasive candidiasis in the human host. We propose that, in addition to the antioxidant mechanisms present in Candida, the moonlighting CWP also confer protection to these pathogens from oxidative stress.

Proteomic analysis of cell wall in four pathogenic species of Candida exposed to oxidative stress.

In order for Candida species to adhere and colonize human host cells they must express cell wall proteins (CWP) and adapt to reactive oxygen species (ROS) generated by phagocytic cells of the human host during the respiratory burst. However, how these pathogens change the expression of CWP in response to oxidative stress (OSR) is not known. Here, fifteen moonlight-like CWP were identified that expressed differentially in four species of Candida after they were exposed to H2O2 or menadione (O2(-)). These proteins included: (i) glycolytic enzymes, such as glyceraldehyde-3-phosphate dehydrogenase (Gapdh), fructose-bisphosphate aldolase (Fba1), phosphoglycerate mutase (Gpm1), phosphoglycerate kinase (Pgk), pyruvate kinase (Pk) and enolase (Eno1); (ii) the heat shock proteins Ssb1 and Ssa2; (iii) OSR proteins such as peroxyredoxin (Tsa1), the stress protein Ddr48 (Ddr48) and glutathione reductase (Glr1); (iv) other metabolic enzymes such as ketol-acid reductoisomerase (Ilv5) and pyruvate decarboxylase (Pdc1); and (v) other proteins such as elongation factor 1-beta (Efb1) and the 14-3-3 protein homolog. RT-PCR revealed that transcription of the genes coding for some of the identified CWP are differentially regulated. To our knowledge this is the first report showing that moonlight-like CWP are the first line of defense of Candida against ROS, and that they are differentially regulated in each of these pathogens.