Mayra Cuéllar-Cruz

Candida species: new insights into biofilm formation

Biofilms of Candida albicans, Candida parapsilosis, Candida glabrata and Candida tropicalis are associated with high indices of hospital morbidity and mortality. Major factors involved in the formation and growth of Candida biofilms are the chemical composition of the medical implant and the cell wall adhesins responsible for mediating Candida-Candida, Candida-human host cell and Candida-medical device adhesion. Strategies for elucidating the mechanisms that regulate the formation of Candida biofilms combine tools from biology, chemistry, nanoscience, material science and physics. This review proposes the use of new technologies, such as synchrotron radiation, to study the mechanisms of biofilm formation. In the future, this information is expected to facilitate the design of new materials and antifungal compounds that can eradicate nosocomial Candida infections due to biofilm formation on medical implants. This will reduce dissemination of candidiasis and hopefully improve the quality of life of patients.

Influence of Culture Media on Biofilm Formation by Candida Species and Response of Sessile Cells to Antifungals and Oxidative Stress

The aims of the study were to evaluate the influence of culture media on biofilm formation by C. albicans, C. glabrata, C. krusei, and C. parapsilosis and to investigate the responses of sessile cells to antifungals and reactive oxygen species (ROS) as compared to planktonic cells. For biofilm formation, the Candida species were grown at different periods of time in YP or YNB media supplemented or not with 0.2 or 2% glucose. Sessile and planktonic cells were exposed to increasing concentrations of antifungals, H2O2, menadione or silver nanoparticles (AgNPs). Biofilms were observed by scanning electron microscopy (SEM) and quantified by the XTT assay. C. albicans formed biofilms preferentially in YPD containing 2% glucose (YPD/2%), C. glabrata in glucose-free YNB or supplemented with 0.2% glucose (YNB/0.2%), while C. krusei and C. parapsilosis preferred YP, YPD/0.2%, and YPD/2%. Interestingly, only C. albicans produced an exopolymeric matrix. This is the first report dealing with the in vitro effect of the culture medium and glucose on the formation of biofilms in four Candida species as well as the resistance of sessile cells to antifungals, AgNPs, and ROS. Our results suggest that candidiasis in vivo is a multifactorial and complex process where the nutritional conditions, the human immune system, and the adaptability of the pathogen should be considered altogether to provide an effective treatment of the patient.

Differential Response of Candida albicans and Candida glabrata to Oxidative and Nitrosative Stresses

AbstractInvasive candidiasis is associated with high mortality in immunocompromised and hospitalized patients. Candida albicans is the main pathological agent followed by Candida glabrata, Candida krusei, Candida parapsilosis, and Candida tropicalis. These pathogens colonize different host tissues in humans as they are able to neutralize the reactive species generated from nitrogen and oxygen during the respiratory burst. Among the enzymatic mechanisms that Candida species have developed to protect against free radicals are enzymes with antioxidant and immunodominant functions such as flavohemoglobins, catalases, superoxide dismutases, glutathione reductases, thioredoxins, peroxidases, heat-shock proteins, and enolases. These mechanisms are under transcriptional regulation by factors such as Cta4p, Cwt1p, Yap1p, Skn7p, Msn2p, and Msn4p. However, even though it has been proposed that all Candida species have similar enzymatic systems, it has been observed that they respond differentially to various types of stress. These differential responses may explain the colonization of different organs by each species. Here, we review the enzymatic mechanisms developed by C. albicans and C. glabrata species in response to oxidative and nitrosative stresses. Lack of experimental information for other pathogenic species limits a comparative approach among different organisms.

Detection of 2 immunoreactive antigens in the cell wall of Sporothrix brasiliensis and Sporothrix globosa.

The cell wall of members of the Sporothrix schenckii complex contains highly antigenic molecules which are potentially useful for the diagnosis and treatment of sporotrichosis. In this study, 2 immunoreactive antigens of 60 (Gp60) and 70 kDa (Gp70) were detected in the cell wall of the yeast morphotypes of Sporothrix brasiliensis and Sporothrix globosa.

Identification of Candida albicans heat shock proteins and Candida glabrata and Candida krusei enolases involved in the response to oxidative stress

In the past two decades, Candida species have become the second leading cause of invasive mycosis in immunocompromised patients. In order to colonize their hosts, these microorganisms express adhesins and cell wall proteins that allow them to adhere and neutralize the reactive oxygen species produced by phagocytic cells during the respiratory burst. However, the precise mechanism by which Candida cell wall proteins change their expression in response to oxidative stress has not been described. In an attempt to understand this change in response to oxidative stress, in this study, three Candida species, namely, C. albicans, C. glabrata and C. krusei, were exposed to increasing concentrations of H2O2 and induced cell wall proteins were identified by two-dimensional gel electrophoresis and peptide mass fingerprinting. Sequence analysis of differential proteins led to the identification of two heat-shock proteins in C. albicans, two enolases in C. glabrata and one enolase in C. krusei. Enolases may be involved in the protection of pathogenic cells against oxidative stress as suggested by the decrease in their expression when they were exposed to high concentrations of H2O2. To our knowledge, this is the first demonstration that expression of these proteins changes in response to oxidative stress in different Candida species. This knowledge can eventually facilitate both an early diagnosis and a more efficient treatment of this mycosis.

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.

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.

Effect of oxidative stress on cell wall morphology in four pathogenic Candida species

Candida species are a primary cause of high mortality in immunocompromised and hospitalized patients. The cell wall (CW) of these pathogens is the first structure contacted by the reactive oxygen species (ROS) generated during respiratory bursting in phagocytic cells. The purpose of this work was to evaluate the effect of oxidative stress on CW and some metabolic activities of C. albicans, C. glabrata, C. krusei and C. parapsilosis. Data revealed that C. krusei is more resistant to H2O2, while C. albicans is more resistant to O2•‾ ions. It was also observed that cells exposed to oxidative stress transited from the normal, oval morphology to other aberrant forms that exhibited indentations and protrusions. In addition, oxidative stress caused damage not only to the CW, but also altered a number of cell biological functions, such as lipidic peroxidation, antioxidant capacity, protein carbonylation and catalase activity. The morphological and other cellular changes undergone by Candida cells exposed to ROS in vitro are possibly similar to those occurring in vivo, and therefore may be important during infection and persistence of the pathogen in the human host.

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.

Biosynthesis of micro‐ and nanocrystals of Pb (II), Hg (II) and Cd (II) sulfides in four Candida species: a comparative study of in vivo and in vitro approaches

Nature produces biominerals (biogenic minerals) that are synthesized as complex structures, in terms of their physicochemical properties. These biominerals are composed of minerals and biological macromolecules. They are produced by living organisms and are usually formed through a combination of chemical, biochemical and biophysical processes. Microorganisms like Candida in the presence of heavy metals can biomineralize those metals to form microcrystals (MCs) and nanocrystals (NCs). In this work, MCs and NCs of PbS, HgS or HgCl2 as well as CdS are synthesized both in vitro (gels) and in vivo by four Candida species. Our in vivo results show that, in the presence of Pb2+, Candida cells are able to replicate and form extracellular PbS MCs, whereas in the presence of Hg2+ and Cd2+, they did synthesize intercellular MCs from HgS or HgCl2 and CdS NCs respectively. The MCs and NCs biologically obtained in Candida were compared with those PbS, HgS and CdS crystals synthetically obtained in vitro through the gel method (grown either in agarose or in sodium metasilicate hydrogels). This is, to our knowledge, the first time that the biosynthesis of the various MCs and NCs (presented in several species of Candida) has been reported. This biosynthesis is differentially regulated in each of these pathogens, which allows them to adapt and survive in different physiological and environmental habitats.