Gloria Molero

Phenotypic characterization of a Candida albicans strain deficient in its major exoglucanase

Both alleles of the XOG1 gene of Candida albicans, which encodes a protein with exoglucanase activity, were sequentially disrupted. Enzymic analysis of either cell extracts or culture supernatants of disrupted strains revealed that this gene is responsible for the major exoglucanase activity in C. albicans, although residual exoglucanase activity could still be detected. xog1 null mutants showed similar growth rates in both rich and minimal liquid medium as compared to the wild-type strain, indicating that the enzyme is not essential for C. albicans growth. In addition, no differences were observed between wild-type and xog1 null mutants with respect to their ability to undergo dimorphic transition. However, small but repeatable differences were found between the wild-type and the null mutant with respect to susceptibility to chitin and glucan synthesis inhibitors. Using a murine model of experimental infection, no significant differences in virulence were observed. The xog1 null strain is thus a suitable recipient for studying Candida gene expression using the exoglucanase as a reporter gene.

Proteomic characterization of human proinflammatory M1 and anti‐inflammatory M2 macrophages and their response to Candida albicans

In response to different stimuli, macrophages can differentiate into either a pro‐inflammatory subtype (M1, classically activated macrophages) or acquire an anti‐inflammatory phenotype (M2, alternatively activated macrophages). Candida albicans is the most important opportunistic fungus in nosocomial infections, and it is contended by neutrophils and macrophages during the first steps of the invasive infection. Murine macrophages responses to C. albicans have been widely studied, whereas the responses of human‐polarized macrophages remain less characterized. In this study, we have characterized the proteomic differences between human M1‐ and M2‐polarized macrophages, both in basal conditions and in response to C. albicans, by quantitative proteomics (2DE). This proteomic approach allowed us to identify metabolic routes and cytoskeletal rearrangement components that are the most relevant differences between M1 and M2 macrophages. The analysis has revealed fructose‐1,6‐bisphosphatase 1, a critical enzyme in gluconeogenesis, up‐regulated in M1, as a novel protein marker for macrophage polarization. Regarding the response to C. albicans, an M1‐to‐M2 switch in polarization was observed. This M1‐to‐M2 switch might contribute to Candida pathogenicity by decreasing the generation of specific immune responses, thus enhancing fungal survival and colonization, or instead, may be part of the host attempt to reduce the inflammation and limit the damage of the infection.

Proteomics of RAW 264.7 macrophages upon interaction with heat-inactivated Candida albicans cells unravel an anti-inflammatory response

Murine macrophages (RAW 264.7) were allowed to interact with heat‐inactivated cells of Candida albicans SC5314 during 45 min. The proteomic response of the macrophages was then analyzed using 2‐D gel electrophoresis. Many proteins having differential expression with respect to control macrophages were identified, and their functions were related to important processes, such as cytoskeletal organization, signal transduction, metabolism, protein biosynthesis, stress response and protein fate. Several of these proteins have been described as being involved in the process of inflammation, such as Erp29, Hspa9a, AnxaI, Ran GTPase, P4hb, Clic1 and Psma1. The analysis of the consequences of their variation unravels an overall anti‐inflammatory response of macrophages during the interaction with heat‐inactivated cells. This result was corroborated by the measurement of TNF‐α and of ERK1/2 phosphorylation levels. This anti‐inflammatory effect was contrary to the one observed with live C. albicans cells, which induced higher TNF‐α secretion and higher ERK1/2 phosphorylation levels with respect to control macrophages.

New amphotericin B-gamma cyclodextrin formulation for topical use with synergistic activity against diverse fungal species and Leishmania spp

Amphotericin B (AmB) has a broad antifungal and leishmanicidal activity with low incidence of clinical resistance. Its parenteral administration has high risk of nephrotoxicity that limits its use. In order to treat cutaneous infections, AmB topical administration is a safer therapy because of the low systemic absorption of the drug across mucous membranes. Moreover, in some developing countries both fungal topical infections and cutaneous leishmaniasis are an important health problem. The aim of this work is to formulate a topical amphotericin preparation and test its in vitro antifungal (against 11 different fungal species) and antileishmanial activity. γ-Cyclodextrin (γ-CD) was chosen to solubilise AmB. Furthermore, γ-CD has shown a synergistic effect on membrane destabilization with AmB. Topical novel formulations based on AmB-CD complex have exhibited greater antifungal activity (48%, 28% and 60% higher) when compared to AmB Neo-Sensitabs(®) disks, AmB dissolved in dimethyl sulfoxide (DMSO) and Clotrimazole(®) cream, respectively. Furthermore, AmB-CD methyl cellulose gel has shown significantly higher inhibition activity on biofilm formation, larger penetration through yeast biofilms and higher fungicidal activity on biofilm cells compared to AmB dissolved in DMSO. In addition, AmB-CD gel exhibited both high in vitro leishmanicidal efficacy with wider therapeutic index (between 2 and 8-fold higher than AmB deoxycholate depending on Leishmania spp.) and also in vivo activity in an experimental model of cutaneous leishmaniasis. These results illustrate the feasibility of a topical AmB formulation easy to prepare, physicochemically stable over 6 months, safe and effective against diverse fungal and parasitic cutaneous infections.

Candida albicans induces pro-inflammatory and anti-apoptotic signals in macrophages as revealed by quantitative proteomics and phosphoproteomics.

UNLABELLED Macrophages play a pivotal role in the prevention of Candida albicans infections. Yeast recognition and phagocytosis by macrophages is mediated by Pattern Recognition Receptors (PRRs) that initiate downstream signal transduction cascades by protein phosphorylation and dephosphorylation. We exposed RAW 264.7 macrophages to C. albicans for 3h and used SILAC to quantify macrophage proteins and phosphoproteins by mass spectrometry to study the effects of infection. We identified 53 macrophage up-regulated proteins and 15 less abundant in the presence of C. albicans out of a total of 2071 identified proteins. 922 unique protein phosphorylation sites were identified by phosphopeptide enrichment and mass spectrometry, including 327 previously unidentified mouse protein phosphorylation sites. 126 peptides showed an increase and 70 a decrease in their phosphorylation level. The majority of the differentially expressed and phosphorylated proteins are receptors, mitochondrial ribosomal proteins, cytoskeletal proteins, and transcription factor activators involved in inflammatory and oxidative responses. In addition, we identified 22 proteins and phosphoproteins related to apoptosis. The analysis of apoptotic markers revealed that anti-apoptotic signals prevailed during the interaction of the yeast. Our proteomics study suggests that besides inflammation, apoptosis is a central pathway in the immune defense against C. albicans infection. BIOLOGICAL SIGNIFICANCE This work uses SILAC and SIMAC methodology combined with CPP (+ TiO2) to study protein and phosphopeptide changes in RAW 264.7 macrophages in response to coincubation with Candida albicans for 3h. We show that the presence of C. albicans induces inflammatory responses and inhibits apoptosis in the macrophages. Our phosphoproteomic analysis identified 327 new mouse protein phosphorylation sites.

In vivo distribution and therapeutic efficacy of a novel amphotericin B poly-aggregated formulation

OBJECTIVES The purpose of this investigation is the study of toxicity, in vivo distribution and therapeutic activity against candidiasis of poly-aggregated amphotericin B, in two different formulations: not microencapsulated (P-AMB) or incorporated in albumin microspheres (MP-AMB). METHODS The therapeutic efficacy and toxicity of amphotericin B formulations was studied in an immunocompetent murine model of systemic candidiasis. A pharmacokinetic study was also performed to measure the plasma, kidney, liver and spleen amphotericin B concentrations after administration of the three formulations to mice. RESULTS The acute toxicity of P-AMB in mice is lower than that of the conventional amphotericin B reference formulation (D-AMB). The 50% lethal doses were increased at least eight times. Intravenous bolus administration of doses up to 40 mg/kg of body weight of poly-aggregated amphotericin B, either P-AMB or MP-AMB, did not produce acute symptoms of toxicity. Interestingly, in the pharmacokinetic study, significant (P < 0.05) lower plasma and kidney amphotericin B concentrations and higher liver and spleen amphotericin B concentrations were achieved after poly-aggregated amphotericin B formulation (P-AMB and MP-AMB) administration in relation to the reference formulation (D-AMB). At high amphotericin B doses, no significant differences in efficacy (P > 0.05) were observed among the formulations (D-AMB, P-AMB and MP-AMB). CONCLUSIONS Although the efficacy in the candidiasis treatment was decreased as a consequence of amphotericin B aggregation, it can be compensated by the possibility of increasing the doses with lower nephrotoxicity. Moreover, due to its lower toxicity while maintaining its effectiveness, the poly-aggregated formulations (P-AMB and MP-AMB) have a better therapeutic index than the conventional formulation (D-AMB).

Sub-proteomic study on macrophage response to Candida albicans unravels new proteins involved in the host defense against the fungus ☆

In previous proteomic studies on the response of murine macrophages against Candida albicans, many differentially expressed proteins involved in processes like inflammation, cytoskeletal rearrangement, stress response and metabolism were identified. In order to look for proteins important for the macrophage response, but in a lower concentration in the cell, 3 sub-cellular extracts were analyzed: cytosol, organelle/membrane and nucleus enriched fractions from RAW 264.7 macrophages exposed or not to C. albicans SC5314 for 3 h. The samples were studied using DIGE technology, and 17 new differentially expressed proteins were identified. This sub-cellular fractionation permitted the identification of 2 mitochondrion proteins, a membrane receptor, Galectin-3, and some ER related proteins, that are not easily detected in total cell extracts. Besides, the study of different fractions allowed us to detect, not only total increase in Galectin-3 protein amount, but its distinct allocation along the interaction. The identified proteins are involved in the pro-inflammatory and oxidative responses, immune response, unfolded protein response and apoptosis. Some of these processes increase the host response and others could be the effect of C. albicans resistance to phagocytosis. Thus, the sub-proteomic approach has been a very useful tool to identify new proteins involved in macrophage-fungus interaction. This article is part of a Special Issue entitled: Translational Proteomics.

Two different NO-dependent mechanisms account for the low virulence of a non-mycelial morphological mutant of Candida albicans

Abstract We have previously described the low virulence of a Candida albicans morphological mutant: 92′. We have now used this strain to examine the role of phagocytes in its pathogenesis. Our results show that C. albicans 92′ cannot evade innate host macrophage defence mechanisms as efficiently as the parental strain. In addition to the high susceptibility to phagocytosis by peritoneal macrophages, the NO produced by macrophages is a very important element in the low virulence of this agerminative mutant, a thesis supported by in vivo and in vitro experiments. Whereas the parental strain was able to inhibit macrophage NO production, the mutant was quite inefficient at reducing NO production by macrophages. In addition, the mutant showed high sensitivity to a NO generator. Treatment of mice with aminoguanidine (a preferred inducible NO synthase inhibitor) caused 90% mortality in 92′ systemic infection, thus supporting a role for NO in the low virulence of this strain. Our data show that both the low inhibitory effect of 92′ on macrophage NO production and the higher sensitivity to NO underlie the low virulence of this strain.

The Importance of the Phagocytes' Innate Response in Resolution of the Infection Induced by a Low Virulent Candida albicans Mutant

We have reported that a Candida albicans mkc1Δ/mkc1Δ mutant, deleted in the Mkc1p mitogen‐activated protein kinase, an essential element of the cell integrity signalling pathway, has reduced virulence in a murine model of systemic infection. We analyse here the immunological basis for this feature in view of its failure to vaccinate. Firstly, the influence of the Th response was analysed by infecting different knockout mice, revealing the importance of interferon‐γ in the resolution of mkc1 systemic infection. Secondly, the role of innate immunity was studied. The infection of neutropenic mice revealed that the candidacidal activity of neutrophils is crucial during the first 3 days of infection for the mutant strain. Macrophages played a critical role in the clearance of infection. Although a similar anti‐Candida activity was found for both fungal strains with naïve macrophages, activated macrophages discriminated between both strains. In vitro experiments revealed that the mutant strain displayed a greater susceptibility to nitric oxide (NO), a reduced inhibitory effect on macrophage NO production and an increased capacity of macrophage stimulation by cell wall extracts. The importance of NO in systemic infection with the mutant strain was confirmed by the strong increase in the susceptibility of aminoguanidine (an iNOs inhibitor)‐treated mice.

Genetic Control of Fungal Cell Wall Autolysis

The major structural components of fungal cell walls are usually polysaccharide homopolymers, such as glucans and chitin, with chemical structures simpler than those of bacterial peptidoglycan. However, fungal cell walls are also thick and complex envelopes playing the same role as bacterial cell walls. For a number of years we have been addressing the question of the biological role of fungal autolysins, enzymes that can be recognized biochemically as being capable of degrading components of the producer’s cell wall. The autolytic action of these enzymes on fungal walls has always been envisaged as the controlled hydrolytic modification of the wall polymers in many situations that might require some kind of modification of the wall structure. Therefore fungal autolysins have been proposed as agents that may be critical for fungal morphogenesis, and they must represent basic elements contributing to the dynamics of the wall structure, that involves a series of changes throughout the yeast mitotic cell cycle and apical extension of hyphae, as well as in other processes such as sexual conjugation, meiosis, dimorphism, etc.