Commercial AHAS-inhibiting herbicides are promising drug leads for the treatment of human fungal pathogenic infections.

The increased prevalence of drug-resistant human pathogenic fungal diseases poses a major threat to global human health. Thus, new drugs are urgently required to combat these infections. Here, we demonstrate that acetohydroxyacid synthase (AHAS), the first enzyme in the branched-chain amino acid biosynthesis pathway, is a promising new target for antifungal drug discovery. First, we show that several AHAS inhibitors developed as commercial herbicides are powerful accumulative inhibitors of Candida albicans AHAS (Ki values as low as 800 pM) and have determined high-resolution crystal structures of this enzyme in complex with several of these herbicides. In addition, we have demonstrated that chlorimuron ethyl (CE), a member of the sulfonylurea herbicide family, has potent antifungal activity against five different Candida species and Cryptococcus neoformans (with minimum inhibitory concentration, 50% values as low as 7 nM). Furthermore, in these assays, we have shown CE and itraconazole (a P450 inhibitor) can act synergistically to further improve potency. Finally, we show in Candida albicans-infected mice that CE is highly effective in clearing pathogenic fungal burden in the lungs, liver, and spleen, thus reducing overall mortality rates. Therefore, in view of their low toxicity to human cells, AHAS inhibitors represent a new class of antifungal drug candidates.

Candida albicans SC5314 inhibits NLRP3/NLRP6 inflammasome expression and dampens human intestinal barrier activity in Caco-2 cell monolayer model.

Candida albicans is an opportunistic fungal pathogen that colonizes human gastro-intestinal mucosal tissues. Its effect on the immune response in intestinal epithelial cells and on the intestinal mucosal barrier are not yet fully understood. In this study, we investigated Caco-2 cells, a monolayer model of intestinal epithelial cells, with or without treatment with C. albicans SC5314 (CA) or heat-inactivated CA (CA-inact). RNA sequencing was conducted, and the mRNA and protein levels of NOD-like receptor pyrin domain-containing protein 3 (NLRP3) or NLRP6/ASC/caspase-1 inflammasome signaling pathway components, inflammatory cytokines (interleukin-18 [IL-18] and IL-1ß), anti-microbial peptides (AMPs; ß-defensin-2 [BD-2], BD-3, and LL-37), and tight junction proteins (occludin and zona occludens-1 [ZO-1]) were examined by real-time PCR, western blotting, and/or immunofluorescence microscopy. Lactase dehydrogenase (LDH) activity in the Caco-2 cell supernatant were measured by enzyme kinetics analysis. Our results showed that the NOD-like receptor signaling pathway participates in the CA- and CA-inact-infected Caco-2 cells, as shown by microarray analysis of total mRNA expression. The expression of NLRP3, NLRP6, ASC, BD-2, BD-3, occludin, and ZO-1 were significantly decreased in Caco-2 cells infected with CA and CA-inact compared to that in the untreated control. IL-1ß expression was decreased in the Caco-2 cells in both the CA- and CA-inact-infected groups compared to that in the control. Caspase-1 and IL-18 levels were not markedly affected by CA or CA-inact in Caco-2 cells. Our findings indicate that CA can inhibit the NLRP3 and NLRP6 pathways and dampen human intestinal mucosal barrier activity by decreasing the production of AMPs and tight junction proteins, independent of CA activity.

Identification of Components of the SUMOylation Machinery in Candida glabrata: ROLE OF THE DESUMOYLATION PEPTIDASE CgUlp2 IN VIRULENCE.

Regulation of protein function by reversible post-translational modification, SUMOylation, is widely conserved in the eukaryotic kingdom. SUMOylation is essential for cell growth, division, and adaptation to stress in most organisms, including fungi. As these are key factors in determination of fungal virulence, in this study, we have investigated the importance of SUMOylation in the human pathogen, Candida glabrata We identified the enzymes involved in small ubiquitin-like modifier conjugation and show that there is strong conservation between Saccharomyces cerevisiae and C. glabrata We demonstrate that SUMOylation is an essential process and that adaptation to stress involves changes in global SUMOylation in C. glabrata Importantly, loss of the deSUMOylating enzyme CgUlp2 leads to highly reduced small ubiquitin-like modifier protein levels, and impaired growth, sensitivity to multiple stress conditions, reduced adherence to epithelial cells, and poor colonization of specific tissues in mice. Our study thus demonstrates a key role for protein SUMOylation in the life cycle and pathobiology of C. glabrata.

Chitinase activation in patients with fungus-associated cystic fibrosis lung disease.

BACKGROUND: Chitinases have recently gained attention in the field of pulmonary diseases, particularly in asthma and chronic obstructive pulmonary disease, but their potential role in patients with cystic fibrosis (CF)-associated lung disease remains unclear. OBJECTIVE: The aim of this study was to assess chitinase activity systemically and in the airways of patients with CF and asthma compared with healthy subjects. Additionally, we assessed factors that regulate chitinase activity within the lungs of patients with CF. METHODS: Chitinase activities were quantified in serum and bronchoalveolar lavage fluid from patients with CF, asthmatic patients, and healthy control subjects. Mechanistically, the role of CF airway proteases and genetic chitinase deficiency was assessed. RESULTS: Chitinase activity was systemically increased in patients with CF compared with that in healthy control subjects and asthmatic patients. Further stratification showed that chitinase activity was enhanced in patients with CF colonized with Candida albicans compared with that in noncolonized patients. CF proteases degraded chitinases in the airway microenvironment of patients with CF. Genetic chitinase deficiency was associated with C albicans colonization in patients with CF. CONCLUSION: Patients with CF have enhanced chitinase activation associated with C albicans colonization. Therefore chitinases might represent a novel biomarker and therapeutic target for CF-associated fungal disease.

Chitinase 3-Like 1 Promotes Candida albicans Killing and Preserves Corneal Structure and Function by Controlling Host Antifungal Responses.

Chitinase 3-like 1 (CHI3L1) has been shown to play a role in promoting antibacterial responses, decreasing tissue injury, and enhancing pulmonary repair. This study sought to elucidate the role of CHI3L1 in augmenting the corneal innate immune response to Candida albicans infection in an animal model of fungal keratitis. Flagellin applied topically 24 h prior to C. albicans inoculation significantly protected the corneal from C. albicans and induced CHI3L1 expression in C57BL/6 mouse corneas. CHI3L1, however, played a detectable but minor role in flagellin-induced protection. While C. albicans keratitis was more severe in the corneas treated with Chi3l1 small interfering RNA (siRNA), corneas treated with recombinant CHI3L1 before C. albicans inoculation had markedly ameliorated keratitis, reduced fungal load, and decreased polymorphonucleocyte (PMN) infiltration in an interleukin 13 receptor α2 (IL-13Rα2)-dependent manner. CHI3L1 treatment resulted in the induction of the antimicrobial peptides ß-defensin 3, CRAMP, and chemokine CXCL10 and its receptor CXCR3 in corneal epithelial cells. Importantly, CHI3L1 administered after C. albicans inoculation also had strong protection against fungal keratitis, suggesting a therapeutic window. This is the first report demonstrating that CHI3L1 is induced during fungal infection, where it acts as an immunomodulator to promote fungal clearance and to regulate antifungal innate immune responses in the cornea.

NADPH oxidase-driven phagocyte recruitment controls Candida albicans filamentous growth and prevents mortality.

Candida albicans is a human commensal and clinically important fungal pathogen that grows as both yeast and hyphal forms during human, mouse and zebrafish infection. Reactive oxygen species (ROS) produced by NADPH oxidases play diverse roles in immunity, including their long-appreciated function as microbicidal oxidants. Here we demonstrate a non-traditional mechanistic role of NADPH oxidase in promoting phagocyte chemotaxis and intracellular containment of fungi to limit filamentous growth. We exploit the transparent zebrafish model to show that failed NADPH oxidase-dependent phagocyte recruitment to C. albicans in the first four hours post-infection permits fungi to germinate extracellularly and kill the host. We combine chemical and genetic tools with high-resolution time-lapse microscopy to implicate both phagocyte oxidase and dual-specific oxidase in recruitment, suggesting that both myeloid and non-myeloid cells promote chemotaxis. We show that early non-invasive imaging provides a robust tool for prognosis, strongly connecting effective early immune response with survival. Finally, we demonstrate a new role of a key regulator of the yeast-to-hyphal switching program in phagocyte-mediated containment, suggesting that there are species-specific methods for modulation of NADPH oxidase-independent immune responses. These novel links between ROS-driven chemotaxis and fungal dimorphism expand our view of a key host defense mechanism and have important implications for pathogenesis.

Novel mechanism coupling cyclic AMP-protein kinase A signaling and golgi trafficking via Gyp1 phosphorylation in polarized growth.

The cyclic AMP (cAMP)-protein kinase A (PKA) signaling activates virulence expression during hyphal development in the fungal human pathogen Candida albicans. The hyphal growth is characterized by Golgi polarization toward the hyphal tips, which is thought to enhance directional vesicle transport. However, how the hypha-induction signal regulates Golgi polarization is unknown. Gyp1, a Golgi-associated protein and the first GTPase-activating protein (GAP) in the Rab GAP cascade, critically regulates membrane trafficking from the endoplasmic reticulum to the plasma membrane. Here, we report a novel pathway by which the cAMP-PKA signaling triggers Golgi polarization during hyphal growth. We demonstrate that Gyp1 plays a crucial role in actin-dependent Golgi polarization. Hyphal induction activates PKA, which in turn phosphorylates Gyp1. Phosphomimetic mutation of four PKA sites identified by mass spectrometry (Gyp1(4E)) caused strong Gyp1 polarization to hyphal tips, whereas nonphosphorylatable mutations (Gyp1(4A)) abolished it. Gyp1(4E) exhibited enhanced association with the actin motor Myo2, while Gyp1(4A) showed the opposite effect, providing a possible mechanism for Golgi polarization. A GAP-dead Gyp1 (Gyp1(R292K)) showed strong polarization similar to that seen with Gyp1(4E), indicating a role for the GAP activity. Mutating the PKA sites on Gyp1 also impaired the recruitment of a late Golgi marker, Sec7. Furthermore, proper PKA phosphorylation and GAP activity of Gyp1 are required for virulence in mice. We propose that the cAMP-PKA signaling directly targets Gyp1 to promote Golgi polarization in the yeast-to-hypha transition, an event crucial for C. albicans infection.

Candida albicans secreted aspartic proteases 4-6 induce apoptosis of epithelial cells by a novel Trojan horse mechanism.

Systemic infection by the pathogenic yeast Candida albicans produces high mortality in immune-compromised people. Such infection starts with the penetration of the organism at the mucosal surfaces, facilitated by the secreted aspartic proteases (Saps) 4, 5, and 6. The functional mechanism of these virulence factors is unclear. We discovered that Saps 4-6 each contains amino acid motifs RGD/KGD to bind integrins on epithelial cell A549 and are internalized to endosomes and lysosomes. These processes are inhibited by RGD-containing peptides or by substituting RGD motifs of these Saps. The internalization of Saps 4-6 results in partial permeabilization of lysosomal membranes, measured by the redistribution of the lysosomal tropic dye acridine orange to the cytosol, and the triggering of apoptosis via caspase activation. Sap 2 and mutated Saps 4-6 contain no RGD motif, are ineffective in these processes, and a proteolytic inhibitor abolished Sap 4 activity in lysosome permeabilization. Same results were also seen for human tongue keratinocyte SCC-15 cells. Mucosal lesions from this fundamental new mechanism may permit C. albicans to enter the body and may be used to attack cells in immune defense during systemic infections. RGD-motif may also be incorporated in Sap inhibitors for Candidiasis drugs targeting to lysosomes.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) and TGFß-activated kinase 1 (TAK1) play essential roles in the C-type lectin receptor signaling in response to Candida albicans infection.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) and TGFß-activated kinase 1 (TAK1) are considered as key intermediates in Toll-like receptor (TLR) signaling. However, the role of TRAF6 and TAK1 in C-type lectin receptors (CLRs) in response to fungal infection has not been studied. In this study, we have utilized macrophages derived from TRAF6 knock-out mice and myeloid-specific TAK1-deficient mice and determined the role of TRAF6 and TAK1 in CLR-induced signal transduction events. We demonstrate that TRAF6 and TAK1 are required for NF-κB and JNK activation, and expression of proinflammatory cytokines in response to Candida albicans infection. Our results highlight TRAF6 and TAK1 as key components in the signaling cascade downstream of C-type lectin receptors and as critical mediators of the anti-fungal immune response. Therefore, our studies provide a mechanistic understanding of the host immune response to C. albicans, which has a significant impact for the development of anti-fungal therapeutics and in understanding risk-factors and determining susceptibility to C. albicans infection.

Extracellular aspartic protease SAP2 of Candida albicans yeast cleaves human kininogens and releases proinflammatory peptides, Met-Lys-bradykinin and des-Arg(9)-Met-Lys-bradykinin.

Bradykinin-related peptides, universal mediators of inflammation collectively referred to as the kinins, are often produced in excessive amounts during microbial infections. We have recently shown that the yeast Candida albicans, the major fungal pathogen to humans, can exploit two mechanisms to enhance kinin levels at the sites of candidial infection, one depending on adsorption and activation of the endogenous kinin-generating system of the host on the fungal cell wall and the other relying on cleavage of kinin precursors, the kininogens, by pathogen-secreted proteases. This work aimed at assigning this kininogenase activity to the major secreted aspartic protease of C. albicans (SAP2). The purified SAP2 was shown to cleave human kininogens, preferably the low molecular mass form (LK) and optimally in an acidic environment (pH 3.5-4.0), and to produce two kinins, Met-Lys-bradykinin and its derivative, [Hydroxyproline(3)]-Met-Lys-bradykinin, both of which are capable of interacting with cellular bradykinin receptors of the B2 subtype. Additionally, albeit with a lower yield, des-Arg(9)-Met-Lys-bradykinin, an effective agonist of B1-subtype receptors, was released. The pathophysiological potential of these kinins and des-Arg-kinin was also proven by presenting their ability to stimulate human promonocytic cells U937 to release proinflammatory interleukin 1ß (IL-1ß) and IL-6.

Pathways regulating cytosolic phospholipase A2 activation and eicosanoid production in macrophages by Candida albicans.

Resident tissue macrophages are activated by the fungal pathogen Candida albicans to release eicosanoids, which are important modulators of inflammation and immune responses. Our objective was to identify the macrophage receptors engaged by C. albicans that mediate activation of group IVA cytosolic phospholipase A(2) (cPLA(2)α), a regulatory enzyme that releases arachidonic acid (AA) for production of prostaglandins and leukotrienes. A comparison of peritoneal macrophages from wild type and knock-out mice demonstrates that the ß-glucan receptor Dectin-1 and MyD88 regulate early release of AA and eicosanoids in response to C. albicans. However, cyclooxygenase 2 (COX2) expression and later phase eicosanoid production are defective in MyD88(-/-) but not Dectin-1(-/-) macrophages. Furthermore, C. albicans-stimulated activation of MAPK and phosphorylation of cPLA(2)α on Ser-505 are regulated by MyD88 and not Dectin-1. In contrast, Dectin-1 mediates MAPK activation, cPLA(2)α phosphorylation, and COX2 expression in response to particulate ß-glucan suggesting that other receptors engaged by C. albicans preferentially mediate these responses. Results also implicate the mannan-binding receptor Dectin-2 in regulating cPLA(2)α. C. albicans-stimulated MAPK activation and AA release are blocked by d-mannose and Dectin-2-specific antibody, and overexpression of Dectin-2 in RAW264.7 macrophages enhances C. albicans-stimulated MAPK activation, AA release, and COX2 expression. In addition, calcium mobilization is enhanced in RAW264.7 macrophages overexpressing Dectin-1 or -2. The results demonstrate that C. albicans engages both ß-glucan and mannan-binding receptors on macrophages that act with MyD88 to regulate the activation of cPLA(2)α and eicosanoid production.

Chitotriosidase Activity Is Counterproductive in a Mouse Model of Systemic Candidiasis.

Mammalian cells do not produce chitin, an insoluble polymer of N-acetyl-D-glucosamine (GlcNAc), although chitin is a structural component of the cell wall of pathogenic microorganisms such as Candida albicans. Mammalian cells, including cells of the innate immune system elaborate chitinases, including chitotriosidase (Chit1), which may play a role in the anti-fungal immune response. In the current study, using knockout mice, we determined the role of Chit1 against systemic candidiasis. Chit1-deficient mice showed significant decrease in kidney fungal burden compared to mice expressing the functional enzyme. Using in vitro anti-candidal neutrophil functional assays, the introduction of the Chit1:chitin digestion end-product, chitobiose (N-acetyl-D-glucosamine dimer, GlcNAc2), decreased fungal-induced neutrophil swarming and Candida killing in vitro. Also, a role for the lectin-like binding site on the neutrophil integrin CR3 (Mac-1, CD11b/CD18) was found through physiological competitive interference by chitobiose. Furthermore, chitobiose treatment of wild type mice during systemic candidiasis resulted in the significant increase in fungal burden in the kidney. These data suggest a counterproductive role of Chit1 in mounting an efficient anti-fungal defense against systemic candidiasis.

Cytosolic phospholipase a2 activation by Candida albicans in alveolar macrophages: role of dectin-1.

Candida albicans is an increasingly important pulmonary fungal pathogen. Resident alveolar macrophages are important in host defense against opportunistic fungal infections. Activation of Group IVA cytosolic phospholipase A(2)alpha (cPLA(2)alpha) in macrophages initiates arachidonic acid (AA) release for production of eicosanoids, which regulate inflammation and immune responses. We investigated the ability of C. albicans to activate cPLA(2)alpha in unprimed alveolar macrophages and after priming with granulocyte macrophage colony-stimulating factor (GM-CSF), which regulates alveolar macrophage maturation. AA was released within minutes by GM-CSF-primed but not unprimed alveolar macrophages in response to C. albicans, and was blocked by soluble glucan phosphate (S-GP). The expression of the beta-glucan receptor dectin-1 was increased in GM-CSF-primed macrophages, and AA release from GM-CSF-primed dectin-1(-/-) alveolar macrophages was reduced to basal levels. The enhanced activation of extracellular signal-regulated kinases and phosphorylation of cPLA(2)alpha on Ser-505 that occurred in GM-CSF-primed macrophages were reduced by MEK1 and Syk inhibitors, which also suppressed AA release. At later times after C. albicans infection (6 h), unprimed and GM-CSF-primed macrophages released similar levels of AA. The expression of cyclooxygenase 2 and prostanoid production at 6 hours was higher in GM-CSF-primed macrophages, but the responses were not dependent on dectin-1. However, dectin-1 contributed to the C. albicans-stimulated increase in TNF-alpha production that occurred in GM-CSF-primed macrophages. The results demonstrate that dectin-1 mediates the acute activation of cPLA(2)alpha in GM-CSF-primed alveolar macrophages, but not in the more delayed phase of AA release and GM-CSF-dependent prostanoid production.

Lipase of Candida albicans induces activation of NADPH oxidase and L-arginine pathways on resting and activated macrophages.

Candida albicans secretes various hydrolytic enzymes which are considered to be an integral part in the pathogenesis. However, the role of lipases is far from being completely understood and the direct effects of these fungal enzymes during the host-pathogen interaction remain to be established. We recently isolated and characterized an extracellular C. albicans lipase (CaLIP), and demonstrated the ability of this fungal enzyme to interact directly with macrophages (Mvarphi) and hepatocytes and to operate as a virulence factor. Herein, we explored the effects of CaLIP on Mvarphi functions such as oxidative burst and l-arginine metabolism. The study was performed in cells with different activation status: normal-resting Mvarphis and Mvarphis primed in vivo or in vitro with C. albicans. The ability of this fungal factor to modulate the above-mentioned parameters was dependent on cells status, dose, and microenvironment, where the interaction took place. These results constitute a new finding in the biology of candidiasis and could illustrate an additional evolutive advantage for the fungus in the framework of the bidirectional host-pathogen interaction.

Endothelial nitric oxide synthase limits host immunity to control disseminated Candida albicans infections in mice.

Three isoforms of nitric oxide synthase (NOS) occur in mammals. High levels of NO produced by NOS2/iNOS can protect against bacterial and parasitic infections, but the role of NOS in fungal innate immunity is less clear. Compared to wild type mice, Nos3-/- mice showed significantly higher survival of candidemia caused by Candida albicans SC5314. NOS3/eNOS is expressed by endothelial cells in the kidney, and colonization of this organ was decreased during the sub-acute stage of disseminated candidiasis. Nos3-/- mice more rapidly eliminated Candida from the renal cortex and exhibited more balanced local inflammatory reactions, with similar macrophage but less neutrophil infiltration than in infected wild type. Levels of the serum cytokines IL-9, IL-12, IL-17 and chemokines GM-CSF, MIP1α, and MIP1ß were significantly elevated, and IL-15 was significantly lower in infected Nos3-/- mice. Spleens of infected Nos3-/- mice had significantly more Th2 and Th9 but not other CD4+ T cells compared with wild type. Inflammatory genes associated with leukocyte chemotaxis, IL-1 signaling, TLR signaling and Th1 and Th2 cell differentiation pathways were significantly overexpressed in infected Nos3-/- kidneys, with Nos2 being the most strongly induced. Conversely, the general NOS inhibitor NG-nitro-L-arginine methyl ester increased virulence in the mouse candidemia model, suggesting that iNOS contributes to the protective mechanism in infected Nos3-/- mice. By moderating neutrophil infiltration, the absence of eNOS may reduce the collateral damage to kidney cortex, and Th-9 CD4+ cells may enhance clearance of the infection. These data suggest that selective eNOS inhibition could mitigate candidemia by a combination of systemic and local responses that promote a more effective host immune response.

Targeting SHIP-1 in Myeloid Cells Enhances Trained Immunity and Boosts Response to Infection.

ß-Glucan-induced trained immunity in myeloid cells leads to long-term protection against secondary infections. Although previous studies have characterized this phenomenon, strategies to boost trained immunity remain undefined. We found that ß-glucan-trained macrophages from mice with a myeloid-specific deletion of the phosphatase SHIP-1 (LysMΔSHIP-1) showed enhanced proinflammatory cytokine production in response to lipopolysaccharide. Following ß-glucan training, SHIP-1-deficient macrophages exhibited increased phosphorylation of Akt and mTOR targets, correlating with augmented glycolytic metabolism. Enhanced training in the absence of SHIP-1 relied on histone methylation and acetylation. Trained LysMΔSHIP-1 mice produced increased amounts of proinflammatory cytokines upon rechallenge in vivo and were better protected against Candida albicans infection compared with control littermates. Pharmacological inhibition of SHIP-1 enhanced trained immunity against Candida infection in mouse macrophages and human peripheral blood mononuclear cells. Our data establish proof of concept for improvement of trained immunity and a strategy to achieve it by targeting SHIP-1.

Leukocyte myeloperoxidase deficiency and disseminated candidiasis: the role of myeloperoxidase in resistance to Candida infection.

The neutrophils and monocytes of a patient with disseminated candidiasis were found to lack detectable levels of the lysosomal enzyme myeloperoxidase (MPO), although they had normal levels of other granule-associated enzymes. Leukocytes from one of the patient's sisters also lacked detectable MPO; leukocytes from his four sons contained approximately one-third of mean normal peroxidase levels. Neither the patient nor his affected relatives had experienced frequent or unusual bacterial infections. The phagocytic activity of the patient's MPO-deficient neutrophils was intact, and the cells displayed normal morphologic and metabolic responses to phagocytosis. In contrast to normal leukocytes which killed 30.5+/-7.3% of ingested Candida albicans in 1 hr, however, the patient's neutrophils killed virtually none. His leukocytes also failed to kill the strain of C. albicans recovered from his lesions, as well as other Candida species. These MPO-deficient neutrophils killed Serratia marcescens and Staphylococens aureus 502A at an abnormally slow rate, requiring 3-4 hr to achieve the bactericidal effect attained by normal leukocytes after 45 min. No other abnormalities in his cellular or humoral immune responses were demonstrated. These findings suggest that hereditary MPO deficiency is transmitted as an autosomal recessive characteristic, that the homozygous state conveys enhanced susceptibility to disseminated candidiasis, and that MPO is necessary for candidacidal activity in human neutrophils. Although lending support to the suggested bactericidal role of MPO in leukocytes, the data indicate that alternative bactericidal mechanisms, effective in the absence of MPO, are functionally dominant in the human neutrophil.

Superoxide dismutases in Candida albicans: transcriptional regulation and functional characterization of the hyphal-induced SOD5 gene.

Superoxide dismutases (SOD) convert superoxide radicals into less damaging hydrogen peroxide. The opportunistic human pathogen Candida albicans is known to express CuZnSOD (SOD1) and MnSOD (SOD3) in the cytosol and MnSOD (SOD2) in the mitochondria. We identified three additional CuZn-containing superoxide dismutases, SOD4, SOD5, and SOD6, within the sequence of the C. albicans genome. The transcription of SOD5 was up-regulated during the yeast to hyphal transition of C. albicans, and SOD5 was induced when C. albicans cells were challenged with osmotic or with oxidative stresses. SOD5 transcription was also increased when cells were grown on nonfermentable substrates as the only carbon source. The Rim101p transcription factor was required for all inductions observed, whereas the Efg1p transcription factor was specifically needed for serum-modulated expression. Deletion of SOD5 produced a viable mutant strain that showed sensitivity to hydrogen peroxide when cells were grown in nutrient-limited conditions. Sod5p was found to be necessary for the virulence of C. albicans in a mouse model of infection. However, the sod5 mutant strain showed the same resistance to macrophage attack as its parental strain, suggesting that the loss of virulence in not due to an increased sensitivity to macrophage attack.

Trehalose-6-Phosphate as a Potential Lead Candidate for the Development of Tps1 Inhibitors: Insights from the Trehalose Biosynthesis Pathway in Diverse Yeast Species.

In some pathogens, trehalose biosynthesis is induced in response to stress as a protection mechanism. This pathway is an attractive target for antimicrobials as neither the enzymes, Tps1, and Tps2, nor is trehalose present in humans. Accumulation of T6P in Candida albicans, achieved by deletion of TPS2, resulted in strong reduction of fungal virulence. In this work, the effect of T6P on Tps1 activity was evaluated. Saccharomyces cerevisiae, C. albicans, and Candida tropicalis were used as experimental models. As expected, a heat stress induced both trehalose accumulation and increased Tps1 activity. However, the addition of 125 µM T6P to extracts obtained from stressed cells totally abolished or reduced in 50 and 60 % the induction of Tps1 activity in S. cerevisiae, C. tropicalis, and C. albicans, respectively. According to our results, T6P is an uncompetitive inhibitor of S. cerevisiae Tps1. This kind of inhibitor is able to decrease the rate of reaction to zero at increased concentrations. Based on the similarities found in sequence and function between Tps1 of S. cerevisiae and some pathogens and on the inhibitory effect of T6P on Tps1 activity observed in vitro, novel drugs can be developed for the treatment of infectious diseases caused by organisms whose infectivity and survival on the host depend on trehalose.