Candida albicans suppresses nitric oxide generation from macrophages via a secreted molecule.

Macrophages and neutrophils generate a potent burst of reactive oxygen and nitrogen species as a key aspect of the antimicrobial response. While most successful pathogens, including the fungus Candida albicans, encode enzymes for the detoxification of these compounds and repair of the resulting cellular damage, some species actively modulate immune function to suppress the generation of these toxic compounds. We report here that C. albicans actively inhibits macrophage production of nitric oxide (NO). NO production was blocked in a dose-dependent manner when live C. albicans were incubated with either cultured or bone marrow-derived mouse macrophages. While filamentous growth is a key virulence trait, yeast-locked fungal cells were still capable of dose-dependent NO suppression. C. albicans suppresses NO production from macrophages stimulated by exposure to IFN-γ and LPS or cells of the non-pathogenic Saccharomyces cerevisiae. The NO inhibitory activity was produced only when the fungal cells were in direct contact with macrophages, but the compound itself was secreted into the culture media. LPS/IFNγ stimulated macrophages cultured in cell-free conditioned media from co-cultures showed reduced levels of iNOS enzymatic activity and lower amounts of iNOS protein. Initial biochemical characterization of this activity indicates that the inhibitor is a small, aqueous, heat-stable compound. In summary, C. albicans actively blocks NO production by macrophages via a secreted mediator; these findings expand our understanding of phagocyte modulation by this important fungal pathogen and represent a potential target for intervention to enhance antifungal immune responses.

The fungal pathogen Candida albicans autoinduces hyphal morphogenesis by raising extracellular pH.

UNLABELLED: pH homeostasis is critical for all organisms; in the fungal pathogen Candida albicans, pH adaptation is critical for virulence in distinct host niches. We demonstrate that beyond adaptation, C. albicans actively neutralizes the environment from either acidic or alkaline pHs. Under acidic conditions, this species can raise the pH from 4 to >7 in less than 12 h, resulting in autoinduction of the yeast-hyphal transition, a critical virulence trait. Extracellular alkalinization has been reported to occur in several fungal species, but under the specific conditions that we describe, the phenomenon is more rapid than previously observed. Alkalinization is linked to carbon deprivation, as it occurs in glucose-poor media and requires exogenous amino acids. These conditions are similar to those predicted to exist inside phagocytic cells, and we find a strong correlation between the use of amino acids as a cellular carbon source and the degree of alkalinization. Genetic and genomic approaches indicate an emphasis on amino acid uptake and catabolism in alkalinizing cells. Mutations in four genes, STP2, a transcription factor regulating amino acid permeases, ACH1 (acetyl-coenzyme A [acetyl-CoA] hydrolase), DUR1,2 (urea amidolyase), and ATO5, a putative ammonia transporter, abolish or delay neutralization. The pH changes are the result of the extrusion of ammonia, as observed in other fungi. We propose that nutrient-deprived C. albicans cells catabolize amino acids as a carbon source, excreting the amino nitrogen as ammonia to raise environmental pH and stimulate morphogenesis, thus directly contributing to pathogenesis. IMPORTANCE: Candida albicans is the most important fungal pathogen of humans, causing disease at multiple body sites. The ability to switch between multiple morphologies, including a rounded yeast cell and an elongated hyphal cell, is a key virulence trait in this species, as this reversible switch is thought to promote dissemination and tissue invasion in the host. We report here that C. albicans can actively alter the pH of its environment and induce its switch to the hyphal form. The change in pH is caused by the release of ammonia from the cells produced during the breakdown of amino acids. This phenomenon is unprecedented in a human pathogen and may substantially impact host physiology by linking morphogenesis, pH adaptation, carbon metabolism, and interactions with host cells, all of which are critical for the ability of C. albicans to cause disease.

Carnitine acetyltransferases are required for growth on non-fermentable carbon sources but not for pathogenesis in Candida albicans.

Carbon starvation is a significant stress encountered by the opportunistic fungal pathogen Candida albicans, and mutations in several pathways required to assimilate non-fermentable carbon sources attenuate virulence. These pathways -- beta-oxidation, the glyoxylate cycle and gluconeogenesis -- are compartmentalized in the fungal cell between the peroxisome, mitochondria and cytosol; thus, the cell must transport key intermediates between these organelles. Transport of acetyl-CoA, a particularly important intermediate of carbon metabolism, is catalysed by membrane-associated carnitine acetyltransferases (CATs). We report here the characterization of the three predicted CAT genes in C. albicans, CTN1, CTN2 and CTN3. Strains lacking CTN1 or CTN2 were unable to grow on ethanol or acetate as sole carbon source; additionally, citrate was utilized poorly (Deltactn2) or not at all (Deltactn1) and the Deltactn2 mutant failed to grow on fatty acids as well. In contrast, deletion of CTN3 had no observable phenotype. All three genes were upregulated in the presence of non-fermentable carbon sources and after macrophage phagocytosis. CTN1 and CTN3 were able to complement the corresponding Saccharomyces cerevisiae Deltayat1 and Deltayat2 mutants. However, these mutants had no obvious attenuation in virulence in a mouse model of disseminated candidiasis, in contrast to other carbon metabolism mutants. These findings extend our understanding of nutrient stress in vivo and in vitro and the contribution of metabolic pathways to virulence in C. albicans.

CTA4 transcription factor mediates induction of nitrosative stress response in Candida albicans.

This work has identified regulatory elements in the major fungal pathogen Candida albicans that enable response to nitrosative stress. Nitric oxide (NO) is generated by macrophages of the host immune system and commensal bacteria, and the ability to resist its toxicity is one adaptation that promotes survival of C. albicans inside the human body. Exposing C. albicans to NO induces upregulation of the flavohemoglobin Yhb1p. This protein confers protection by enzymatically converting NO to harmless nitrate, but it is unknown how C. albicans is able to detect NO in its environment and thus initiate this defense only as needed. We analyzed this problem by incrementally mutating the YHB1 regulatory region to identify a nitric oxide-responsive element (NORE) that is required for NO sensitivity. Five transcription factor candidates of the Zn(II)2-Cys6 family were then isolated from crude whole-cell extracts by using magnetic beads coated with this DNA element. Of the five, only deletion of the CTA4 gene prevented induction of YHB1 transcription during nitrosative stress and caused growth sensitivity to the NO donor dipropylenetriamine NONOate; Cta4p associates in vivo with NORE DNA from the YHB1 regulatory region. Deletion of CTA4 caused a small but significant decrease in virulence. A CTA4-dependent putative sulfite transporter encoded by SSU1 is also implicated in NO response, but C. albicans ssu1 mutants were not sensitive to NO, in contrast to findings in Saccharomyces cerevisiae. Cta4p is the first protein found to be necessary for initiating NO response in C. albicans.

MinC mutants deficient in MinD- and DicB-mediated cell division inhibition due to loss of interaction with MinD, DicB, or a septal component.

The min locus encodes a negative regulatory system that limits formation of the cytokinetic Z ring to midcell by preventing its formation near the poles. Of the three Min proteins, MinC is the inhibitor and prevents Z-ring formation by interacting directly with FtsZ. MinD activates MinC by recruiting it to the membrane and conferring a higher affinity on the MinCD complex for a septal component. MinE regulates the cellular location of MinCD by inducing MinD, and thereby MinC, to oscillate between the poles of the cell, resulting in a time-averaged concentration of MinCD on the membrane that is lowest at midcell. MinC can also be activated by the prophage-encoded protein DicB, which targets MinC to the septum without recruiting it first to the membrane. Previous studies have shown that the C-terminal domain of MinC is responsible for the interaction with MinD, DicB, and the septal component. In the present study, we isolated mutations in the C-terminal domain of MinC that affected its interaction with MinD, DicB, and the septal component. Among the mutations isolated, R133A and S134A are specifically deficient in the interaction with MinD, E156A is primarily affected in the interaction with DicB, and R172A is primarily deficient in the interaction with the septum. These mutations differentiate the interactions of MinC with its partners and further support the model of MinCD- and MinC-DicB-mediated cell division inhibition.

Analysis of MinD mutations reveals residues required for MinE stimulation of the MinD ATPase and residues required for MinC interaction.

The MinD ATPase is critical to the oscillation of the Min proteins, which limits formation of the Z ring to midcell. In the presence of ATP, MinD binds to the membrane and recruits MinC, forming a complex that can destabilize the cytokinetic Z ring. MinE, which is also recruited to the membrane by MinD, displaces MinC and stimulates the MinD ATPase, resulting in the oscillation of the Min proteins. In this study we have investigated the role of lysine 11, present in the deviant Walker A motif of MinD, and the three residues in helix 7 (E146, S148, and D152) that interact electrostatically with lysine 11. Lysine 11 is required for interaction of MinD with the membrane, MinC, MinE, and itself. In contrast, the three residues in helix 7 that interact with lysine 11 are not required for binding to the membrane or activation of MinC. They are also not required for MinE binding; however, they are required for MinE to stimulate the MinD ATPase. Interestingly, the D152A mutant self-interacts, binds to the membrane, and recruits MinC and MinE in the presence of ADP as well as ATP. This mutant provides evidence that dimerization of MinD is sufficient for MinD to bind the membrane and recruit its partners.

The switch I and II regions of MinD are required for binding and activating MinC.

MinD and MinC cooperate to form an efficient inhibitor of Z-ring formation that is spatially regulated by MinE. MinD activates MinC by recruiting it to the membrane and targeting it to a septal component. To better understand this activation, we have isolated loss-of-function mutations in minD and carried out site-directed mutagenesis. Many of these mutations block MinC-MinD interaction; however, they also prevent MinD self-interaction and membrane binding, suggesting that they affect nucleotide interaction or protein folding. Two mutations in the switch I region (MinD box) and one mutation in the switch II region had little affect on most MinD functions, such as MinD self-interaction, membrane binding, and MinE stimulation; however, they did eliminate MinD-MinC interaction. Two additional mutations in the switch II region did not affect MinC binding. Further study revealed that one of these allowed the MinCD complex to target to the septum but was still deficient in blocking division. These results indicate that the switch I and II regions of MinD are required for interaction with MinC but not MinE and that the switch II region has a role in activating MinC.

Membrane binding by MinD involves insertion of hydrophobic residues within the C-terminal amphipathic helix into the bilayer.

MinD binds to phospholipid vesicles in the presence of ATP and is released by MinE, which stimulates the MinD ATPase. Membrane binding requires a short conserved C-terminal region, which has the potential to form an amphipathic helix. This finding has led to a model in which the binding of ATP regulates the formation or accessibility of this helix, which then embeds in the membrane bilayer. To test this model, we replaced each of the four hydrophobic residues within this potential helix with tryptophan or a charged residue. Introduction of a negatively charged amino acid decreased membrane binding of MinD and its ability to activate MinC. In contrast, mutants with tryptophan substitutions retained the ability to bind to the membrane and activate MinC. Fluorescence emission spectroscopy analysis of the tryptophan mutants F263W, L264W, and L267W confirmed that these tryptophan residues did insert into the hydrophobic interior of the bilayer. We conclude that membrane binding by MinD involves penetration of the hydrophobic residues within the C-terminal amphipathic helix into the hydrophobic interior of the bilayer.