Candida albicans is the most important human-associated fungus, existing as both an integral component of the microbiota and as a pathogen of multiple organ systems. C. albicans causes infections ranging from easily treatable mucosal infections (thrush, vaginitis) to refractory and frequently fatal systemic infections. Disseminated infections are often a result of defects in innate immunity and this has motivated studies of C. albicans-phagocyte interactions. Phagocytosis by macrophages induces a dynamic and complex response in the fungus, including a dramatic metabolic shift to a gluconeogenic mode of growth, and we have shown that many aspects of this shift are required for full virulence. Modeling this system in vitro has uncovered a previously unknown ability of this organism to radically change extracellular pH as a byproduct of these metabolic changes. Amino acids are predicted to be plentiful in the host, and their use to satisfy cellular carbon requirements result in a dramatic rise in extracellular pH, driven by the export of ammonia, and perhaps other basic nitrogenous compounds. We propose a model in which this ammonia derives from the amino or side-chain amine groups of the amino acids as a metabolic byproduct. While this is a potential consequence of amino acid catabolism in most organisms, Candida species (C. albicans in particular) show a far more robust pH change than has been previously described. Understanding this process is part of my long-standing interest in how this organism has adapted basic metabolic functions to support it as a successful commensal and pathogen. Genetic analysis has identified non-alkalinizing mutants, which are enriched for amino acid import and catabolic functions. The most severely deficient mutant is in STP2, encoding a transcription that regulates amino acid permease expression. Mutants lacking STP2 are impaired during contact with host cells: they germinate poorly and are both more susceptible to killing by macrophages and cause less damage to macrophages than wild-type controls. Moreover, stp2? cells are found in acidic phagolysosomes, as are killed cells. In contrast, wild-type cells occupy a more neutral phagolysosome, suggesting they have a mechanism to alter intracellular pH lacking in stp2? cells. Others have shown that stp2? mutants are attenuated in a fly model of candidiasis;two other non-alkalinizing mutant, csh3?, encoding an ER chaperone for amino acid permeases, and dur1,2?, encoding the amino acid catabolic enzyme urea amidolyase, are avirulent in mice. Thus, there is strong preliminary evidence that factors that regulate alkalinization also alter the host-pathogen interaction, though the precise connection between the two has not been firmly established. We have recently discovered a second alkalinization mechanism during growth on dicarboxylic acid intermediates of the TCA cycle, and will characterize this mechanism as well. This application proposes to test the hypothesis that alkalinization of key host niches, including the phagolysosome, promotes virulence of C. albicans by inhibiting the host immune response. We will do so by testing our model for the metabolic adaptations that make this phenomenon much more vigorous in C. albicans than in other fungi, including determining the source of the ammonia (and other potential basic metabolites) released. We will also determine whether alkalinization occurs within the phagocyte and if this is the key signal that induces hyphal morphogenesis. Finally, we will use live cell imaging to dissect the intracellular trafficking of C. albicans in the macrophage and determine the role of alkalinization and ammonia release in modulating endocytic maturation.

Public Health Relevance

Candida albicans is a normal part of the population of microbes that live in the human gastrointestinal tract. In immunocompromised patients, however, it can cause bloodstream infections (sepsis) that are often fatal. We have studied the interaction of C. albicans with macrophages, an immune system cell type critical for normal defense against fungal infections, revealing a complex and dynamic response to the macrophage. As part of these studies, we have shown that C. albicans can actively alter its environment, raising the pH outside the cell. We have published a mechanism that involves excretion of ammonia derived from the breakdown of amino acids, but recently discovered a second mechanism as well. In culture medium, this induces the cell to switch from a round yeast form to an invasive, elongated hyphal form, a switch known to be required for disease progression in animals. This application proposes experiments to further understand the metabolic reactions that drive this pH change and the impact of this phenomenon on interactions with macrophages and in animal models.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
5R01AI075091-07
Application #
8666707
Study Section
Pathogenic Eukaryotes Study Section (PTHE)
Program Officer
Duncan, Rory A
Project Start
Project End
Budget Start
Budget End
Support Year
7
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of Texas Health Science Center Houston
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
City
Houston
State
TX
Country
United States
Zip Code
77225
Collette, John R; Zhou, Huaijin; Lorenz, Michael C (2014) Candida albicans suppresses nitric oxide generation from macrophages via a secreted molecule. PLoS One 9:e96203
Vylkova, Slavena; Lorenz, Michael C (2014) Modulation of phagosomal pH by Candida albicans promotes hyphal morphogenesis and requires Stp2p, a regulator of amino acid transport. PLoS Pathog 10:e1003995
Cruz, Melissa R; Graham, Carrie E; Gagliano, Bryce C et al. (2013) Enterococcus faecalis inhibits hyphal morphogenesis and virulence of Candida albicans. Infect Immun 81:189-200
Jain, Charu; Pastor, Kelly; Gonzalez, Arely Y et al. (2013) The role of Candida albicans AP-1 protein against host derived ROS in in vivo models of infection. Virulence 4:67-76
Marshall, Alexandra N; Montealegre, Maria Camila; Jimenez-Lopez, Claudia et al. (2013) Alternative splicing and subfunctionalization generates functional diversity in fungal proteomes. PLoS Genet 9:e1003376
Jimenez-Lopez, Claudia; Collette, John R; Brothers, Kimberly M et al. (2013) Candida albicans induces arginine biosynthetic genes in response to host-derived reactive oxygen species. Eukaryot Cell 12:91-100
Vylkova, Slavena; Carman, Aaron J; Danhof, Heather A et al. (2011) The fungal pathogen Candida albicans autoinduces hyphal morphogenesis by raising extracellular pH. MBio 2:e00055-11
Collette, John R; Lorenz, Michael C (2011) Mechanisms of immune evasion in fungal pathogens. Curr Opin Microbiol 14:668-75
Lorenz, Michael C (2010) Host-microbe interactions: fungi. Curr Opin Microbiol 13:389-91
Palanisamy, Suresh K A; Ramirez, Melissa A; Lorenz, Michael et al. (2010) Candida albicans PEP12 is required for biofilm integrity and in vivo virulence. Eukaryot Cell 9:266-77

Showing the most recent 10 out of 12 publications