Citation: Vylkova S (2017) Environmental pH modulation by pathogenic fungi as a strategy to conquer the host. PLoS Pathog 13(2): e1006149. https://doi.org/10.1371/journal.ppat.1006149
Editor: Deborah A. Hogan, Geisel School of Medicine at Dartmouth, UNITED STATES
Published: February 23, 2017
Copyright: © 2017 Slavena Vylkova. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported the German Ministry for Education and Science in the program Unternehmen Region (BMBF 03Z2JN11). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The author has declared that no competing interests exist.
The ability of microorganisms to sense and adapt to changes in the environment is essential to their survival. This is particularly important for species with an intimate association with host organisms, such as pathogens, symbionts, and commensals. Host environments vary greatly in pH, ranging from highly acidic in the stomach (pH < 2) to mildly acidic on the skin and plant surfaces (6.5 < pH < 4.5), neutral in the blood (pH 7.4), and basic in parts of the intestine (pH < 8.5) [1–4], and fungi have developed multiple mechanisms to adapt to pH variations. This Pearl will focus on the ability of pathogenic fungi to respond to and actively modulate the host’s pH.
Fungal adaptation to changes in ambient pH
In fungi, the adaptive responses induced by changes in ambient pH have been extensively studied in model organisms. For example, the response to weak acid stress has been characterized in Saccharomyces cerevisiae , while the role of the Pal/Rim alkaline response pathway, one of the most specialized and conserved signaling cascades in fungi, has been delineated in S. cerevisiae, Aspergillus nidulans, Yarrowia lipolytica, and several fungal pathogens [6–8]. The mechanisms of pH sensing and adaptation in fungi have been reviewed elsewhere [9–11].
Fungal pathogens can modulate the pH of their host
Another aspect of pH regulation is the ability of microorganisms to actively modify the pH of their environment. Fungi can achieve this by secreting acids or alkali. The ability of fungi to secrete natural organic acids (such as butyrate, oxalate, malate, citrate, gluconate, and succinate) is well utilized in the industry, particularly with nonpathogenic Aspergillus sp. and Rhizopus sp. The magnitude of pH change depends on the nutrient availability, the organic acids being produced, and on the ability of the fungus to remove ammonium ions from ammonium sulfate salt or to excrete H+-ions as a byproduct of NH4+ assimilation [12, 13]. Acidifying fungi can also raise extremely low pH levels to a favorable level.
Certain pathogenic fungi acidify the environment as a strategy to damage host tissues. Many plant-necrotizing fungi secrete significant amounts of acid: Sclerotinia sclerotiorum and Butrytis sp. produce oxalic acid , while Pennicilium sp. and Aspergillus sp. secrete mainly gluconic and citric acids [15, 16]. The produced acids not only acidify the tissues but can also lower the activity of reactive oxygen species produced by the host . Fusaric acid produced by Fusarium oxysporum acidifies plant surfaces and activates the membrane H+-ATPase, a pH-regulated process that leads to the expression of proteases and subsequent tissue invasion . Similarly, the human pathogen Candida albicans acidifies the environment in a carbohydrate-dependent fashion, allowing production of aspartyl proteases, which are potent virulence factors .
Ammonia as a key player in environmental alkalinization
Environmental alkalinization in fungi is common yet not a well-understood phenomenon. Often, this process is mediated by ammonia, a multifunctional biological molecule with diverse roles in eukaryotes. In fungi, it supports communication between colonies in S. cerevisiae , aging of surface-ripened cheeses by Y. lipolytica and Debaryomyces hansenii , and expression of pectin lyase, a key virulence factor in Colletotrichum sp. , among other functions.
Ammonia is generated either extracellularly or within the fungal cell as a byproduct of protein and amino acid catabolism, common nutrients in many host niches [23, 24]. Excess intracellular ammonia is secreted or exported from the cell or exported as urea and subsequently converted to NH4+ by secreted ureases. Accumulation of this highly basic compound in the immediate environment raises the pH. An excellent example of this process is found in the phytopathogen Colletotrichum gloeosporioides, a cause of anthracnose fruit rot. The fungus utilizes L-glutamate or glutamine to produce ammonia, which elevates the environmental pH of healthy fruit from 5.6 to 8.5. This results in the activation of fungal pathogenicity factors, such as production of pectate lyase, induction of appressorium formation during host penetration, and stimulation of host cell death mechanisms  (Fig 1). In other fungal species, the alkalinized environment also activates the expression of virulence traits: production of asexual spores and secretion of lytic enzymes in Magnaporthe oryzae, melanin formation and capsule production in Cryptococcus neoformans, and hyphal morphogenesis, adhesion, and invasion in C. albicans, among others [25–29].
C. gloeosporioides infects the tomato fruit in a process initiated upon attachment of the fungal conidia to the plant surface. During the quiescent stage of infection, fruit physiological factors such as nutrient availability, acidic pH, and surface waxes determine the rate of fungal growth and germination. As the fruit ripens, conidia germinate into a specialized structure, named appressorium, which eventually becomes melanized. Melanin alters the permeability of the plant cell wall, creating a hypertonic environment that allows the fungus to penetrate the host epidermis using turgor pressure. This process is accompanied by active metabolism of amino acids, such as glutamate and glutamine, and gradual environmental alkalization. The fungus transitions into the necrotrophic stage, characterized by a dramatic shift in fungal metabolism and activation of pathogenicity factors, such as proteases and lyases, resulting in anthracnose fruit rot.
Typically, fungi increase the environmental pH at a steady but slow pace. A clear exception is C. albicans, which is capable of ammonia-driven alkalinization at a remarkable rate. Upon growth on amino acids as the sole carbon source, this human pathogen can modulate the environmental pH from 4 to ~7.5 within few hours, a process also driven (albeit more slowly) by other Candida sp. [23, 30] The alkalinization mechanism has been studied extensively and includes sensing of amino acids from the extracellular milieu via the SPS (Ssy1, Ptr3, and Ssy5) sensor system, followed by activation of the transcription factor Stp2p in an SPS-dependent manner and induction of amino acid influx . As a result, ammonia is generated and exported via ammonium transporters to raise the environmental pH and allow fungal transition to the more virulent hyphal form [32, 33]. Deletion of genes involved in any step of this mechanism leads to impaired generation of ammonia and neutralization of the medium in response to these nutrients. Thus, metabolism of amino acids is critical for pH modulation by this fungus.
C. albicans is closely associated with the host and has evolved to utilize a variety of nonpreferred carbon sources available in different anatomical sites. Metabolism of organic acids and N-acetylglucosamine also results in environmental alkalinization [23, 34, 35]. However, genes required for alkalinization on amino acids do not affect growth or pH changes on these nutrients, suggesting different mechanisms for pH modulation . Most importantly, C. albicans cells grown on organic acids do not generate ammonia. How C. albicans generates a basic extracellular environment under these conditions is currently unknown. It is also not clear if other fungal species share the ability to neutralize the environment upon utilization of these nutrients.
Regulation of ammonia production
Fungi regulate the production of ammonia depending on environmental cues. Ammonia production by M. anisopliae is tightly regulated by amino acids, a signal for the presence of proteinaceous nutrients in the environment. M. anisopliae grown in media containing low levels of single amino acids yields higher levels of ammonia than when amino acids are abundant, implying either induction of catabolite repressible enzyme(s) or regulation of enzyme activity via substrate inhibition . Generation of ammonia by N. crassa and A. fumigatus is a loosely regulated process triggered by nutrient deprivation . Presence of glucose in the environment represses the process, presumably due to the metabolic switch from gluconeogenesis to glycolysis or repression of deaminases and ammonia transporters. It is also possible that the higher growth rate in the presence of glucose allows for complete utilization of ammonia released from amino acid catabolism.
Environmental alkalinization as a virulence factor
Many fungal pathogens modulate environmental pH as a means to escape host immune responses, facilitate destruction of the host tissues, and/or stimulate reproduction. Most fungi inhabit mildly acidic environments, such as soil, plant, and animal surfaces. On the other hand, for some fungi, such as the phytopathogens C. gloeosporioides and M. oryzae, acidic pH favors fungal colonization and invasion [12, 28]. The mildly acidic pH of the plant surface favors both germination of attached conidia and rapid differentiation of the germ tube into a specialized cell named appressorium. Once the appressorium penetrates the plant tissues, the fungus switches to necrotrophic development, associated with rapid ammonia release and increase in environmental pH, which triggers the expression of virulence factors [13, 22, 28, 37]. Thus, the acidic environment serves as a signal in this fungus to switch from saprotrophic to necrotrophic growth and damage the host (Fig 1).
Neutralization of acidic niches is a very common microbial strategy to evade host immune responses. For example, C. albicans neutralizes the macrophage phagosome, a process essential for germination and escape from the immune cell [31–33]. C. albicans genes essential for pH modulation in vitro fail to induce macrophage damage, highlighting the importance of this process in immune evasion (Fig 2) [31–33]. Similarly, Candida glabrata mutants with alkalinization defect in vitro, such as cells lacking functional mannosyltransferases, fail to effectively modify phagosomal pH and damage the macrophage . The spherules of Coccidioides spp., causative agents of coccidioidomycosis, release enzymatically active urease and ammonia to raise the environmental pH and destroy the host tissue [39, 40]. Disruption of the urea degradation pathway in this organism significantly attenuates fungal virulence and survival in mice . Thus, fungal pathogens can alkalinize the host environment to modulate their virulence, underlining the importance of this process on microbial and human physiology.
Upon phagocytosis by the macrophages, C. albicans responds to the presence of amino acids and other alternative carbon sources abundant in the phagosomal milieu. Amino acids (AA) in particular are sensed via the SPS sensor system (orange), leading to activation of the transcription factor Stp2p, which induces the expression of genes encoding for amino acid permeases (green rectangles) and ammonium transporters (purple rectangles). This results in uptake of amino acids into and release of NH4+ from the fungal cell and increase of phagosomal pH. Hyphal growth causes physical damage to the macrophage membranes, leading to leakage of cellular content and death. C. albicans cells defective in utilization of amino acids and/or extrusion of NH4+, such as the stp2Δ mutant, fail to modulate the pH of the phagosome and are readily cleared by the immune cells.
In summary, adaptation of fungi to pH variations in the host is critical for their survival, and fungal pathogens are capable of actively modulating the environmental pH. Acidification of the host tissues promotes expression and activity of fungal proteases. Many fungi utilize nitrogen or carbon metabolism pathways to generate ammonia, which is released from the cell to raise the extracellular pH. Generation of alkaline pH favors morphogenetic and reproductive processes in fungi, such as germination, hyphal growth, and formation of fruiting bodies, all critical for disease progression. The alkaline pH increases fungal virulence by facilitating penetration into host surfaces and hindering or evasion of immune responses. Thus, the ability to control extracellular pH is an important aspect of fungal physiology that contributes to fitness within the host.
I am grateful to M. C. Lorenz for the constructive criticism and the helpful suggestions.
- 1. Arbenz H. [Studies on pH values of the surface of the normal skin]. Dermatologica. 1952;105(6):333–53. pmid:13043261
- 2. Krahulec IL, Poncova-Kozouskova V. [Determination of pH in the oral cavity]. Cesk Stomatol. 1951;51(6–7):210–22. pmid:14887035
- 3. Dubois D. A GLASS ELECTRODE FOR TESTING THE pH OF BLOOD. Science. 1932;76(1976):441–3. pmid:17744347
- 4. Lang WR. Vaginal acidity and pH; a review. Obstet Gynecol Surv. 1955;10(4):546–60. pmid:13244967
- 5. Piper P, Calderon CO, Hatzixanthis K, Mollapour M. Weak acid adaptation: the stress response that confers yeasts with resistance to organic acid food preservatives. Microbiology. 2001;147(Pt 10):2635–42. pmid:11577142
- 6. Blanchin-Roland S, Da Costa G, Gaillardin C. ESCRT-I components of the endocytic machinery are required for Rim101-dependent ambient pH regulation in the yeast Yarrowia lipolytica. Microbiology. 2005;151(Pt 11):3627–37. pmid:16272384
- 7. Penalva MA, Arst HN Jr. Regulation of gene expression by ambient pH in filamentous fungi and yeasts. Microbiol Mol Biol Rev. 2002;66(3):426–46, table of contents. PubMed Central PMCID: PMC120796. pmid:12208998
- 8. Xu W, Smith FJ Jr., Subaran R, Mitchell AP. Multivesicular body-ESCRT components function in pH response regulation in Saccharomyces cerevisiae and Candida albicans. Mol Biol Cell. 2004;15(12):5528–37. PubMed Central PMCID: PMC532031. pmid:15371534
- 9. Maeda T. The signaling mechanism of ambient pH sensing and adaptation in yeast and fungi. FEBS J. 2012;279(8):1407–13. pmid:22360598
- 10. Davis D. Adaptation to environmental pH in Candida albicans and its relation to pathogenesis. Curr Genet. 2003;44(1):1–7. pmid:12819929
- 11. Bignell E. The Molecular Basis of pH Sensing, Signaling, and Homeostasis in Fungi. Adv Appl Microbiol. 2012;79:1–18. pmid:22569515
- 12. Prusky D, McEvoy JL, Leverentz B, Conway WS. Local modulation of host pH by Colletotrichum species as a mechanism to increase virulence. Mol Plant Microbe Interact. 2001;14(9):1105–13. pmid:11551075
- 13. Bi F, Barad S, Ment D, Luria N, Dubey A, Casado V, et al. Carbon regulation of environmental pH by secreted small molecules that modulate pathogenicity in phytopathogenic fungi. Mol Plant Pathol. 2016;17(8):1178–95. pmid:26666972
- 14. Manteau S, Abouna S, Lambert B, Legendre L. Differential regulation by ambient pH of putative virulence factor secretion by the phytopathogenic fungus Botrytis cinerea. FEMS Microbiol Ecol. 2003;43(3):359–66. pmid:19719667
- 15. Ruijter GJ, Visser J. Characterization of Aspergillus niger phosphoglucose isomerase. Use for quantitative determination of erythrose 4-phosphate. Biochimie. 1999;81(3):267–72. pmid:10385009
- 16. Prusky D, Yakoby N. Pathogenic fungi: leading or led by ambient pH? Mol Plant Pathol. 2003;4(6):509–16. pmid:20569410
- 17. Chen C, Dickman MB. Proline suppresses apoptosis in the fungal pathogen Colletotrichum trifolii. Proc Natl Acad Sci U S A. 2005;102(9):3459–64. PubMed Central PMCID: PMC552905. pmid:15699356
- 18. Brandao RL, Castro IM, Passos JB, Nicoli JR, Thevelein JM. Glucose-induced activation of the plasma membrane H(+)-ATPase in Fusarium oxysporum. J Gen Microbiol. 1992;138 Pt 8:1579–86.
- 19. Naglik JR, Challacombe SJ, Hube B. Candida albicans secreted aspartyl proteinases in virulence and pathogenesis. Microbiol Mol Biol Rev. 2003;67(3):400–28, table of contents. PubMed Central PMCID: PMC193873. pmid:12966142
- 20. Palkova Z, Janderova B, Gabriel J, Zikanova B, Pospisek M, Forstova J. Ammonia mediates communication between yeast colonies. Nature. 1997;390(6659):532–6. pmid:9394006
- 21. Gori K, Mortensen HD, Arneborg N, Jespersen L. Ammonia production and its possible role as a mediator of communication for Debaryomyces hansenii and other cheese-relevant yeast species. J Dairy Sci. 2007;90(11):5032–41. pmid:17954742
- 22. Shnaiderman C, Miyara I, Kobiler I, Sherman A, Prusky D. Differential activation of ammonium transporters during the accumulation of ammonia by Colletotrichum gloeosporioides and its effect on appressoria formation and pathogenicity. Mol Plant Microbe Interact. 2013;26(3):345–55. pmid:23387470
- 23. Vylkova S, Carman AJ, Danhof HA, Collette JR, Zhou H, Lorenz MC. The fungal pathogen Candida albicans autoinduces hyphal morphogenesis by raising extracellular pH. MBio. 2011;2(3):e00055–11. PubMed Central PMCID: PMC3101780. pmid:21586647
- 24. Schindler J, Sussman M. Ammonia determines the choice of morphogenetic pathways in Dictyostelium discoideum. J Mol Biol. 1977;116(1):161–9. pmid:563469
- 25. Sun Y, Cao C, Jia W, Tao L, Guan G, Huang G. pH Regulates White-Opaque Switching and Sexual Mating in Candida albicans. Eukaryot Cell. 2015;14(11):1127–34. PubMed Central PMCID: PMC4621312. pmid:26342021
- 26. Nobile CJ, Solis N, Myers CL, Fay AJ, Deneault JS, Nantel A, et al. Candida albicans transcription factor Rim101 mediates pathogenic interactions through cell wall functions. Cell Microbiol. 2008;10(11):2180–96. PubMed Central PMCID: PMC2701370. pmid:18627379
- 27. Davis D, Edwards JE Jr., Mitchell AP, Ibrahim AS. Candida albicans RIM101 pH response pathway is required for host-pathogen interactions. Infect Immun. 2000;68(10):5953–9. PubMed Central PMCID: PMC101559. pmid:10992507
- 28. Landraud P, Chuzeville S, Billon-Grande G, Poussereau N, Bruel C. Adaptation to pH and role of PacC in the rice blast fungus Magnaporthe oryzae. PLoS ONE. 2013;8(7):e69236. PubMed Central PMCID: PMC3712939. pmid:23874922
- 29. Vecchiarelli A, Pericolini E, Gabrielli E, Kenno S, Perito S, Cenci E, et al. Elucidating the immunological function of the Cryptococcus neoformans capsule. Future Microbiol. 2013;8(9):1107–16. pmid:24020739
- 30. Priest SJ, Lorenz MC. Characterization of Virulence-Related Phenotypes in Candida Species of the CUG Clade. Eukaryot Cell. 2015;14(9):931–40. PubMed Central PMCID: PMC4551586. pmid:26150417
- 31. Miramon P, M CL . The SPS amino acid sensor mediates nutrient acquisition and immune evasion in Candida albicans. Cell Microbiol. 2016.
- 32. Danhof HA, Lorenz MC. The Candida albicans ATO Gene Family Promotes Neutralization of the Macrophage Phagolysosome. Infect Immun. 2015;83(11):4416–26. PubMed Central PMCID: PMC4598414. pmid:26351284
- 33. Vylkova S, Lorenz MC. Modulation of phagosomal pH by Candida albicans promotes hyphal morphogenesis and requires Stp2p, a regulator of amino acid transport. PLoS Pathog. 2014;10(3):e1003995. PubMed Central PMCID: PMC3953444. pmid:24626429
- 34. Naseem S, Araya E, Konopka JB. Hyphal growth in Candida albicans does not require induction of hyphal-specific gene expression. Mol Biol Cell. 2015;26(6):1174–87. PubMed Central PMCID: PMC4357515. pmid:25609092
- 35. Danhof HA, Vylkova S, Vesely EM, Ford AE, Gonzalez-Garay M, Lorenz MC. Robust Extracellular pH Modulation by Candida albicans during Growth in Carboxylic Acids. MBio. 2016;7(6).
- 36. Wiame JM, Grenson M, Arst HN Jr. Nitrogen catabolite repression in yeasts and filamentous fungi. Adv Microb Physiol. 1985;26:1–88. pmid:2869649
- 37. Alkan N, Davydov O, Sagi M, Fluhr R, Prusky D. Ammonium secretion by Colletotrichum coccodes activates host NADPH oxidase activity enhancing host cell death and fungal virulence in tomato fruits. Mol Plant Microbe Interact. 2009;22(12):1484–91. pmid:19888814
- 38. Kasper L, Seider K, Gerwien F, Allert S, Brunke S, Schwarzmuller T, et al. Identification of Candida glabrata genes involved in pH modulation and modification of the phagosomal environment in macrophages. PLoS ONE. 2014;9(5):e96015. PubMed Central PMCID: PMC4006850. pmid:24789333
- 39. Mirbod-Donovan F, Schaller R, Hung CY, Xue J, Reichard U, Cole GT. Urease produced by Coccidioides posadasii contributes to the virulence of this respiratory pathogen. Infect Immun. 2006;74(1):504–15. PubMed Central PMCID: PMC1346605. pmid:16369007
- 40. Wise HZ, Hung CY, Whiston E, Taylor JW, Cole GT. Extracellular ammonia at sites of pulmonary infection with Coccidioides posadasii contributes to severity of the respiratory disease. Microb Pathog. 2013;59–60:19–28. PubMed Central PMCID: PMC3656146.