Candida albicans is a commensal yeast of humans and colonizes ≈70% of the human population. It is common for C. albicans to cause self-limiting infections, but severe infections can be life-threatening. Invasive candidiasis occurs in patients with a compromised immune system, such as anti-cancer therapies. Even if antifungal drugs exist, ≈3 million people acquire life-threatening fungal infections every year, 700,000 of which are invasive candidiasis. Despite this, fungi remain underestimated as pathogens.

Fig. 1. (A) C. albicans undergoes yeast-to-hyphal transition inside the macrophage phagosome.
(B) C. albicans secretes NH3 into the acidic phagosome. However, the influx of H+ via V-ATPases exceeds NH3 production by ~100-fold, and NH3 leakage from the phagosome exceeded the rate of NH3 production by ~10,000-fold.
(C) We propose that the observed increase in phagosomal pH is a consequence of membrane rupture and H+ leakage, not NH3 production.

Unlike many other pathogenic microbes, C. albicans is polymorphic and grows as budding yeast or filamentous hypha (Fig. 1A). This switch in morphology, termed yeast-to-hypha transition, occurs when the fungus is confronted by stressful conditions, including contact with immune cells, elevated temperatures, and changes in nutrient availability. Engulfment and destruction of fungi by professional phagocytes (Greek for “cells that eat”) is critical to the innate immune response. One important phagocyte known as “macrophage” is a type of white blood cell patrolling the body. As the name describes (macrophage meaning “large eater”), macrophages can effectively sense and internalize fungi by a process termed phagocytosis. The newly formed phagosome contains the engulfed C. albicans yeast, and thereafter undergoes a series of events termed “phagosome maturation”, where the phagosome acquires a hostile, acidic environment with low nutrient availability. The acidic phagosomal environment is critical to limit fungal growth. Many enzymes inside this fungus-containing phagosome require low pH to function properly. Phagosome acidification is achieved by influx of protons (H⁺) via pumps named V-ATPases (Fig. 1B). It is currently believed that the yeast-to-hypha transition is inhibited within acidic phagosomes. Consequently, C. albicans is thought to increase the phagosomal pH prior to yeast-to-hypha transition. Thus, the ability of C. albicans to increase pH inside the phagosome is considered crucial for its survival and escape from macrophages.

C. albicans was shown to form hyphal filaments inside the macrophage phagosome, which leads to its escape and sustained infection. Recent studies have proposed that C. albicans increases the phagosomal pH by producing and secreting ammonia (NH₃) inside the phagosomal space. NH₃ can in principle elevate the pH of the phagosome by binding free H⁺ inside the phagosome to form ammonium (NH₄⁺) (Fig. 1B). We revisited this hypothesis with the following argumentation: to effectively increase phagosomal pH, NH₃ production by C. albicans has to exceed the rate of H⁺ pumping into the phagosome by the V-ATPases. Most importantly, the rate of fungal NH₃ generation has to exceed the rate at which NH₃diffuses out of the phagosome (Fig. 1B). In this regard, it is noteworthy that NH₃ diffuses rapidly through most mammalian membranes.

We used a quantitative pH imaging approach to measure the rate of H⁺ pumping into the phagosome, and NH₃ leakage across the phagosomal membrane. Our results indicate that the influx of H⁺ into the phagosome exceeded NH₃ production by ≈100-fold, and that NH₃ leakage from the phagosome exceeded the rate of NH₃ production by ≈10,000-fold. This suggests that NH₃ production cannot account for the observed changes in phagosomal pH. C. albicans would have to produce at least 10⁶ times more NH₃ to alkalinize the phagosome. Instead, we show that the yeast-to-hyphal transition occurs inside acidic phagosomes. C. albicans grows for 3-4 hours before the extending hypha ruptures the phagosomal membrane, leading to H⁺ leakage out of the phagosome and consequent phagosomal alkalinization (Fig. 1C). In conclusion, we propose that the yeast-to-hypha transition is initiated (at a reduced rate) inside acidic phagosomes and that alkalinization results from phagosomal membrane rupture.

Johannes Westman
Program in Cell Biology, The Hospital for Sick Children, Toronto, Canada

Publication

Candida albicans Hyphal Expansion Causes Phagosomal Membrane Damage and Luminal Alkalinization.
Westman J, Moran G, Mogavero S, Hube B, Grinstein S
mBio. 2018 Sep 11

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