Candida albicans Hyphal Morphogenesis within Macrophages Does Not Require Carbon Dioxide or pH-Sensing Pathways

Abstract

The opportunistic fungal pathogen Candida albicans has evolved a variety of mechanisms for surviving inside and escaping macrophages, including the initiation of filamentous growth. Although several distinct models have been proposed to explain this process at the molecular level, the signals driving hyphal morphogenesis in this context have yet to be clarified. Here, we evaluate the following three molecular signals as potential hyphal inducers within macrophage phagosomes: CO₂, intracellular pH, and extracellular pH. Additionally, we revisit previous work suggesting that the intracellular pH of C. albicans fluctuates in tandem with morphological changes in vitro. Using time-lapse microscopy, we observed that C. albicans mutants lacking components of the CO₂-sensing pathway were able to undergo hyphal morphogenesis within macrophages. Similarly, a rim101Δ strain was competent in hyphal induction, suggesting that neutral/alkaline pH sensing is not necessary for the initiation of morphogenesis within phagosomes either. Contrary to previous findings, single-cell pH-tracking experiments revealed that the cytosolic pH of C. albicans remains tightly regulated both within macrophage phagosomes and under a variety of in vitro conditions throughout the process of morphogenesis. This finding suggests that intracellular pH is not a signal contributing to morphological changes.

Keywords: Candida; host-pathogen interactions; macrophages; morphogenesis.

FIG 1

CO₂ sensing is dispensable for C. albicans hyphal induction inside macrophage phagosomes. (A) The CO₂-sensing hyphal induction pathway in C. albicans. Environmental CO₂ is converted to bicarbonate by carbonic anhydrase (Nce103), which then binds to and activates adenylyl cyclase (Cyr1) to activate the hyphal-inducing protein kinase A pathway. (B) Distributions of C. albicans cell length measurements postphagocytosis for the indicated strains at three time points. Mutants of interest elongate at rates comparable to the wild-type strain inside macrophages. The max Feret diameter is defined as the maximum distance between two parallel planes enclosing an object and is thus a proxy for quantifying hyphal growth inside phagosomes. Hyphal measurements were acquired from images of fixed coculture samples (n = 20 fungal cells per strain per time point), and statistical analysis was performed using an unpaired two-tailed t test. (C) Hyphal morphogenesis initiation rates postphagocytosis for the indicated strains. Hyphal initiation data (n of >25 per strain per experiment) were acquired via live imaging microscopy. Phagocytosed fungal cells were followed over time to determine the time point at which germ tubes were visible. Statistical analysis was performed using a two-way ANOVA. (D) Representative images of the C. albicans mutants of interest forming hyphae inside J774A.1 macrophages at 1 h postinfection. Data represent three biological experiments. ns, not significant; *, P < 0.05; ***, P < 0.001 (unpaired t test in panel B). Scale bar, 10 μm. Images were acquired using an Olympus IX-83 microscope as described in the Materials and Methods section.

FIG 2

The intracellular pH of C. albicans is maintained at near neutral throughout hyphal morphogenesis in Lee’s medium. (A) Representative time-lapse images of C. albicans cells expressing pHluorin2 forming hyphae in response to Lee’s medium at 37°C. (B) Single-cell pH measurements acquired during the course of one hyphal induction experiment in Lee’s medium. Each line represents a single cell undergoing morphogenesis within the 180-minute time frame. (C) Shown is the average pH over time (compiled averages from three biological experiments), n = 20 cells for each individual experiment. (D) The average deviation of cytosolic pH of individual cells from C relative to t = 0 is plotted over time (compiled averages from three biological experiments). (E) The time-lapse pH measurements for each cell were normalized to focus on the measurements at 15 min before, during, and 15 min after germ tube emergence for all cells. The graph represents averages from three biological experiments. Scale bar, 20 μm. Images were acquired every 15 min using an Olympus IX-83 microscope as described in the Materials and Methods section.

FIG 3

The intracellular pH of C. albicans is maintained at near neutral throughout hyphal morphogenesis in a variety of conditions in vitro. (A) Single-cell pH measurements acquired during the course of one hyphal induction experiment (one biological experiment) in 6 different hyphal-inducing media at 37°C. Each line represents a single cell that germinated within the 180-minute time frame. (B and C) pH-tracking data compiled from three biological experiments (averaged) showed no significant trend upward or downward for intracellular pH during morphogenesis under a variety of conditions. (D) The time-lapse pH measurements for each cell were normalized to focus on the measurements 15 min before, during, and 15 min after germ tube emergence for all cells. The graph represents averages from three biological experiments. Images were acquired every 15 min using an Olympus IX-83 microscope as described in the Materials and Methods section. n = 20 cells per condition for each of 3 biological experiments.

FIG 4

The intracellular pH of C. albicans is maintained at near neutral throughout hyphal morphogenesis ex vivo. (A) Representative time-lapse coculture images of SC5314 expressing pHluorin2 within macrophages. (B) Single-cell intracellular pH measurements of phagocytosed fungal cells. Each line represents a single C. albicans cells undergoing hyphal morphogenesis within a phagosome. (C and D) pH-tracking data compiled from three biological experiments (averaged) showed no trend upward or downward for intracellular pH within the time frame of morphogenesis. (E) pH measurements compiled from three experiments were normalized to show the average pH at 10 min before, during, and 10 min after germ tube emergence for all cells. Scale bar, 10 μm. Images were acquired using an Olympus IX-83 microscope as described in the Materials and Methods section. n = 30 C. albicans cells for each of three biological experiments.

FIG 5

Neutral/alkaline pH sensing is dispensable for C. albicans hyphal induction inside macrophage phagosomes. (A) Representative images of SC5314 and rim101Δ cells forming hyphae inside J774A.1 macrophages at 1 h postinfection. (B) Hyphal morphogenesis initiation rates postphagocytosis for the indicated strains were acquired via live imaging microscopy, and statistical analysis was performed using a two-way ANOVA. Data represent three biological experiments (n of >50 per experiment). Scale bar, 15 μm. Images were acquired using an Olympus IX-83 microscope as described in the Materials and Methods section.