| dc.description.abstract | Candida albicans is an opportunistic human fungal pathogen, capable of causing severe
disease in immunocompromised individuals. In addition, the fungus causes millions
of mucosal infections in predisposed people, particularly women of child-bearing age.
Treatment options for such diseases are limited due to the increasing number of strains
resistant to antifungal drug treatment, and the ability of the fungus to evade clearance
by the innate immune system. The ability of C. albicans to switch to polarised hyphal
growth, in addition to responding to external stresses induced by drug treatment and the
immune response, allows C. albicans to survive and prolong infection. How such stress
signals are sensed in fungi, however, is not fully understood.
Calcium (Ca2+) is an essential trace element required in all eukaryotic organisms and
is used a second messenger to activate intracellular signalling pathways. In fungi,
however, Ca2+ dynamics, and its role in stress adaptation and cell biology, are not
fully understood due to the lack of suitable Ca2+ reporters that can accurately visualise
Ca2+ dynamics at the single cell level. To overcome this problem, we engineered the
single cell Ca2+ reporter, GCaMP6f, to visualise cytosolic Ca2+ dynamics in C. albicans
at the single cell level for the first time. Chapter 3 describes the general optimisation
of the GCaMP6 reporter in C. albicans, which included generating a panel of GCaMP
expressing strains for future work in the thesis. Optimal experimental conditions were
established, where GCaMP outputs were found to be both dependent on the pH and
Ca2+ concentration of the external medium. By also imaging an empty vector control
strain in a microfluidics system, GCaMP dependent signals were characterised using
bespoke software, which were termed Ca2+ GCaMP spikes due to their transient profile
within individual cells. By imaging Ca2+ GCaMP spikes at higher speeds, complete spike
signatures were characterised in C. albicans, which lasted for ∼ 5 s.
In Chapter 4, we used common stress compounds (0.05 % SDS, 1 M NaCl and 5
mM H2O2), at concentrations previously established in the fungal literature, to visualise
immediate GCaMP responses to stress, as well as longer term adaptation responses
over repeated exposures. Exposure to osmotic shock caused no adaptive response,
however 1 M NaCl led to an inhibition of Ca2+ GCaMP spiking, whereas an equimolar
level of 0.6 M CaCl2 caused increased levels of spiking, but with the same level of cell
shrinkage. Treatment with 0.05 % SDS elicited a Ca2+ GCaMP dependent spike burst
across the cell population, as well as a more prolonged increase in GCaMP fluorescence,
which was associated with an increase in cytosolic Ca2+, followed by auto-fluorescent
signals that indicated high levels of cell death. Surviving cells adapted over repeated
exposures, which required calcineurin; increased protection to SDS was also provided
by removing external Ca2+, suggesting adaptation to SDS was caused by remodelling
of the damaged plasma membrane to prevent unregulated Ca2+ influx. Treatment
with 5 mM H2O2 suppressed Ca2+ GCaMP spiking and caused population wide auto
6fluorescence, however cells adapted to repeated exposures. Adaptation was dependent
on Cap1, Cch1 and calcineurin, but not the calcineurin target, Crz1, Hog1, or Yvc1.
This suggested a pre-adapted state in the absence of these proteins, potentially due to
membrane permeability altering the diffusion of H2O2 across the plasma membrane into
the cytosol.
In Chapter 5, the validated GCaMP reporter was used to investigate Ca2+ homeostasis
in C. albicans, by characterising a novel TRP-like channel, Fgr29, that plays a role in
Ca2+ homeostasis. Novel AlphaFold software predicted a homo-tetrameric structure for
Fgr29, similar to other characterised TRP channels, such as Yvc1. Ca2+ GCaMP signals
in the fgr29∆ strain were consistently higher than in WT cells, suggesting that Fgr29
regulates cytosolic Ca2+ homeostasis. The fgr29∆ strain displayed alternative stress
response signatures, compared to WT, which were consistent with constitutive activation
of stress response pathways, such as the calcineurin and CWI pathways, and resistance
to stress. Phenotypic assays revealed that the absence of Fgr29 promoted resistance
to myriocin, an inhibitor of the sphingolipid biosynthesis pathway. Fgr29 may therefore
localise to the ER, and mutants lacking Fgr29 may have an alternate sphingolipid profile
in the plasma membrane.
In Chapter 6, GCaMP6 was imaged in C. albicans hyphae, the invasive form of
C. albicans, often associated with disease and toxin production. Ca2+ GCaMPsignals
in hyphae were more prolonged than the spikes observed in yeast cells, and occurred
independently within individual hyphal compartments. The signals were also biased
towards the apical hyphal cell suggesting an increased requirement for Ca2+ in the
metabolically active cell, perhaps for Golgi-localised enzymes, calcineurin dependent
cell cycle effectors, and actin polymerisation. Examination of Ca2+ flux during
contact-associated events in hyphal cells was limited due to growth being limited by
the system and also high frame rate imaging.
Overall, the development of the GCaMP6 reporter has allowed the investigation of Ca2+
dynamics at the single cell level in C. albicans for the first time. Compared to previous
approaches, the use of GCaMP6 has revealed the dynamics of Ca2+ influx and regulation
and the role in temporal stress response signatures and adaptation. | en_GB |
