1. Introduction
Morphogenesis in
C. albicans is controlled by multiple sensing and signaling pathways including the Rim101, the protein kinase A (PKA), and the mitogen-activated protein kinase (MAPK) pathways. These last two pathways are activated by many external stimuli, such as amino acids, CO
2 levels, quorum sensing molecules, or glucose [
1,
2,
3,
4,
5]. The MAPK pathway has different parallel pathways which are all activated by specific stimuli, such as cell wall stress and pheromones [
6]. One of these mechanisms is the activation of Cdc42 by Ras1, both GTPases. This results in a signaling cascade including Cst20 (MAPKKKK), Ste11 (MAPKKK), Hst7 (MAPKK), and Cek1 (MAPK), and finally the activation of transcription factor Cph1 [
6]. Cph1 is involved in hyphae formation on solid medium, mating, and biofilm formation [
6,
7]. On the other hand, activation of the PKA pathway by glucose is mediated via Cdc25 and Ras1. Activation results in the activation of Cyr1, resulting the production of cAMP [
8,
9]. cAMP then binds to the regulatory subunits of PKA, releasing the catalytic subunits which then phosphorylate different effector proteins, including the transcription factor Efg1 [
1,
10]. Expression of Efg1 results in morphogenesis and the expression of various hydrolases [
7,
11]. Since PKA is not only involved in morphogenesis, but also influences other virulence traits, such as e.g., white-opaque switching or expression of several cell wall proteins, it is an important signaling pathway for
C. albicans [
1]. Despite a lot of research, the upstream sensing part and more particularly the role of Ras1 and Ras2 in the different virulence traits remain elusive. In this paper, we will focus on the role of Cdc25, Ras1, and Ras2 for their contribution to several in vitro and ex vivo virulence characteristics.
Cdc25 consists of 1333 amino acids and contains a highly conserved C-terminal region that it is part of the GDP-GTP exchange factor domain towards Ras1 [
12,
13]. A
cdc25 mutant is still viable, although it has a slower growth rate compared to the WT strain and is unable to form hyphae on a serum-containing medium [
14].
Ras1 consists of 291 amino acids and is important for both cell viability and virulence [
15]. It is localized near the plasma membrane via its C-terminal motif and shows high similarity with its ortholog in
S. cerevisiae [
16,
17,
18]. Ras1 is very important for cAMP production by Cyr1 as a
ras1 mutant produces 20 times lower cAMP levels compared to a WT strain [
18]. However, Ras1 is not involved in maintaining basal levels of cAMP in the cells, since
ras1 mutants still contain basal cAMP levels [
15,
18]. Due to its effect on cAMP levels and hence PKA activation, Ras1 is involved in the regulation of different virulence factors. A
ras1 mutant is unable to form hyphae on serum containing media and upon phagocytosis by macrophages [
16,
19]. As the
ras1 mutant is also sensitive to oxidative stress, this strain shows a reduced survival rate after coculturing with macrophages. The mutant shows a reduced virulence during a systemic infection in mice, with no hyphae but only yeast-like cells found in the kidneys [
19,
20]. However, a
ras1 mutant shows higher survival in the presence of neutrophils compared to the WT strain [
21]. Furthermore, it has a reduced toxicity towards oral epithelial cells and a delayed apoptosis after addition of acetic acid and hydrogen peroxide [
22,
23,
24].
Ras2 is an atypical Ras protein that shows low similarity in sequence with other Ras proteins [
18]. However, it contains the typical CCIIT sequence for membrane anchoring, suggesting localization of this protein at the plasma membrane [
18,
25]. Ras2 has a negative effect on Cyr1 activity as a deletion of
RAS2 in a
ras1 background restores the cAMP levels to 30% of the WT level after glucose addition [
18]. Furthermore, Ras2 seems to have a small effect on morphogenesis, but this is masked by Ras1 as deletion of
RAS2 in a
ras1 background worsens the hyphal defect [
18]. The synergistic effect of Ras1 and Ras2 on morphogenesis and their antagonistic effect on cAMP levels suggest that these proteins affect the cell morphology both via a cAMP-dependent and independent mechanism [
15]. A
ras2 mutant also shows lower resistance towards hydrogen peroxide and heavy metal Co
2+ which is opposite to a
ras1 mutant [
18].
As mentioned above, a lot is known about the role of Ras1 during virulence; however, we decided to include the
ras1 mutant in this study, since some of the previous experiments reported were performed in different background strains and previous studies indicated the importance of the background strain in regulatory networks [
26]. Furthermore, we wanted to compare our results for Cdc25 and Ras2 with the results for Ras1. We suggest that, apart from Ras1, Cdc25 also has an effect on in vitro and ex vivo virulence characteristics via both a PKA-dependent and PKA-independent mechanism. We show that a deletion of
CDC25 or
RAS1 results in less toxicity towards oral epithelial cells. This is expected as these deletions result in a morphogenesis defect and hyphal formation is important during the invasion of epithelial cells. However, the toxicity towards cervical cells increases in a
cdc25 and
ras2 mutant while decreases in a
ras1 mutant compared to the WT. Furthermore, we found that a
cdc25 and
ras1 mutant have a higher survival rate in the presence of primary macrophages compared to the WT. This indicates a negative effect of PKA on this virulence trait.
2. Materials and Methods
2.1. Culturing Conditions of C. albicans
Cells were grown at 30 °C in YP or SC medium supplemented with 100 mM glucose, unless stated otherwise. YP medium contains 1% yeast extract and 2% bacteriological peptone. SC medium contains 0.079% complete CSM (MP biomedicals, Santa Ana, CA, USA), 0.17% yeast nitrogen base without amino acids or ammonium sulfate ((NH4)2SO4; Difco), and 0.5% (NH4)2SO4. 1.5% agar was added to obtain solid medium. Both YP and SC medium could be supplemented with 5 mM, 50 mM, or 100 mM glucose, fructose, galactose, or glycerol. During the transformations, nourseothricin (NAT) was added to the medium to a final concentration of 200 µg/mL to select for cells which contained the NAT marker. To flip out this marker, cells were grown at 30 °C on YP 100 mM maltose overnight. Afterwards, the culture was plated for single colony and cells were restreaked on NAT containing solid medium to check for growth. RPMI 1640 with L-glutamine (Sigma-Aldrich, Saint Louis, MO, USA) was used to test the growth of the strain in physiological relevant medium. This medium was buffered with 0.165 M morpholinepropanesulfonic acid (MOPS) to pH 7. For the filamentation check, different hyphal inducing solid media were used: YP with 100 mM glucose, YP with 100 mM glucose and 10% fetal bovine serum (FBS), Spider (1% nutrient broth, 1% mannitol and 0.2% potassium phosphate (KH2PO4)), SLAD (0.17% yeast nitrogen based without amino acids and (NH4)2SO4, 6.6 × 10−4% (NH4)2SO4 and 100 mM glucose), SLD (0.17% yeast nitrogen based without amino acids and (NH4)2SO4, 0.5% (NH4)2SO4 and 0.1%. glucose), and Lee (0.5% (NH4)2SO4, 0.02% magnesium sulfate (MgSO4), 0.025% KH2PO4, 0.5% sodium chloride (NaCl), 7.16 × 10−2% ornithine, 0.05% alanine, 0.3% leucine, 0.1% lysine, 0.05% phenylalanine, 0.05% proline, 0.05% threonine, 0.01% methionine 0.1% biotin, 62.5 mM glucose).
2.2. Culturing Conditions of L-929 Cells
The supernatants of L-929 cells were collected and filter sterilized after culturing the cells for 1 week to obtain L-conditioned medium. Cells were incubated at 37 °C and 5% CO2 in L-cell medium (Iscove’s Modified Dulbecco’s medium (IMDM; Gibco, Waltham, MA, USA), 10% heat-inactivated Fetal calf serum (FCS; Gibco), 50,000 U Penicillin-Streptomycin (Gibco), 1% non-essential amino acids (Gibco), and 1% Sodium pyruvate (Gibco).
2.3. Cultivation of Primary Bone Marrow Derived Macrophages
Bone marrow from female C57BL/6 mice of 12 weeks was differentiated into macrophages by using L-conditioned medium. Femur and tibia were removed from mice and bone marrow cells were collected. Next, the bone marrow-derived macrophages (BMDMs) were cultured for 10 days in differentiation medium (IMDM medium, 10% heat-inactivated FCS (Gibco), 50,000 U Penicillin-Streptomycin (Gibco), and 30% L-conditioned medium) before they were used during experiments.
2.4. Cultivation of HeLa Cells
A vial of HeLa cells was taken from the liquid nitrogen tank and the cells were thawed for 2 min at 37 °C. The vial was cleaned with 70% ethanol and the content was added to 9 mL growth medium (Dulbecco’s Modified Eagle medium (DMEM; Gibco) supplemented with 1× Glutamax (Gibco), antibiotics (Gibco) and 10% FCS). The cells were centrifuged for 10 min at 100 g and the supernatants was discarded. Then, 2 mL of pre-warmed fresh growth medium was added to the cells and the cells were transferred to a culture flask prefilled with 20 mL growth medium. After three days, the cells were further cultured by removing the medium and washing the cells two times with 1× PBS (Gibco). Next, 1× trypsin was added to the cells and incubated for 5 min at 37 °C. Afterwards, 15 mL of fresh growth medium was added to the cells and the cells were counted by making use of a counting chamber. Cells were seeded at 100,000 to 200,000 cells/mL and incubated at 37 °C and 5% CO2.
2.5. Cultivation of TR146 Cells
A vial of TR146 cells was taken from the liquid nitrogen tank and the cells were thawed for 2 min at 37 °C. The vial was cleaned with 70% ethanol and the content was added to 9 mL of TR146 medium (DMEM/Nutrient Mixture F-12 (Gibco) supplemented 10% heat-inactivated FCS and 50,000 U Penicillin-Streptomycin (Gibco)). The cells were centrifuged for 10 min at 100 g and the supernatants was discarded. Then, 2 mL of fresh TR146 medium was added to the cells and the cells were transferred to a culture flask prefilled with 20 mL TR146 medium. After three days, the cells were further cultured by removing the medium and washing the cells two times with 1× PBS (Gibco). Next, 5× trypsin was added to the cells and this was incubated for 5 min at 37 °C. Afterwards, 15 mL of fresh TR146 medium was added to the cells and the cells were counted by making use of a counting chamber. Cells were seeded at 750,000 to 2,000,000 cells/flask and incubated at 37 °C and 5% CO2.
2.6. Construction of the Deletion Collection
A CRISPR genome editing method with a cloning free approach to obtain different components of this system was used [
27]. Fragment A was amplified via PCR from plasmid pADH110 and contained the second part of the NAT marker. Fragment B was amplified from plasmid pADH147 and contained a specific gRNA for the gene of interest. To obtain fragment C, fragment A and B were linked with each other via stitching PCR. The CAS 9 gene and the first part of the NAT marker were present on plasmid pADH99 which was cut at the MssI site to obtain a linear fragment. Both fragment C and the cut pADH99 plasmid were transformed into the strain of interest to delete a gene. The obtained colonies were checked via PCR and the ones with the deleted gene were cultured in YP maltose (100 mM) to lose the
CAS9 gene and the NAT marker. The transformation was done by using the Gietz method. At least three independent transformants of each strain were created to work further with (
Table 1).
2.7. Growth Curve
Strains were grown overnight in YP glucose (100 mM). Cells were collected and washed twice with sterile Milli-Q water. Samples were diluted to OD600 0.1 in sterile Milli-Q water. Then, 20 µL of the cell suspension was added to 180 µL of SC medium supplemented with the indicated carbon source or RMPI medium in a sterile 96-well plate. The OD600 was measured every half hour for 72 h in a Multiskan (Thermo Fisher, Waltham, MA, USA). Data analysis was performed in Graphpad Prism by using an ANOVA test with Bonferroni correction.
2.8. Spot Assay
Strains were grown overnight in YP glycerol (100 mM). Cells were collected and washed twice with sterile Milli-Q water. Samples were diluted to OD600 0.1 in sterile Milli-Q water. A three-time dilution series of 1/10 was made to have in total four dilutions of one culture. Then, 5 µL of each dilution was spotted on SC medium supplemented with glucose, fructose, galactose, and glycerol. The plates were incubated at 30 °C and pictures were taken after 24 h and 48 h.
2.9. Macrophage Survival Experiment
BMDMs were seeded in a Nunc™ F96 MicroWell™ plate (1 × 105 cells/well) in differentiation medium and infected with C. albicans cells (1 × 104 cells/well) in triplicates. Three hours after co-culturing, 4% Triton-X 100/PBS solution was added to lyse the macrophages. C. albicans cells were collected, plated, and colony forming units (CFUs) were counted. Data analysis and statistical analysis were performed in Graphpad Prism by using an ANOVA test with Bonferroni correction.
2.10. HeLa Cell Toxicity Assay
HeLa cells were collected and counted as described earlier. The cells were diluted to a concentration of 105 cells/mL in growth medium, 100 µL of this suspension was added to the wells of a Nunc™ F96 MicroWell™ plate (Thermo fisher), and the plate was put for 24 h at 37 °C and 5% CO2. The next day, C. albicans cells of an overnight culture were collected and were washed three times with 1× PBS. The OD600 was measured, and the cells were diluted to a final concentration of OD600 10. For the experiments with the additional dibutyryl cAMP (Sigma), cells were diluted in growth medium supplemented with the specific dibutyryl cAMP concentrations. A measure of 10 µL of the cell suspension was added to the HeLa cells and incubated for 24 h at 37 °C and 5% CO2. At day three of the experiment, the cytotoxicity of the C. albicans cells towards the HeLa cells was measured by making use of the CyQUANTUM LDH Cytotoxicity Assay Kit (Invitrogen, Waltham, MA, USA). First, 10 µL of lysis buffer was added to the positive control wells which only contained HeLa cells which was incubated for 45 min 37 °C and 5% CO2. Next, 50 µL supernatants of the wells was transferred to a new 96-well plate, 50 µL of reaction buffer was added, and the mixtures were mixed by tapping gently to the wells. After 30 min of incubation in the dark at room temperature, 50 µL of stop solution was added to the wells and the absorbance was measured at 490 nm and 680 nm by making use of the Synergy (BioTek, Winooski, VT, USA). Data analysis and statistical analysis was performed in Graphpad Prism by using an ANOVA test with Bonferroni correction.
2.11. TR146 Cell Toxicity Assay
TR146 cells were collected and counted as described earlier. The cells were diluted to a concentration of 105 cells/mL in TR146 medium, 100 µL of this suspension was added to the wells of a Nunc™ F96 MicroWell™ plate (Thermo fisher), and the plate was put for 24 h at 37 °C and 5% CO2. The next day, C. albicans cells of an overnight culture were collected and were washed three times with 1× PBS. The OD600 was measured, and the cells were diluted to a final concentration of OD600 1. Then, 10 µL of the cell suspension was added to the TR146 cells and incubated for 24 h at 37 °C and 5% CO2. At day three of the experiment, the cytotoxicity of the C. albicans cells towards the TR146 cells was measured by making use of the CyQUANTUM LDH Cytotoxicity Assay Kit (Invitrogen). First, 10 µL of lysis buffer was added to the positive control wells, only containing TR146 cells, which were incubated for 45 min 37 °C and 5% CO2. Next, 50 µL supernatants of the wells was transferred to a new 96-well plate, 50 µL of reaction buffer was added, and the mixtures were mixed by tapping gently to the wells. After 30 min of incubation in the dark at room temperature, 50 µL of stop solution was added to the wells and the absorbance was measured at 490 nm and 680 nm by making use of the Synergy. Data analysis and statistical analysis was performed in Graphpad Prism by using an ANOVA test with Bonferroni correction.
4. Discussion
The Ras1/cAMP/PKA pathway is important for cell growth and virulence in
C. albicans [
1]. Such an important and central pathway requires extensive research to check the effect of its different components, such as for example Ras1, on virulence. However, much of this pathway remains unknown, such as the role of its upstream activator, Cdc25, and the other Ras component in the cell, Ras2. Therefore, we wanted to see the effect of Cdc25 and Ras2 on growth and virulence of
C. albicans towards mammalian cells and thus see which virulence factors in these settings are regulated via PKA activation and which are regulated via other mechanisms. We also included our
ras1 mutant in this study to have a complete picture so that we can compare the different strains in one experiment. Furthermore, most of the experiments with
ras1 mutants found in literature are performed with a CAI4 background strain which by itself already has different phenotypes compared to the SC5314 background strain [
29].
Both Cdc25 and Ras1 are important for a normal growth rate on all tested carbon sources. Deletion of
CDC25 results in a significant decreased growth compared to the WT (except for 100 mM fructose), and a
ras1 mutant shows an even more significant affected growth. However, a growth defect of the
cdc25 and
ras1 mutant on 5 mM glucose and RPMI is absent compared to higher sugar concentrations. This can be explained by the fact that cells generally grow slower at these low sugar concentrations. Therefore, the growth defect is not clearly pronounced, and it is harder to distinguish the growth of the mutant compared to the growth of the WT strain. Since the growth defect is observed in both a
cdc25 and
ras1 mutant, this defect can be caused by inactivation of the PKA pathway. The difference between the growth rate of these two mutants can be explained by the fact that Cdc25 activates Ras1, but basal activated Ras1 levels are also present without activation by Cdc25. We also see a significant growth defect of the
cdc25 and
ras1 mutant on carbon sources such as fructose, galactose, and glycerol during growth on solid medium [
1,
2,
4]. These results are unexpected as these three carbon sources do not activate PKA. Therefore, we did not expect an effect of a
CDC25 or
RAS1 deletion on the growth on fructose, galactose, and glycerol as we assign the growth defect of these two mutant strain to inactivation of the PKA pathway. However, we also show that the
cdc25 and
ras1 mutant have a different growth pattern compared to the WT on solid SC medium without a carbon source. This indicates that the growth defect of the
cdc25 and
ras1 mutant is not caused by inactivation of PKA due to these deletions otherwise we would only observe a growth defect on carbon sources that are able to activate PKA. It is possible that these two proteins influence cell growth independently of PKA and thus we see this growth defect during all tested conditions.
Different steps are involved in damaging human epithelial cells by
C. albicans: adhesion, penetration, and lysis [
32]. Since PKA regulates different genes involved in this process, it is suggested that inhibiting this pathway can result in a reduced toxicity. This would indicate that deletion of
CDC25 and
RAS1 also decreases the toxicity. However, this is not observed; the
cdc25 mutant shows an increased toxicity, while the
ras1 mutant shows decreased toxicity. As these two mutants show antagonistic phenotypes and the addition of extra dibutyryl cAMP does not rescue this
RAS1 deletion phenotype, it suggests that this effect is independent from PKA. In contrast, an
efg1 mutant, a downstream transcription factor of PKA, a
cdc25 efg1 mutant, and a
ras1 efg1 mutant show the same reduced toxicity phenotype as the
ras1 mutant [
33]. To conclude, we hypothesize that the phenotype of the
ras1 mutant is not caused by inactivation of PKA. Since Ras1 works upstream and Efg1 downstream of PKA, we obtain similar phenotypes with these mutants, but the molecular mechanisms behind can be completely different [
33].
Since we hypothesize that PKA is not the main pathway in the toxicity towards HeLa cells, we investigated the role of the MAPK pathway, as this pathway is also involved in virulence [
6]. Our data suggest that Cph1 has a negative effect on epithelial damage as a
cph1 mutant shows an increased toxicity compared to the WT. Recently, it was shown that activation of Cph1 results in the unmasking of β-(1,3)-glucan which results in recognition by the HeLa cells [
34,
35]. It is possible that due to the deletion of
CPH1, less β-(1,3)-glucan unmasking occurs and consequently
C. albicans has an increased toxicity towards these epithelial cells as the pathogen is not recognized. Furthermore, to influence the pathogen, the epithelial cells need to produce different cytokines to recruit immune cells which will clear the infection [
34]. In our experimental setup, only HeLa cells were present, so the difference in β-(1,3)-glucan masking between the WT and mutant could not result in a different amount of recruited immune cells as they were not present. Interestingly, the
cdc25,
ras2, and
cph1 mutants show the same phenotype in cell damage towards HeLa cells. It is possible that these three proteins are working in the same mechanism that negatively regulates toxicity. We hypothesize that Cdc25 activates Ras2 which on its turn is responsible for the activation of the MAPK pathway and finally Cph1, instead of Ras1. This can explain the similar phenotype of the
cdc25,
ras2, and
cph1 mutant towards cervical cell toxicity. If this is indeed the mechanism, we hypothesize that Cdc25 is activated via multiple stimuli which results in a specific conformational change dependent on the stimulus. A specific conformation can result in either activation of Ras1 or Ras2 which consequently leads to the activation of the PKA or MAPK pathway, respectively. The double mutant
cdc25 cph1 shows the same phenotype as the WT which is unexpected. On the other hand, the double mutant
ras2 cph1 shows the same phenotype as the
ras2 and
cph1 single mutants which strengthens our hypothesis that these two proteins work in the same mechanism. However, previous research showed that a
ras1 and
cph1 mutant give the same morphogenesis defect so they assume that Ras1 is the upstream activator of Cph1 [
1]. Another explanation for the increased toxicity towards cervical cells in the
ras2 mutant can be found in the effect of Ras2 on cAMP levels. Literature shows that Ras2 has a negative effect on the cAMP levels in the cell [
18]. By deleting
RAS2, higher cAMP levels can be present which can result in overactivation of PKA and consequently a higher toxicity towards cervical cells.
Glucose is present in high concentrations in the mouth of the human body after food uptake [
36]. Therefore, glucose can be important for
C. albicans cells present in the oral cavity as it is the main and preferred energy source for this pathogen, and it can activate the PKA pathway. Furthermore, glucose activation of the PKA pathway in this niche is a key factor since this pathway regulates different genes involved in epithelial cell damage [
32]. Both a
cdc25 and
ras1 mutant show a decreased toxicity in comparison to the WT. These findings are strengthened by the phenotype of the
efg1 mutant which is already shown in literature [
37]. Due to the deletion of
EFG1, the expression of
SAP1 and
SAP3, genes encoding proteases involved in the degradation of specific host cell components, is reduced which results in a decreased toxicity towards oral epithelial cells [
37]. Therefore, the reduced toxicity observed in the
cdc25 and
ras1 mutant strains is probably due to inactivation of PKA and consequently Efg1 which results in reduced expression of
SAP1 and
SAP3. Probably, other genes encoding proteins involved in cell damage are affected by these deletions and contribute to these phenotypes. This hypothesis is strengthened by the phenotype of the
cdc25 efg1 and
ras1 efg1 double deletion strain which shows that Efg1 has a function downstream of Cdc25 and Ras1. The difference in toxicity between the
cdc25 and
ras1 mutants is quite big, indicating that basal Ras1 activation seems sufficient for partial toxicity. However, it is also possible that Ras1 has an extra function in the toxicity towards TR146 cells besides Cyr1 activation, such as a cAMP-independent effect on morphogenesis of Ras1 as described earlier in literature [
18]. Therefore, due to inhibition of PKA and these possible extra functions, the
ras1 mutant has a more drastic decreased toxicity compared to the
cdc25 mutant. In addition, the MAPK pathway does not seem to play a major role in the infection of oral epithelial cells as the
cph1 mutant and the WT strain or the
cdc25 mutant and the
cdc25 cph1 mutant show the same toxicity. The same is true for the
ras2 mutant as Ras2 has a negative effect on cAMP levels, so its deletion does not result in PKA inactivation [
18].
Different niches in the human body have widely differing chemical characteristics and nutrient composition, such as pH, the presence of glucose, or oxygen levels. This influences the activation of specific proteins and pathways, like the Rim101 or PKA pathway [
1]. Efg1 is a stimulator of morphogenesis but under certain conditions with limited oxygen access and embedded in agar, Efg1 is a repressor of morphogenesis [
11]. When we compare our toxicity results obtained using HeLa and TR146 cells, the significantly different results may originate from the fact that different media are used to culture the different cell lines. As these media contain different glucose concentrations, it is possible that the toxicity difference towards the different cell lines is caused by a difference in glucose concentration. Furthermore, it is possible that
C. albicans receives specific stimuli from these oral epithelial cells, which are absent in cervical cells, and activate PKA.
Macrophages are an important part of the innate immune system against
C. albicans, so this fungus has different mechanisms to cope with these macrophages to survive. For example, they neutralize the ROS molecules inside immune cells and form hyphae to break out [
31]. Since Ras1 is involved in morphogenesis, partially via activation of PKA, it is logical that a deletion of
RAS1 results in a decreased survival in the presence of macrophages compared to the WT, as is shown by the group of Schröppel [
16,
19]. However, this is not what we observed in our experiments. We showed that both the
cdc25 and
ras1 mutant have an increased survival during coculturing with macrophages compared to the WT strain. As both mutants give a similar phenotype, it is hypothesized that this effect is caused by inactivation of the PKA pathway. The main question is why we obtain different results compared to the other research group. First, it is possible that a difference in strain background between the two studies affects the results. We made the mutants in a SC5314 background strain whilst the other group made their strains in the CAI4 background strain. It is known from previous studies that this CAI4 background gives different phenotypes compared to the prototrophic SC5314 strain due to differences in
URA3 expression [
29]. This indicates that the decreased survival of the
ras1 mutant in the CAI4 strain can be caused by the strain background and not by the deletion itself. A second explanation can be the use of different macrophages in the different studies. We used macrophages derived from bone marrow whilst the other group used inflammatory peritoneal exudate-derived macrophages [
19]. Therefore, it is possible that these different types react differently on the presence of
C. albicans cells.
The PKA pathway regulates different processes involved in macrophage infections, like morphogenesis and adhesion [
15]. Therefore, as previously mentioned, the increased survival of the
cdc25 and
ras1 mutant is unexpected. However, several hypotheses and explanations, involving recognition, uptake, and survival within the immune cells, can be formulated. First, the pathogen needs to be recognized through PAMP-PRR interactions with the cell wall displaying the different PAMPs to be recognized. It is known that the cell wall components are partially regulated via the PKA pathway [
38,
39]. Therefore, it is possible that by deleting
CDC25 and
RAS1 and hence inactivation of PKA, the cell wall and its constituents are altered, resulting in lower fungal cell recognition. Secondly, these two mutants are not able to form hyphae which is important to escape from inside the macrophages [
16,
31]. However, this does not result in a decreased survival rate inside macrophages. The observed increase in survival rate of the two mutants can be explained by the fact that due to the inability to form hyphae, the cells are recognized to a lesser extent since cells with a difference in morphology also display different cell wall proteins, such as adhesins. Another explanation can be found in counteracting ROS molecules to survive inside these macrophages [
40]. It is possible that PKA plays a negative role in this process. This would explain the increased survival we obtain in the
cdc25 and
ras1 mutant as they have no active PKA. It is likely that this is the main reason why we observe this increased survival rate of these two mutant strains compared to the WT. Previously, it was shown that deletion of
RAS1 results in resistance to oxidative and nonoxidative killing by neutrophils [
21]. Therefore, probably, the
cdc25 and
ras1 mutant have a higher resistance towards the ROS molecules produced by the macrophages compared to the WT.