Carbon substrates promotes stress resistance and drug tolerance in clinical isolates of Candida tropicalis

Candida tropicalis is a human pathogen and one of the most prevalent non-Candida albicans Candida (NCAC) species causing invasive infections. Azole antifungal resistance in C. tropicalis is also gradually increasing with the increasing incidence of infections. The pathogenic success of C. tropicalis depends on its effective response in the host microenvironment. To become a successful pathogen, cellular metabolism, and physiological status determine the ability of the pathogen to counter diverse stresses inside the host. However, to date, limited knowledge is available on the impact of carbon substrate metabolism on stress adaptation and azole resistance in C. tropicalis . In this study, we determined the impact of glucose, fructose, and sucrose as the sole carbon source on the fluconazole resistance and osmotic (NaCl), oxidative (H 2 O 2 ) stress adaptation in C. tropicalis clinical isolates. We confirmed that the abundance of carbon substrates influences or increases drug resistance and osmotic and oxidative stress tolerance in C. tropicalis . Additionally, both azole-resistant and susceptible isolates showed similar stress adaptation phenotypes, confirming the equal efficiency of becoming successful pathogens irrespective of drug susceptibility profile. To the best of our knowledge, our study is the first on C. tropicalis to demonstrate the direct relation between carbon substrate metabolism and stress tolerance or drug resistance.

Azole antifungals are commonly used to treat Candida infection, among which fluconazole is most widely used due to its low cost and limited side effects (Ou et al., 2017;Wiederhold, 2017).
With the increasing incidence of C. tropicalis infections, a parallel rise in fluconazole resistance was also observed (Chakrabarti et al., 2015;Fan et al., 2017;Wu et al., 2017).The SENTRY global surveillance report on 31 countries showed that the rate of fluconazole resistance in C. tropicalis was 11.6%.Azole resistance in C. tropicalis has increased in the last two decades due to log-term antifungal treatment, inappropriate antifungal use, indiscriminate use of agricultural antifungals, and veterinary medicines (Chakrabarti et al., 2015;Dos Santos & Ishida, 2023).
The efficiency of yeast responding effectively to the microenvironment determines its pathogenic success (Rodaki et al., 2009).To cause disease, fungal pathogens need to uptake nutrients, survive and multiply.Pathogens need flexibility in adaptation to frequently changing environments for the short term and for the long-term evolution of different mechanisms provide this flexibility (Brown, Brown, et al., 2014).Cellular metabolism and physiological status strongly influence the ability of a fungus to counter stress in the host microenvironment and host defenses (Brown, Brown, et al., 2014).The pathogenicity and virulence of Candida spp.depends on the frequent changes in nutrient availability in the host (Dos Santos & Ishida, 2023).
Diabetes is one of the predisposing factors for candidiasis (de Leon et al., 2002).Increasing risk of C. albicans infection in diabetic patients partially due to elevated glucose levels in serum (Martinez et al., 2013).Glucose has already been reported to induce C. albicans growth, as well as increase resistance to osmotic and oxidative stress, and elevate antifungal drug resistance (Ene, Adya, et al., 2012;Mandal et al., 2014;Rodaki et al., 2009).Additionally, glucose can also induce drug efflux pumps Cdr1p in C. albicans (Szczepaniak et al., 2015).Studies also confirmed higher fructose concentrations in diabetic patients and in different oncological diseases (Hui et al., 2009;Kawasaki et al., 2002).Fructose has been reported to increase fluconazole resistance through the activation of Mdr1 and Cdr1 transporters (Suchodolski & Krasowska, 2021).
C. tropicalis can assimilate and ferment glucose, sucrose, maltose, and other sugar utilizing the oxidative pathway, where glucose is the most preferred carbon source (Dos Santos & Ishida, 2023;Zuza-Alves et al., 2017).Additionally, C. tropiclis is intrinsically osmotolerant, which provides an advantage in high-saline environments (Zuza- Alves et al., 2017).This property confirms the variation in biology of each species of the genus associated with infection and pathogenesis (Papon & Naglik, 2021).Although studies showed similarities in carbon metabolism between C. albicans and C. tropicalis, but it does not happen always (Alves et al., 2020;Naseem et al., 2017).Limited knowledge is available on the specific processes of nutrient acquisition in C. tropicalis (Dos Santos & Ishida, 2023).However, to date, no study available on the impact of glucose, fructose, and sucrose metabolism on either azole resistance, osmotic or oxidative stress resistance in C. tropicalis.
This study used azole-resistant and susceptible isolates to understand the metabolic diversity based on drug susceptibility patterns.This study also highlighted that glucose, fructose, and sucrose increase fluconazole resistance and NaCl-induced osmotic and H2O2-induced oxidative stress tolerance in clinical islets of C. tropicalis.Additionally, similar stress adaptation phenotypes in resistant and susceptible isolates of C. tropicalis confirmed equal ability to cause infection irrespective of susceptibility profile.

Details of isolates used
Stored and frozen C. tropicalis isolates were obtained from our institute's National Culture Collection of Pathogenic Fungi facility.This study used C. tropicalis ATCC 750 and eight other azole antifungal resistant and susceptible isolates collected from invasive infections.Isolates were inoculated on yeast extract peptone dextrose (YPD) agar media (Himedia, India) and were grown at 30C for 24 hours.Our previous studies also used these isolates (Paul et al., 2021;Paul et al., 2022;Paul et al., 2020).For better understanding, we provided the details of the clinical isolates in Table S1.Our institutional ethics committee approved this study, and informed consent was obtained from the patients before collecting the samples.

Preparation of caron supplements and stressors
We used five different carbon supplements for this study.We chose hexose monosaccharide [(glucose and fructose) (Himedia, India)], disaccharide sucrose (Qualigens, India), and pentose monosaccharide [(ribose and arabinose)(Sisco Research Laboratories, India) for carbon metabolism assay.For each experiment, we used freshly made yeast nitrogen base (YNB)(Sigma-Aldrich, Germany) media without glucose.We supplemented the media with 1, 5, 20, and 50 mg/mL of any one of the previously mentioned carbon substrates and filter sterilized it.This study selected fluconazole (Sigma-Aldrich, Germany) as drug-induced stress, NaCl (Sisco Research Laboratories, India) as osmotic, and H2O2 (Merck Life Science, India) as an oxidative stressor.
Depending on the experiment setup, the variable concentrations of each stressor were mixed with freshly made YNB supplemented with carbon substrates.

Preparation cells for carbon metabolism
Isolated colonies from freshly revived C. tropicalis isolates were inoculated in YNB liquid media and incubated at 30C overnight in an incubator-shaker at 200 rpm.Cells were harvested by centrifuging at 7000 rpm for 5 min and washed twice (7000 rpm, 5 min) with phosphate buffer saline (PBS).Absorbance at 600 nm was measured, and the final cell concentration was adjusted to 10 7 cells/mL in YNB without carbon substrates.Ten microliters of the adjusted cell suspension was added into 100 L of YNB supplemented with variable concentrations of different carbon substrates in a 96-well plate.The OD at 600 nm was kinetically measured for 24 to 36 hours, depending on the experiment with a microplate reader (BioTek Instruments, USA).

Preparation of cells with different stressors
The cell concentration was adjusted in the same way as mentioned.To determine the stress response, 10 L cell suspension was mixed with 100 L of YNB supplemented with variable concentrations of different carbon substrates with respective stressors (fluconazole, NaCl, or H2O2).The rest of the process is the same as the previous one.

Statistical analysis
The one-way ANOVA and two-tailed tests were used for statistical analysis, as indicated in figure legends.All graphs in this study were made using GraphPad Prism version 10. Figures were organized and finalized in MS PowerPoint.

Results:
Abundance of carbon supplements modulates cellular growth.
We initially used C. tropicalis ATCC750 as the reference strain for an initial understanding of carbon metabolism.To test the effect of different carbon supplements on growth, we selected 1, 5, 20, and 50 mg/mL concentrations of each sugar tested.The Hexose sugars (glucose, fructose, and sucrose) influence cellular growth in a concentration-dependent manner (Fig. 1A, B, C).In the presence of 1 mg/mL, we noticed significantly less growth when compared with the optimum growth of 20 or 50 mg/mL.We also screened growth in the presence of two pentose sugar ribose and arabinose.We noticed reduced growth in the presence of pentoses (Fig. 1D, E).However, a significant growth difference was observed when the lowest and highest sugar concentrations were compared.Therefore, we exclude pentoses from our further study.Like the previous study, we didn't find any noticeable variation in growth when comparing the growth at 8, 16, and 24 hours with indicated concentrations of each carbon supplement (Fig. 1F) (Suchodolski & Krasowska, 2021).

Sugar metabolism elevates drug tolerance.
To understand the effect of carbon metabolism on drug tolerance, we checked the growth of the ATCC750 strain in media supplemented with different concentrations of each carbon supplement and different concentrations of fluconazole (1, 2, 4, and 8 g/mL).We noticed that in each drug concentration tested, cells could overcome the drug suppression with the increasing concentration of the carbon supplements (Fig. 2A-F and Fig. S1).A previous study confirmed that cells grown in the media with fructose showed higher fluconazole tolerance than those grown with glucose (Suchodolski & Krasowska, 2021).We noticed no variation in growth in the presence of glucose and fructose, whereas the growth was comparatively less in the presence of sucrose.Additionally, less growth was observed in the presence of 4 and 8 g/mL compared to the lower concentrations.
To understand the growth variation in each conduction tested, we checked the mean growth in the form of OD at 8, 16, 24, and 32 hours.At 8 hours, less growth was observed in the presence of three sugars tested, irrespective of their concentrations and the concentrations of fluconazole.
Meanwhile, at subsequent time points, we noticed elevated OD depending on the concentration of fluconazole and carbon supplements.We also noticed an extended lag phase in the presence of all the fluconazole concentrations used.Based on the results, the growth was comparatively less with sucrose than with glucose or fructose (Fig. 2G-I).All experiments were repeated in biological triplicate and shown as mean OD.

Sugar metabolism is important to overcome osmotic stress.
We wanted to know if carbon metabolism by the cells can overcome osmotic stress.Therefore, we grow the standard strain in media supplemented with 1.5 M NaCl and indicated concentrations of each carbon source.We observed that 20 and 50 mg/mL of glucose and fructose supplements were able to promote growth when compared with 5 mg/mL or lower concentrations (Fig. 3A, B).We also noticed that in the presence of 50 mg/mL glucose and fructose, the log phase extended when compared with 20 mg/mL.Although higher sucrose concentrations promote growth, the growth rate was way lower than glucose or fructose (Fig. S2).When we determined the growth rate at different time points with each carbon supplement, we noticed a similar growth rate with glucose and fructose in compared to the reduced growth with sucrose (Fig. 3C).Carbon metabolism can help to overcome oxidative stress.
Cells were treated with 1 and 2.5 mM H2O2 to understand oxidative stress tolerance.In the presence of 1 mM H2O2, cells with glucose showed slow and suppressed growth with a prolonged lag phase.However, 2.5 mM concentration completely inhibits cellular growth even at 24 hours (Fig. 4A).
We noticed that fructose and sucrose failed to support growth in the presence of both the H2O2 concentrations tested (Fig. 4B, C).We also determine the growth variability in the presence of each stressor with the highest concentration of each sugar tested.The growth in the presence of the lowest concentration of every stressor (Fluconazole, NaCl, and H2O2) was significantly lower than the control setup (with each sugar without any stressor).Compared to the control, we noticed a lengthier lag phase, suppressed log, and stationary phase (Fig. 4D-F).

Resistant and susceptible clinical isolates can metabolize carbon substrates similarly.
After determining that the availability of carbon substrates influences the cellular growth in the standard isolate, we sought to understand the metabolic diversity in clinical isolates.To do so, we took 4 fluconazole susceptible (0.5 g/mL) and 4 fluconazole-resistant isolates (64-256 g/mL)(Table S1).Additionally, the selected isolates represent diverse patient groups (male, female, child, adult, and old).Resistant and susceptible isolates were grown in the media supplemented with either glucose, fructose, or sucrose.The cellular growth level was determined as OD600 at 8, 16, and 24-hour time points.All resistant or susceptible isolates clustered nicely based on the growth level.Irrespective of the susceptibility pattern, both resistant and susceptible isolates showed similar growth patterns at each time point with each carbon substrate tested (Fig. 5A-C).

Carbon metabolism influences drug tolerance in an isolate-dependent manner.
To understand the drug adaptation of clinical isolates in the presence of different concentrations of each substrate tested, we used the four susceptible isolates, and 2 mg/mL fluconazole was the concentration of the drug.We noticed a concentration-dependent drug tolerance induction in each susceptible isolate with the carbon substrate used.The drug tolerance was comparatively less in sucrose than in glucose or fructose.Although the drug adaptation significantly increased with the increasing concentration of carbon substrates.Each isolate has its unique adaptation pattern in the presence of the carbon substrates (Fig. 6A-I and Fig. S3).

Carbon metabolism induces osmotic and oxidative stress tolerance.
Both resistant and susceptible isolates were used to understand the osmotic and oxidative stress adaptation.We used 1.5 M NaCl as osmotic and 2.5 mM H2O2 as an oxidative stressor.Stress adaptation with different concentrations of each carbon substrate was measured as OD600 at 8, 16, 24, and 32-hour time points.Both resistant and susceptible isolates took at least 16 hours to adapt to the NaCl stress before they started growing exponentially.Increasing concentrations of glucose or fructose support better growth support when compared with sucrose.Each isolate has a unique level of osmotic stress adaptation, although the increasing sugar concentrations helped for better adaptation (Fig. 7A-C).
Cells could overcome the H2O2-induced oxidative stress comparatively faster than osmotic stress.Both resistant and susceptible isolates overcome oxidative stress.The three carbon substrates successfully promote growth in a concentration-dependent manner (Fig. 7D-F).Finally, irrespective of the susceptibility pattern, the abundance of sugar determines the osmotic and oxidative stress tolerance in C. tropicalis.

Substrate concentration determines the level of stress tolerance.
To understand how the sugar concentration regulates stress tolerance, we checked the level of growth at the endpoint of each experiment in the presence of the lowest (1 mg/mL) and highest (50 mg/mL) concentrations of the three carbon substrates used.Both resistant and susceptible isolates showed noticeably higher drug, oxidative, and osmotic stress tolerance in the presence of 50 mg/mL of each substrate compared with the 1 mg/mL (Fig. 8A-I).Interestingly, we noticed that each isolate had a unique tolerance level for each stressor, and each carbon substrate promotes isolate-dependent stress adaptation.
Fig. 8 The amount of carbon in the growth environment is critical for elevated stress tolerance C. tropicalis.Resistant (R) and susceptible (S) isolates were grown in the media supplemented with glucose, fructose, and sucrose along with 2 µg/mL fluconazole, and 1.5 M NaCl, and 2.5 mM H2O2.Growth variations with the lowest (1 mg/mL)1 and highest (50 mg/mL) concentrations of each carbon substrates was determined by measuring the endpoint OD600.

Higher substrate concentration reduces the generation time.
We were also curious to know the relation between the abundance of carbon substrates and cell doubling time or generation time to confirm the stress tolerance.To do so, we used the starting and endpoint OD in the presence of the lowest (1 mg/mL) and highest (50 mg/mL) concentration of each carbon substrate (glucose, fructose, and sucrose) with each stressor (fluconazole, NaCl and H2O2).To determine the doubling time, we used the formula td = t x [ln(2)/ln(Nt/N0)], adopted from the Omni Calculator online portal (omnicalculator.com).In this formula, td: generation time or doubling time, t: Elapsed time, Nt: Population as OD at end time, N0: Population as OD at starting time.When we examined the fluconazole-induced stress adaptation, the doubling time of 50 mg/mL of each substrate was almost half of 1 mg/mL (Fig. 9A).In osmotic stress, the highest glucose or fructose concentration significantly reduced the doubling time.Meanwhile, fructose didn't show significant variation in generation time (Fig. 9B).Interestingly, sucrose significantly reduced the doubling time in resistant and susceptible isolates in the presence of H2O2.At the same time, glucose decreased the doubling time only for susceptible isolates.However, no noticeable variation was observed in the presence of both lower and higher fructose concentrations (Fig. 9C).

Discussion:
Among the non-albicans Candida species, C. tropicalis has emerged as one of the most predominantly isolated species in Asia and South America (Dos Santos & Ishida, 2023).It can also cause non-invasive infections in the gastrointestinal, urinary, and respiratory tract (Wang et al., 2021).Studies showed that complex commensalism depends on the survival ability of C. albicans in diverse host niches, where fitness is determined by nutrient accessibility with other host factors (Gallo et al., 2022).However, C. albicans adapted to uptake various carbon substrates simultaneously from the host, as the nutrient availability varies a lot (Suchodolski & Krasowska, 2021).Many studies confirmed that carbon metabolism enhances cellular resistance to oxidative, osmotic, and drug-induced stress (Brown, Brown, et al., 2014;Ene, Adya, et al., 2012;Ene, Heilmann, et al., 2012;Rodaki et al., 2009).A study by Suchodolski et al. showed that fructose can increase fluconazole tolerance in C. albicans, and different cellular transporters are involved in this process (Suchodolski & Krasowska, 2021).Rodaki et al. confirmed that glucose can elevate drug-induced stress and oxidative and osmotic stress resistance in C. albicans (Rodaki et al., 2009).
Limited knowledge about the variation in carbon substrate metabolism and their effect on drug, osmotic, and oxidative stress tolerance in C. tropicalis is available.Here, we sought to evaluate the impact of glucose, fructose, and sucrose, the carbon sources naturally occurring in the human body, on the sensitivity of C. tropicalis to fluconazole (drug), osmotic (NaCl), and oxidative (H2O2) stress.
Fitness attributes like the capacity to uptake nutrients from the host that supports cell division, the ability to tolerate physiological stress in the host microenvironments, and cell wall synthesis ability determine the pathogenicity in C. albicans (Brown, Brown, et al., 2014).Among the carbon substrates, glucose and fructose are among the most preferred carbon sources for cellular energy metabolism in yeast (Van Ende et al., 2019).The sensing and transport mechanisms for glucose and fructose in C. albicans are similar (Van Ende et al., 2019).In Saccharomyces cerevisiae, the Gpa2 receptor senses glucose and sucrose and further activates the downstream metabolic signaling pathways (Lemaire et al., 2004).We noticed a similar growth profile in either glucose, fructose, or sucrose.This might be due to the similar types of sensing and transport mechanisms of the sugars, as previously mentioned in (Lemaire et al., 2004;Van Ende et al., 2019).
Rodaki et al. examined the impact of a wide range of glucose levels on central metabolic genes and showed that even 0.01% glucose regulates central metabolic genes in C. albicans.This suggests that blood glucose concentrations (approximately 0.05-0.1%)significantly effect gene regulation (Rodaki et al., 2009).Similarly, for the standard strain and clinical isolates, we noticed a concentration-dependent growth variation in the presence of the carbon substrates because of different gene regulation and levels of cell division.We choose resistant and susceptible clinical isolates to understand the metabolic diversity among them based on their susceptibility profile and the background of the patients from where they were collected.Interestingly, the resistant and susceptible isolates showed similar growth profiles.All clinical isolates used were obtained from invasive infections, and they might be equally efficient in the uptake of nutrients from the host microenvironments, irrespective of their drug susceptibility patterns (Brown, Brown, et al., 2014).
Previous studies reported that the yeast could metabolize ribose and arabinose through the pentose phosphate pathway (PPP) (Ruchala & Sibirny, 2021;van Zyl et al., 1993;Wisselink et al., 2007).Therefore, along with the central carbon metabolism, we were curious to know the role of PPP in energy metabolism by using ribose and arabinose as the sole carbon source.We noticed minimal growth in the presence of either ribose or arabinose.This is likely due to limited uptake of the pentose sugars.These findings suggest that C. tropicalis has limited ability to metabolize ribose and arabinose.Therefore, we excluded ribose and arabinose from our stress adaptation study.
Metabolism promotes virulence by increasing stress adaptation and reducing the vulnerability to environmental stresses in host niches (Arana et al., 2007;Brown, Brown, et al., 2014;Patterson et al., 2013).Rodaki et al. showed glucose exposure can rapidly increase the transcript levels of the antifungal drug resistance genes in C. albicans.Additionally, compared to control (no glucose), glucose exposure provided significantly higher protection against miconazole, an azole antifungal drug (Rodaki et al., 2009).Further, a study by Suchodolsk et al.
confirmed that fructose can increase fluconazole resistance in C. albicans.This resistance is mediated by activating drug transporters Mdr1p and Cdr1p (Suchodolski & Krasowska, 2021).
Based on previous findings, we wanted to know if glucose, fructose, and sucrose can induce fluconazole tolerance in the standard strain and fluconazole-susceptible clinical isolates.We noticed that 50 mg/mL of either glucose, fructose, or sucrose significantly increased fluconazole tolerance compared to 1 mg/mL.This confirms that the carbon substrate concentration is critical for fluconazole-induced drug stress tolerance.Additionally, higher glucose concentration provided noticeably higher protection against fluconazole might be due to the direct relation between central carbon metabolism and increased expression of drug-resistance genes (Rodaki et al., 2009;Suchodolski & Krasowska, 2021).We noticed that the protection in the presence of glucose or sucrose was higher than sucrose.Succors is a disaccharide, that undergoes enzymatic hydrolysis to produce monosaccharide before entering the metabolic pathway, which might be a reason for getting competitively less protection of sucrose against fluconazole (Sabina & Brown, 2009).A review article highlighted the discoveries on the reason for metabolic variations within the clonal microbial population.The most important factors involved are inherent dynamics, ecological factors, and molecular noise (Takhaveev & Heinemann, 2018).Another study confirmed genomic and phenotypic variations among clinical C. albicans isolates (Hirakawa et al., 2015).These studies support our finding of getting variation in fluconazole-induced stress adaptation in an isolate-dependent manner.This might be due to the clinical isolates being from different backgrounds (from different patients) or clonal variation among the isolates.
Several virulence factors are expressed by fungi in a stressful environment, and some yeast can tolerate high salt concentrations or neutralize the effect of osmotic stress (Beales, 2004;Brown, Budge, et al., 2014;Zuza-Alves et al., 2017).C. tropicalis can grow in the presence of 10-15% NaCl and was also isolated from the hypersaline Dead Sea and Amazon Forest samples (Bastos et al., 2000;Butinar et al., 2005).Garcia et al. reported that the hypersaline environment activates Na + /K + -ATPase transporters, which restore the cellular osmotic equilibrium by rapid efflux of ions (Garcia et al., 1997).C. tropicalis is widely used in the food industry and bioremediation processes because of its high salt tolerance (Al-Otibi et al., 2022;Kwon et al., 2006;Zuza-Alves et al., 2017).Zuza-Alves et al. showed that C. tropicalis strains isolated from coastal environments presented high virulence activity with high salt tolerance and higher MICs against different antifungals.These confirm that the pathogenic potential of environmental strains and the overexpression of efflux pumps partially explain the reason for higher drug resistance in C. tropicalis without prior exposure to antifungal drugs (Zuza- Alves et al., 2016).Rodaki et al. also reported that glucose can increase osmotic stress resistance via induction of C. albicans osmotic stress-related genes.This study showed that glucose-exposed cells significantly increase NaCl resistance compared to glucose-untreated control (Rodaki et al., 2009).Another study showed that carbon adaptation influences osmotic stress tolerance, which is mediated by cell wall remodeling (Brown, Brown, et al., 2014;Ene, Adya, et al., 2012).Based on previous findings, we were interested to know the effect of glucose, fructose, and sucrose on NaCl-induced osmotic stress tolerance.Higher glucose and fructose concentrations significantly induced oxidative stress tolerance compared to glucose-limited conditions.This might be due to the higher abundance of carbon substrates inducing the central carbon metabolism, which might be directly linked to the overexpression of stress-related genes and elevated activation efflux pumps, as previously observed (Garcia et al., 1997;Rodaki et al., 2009;Zuza-Alves et al., 2016).Sucrose showed way lower osmotic stress adaptation, possibly due to the comparatively lower uptake and metabolism of sucrose by the cells when compared with either glucose or fructose.Further investigation is needed for a more in-depth understanding of the oxidative stress tolerance in C. tropicalis.
Along with other stresses, oxidative stress increases when macrophages and neutrophils engulf cells.C. albicans and other yeasts showed oxidative stress adaptation mediated by several adaptive pathways (Brown, Budge, et al., 2014).A study by Rodaki et al. reported that transient glucose exposure increases oxidative stress resistance in C. albicans, and glucose induces the transcription of oxidative stress-related genes (Brown, Brown, et al., 2014;Rodaki et al., 2009).This oxidative stress adaptation in C. albicans is medicated by a glucose-sensing pathway by which, over evolutionary time, cells learned to escape from phagocytic attack after entering the bloodstream (Brown, Budge, et al., 2014;Mitchell et al., 2009).Surprisingly, in the evolutionary process C. albicans and its closely related yeast S. cerevisiae, showed contrasting stress responses to glucose.Glucose induces oxidative stress tolerance in C. albicans, whereas glucose decreases resistance in S. cerevisiae (Garreau et al., 2000;Gasch et al., 2000).This opposite effect of glucose is due to the differential regulation of transcription factors in different yeast species (Gasch et al., 2000;Ramsdale et al., 2008;Roetzer et al., 2008).Therefore, we were curious about the impact of glucose, fructose, and sucrose on the oxidative stress resistance in C. tropicalis.These three carbon substrates induced the H2O2-induced oxidative stress resistance among the clinical isolates in a concentration-dependent manner, which was quite similar to the previous findings on C. albicans (Rodaki et al., 2009).Surprisingly, we noticed that the standard strain showed very little oxidative stress adaptation even at 24 hours with all the carbon substrates used, which contrasts with the stress adaptation of the clinical isolates.We noticed a flat growth curve, even the highest concentration of each carbon substrate.We don't know the exact reason for the contrasting differences between standard strain and clinical isolates.This might be due to the isolate-specific metabolic variation and differences in the level of stress adaptation.Further studies are warranted to address the mechanism behind these opposite phenotypes.
Studies showed that carbon substrates can significantly increase drug-induced stress and oxidative and osmotic stress resistance (Rodaki et al., 2009;Suchodolski & Krasowska, 2021).We determine the stress adaptation in glucose-limited and high-glucose conditions.Like previous findings, glucose, fructose, or sucrose drastically increase fluconazole, NaCl, and H2O2 stress tolerance in C. tropicalis (Fig. 8).We also noticed that drug, osmotic, and oxidative stress extend the lag phase as examined in the standard strain (Fig. 4D-F).A similar phenotype was also observed in clinical isolates (Data not shown).This is because the cells took a comparatively longer time to adapt to the stress environment and then started doubling.To reconfirm the effect of carbon substrate availability and level of stress adaptation, we determined the doubling time using the starting and endpoint OD600.We noticed that the generation time or doubling time with 50 mg/mL carbon substrates was almost half of 1 mg/mL.Therefore, carbon substrate availability is critical for cell division and stress adaptation in C. tropicalis.
In conclusion, the present study provides promising information about the effect of carbon substrates on the drug, osmotic, and oxidative stress resistance.This study highlighted for the first time that sugar metabolism induced C. tropicalis stress tolerance in standard strains and clinical islets obtained from invasive infections with different patient populations.This study also used azole-resistant and susceptible isolates to understand the metabolic diversity based on drug susceptibility patterns.Our findings demonstrate that glucose, fructose, and sucrose as carbon sources induce stress resistance in C. tropicalis.Both resistant and susceptible isolates showed similar stress adaptation phenotypes, confirming the equal pathogenic ability irrespective of susceptibility profile.However, given the rising invasive infections and azole resistance in C. tropicalis, extensive further studies are warranted to elucidate the stress resistance mechanisms for managing infections fully.

Fig. 2
Fig. 2 Sugar metabolism can enhance fluconazole tolerance in C. tropicalis.ATCC750 standard strain was grown in the presence of different concentrations of carbon substrates and fluconazole, (A and D) Glucose + Fluconazole, (B and E) Fructose + Fluconazole, (C and F) Sucrose + Fluconazole.Growth was kinetically measured in each hour by obtaining OD600.All experiments were repeated in biological triplicate and shown as mean ± SEM.Significance was assessed using One-way ANOVA *, p<0.05; **, p< 0.01; ****, p< 0.0001; ns, not significant.Heat Map representing growth as OD600 in the presence of different concentrations of (G) glucose + fluconazole, (H) fructose + fluconazole and (I) sucrose + fluconazole at 8, 16, 24, and 32 hours.

Fig. 3
Fig. 3 Hexose metabolism can influence osmotic stress response in C. tropicalis.Standard strain was grown in the presence of media supplemented with 1.5 M NaCl and different concentrations of (A) glucose and (B) Fructose.Growth was kinetically measured in each hour by obtaining OD600.All experiments were repeated in biological triplicate and shown as mean ± SEM.

Fig. 4
Fig. 4 Increased Carbon metabolism is crucial for oxidative stress tolerance.(A, B, and C) Heat Map representing growth of standard strain as OD600 in the presence of 1 and 2.5 mM H2O2 with different concentrations of glucose, fructose, and sucrose at time points 8, 16, and 24 hours.All experiments were repeated in biological triplicate and shown as mean OD. (D, E and F) Cells were grown in the presence of media supplemented with highest concentration of each carbon substrate (glucose/ fructose/ sucrose) and either 1 µg/mL fluconazole or 1.5 M NaCl or 1 mM H2O2.Growth was kinetically measured in each hour by obtaining OD600.All experiments were repeated in biological triplicate and shown as mean ± SEM.Significance was assessed using Oneway ANOVA *, p<0.05; ***p<0.001;****, p< 0.0001; ns, not significant.

Fig. 5
Fig. 5 Irrespective of the susceptibility profile, the carbon metabolism in clinical isolates is quite similar.C. tropicalis resistant and susceptible clinical isolates were grown in the presence of 1, 5, 20, and 50 mg/mL of (A) Glucose, (B) Fructose, (C) Sucrose and variations in growth among resistant and susceptible isolates, at 8, 16 and 24 hours determined by comparing the OD600.All experiments were repeated in biological triplicate and shown as mean ± SEM.

Fig. 6
Fig. 6 Carbon metabolism can elevate drug tolerance in clinical isolates.C. tropicalis susceptible clinical isolates were grown in the presence 2 µg/mL fluconazole along with 1, 5, 20, and 50 mg/mL of (A, D, G) Glucose, (B, E, H) Fructose, (C, F, I) Sucrose and growth was kinetically measured in each hour by obtaining OD600.All experiments were repeated in biological triplicate data represented as area fill plot and shown as mean ± SEM.Significance was

Fig. 7
Fig. 7 Clinical isolates can tolerate osmotic and oxidative stress by inducing carbon metabolism.Resistant and susceptible isolates were grown in the media supplemented with different concentrations of carbon substrates with either 1.5 M NaCl or 2.5 mM H2O2.Heat Map representing growth as OD600 in the presence of (A) Glucose + NaCl, (B) Fructose + NaCl, (C) Sucrose + NaCl, (D) Glucose + H2O2, (E) Fructose + H2O2, and (F) Sucrose + H2O2.All experiments were repeated in biological triplicate and shown as mean OD.

Fig. 9
Fig. 9 In stress condition abundance of carbon substrate determines the generation time.Resistant (R) and susceptible (S) isolates were grown in the media supplemented with different concentrations of glucose, fructose, and sucrose along with 2 µg/mL fluconazole, 1.5 M NaCl, and 2.5 mM H2O2.Variations in generation time with lowest (1 mg/mL)1 and highest (50 mg/mL) concentrations of each carbon substrates along with (A) Fluconazole, (B) NaCl, and (C) H2O2 was determined.All experiments were repeated in biological triplicate and shown as mean ± SEM.Significance was assessed using t tests *, p<0.05; ***p<0.001;****, p< 0.0001; ns, not significant.