In vitro effects of selective serotonin reuptake inhibitors on Cryptococcus gattii capsule and biofilm

Abstract Infections caused by Cryptococcus gattii mainly affect immunocompetent individuals and the treatment presents important limitations. This study aimed to validate the efficacy of selective serotonin reuptake inhibitors (SSRI), fluoxetine hydrochloride (FLH), and paroxetine hydrochloride (PAH) in vitro against C. gattii. The antifungal activity of SSRI using the microdilution method revealed a minimal inhibitory concentration (MIC) of 31.25 µg/ml. The combination of FLH or PAH with amphotericin B (AmB) was analyzed using the checkerboard assay and the synergistic effect of SSRI in combination with AmB was able to reduce the SSRI or AmB MIC values 4–8-fold. When examining the effect of SSRI on the induced capsules, we observed that FLH and PAH significantly decreased the size of C. gattii capsules. In addition, the effects of FLH and PAH were evaluated in biofilm biomass and viability. The SSRI were able to reduce biofilm biomass and biofilm viability. In conclusion, our results indicate the use of FLH and PAH exhibited in vitro anticryptococcal activity, representing a possible future alternative for the cryptococcosis treatment.


Introduction
According to the Global Action Fund for Fungal Infections (GAFFI), mycoses globally affect > 300 million people, and ∼25 million are at serious risk of death (Rodrigues and Nosanchuk 2020 ).Aspergillus spp., Candida spp., Cryptococcus spp., and Paracoccidioides spp.are the main ones responsible for invasive fungal diseases (Gow et al. 2022 ).
Cryptococcus gattii, an emerging fungal pathogen, has demonstrated a notable potential to cause infections in immunocompetent individuals (Springer and Chaturvedi 2010 ).The treatment of cryptococcosis involves the antifungals amphotericin B (AmB) and fluconazole (Flz).Amphotericin B (AmB), introduced in the late 1950s, has long been considered the gold standard for se v er e cases (Saag et al. 2000 ).Fluconazole, a triazole antifungal agent, is used for both induction and maintenance ther a p y (P erfect et al. 2010 ).Conv entional tr eatments for cryptococcosis face challenges such as fungal resistance development, notable host toxicity, and limited central nervous system (CNS) penetration for certain medications (Zhai et al. 2012 ).Consequentl y, ther e is a pr essing need for the de v elopment of alternativ es to addr ess systemic mycoses treatment.
Fluoxetine hydr oc hloride (FLH) and par oxetine hydr oc hloride (PAH), both from the class of selectiv e ser otonin r euptake inhibitors (SSRI) drugs , ha v e r eceiv ed a ppr ov al as antidepr essants for the management of depression and anxiety disorders (Cruz et al. 2020, Murphy et al. 2021 ).The antifungal potential of SSRI against Cryptococcus neoformans has recently been demonstrated at fungicidal concentrations below 10 μg/ml (Per eir a et al. 2021 ) and further, these drugs can act syner gisticall y or ad diti v el y with Flz in vivo , reducing the fungal burden in the brain, kidney, and spleen (Zhai et al. 2012 ).In this study, we aimed to investigate in vitro and biofilm antifungal effects of FLH and PAH against C. gattii.

Strains and growth conditions
Cryptococcus gattii ATCC 56990 and C. gattii clinical isolate 5 ( C. gattii 5 ) obtained from the collection from the Oral Microbiology and Imm unology Labor atory of the Institute of Science and Tec hnology of São José dos Campos/UNESP wer e used.Both str ains wer e maintained in Sabouraud dextrose agar (SDA; Difco Laboratories, Detroit, MI, USA) at 37 • C. For the assa ys , y easts w er e gr own in Sabour aud br oth (Difco Labor atories) incubated at 37 • C (150 r pm) for 48 h.

Drugs and antifungals
The SSRI drugs FLH (Fa gr on, Bologna, Ital y), PAH (Infinity Pharma, Hong Kong, China), and the antifungal AmB (Sigma-Aldrich, Saint Louis, MO, USA) were used in this study.A fresh solution of each drug was pr epar ed befor e eac h assay using a maxim um concentration of 1% dimethyl sulfoxide (Sigma-Aldrich) as a co-solvent to maximize solubility.The intermediate solution was pr epar ed in RPMI 1640 medium (Sigma-Aldrich) for the assa ys .

Antimicrobial susceptibility testing
To e v aluate the antifungal activity of the SSRI drugs the broth microdilution assay was performed according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (Arendrup et al. 2017 ).Fluoxetine hydrochloride (FLH) and PAH at concentr ations fr om 1.95 to 1000 μg/ml, and AmB at concentr ations from 0.03 to 8 μg/ml were prepared.Then, C .gattii (2.5 × 10 5 cells/ml) were added to 96-well plates containing the drugs for treatment and incubated for 48 h for 37ºC.Spectrophotometric readings at 530 nm were performed and the minimal inhibitory concentration (MIC) was defined as the lowest drug concentration that did not allow fungal growth.The minimal fungicidal concentration (MFC) was determined by transferring an inoculum from the wells of microdilution assay to SDA plates using sterile wood picks and further incubation at 37ºC for 48 h.The MFC was defined as the lo w est drug concentration that did not permit visual growth of any fungal colonies.

Ev alua tion of synergistic effects of the SSRI drugs with AmB
The synergistic effects of SSRI drugs and AmB were assessed using the c hec kerboard assay, based on the broth microdilution method as described in pr e vious studies (Odds 2003, Arendrup et al. 2017 ).Concentrations of FLH and PAH r anging fr om 0.97 to 500 μg/ml, and AmB at concentrations ranging from 0.06 to 8 μg/ml were used.Cryptococcus gattii suspensions at 5 × 10 5 cells/ml were used and subsequently, the plates were incubated at 37ºC for 48 h.Spectr ophotometric r eadings at 530 nm wer e conducted.To assess synergistic activity, the fractional inhibitory concentration index (FICI) was determined using the formula: FIC = FIC A + FIC B , wher e FIC r epr esents the r atio of the MIC of the drug in combination with the MIC when used alone.Next, FICI was categorized as follows: "synergistic effect" if FICI is ≤ 0.5 and "indifferent" if FICI is > 1 and ≤ 4, and "antagonistic" relationship was defined as FIC index > 4.0 (Odds 2003 ).

Analyses of capsule size
Cryptococcus gattii capsules w ere induced, accor ding to Zar a goza and Casade v all ( 2004 ).For this, C. gattii at 5 × 10 6 cells/ml was inoculated in the induction medium (10% Sabour aud br oth in 50 mM MOPS, pH 7.4) and incubated for 24 h at 30ºC.Subsequently, cells with induced capsules were removed from the induction medium and treated with subinhibitory drug concentrations (sub-MIC: FLH and PAH = 15.6 μg/ml and AmB = 0.25 μg/ml) in RPMI.The untreated cells were used as the control.The treatment was performed for 24 h at 37ºC.Cells were stained with India ink and analyzed with optical microscopy Axioplan 2 (Zeiss, Germany).Capsule size was measured using the Ima geJ softwar e (Rueden et al. 2016 ) by calculating the difference between the whole cell and the cell body size.

Biofilm formation and treatment
Biofilm formation was performed as described by Martinez and Casade v all ( 2006 ).For biofilm preadhesion, 100 μL of 10 8 cells/ml of C. gattii pr epar ed in RPMI medium supplemented with 2% glucose was added to 96-well plates and incubated at 37ºC without agitation for 24 h.The wells were washed with phosphatebuffered saline (PBS) to remove nonadherent yeasts.Subsequently, 100 μL of the medium was added to the wells and the plate was incubated at 37ºC for 48 h without agitation.After this, w ells w ere w ashed with PBS to r emov e nonadher ent yeasts.Adherent fungal cells were considered mature biofilms.To evaluate the susceptibility of C. gattii biofilms to FLH and PAH, biofilms were treated with 10x of SSRI drugs MIC (312.5 μg/ml), 20 × SSRI drugs MIC (625 μg/ml), or AmB 10x MIC (5 μg/ml) and incubated at 37ºC for 24 h.

Effect of SSRI drug treatment on C. gattii biofilms biomass
Effects of SSRI drug treatment on C. gattii biofilms biomass were analyzed using the crystal violet method according to Peeters et al. ( 2008 ).After treatments, wells with biofilms were washed with PBS and fixed with absolute ethanol for 15 min.Next, 100 μL of 0.5% crystal violet was added to each well and incubated for 20 min.The excess dye was r emov ed and the wells were washed with PBS.Further, 100 μL of absolute ethanol was added to dilute the dye.The absorbance was measured at 570 nm and the results were expressed as a percentage reduction.Cryptococcus gattii biofilms formed after 48 h without treatment were used as the control and the corresponding absorbance values were re presentati ve of 100% biomass.

Effect of SSRI drug treatment on biofilm cell viability
Biofilms were formed as described abo ve , and the effect of SSRI drugs on C. gattii biofilm viability was measured by tetrazolium salt (XTT) assay (Martinez and Casade v all 2006 ).After biofilm formation and drug treatment, wells were washed with 100 μL of PBS and subsequently inoculated with a solution of 50 μL of 1 mg/ml XTT (Sigma-Aldrich) and 4 μL of 1 mM menadione (Sigma-Aldrich).After 1 h of incubation in the dark at 37 • C, 50 μL of solution was tr ansferr ed fr om eac h well to another plate and its absorbance was recorded at 490 nm.

Sta tistical anal yses
Statistical analyses were performed using the GraphPad Prism 5.0 softwar e (Gr a phP ad Softwar e Inc., La J olla, C A, USA).The data obtained wer e anal yzed using Kruskal-Wallis and Dunn´s.For all tests, the significance le v el adopted was P < 0.05.

Susceptibility of C. gattii to SSRI drugs and synergistic effect with AmB
Fluoxetine hydr oc hloride (FLH) and PAH wer e activ e a gainst C. gattii ATCC 56990 and C. gattii 5 with a MIC value of 31.25 μg/ml.Notably, the MFC for both drugs corresponded to the MIC.For C. gattii ATCC 56990, FLH + AmB combination resulted in two syner gistic concentr ations (FLH 3.9 μg/ml + AmB 0.125 μg/ml and FLH 7.8 μg/ml + AmB 0.125 μg/ml) with a 4-fold to 8-fold reduction in MIC v alues.Additionall y, one syner gistic combination for PAH + AmB (PAH 7.8 μg/ml + AmB 0.125 μg/ml) with a 4-fold reduction in MIC value was observed when combined with SSRI drugs (Table 1 ).For the C. gattii 5, an indiffer ent r esult to the combinations was observed.

Effects of SSRI drugs on biofilm viability
The effect of the SSRI drugs on C. gattii biofilms was e v aluated at 10x and 20x MIC and AmB at 10x MIC.For C. gattii ATCC 56990 the treatment with FLH revealed a decreased biofilm viability by 39.05% ( P = 0.0109) and 78.94% ( P = 0.0082) at 10x MIC and 20 × MIC, r espectiv el y.The biofilm viability percenta ge with tr eatment with PAH was 56.99% ( P < 0.0001) and 67.64% ( P < 0.0001) for 10x MIC and 20 × MIC, r espectiv el y.AmB 10x MIC used as a contr ol, decr eased biofilm viability by 42.24% ( P = 0.0037) (Fig. 3 A).

Discussion
Fungal infections have shown a significant increase in recent years, with cryptococcosis being notabl y pr e v alent among both imm unocompr omized and immunocompetent individuals (Perfect et al. 2010, Spitzer et al. 2017 ).Resistance to conventional treatments has been reported for Cryptococcus spp., coupled with the scarcity of antifungal options, ele v ated costs, and significant host toxicity, highlighting the imper ativ e need for treatment alternativ es (Roder o et al. 2003, Scorzoni et al. 2017 ).
Drug repositioning is a promising alternative to conventional antifungal tr eatment (Katr a gk ou et al. 2016, Pushpak om et al. 2019 )  SSRI drugs have a proven inhibitory effect on Candida spp.by reducing biofilm formation and causing cell death by apoptosis.In addition, FLH was shown to inhibit fungal growth at concentrations of 127-508 μg/ml, and PAH at concentrations of 80-320 μg/ml (Costa Silva et al. 2017, Oliveira et al. 2018 ) Ther efor e, we e v aluated the antibiofilm activity of the SSRI, FLH, and PAH on C. gattii.Our results indicate a significant reduction of biofilm biomass and viability by both drugs at 10x and 20x MIC.Studies with drugs from other classes, such as the anthelmintic benzimidazoles , ha v e r e v ealed a r eduction in biofilms against C. neoformans , also proving active for C. gattii (Joffe et al. 2017 ).
The mechanisms action of the effect of SSRI on C. gattii is poorly described.Sertraline has been shown to affect intracellular membr ane or ganization, tr anslation, and v esicle tr ansport (Zhai et al. 2012 ), given that both drugs are from SSRI, with a comparable mechanism of action, FLH and PAH could be acting similarly.
The treatment of systemic mycoses is consider abl y complicated by the limited number of antifungal drugs and the poor penetration of antifungals in the CNS due to the blood-brain barrier, onl y a fe w fungistatic drugs show r easonable penetr ation into the CNS.On the other hand, when the concentrations of SSRI drugs such as sertraline were analyzed in the cerebrospinal fluid and br ain, they wer e ∼20-40 times higher compared to plasma levels (Lass-Florl 2001, Marchetti et al. 2004 ).Sertraline exhibited potent antifungal activity against C. neoformans , the main etiological agent of cryptococcal meningitis (Zhai et al. 2012 ).In addition, sertr aline decr eased the fungal burden in the brain and spleen of mice using murine model of cryptococcosis (Treviño-Rangel et al. 2016 ).Additionally, according to the British Pharmacopeia, lethal dose 50 (DL50) of SSRI drugs her e e v aluated ar e 374 mg/kg (PAH) and 452 mg/kg (FLH) when administr ated or all y in r ats .T her efor e, in vivo studies with SSRIs are needed to e v aluate the efficiency of these drugs.
In summary, this study provides evidence of the potent anticryptococcal activity of FLH and PAH in vitro and its effects on C. gattii capsules and biofilm.Ho w ever, future in vivo investigations to better describe these activities ar e r equir ed to demonstr ate the r ele v ance of FLH and PAH as antifungal agents for cryptococcosis.
and drugs from the SSRI class have shown activity against fungal agents, including Candida spp, Aspergillus spp.and C. neoformans (Lass-Florl 2001 , Oliv eir a et al. 2014 , Per eir a et al. 2021 ).Ho w e v er, ther e ar e no r e ports of SSRI drugs acti vities on C. gattii.

Table 1 .
Antifungal susceptibility assay and combinatory effect of SSRI and AmB against C. gattii.
. For C.neoformans, the concentrations able to inhibit fungal growth were 9.6 μg/ml for FLH and 41 μg/mLlfor PAH (Per eir a et al. 2021 ).In this study, both drugs, FLH and PAH sho w ed a MIC of 31.25 μg/ml for C. gattii ATCC 56990 and C. gattii 5 .When combined effects of SSRI drugs with AmB wer e e v aluated, thr ee syner gistic combinations wer e found for C. gattii ATCC 56990, which reduced the MIC values by 4-8-fold.Cryptococcus spp.capsule is a virulence mechanism essential for the pathogenicity of this yeast.It hinders phagocytosis and modulates host responses, contributing to disease pr ogr ession.Understanding the capsule's molecular mechanisms is essential for dev eloping effectiv e cryptococcosis tr eatments (Zar a goza et al. 2008 , Vecc hiar elli et al. 2013 ).In this study, we demonstrated that subinhibitory concentration of FLH and PAH (15.62 μg/ml) reduced cap-sule size by up to 37.6%.In C. neoformans, at sub-MIC concentrations, FLH (4.8 μg/ml) and PAH (20 μg/ml) markedly decreased capsule size by up to 63% (Per eir a et al. 2021 ).