Antifungal and antibiofilm effect of duloxetine hydrochloride against Cryptococcus neoformans and Cryptococcus gattii.

Cryptococcosis is an invasive mycosis caused mainly by Cryptococcus gattii and C. neoformans and is treated with amphotericin B (AMB), fluconazole and 5-fluorocytosine. However, antifungal resistance, limited and toxic antifungal arsenal stimulate the search for therapeutic strategies such as drug repurposing. Among the repurposed drugs studied, the selective serotonin reuptake inhibitors (SSRIs) have shown activity against Cryptococcus spp. However, little is known about the antifungal effect of duloxetine hydrochloride (DH), a selective serotonin and norepinephrine reuptake inhibitor (SSNRI), against C. neoformans and C. gattii. In this study, DH inhibited the growth of several C. neoformans and C. gattii strains at concentrations ranging from 15.62 to 62.50 µg/mL. In addition, DH exhibited fungicidal activity ranging from 15.62 to 250 µg/mL. In biofilm, DH treatment reduced Cryptococcus spp. biomass at a level comparable to AMB, with a significant reduction (85%) for C. neoformans biofilms. The metabolic activity of C. neoformans and C. gattii biofilms decreased significantly (99%) after treatment with DH. Scanning electron micrographs confirmed the anti-biofilm activity of DH, as isolated cells could be observed after treatment. In conclusion, DH showed promising antifungal activity against planktonic cells and biofilms of C. neoformans and C. gattii, opening perspectives for further studies with DH in vivo.

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

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.

Synergistic effect of the verapamil and amphotericin B against Cryptococcus neoformans.

Cryptococcus neoformans is an encapsulated yeast that can cause cryptococcosis and cryptococcal meningitis, which conventional treatment involves antifungal drugs such as polyenes, flucytosine, azoles, and their combinations. However, the high cost, toxicity, and increase in fungi resistance to antifungal agents stimulate the search for therapeutic strategies such as drug repurposing and combination therapy. This study evaluated the activity of the antihypertensive verapamil (VEH) alone and combined with amphotericin B (AmB) against C. neoformans. VEH exhibited antifungal activity against C. neoformans with minimum inhibitory concentration and minimum fungicidal concentration of 118 µg per mL. The combination of VEH and AmB exhibited synergism, reducing at least eightfold both drugs' concentrations. Moreover, the combination decreased the size and glucuronoxylomannnan content of C. neoformans capsule. However, no difference was observed in ergosterol levels of C. neoformans after treatment with VEH and AmB in combination. Altogether, VEH in combination with AmB exhibits potential as a candidate as for the development of anti-cryptococcal drug.

Antimicrobial action of four herbal plants over mixed-species biofilms of Candida albicans with four different microorganisms.

This study aimed to evaluate the antimicrobial effect of four herbal plants glycolic extracts over mixed-species biofilm composed of Candida albicans (C. albicans) and another pathogenic bacterium as alternative therapy to be investigated. Four plants extract of Pfaffia paniculata roots; Hamamelis virginiana leaf, Stryphnodendron barbatiman tree bark and Gymnema sylvestre stem and leaves were tested over multi-species biofilm of C. albicans (ATCC 18804) and Streptococcus mutans (ATCC 35688), Staphylococcus aureus (ATCC 6538), Enterococcus faecalis (ATCC 4083) or Pseudomonas aeruginosa (ATCC 15442) for 5 min and 24 h and colony forming units per millilitre was calculated. The data were analysed using Kruskal-Wallis with Dunn's test (p ≤ 0.05). All tested extracts showed antimicrobial action over the mixed-species biofilms after 24 h. Some extracts eliminated totally the biofilms. The glycolic extract of P. paniculata, H. virginiana, S. barbatiman and G. sylvestre are effective over mixed-species biofilms and may be indicated as endodontic irrigant or intracanal medication.

Synergistic combination of duloxetine hydrochloride and fluconazole reduces the cell growth and capsule size of Cryptococcus neoformans.

This study aimed to evaluate the effect of duloxetine hydrochloride (DH) on Cryptococcus neoformans. DH minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) were 18.5 µg/mL, and the combination with fluconazole (FLZ) reduced the MIC value by 16-and 4-fold for DH and FLZ, respectively. The capsule size decreased by 67% ​​and 16% when treated with DH and DH with FLZ, respectively. Therefore, this study showed that DH is active against C. neoformans alone and in combination with FLZ, leading to the reduction of the capsule size of this yeast.

Cryptococcus spp. and Cryptococcosis: focusing on the infection in Brazil.

Cryptococcosis is a global fungal infection caused by the Cryptococcus neoformans/Cryptococcus gattii yeast complex. This infection is acquired by inhalation of propagules such as basidiospores or dry yeast, initially causing lung infections with the possibility of progressing to the meninges. This infection mainly affects immunocompromised HIV and transplant patients; however, immunocompetent patients can also be affected. This review proposes to evaluate cryptococcosis focusing on studies of this mycosis in Brazilian territory; moreover, recent advances in the understanding of its virulence mechanism, animal models in research are also assessed. For this, literature review as realized in PubMed, Scielo, and Brazilian legislation. In Brazil, cryptococcosis has been identified as one of the most lethal fungal infections among HIV patients and C. neoformans VNI and C. gattii VGII are the most prevalent genotypes. Moreover, different clinical settings published in Brazil were described. As in other countries, cryptococcosis is difficult to treat due to a limited therapeutic arsenal, which is highly toxic and costly. The presence of a polysaccharide capsule, thermo-tolerance, production of melanin, biofilm formation, mechanisms for iron use, and morphological alterations is an important virulence mechanism of these yeasts. The introduction of cryptococcosis as a compulsory notification disease could improve data regarding incidence and help in the management of these infections.

Antifungal activity and toxicity of an octyl gallate-loaded nanostructured lipid system on cells and nonmammalian animals.

Aim: Octyl gallate (OG) loaded into a nanostructured lipid system (NLS) was tested for antifungal activity and in vitro and in vivo toxicity. Methods & Results: The features of NLS-OG were analyzed by dynamic light scattering and showed adequate size (132.1 nm) and homogeneity (polydispersity index = 0.200). OG was active against Paraccoccidioides spp., and NLS-OG did not affect antifungal activity. NLS-OG demonstrated reduced toxicity to lung cells and zebrafish embryos compared with OG, whereas NLS was toxic to hepatic cells. OG and NLS-OG did not show toxicity in a Galleria mellonella model at 20 mg/kg. All toxic concentrations were superior to MIC (antifungal activity). Conclusion: These results indicate good anti-Paracoccidioides activity and low toxicity of NLS-OG.

Plain language summary Drugs for the treatment of fungal diseases are limited in number and present side effects, drug interactions, risks for pregnant women and fungal resistance. The authors produced a derivative compound from plants called octyl gallate (OG) and then incorporated it into a nanoparticle lipid system (NLS) for better distribution in biological fluids. NLS-OG was tested against a fungus called Paracoccidioides, which causes lung infections. The toxicity profile of NLS-OG was also evaluated in lung and hepatic cells as well as novel animal models. NLS-OG presented good antifungal activity and low toxicity in lung cells and embryos.

Current and promising pharmacotherapeutic options for candidiasis.

Introduction: Candida spp. are commensal yeasts capable of causing infections such as superficial, oral, vaginal, or systemic infections. Despite medical advances, the antifungal pharmacopeia remains limited and the development of alternative strategies is needed.Areas covered: We discuss available treatments for Candida spp. infections, highlighting advantages and limitations related to pharmacokinetics, cytotoxicity, and antimicrobial resistance. Moreover, we present new perspectives to improve the activity of the available antifungals, discussing their immunomodulatory potential and advances on drug delivery carriers. New therapeutic approaches are presented including recent synthesized antifungal compounds (Enchochleated-Amphotericin B, tetrazoles, rezafungin, enfumafungin, manogepix and arylamidine); drug repurposing using a diversity of antibacterial, antiviral and non-antimicrobial drugs; combination therapies with different compounds or photodynamic therapy; and innovations based on nano-particulate delivery systems.Expert opinion: With the lack of novel drugs, the available assets must be leveraged to their best advantage through modifications that enhance delivery, efficacy, and solubility. However, these efforts are met with continuous challenges presented by microbes in their infinite plight to resist and survive therapeutic drugs. The pharmacotherapeutic options in development need to focus on new antimicrobial targets. The success of each antimicrobial agent brings strategic insights to the next phased approach in treatingCandida spp. infections.

Antifungal effects of Streptococcus mutans extract on Candida strains susceptible and resistant to fluconazole: An in vivo study.

Previous studies showed that the crude extract obtained from Streptococcus mutans inhibited the growth of Candida albicans reference strains. In this study, we evaluated whether the antifungal effects of S. mutans extract can be extended to clinical Candida isolates, including C. albicans and non-abicans strains with different susceptibilities to fluconazole. We verified that S. mutans extract increased the survival of Galleria mellonella larvae infected with C. albicans and C. glabrata and inhibited the fungal cells in hemolymph. These antifungal effects occurred for both fluconazole-susceptible and fluconazole-resistant strains. However, larvae infected by C. krusei were not affected by S. mutans extract. LAY SUMMARY: Streptococcus mutans crude extract shows antifungal effects on clinical Candida strains susceptible and resistant to fluconazole in Galleria mellonella model.

In vitro synergistic effects of fluoxetine and paroxetine in combination with amphotericin B against Cryptococcus neoformans.

Cryptococcus neoformans is a yeast that mainly affects immunocompromised individuals and causes meningoencephalitis depending on the immune status of the host. The present study aimed to validate the efficacy of selective serotonin reuptake inhibitors, fluoxetine hydrochloride (FLH) and paroxetine hydrochloride (PAH), alone and in combination with amphotericin B (AmB) against C. neoformans. Susceptibility tests were conducted using the broth microdilution method and synergistic effects of combining FLH and PAH with AmB were analyzed using the checkerboard assay. Effects of minimum inhibitory concentration (MIC) and synergistic concentration were evaluated in biofilms by quantifying the biomass, measuring the viability by counting the colony-forming units (CFU/mL) and examining the size of the induced capsules. Cryptococcus neoformans was susceptible to FLH and PAH and the synergistic effect of FLH and PAH in combination with AmB reduced the MIC of AmB by up to 8-fold. The isolated substances and combination with AmB were able to reduce biofilm biomass and biofilm viability. In addition, FLH and PAH alone or in combination with AmB significantly decreased the size of the yeast capsules. Collectively, our results indicate the use of FLH and PAH as a promising prototype for the development of anti-cryptococcal drugs.

In Vitro and In Vivo Effect of Peptides Derived from 14-3-3 Paracoccidioides spp. Protein.

BACKGROUND: Paracoccidioidomycosis (PCM) is a chronic disease that causes sequelae and requires prolonged treatment; therefore, new therapeutic approaches are necessary. In view of this, three peptides from Paracoccidioides brasiliensis 14-3-3 protein were selected based on its immunogenicity and therapeutic potential. METHODS: The in vitro antifungal activity and cytotoxicity of the 14-3-3 peptides were evaluated. The influence of the peptides in immunological and survival aspects was evaluated in vivo, using Galleria mellonella and the expression of antimicrobial peptide genes in Caenorhabditis elegans. RESULTS: None of the peptides were toxic to HaCaT (skin keratinocyte), MRC-5 (lung fibroblast), and A549 (pneumocyte) cell lines, and only P1 exhibited antifungal activity against Paracoccidioides spp. The peptides could induce an immune response in G. mellonella. Moreover, the peptides caused a delay in the death of Paracoccidioides spp. infected larvae. Regarding C. elegans, the three peptides were able to increase the expression of the antimicrobial peptides. These peptides had essential effects on different aspects of Paracoccidioides spp. infection showing potential for a therapeutic vaccine. Future studies using mammalian methods are necessary to validate our findings.

Antifungal and anti-biofilm effect of the calcium channel blocker verapamil on non-albicans Candida species.

Candida is a human fungal pathogen that causes a wide range of diseases. Candida albicans is the main etiologic agent in these diseases; however, infections can be caused by non-albicans Candida species. Virulence factors such as biofilm production, which protect the fungus from host immunity and anti-fungal drugs, are important for the infection. Therefore, available antifungal drugs for candidiasis treatment are limited and the investigation of new and effective drugs is needed. Verapamil is a calcium channel blocker with an inhibitory effect on hyphae development, adhesion, and colonization of C. albicans. In this study, we investigated the effect of verapamil on cell viability and its antifungal and anti-biofilm activity in non-albicans Candida species. Verapamil was not toxic to keratinocyte cells; moreover, C. krusei, C. parapsilosis, and C. glabrata were susceptible to verapamil with a minimal inhibitory concentration (MIC) of 1250 µM; in addition, this drug displayed fungistatic effect at the evaluated concentrations. After treatment with verapamil, reduced viability, biomass, and mitochondrial activity were observed in biofilms of the non-albicans Candida species C. krusei, C. glabrata, and C. parapsilosis. These findings highlight the importance of the study of verapamil as an alternative treatment for infections caused by non-albicans Candida species.

Brazilian Copaifera Species: Antifungal Activity against Clinically Relevant Candida Species, Cellular Target, and In Vivo Toxicity.

Plants belonging to the genus Copaifera are widely used in Brazil due to their antimicrobial properties, among others. The re-emergence of classic fungal diseases as a consequence of antifungal resistance to available drugs has stimulated the search for plant-based compounds with antifungal activity, especially against Candida. The Candida-infected Caenorhabditis elegans model was used to evaluate the in vitro antifungal potential of Copaifera leaf extracts and trunk oleoresins against Candida species. The Copaifera leaf extracts exhibited good antifungal activity against all Candida species, with MIC values ranging from 5.86 to 93.75 µg/mL. Both the Copaifera paupera and Copaifera reticulata leaf extracts at 46.87 µg/mL inhibited Candida glabrata biofilm formation and showed no toxicity to C. elegans. The survival of C. glabrata-infected nematodes increased at all the tested extract concentrations. Exposure to Copaifera leaf extracts markedly increased C. glabrata cell vacuolization and cell membrane damage. Therefore, Copaifera leaf extracts are potential candidates for the development of new and safe antifungal agents.

Streptococcus mutans Secreted Products Inhibit Candida albicans Induced Oral Candidiasis.

In the oral cavity, Candida species form mixed biofilms with Streptococcus mutans, a pathogenic bacterium that can secrete quorum sensing molecules with antifungal activity. In this study, we extracted and fractioned culture filtrate of S. mutans, seeking antifungal agents capable of inhibiting the biofilms, filamentation, and candidiasis by Candida albicans. Active S. mutans UA159 supernatant filtrate components were extracted via liquid-liquid partition and fractionated on a C-18 silica column to resolve S. mutans fraction 1 (SM-F1) and fraction 2 (SM-F2). We found anti-biofilm activity for both SM-F1 and SM-F2 in a dose dependent manner and fungal growth was reduced by 2.59 and 5.98 log for SM-F1 and SM-F2, respectively. The SM-F1 and SM-F2 fractions were also capable of reducing C. albicans filamentation, however statistically significant differences were only observed for the SM-F2 (p = 0.004). SM-F2 efficacy to inhibit C. albicans was confirmed by its capacity to downregulate filamentation genes CPH1, EFG1, HWP1, and UME6. Using Galleria mellonella as an invertebrate infection model, therapeutic treatment with SM-F2 prolonged larvae survival. Examination of the antifungal capacity was extended to a murine model of oral candidiasis that exhibited a reduction in C. albicans colonization (CFU/mL) in the oral cavity when treated with SM-F1 (2.46 log) and SM-F2 (2.34 log) compared to the control (3.25 log). Although both SM-F1 and SM-F2 fractions decreased candidiasis in mice, only SM-F2 exhibited significant quantitative differences compared to the non-treated group for macroscopic lesions, hyphae invasion, tissue lesions, and inflammatory infiltrate. Taken together, these results indicate that the SM-F2 fraction contains antifungal components, providing a promising resource in the discovery of new inhibitors for oral candidiasis.

Interaction between Lactobacillus reuteri and periodontopathogenic bacteria using in vitro and in vivo (G. mellonella) approaches.

Periodontitis is a multifactorial inflammatory disease, and the major cause of tooth loss in adults. New therapies have been proposed for its treatment, including the use of probiotics such as Lactobacillus reuteri. The objective of this study was to evaluate the antimicrobial effects of L. reuteri: live, heat-killed and culture filtrate (cell-free supernatant), on periodontopathogenic bacteria (Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans) in vitro, as well as the in vivo survival curve, hemocyte density and microbial recovery using Galleria mellonella. For in vitro assays, all preparations reduced colony forming units of F. nucleatum, while only live L. reuteri reduced the growth of A. actinomycetemcomitans. All treatments reduced periodontopathogenic bacteria growth in vivo. The treatment with the supernatant increased the survival of larvae infected with F. nucleatum more than the treatment with live L. reuteri, and none of the treatments altered the survival of A. actinomycetemcomitans-infected larvae. In addition, the treatment with L. reuteri preparations did not alter the hemocyte count of F. nucleatum- and A. actinomycetemcomitans-infected larvae. This study demonstrated that L. reuteri preparations exerted antimicrobial effects and increased the survival of G. mellonella infected by F. nucleatum, although only live L. reuteri was able to reduce the growth of A. actinomycetemcomitans in vitro.

Incorporation of Nonyl 3,4-Dihydroxybenzoate Into Nanostructured Lipid Systems: Effective Alternative for Maintaining Anti-Dermatophytic and Antibiofilm Activities and Reducing Toxicity at High Concentrations.

Dermatophytosis is the most common mycosis worldwide, affecting approximately 20 to 25% of the population, regardless of gender, race, color, and age. Most antifungal agents used for the treatment of dermatophytosis belong to the azole and allylamine classes. Dermatophytes are reported to be resistant to most commercial drugs, especially microbial biofilms, in addition to their considerable toxicity. It should be emphasized the importance of looking for new molecules with reduced toxicity, as well as new targets and mechanisms of action. This work aims to incorporate nonyl 3,4-dihydroxybenzoate, a potent fungicide compound against planktonic cells and dermatophyte biofilms in nanostructured lipid systems (NLS), in order to reduce toxicity in high concentrations, improve its solubility and maintain its effectiveness. The compound was incorporated into NLS constituted by cholesterol, mixture of polyoxyethylene (23) lauryl ether (Brij®98) and soybean phosphatidylcholine (Epikuron® 200)], 2: 1 ratio and PBS (phosphate-buffered saline). The characterization of the incorporation was performed. Susceptibility tests were conducted according to document M38-A2 by CLSI (2008). The toxicity of the NLS compound was evaluated in HaCaT cell lines by the sulforhodamine B method and in alternative models Caenorhabditis elegans and zebrafish. Finally, its efficacy was evaluated against the mature Trichophyton rubrum and Trichophyton mentagrophytes biofilms. NLS and nonyl 3,4-dihydroxybenzoate loaded into NLS displayed sizes ranging from 137.8 ± 1.815 to 167.9 ± 4.070 nm; the polydispersity index (PDI) varying from 0.331 ± 0.020 to 0.377 ± 0.004 and zeta potential ranging from -1.46 ± 0.157 to -4.63 ± 0.398 mV, respectively. Polarized light microscopy results confirmed the formation of NLS of the microemulsion type. Nonyl incorporated into NLS showed minimum inhibitory concentration (MIC) values, ranging from 2 to 15.6 mg/L. The toxicity tests presented cell viability higher than 80% in all tested concentrations, as well as, a significantly increased of the survival of Caenorhabditis elegans and zebrafish models. Anti-biofilm tests proved the efficacy of the incorporation. These findings contribute significantly to the search for new antifungals and allow the systemic administration of the compound, since the incorporation can increase the solubility of non-polar compounds, improve bioavailability, effectiveness and reduce toxicity.

Candida Biofilms: An Update on Developmental Mechanisms and Therapeutic Challenges.

Fungi of the genus Candida are important etiological agents of superficial and life-threatening infections in individuals with a compromised immune system. One of the main characteristics of Candida is its ability to form highly drug tolerance biofilms in the human host. Biofilms are a dynamic community of multiple cell types whose formation over time is orchestrated by a network of transcription regulators. In this brief review, we provide an update of the processes involved in biofilm formation by Candida spp. (formation, treatment, and control), as well as the transcriptional circuitry that regulates its development and interactions with other microorganisms. Candida albicans is known to build mixed species biofilms with other Candida species and with various other bacterial species in different host niches. Taken together, these properties play a key role in Candida pathogenesis. In addition, this review gathers recent studies with new insights and perspectives for the treatment and control of Candida biofilms.

Heat-killed Lactobacillus reuteri and cell-free culture supernatant have similar effects to viable probiotics during interaction with Porphyromonas gingivalis.

BACKGROUND AND OBJECTIVE: In the last decade, numerous studies have been published to clarify the role of probiotics, especially Lactobacillus reuteri, as an adjunct to conventional periodontal treatment. Although the health benefits of probiotics are numerous, they are live bacteria, and the administration of live organisms is not risk-free. We evaluated the antimicrobial effect of L reuteri and its cell-free culture supernatant on Porphyromonas gingivalis, a keystone periodontal pathogen, in vitro. We also evaluated the influence of this probiotic in its live, heat-killed (HKL, paraprobiotic) form and its supernatant on the Galleria mellonella invertebrate model after infection by P gingivalis. METHODS: The interaction assay was conducted with P gingivalis and L reuteri preparations (live cells and supernatant preparation). For this, P gingivalis and L reuteri preparations were added to tubes containing Brain Heart Infusion broth and incubated for 3 days. The suspensions were then seeded onto appropriate culture media for the calculation of colony-forming units per mL (CFU/mL). An in vivo assay with the G mellonella model was also performed. Live L reuteri, HKL, or supernatant was inoculated 2 hours prior to infection with P gingivalis. Survival was evaluated over 7 days, and the number of hemocytes in the hemolymph was estimated 3 hours after P gingivalis infection. Data were then subjected to statistical testing (α = 5%). RESULTS: Both live L reuteri and its supernatant had antimicrobial activity against P gingivalis (CFU reduction up to 86%, P < .05). Moreover, treatment with live and HKL had similar effects on G mellonella survival (increased survival up to 46%, P < .05). However, only live L reuteri was able to significantly increase the hemocyte density in this invertebrate model. CONCLUSION: Lactobacillus reuteri antimicrobial activity against P gingivalis and its effects on G mellonella survival after infection with a periodontopathogen do not depend on cell viability. This allows the development of products without live bacterium while maintaining similar effects.

Galleria mellonella as an experimental model for studying periodontopathogens.

In the present study, Galleria mellonella was evaluated as a potential infection model for periodontal bacteria, more specifically, Porphyromonas gingivalis, Fusobacterium nucleatum, and Aggregatibacter actinomycetemcomitans. All the bacteria evaluated were pathogenic to G. mellonella, causing their death in a concentration-dependent manner, and a decrease in their hemocyte count. Moreover, it was possible to recover the bacteria from the larvae hemolymph and determine the colony-forming units per larvae. G. mellonella is an effective model that may help to better understand the host-microbe interactions in periodontics.

Down-regulation of TUFM impairs host cell interaction and virulence by Paracoccidioides brasiliensis.

The genus Paracoccidioides consist of dimorphic fungi geographically limited to the subtropical regions of Latin America, which are responsible for causing deep systemic mycosis in humans. However, the molecular mechanisms by which Paracoccidioides spp. causes the disease remain poorly understood. Paracoccidioides spp. harbor genes that encode proteins involved in host cell interaction and mitochondrial function, which together are required for pathogenicity and mediate virulence. Previously, we identified TufM (previously known as EF-Tu) in Paracoccidioides brasiliensis (PbTufM) and suggested that it may be involved in the pathogenicity of this fungus. In this study, we examined the effects of downregulating PbTUFM using a silenced strain with a 55% reduction in PbTUFM expression obtained by antisense-RNA (aRNA) technology. Silencing PbTUFM yielded phenotypic differences, such as altered translation elongation, respiratory defects, increased sensitivity of yeast cells to reactive oxygen stress, survival after macrophage phagocytosis, and reduced interaction with pneumocytes. These results were associated with reduced virulence in Galleria mellonella and murine infection models, emphasizing the importance of PbTufM in the full virulence of P. brasiliensis and its potential as a target for antifungal agents against paracoccidioidomycosis.