Paracoccidioides spp.: the structural characterization of extracellular matrix, expression of glucan synthesis and associated genes and adhesins during biofilm formation.

The genus Paracoccidioides includes Paracoccidioides lutzii and the Paracoccidioides brasiliensis complex, which comprises four phylogenetic species. A key feature distinguishing planktonic growth from biofilm is the presence of a 3D extracellular matrix (ECM). Therefore, in this study, we analyzed biofilm formation in different species of Paracoccidioides yeast phase, characterized the structural elements of the matrix of P. brasiliensis (Pb18), P. lutzii (Pl01 and 8334) and P. restrepiensis (339 and 192) and evaluated the expression of glucan genes, according to the stage of biofilm evolution for P. brasiliensis. The strains were cultivated in planktonic and biofilm form for 24-144 h. The fungi biomass and metabolic activity were determined by crystal violet and tetrazolium salt reduction (XTT) tests and colony-forming unit (CFU) by plating. The biofilm structure was designed using scanning electron microscopy and confocal laser scanning microscopy techniques. The extracellular matrix of P. brasiliensis and P. lutzii biofilms was extracted by sonication, and polysaccharides, proteins, and extracellular DNA (eDNA) were quantified. The RNA was extracted with the Trizol® reagent and quantified; then, the cDNA was synthesized to analyze the enolase expression, 14-3-3, FKS1, AGS1, GEL3, and KRE6 genes by real-time PCR. All strains of Paracoccidioides studied form a biofilm with more significant metabolic activity and biomass values in 144 h. The extracellular matrix of P. brasiliensis and P. lutzii had a higher content of polysaccharides in their composition, followed by proteins and eDNA in smaller quantities. The P. brasiliensis biofilm kinetics of formation showed greater expression of genes related to glucan's synthesis and its delivery to the external environment in addition adhesins during the biofilm's adhesion, initiation, and maturation. The GEL3 and enolase genes increased in expression within 24 h and during the biofilm maturation period, there was an increase in 14-3-3, AGS1, and FKS1. Furthermore, at 144 h, there was a decrease in KRE6 expression and an increase in GEL3. This study highlights the potential for biofilm formation for three species of Paracoccidioides and the main components of the extracellular matrix that can contribute to a better understanding of biofilm organization.

Mycobacterium tuberculosis and Paracoccidioides brasiliensis Formation and Treatment of Mixed Biofilm In Vitro.

Co-infection of Mycobacterium tuberculosis and Paracoccidioides brasiliensis, present in 20% in Latin America, is a public health problem due to a lack of adequate diagnosis. These microorganisms are capable of forming biofilms, mainly in immunocompromised patients, which can lead to death due to the lack of effective treatment for both diseases. The present research aims to show for the first time the formation of mixed biofilms of M. tuberculosis and P. brasiliensis (Pb18) in vitro, as well as to evaluate the action of 3'hydroxychalcone (3'chalc) -loaded nanoemulsion (NE) (NE3'chalc) against monospecies and mixed biofilms, the formation of mixed biofilms of M. tuberculosis H37Rv (ATCC 27294), 40Rv (clinical strains) and P. brasiliensis (Pb18) (ATCC 32069), and the first condition of formation (H37Rv +Pb18) and (40Rv + Pb18) and second condition of formation (Pb18 + H37Rv) with 45 days of total formation time under both conditions. The results of mixed biofilms (H37Rv + Pb18) and (40Rv + Pb18), showed an organized network of M. tuberculosis bacilli in which P. brasiliensis yeasts are connected with a highly extracellular polysaccharide matrix. The (Pb18 + H37Rv) showed a dense biofilm with an apparent predominance of P. brasiliensis and fragments of M. tuberculosis. PCR assays confirmed the presence of the microorganisms involved in this formation. The characterization of NE and NE3'chalc displayed sizes from 145.00 ± 1.05 and 151.25 ± 0.60, a polydispersity index (PDI) from 0.20± 0.01 to 0.16± 0.01, and zeta potential -58.20 ± 0.92 mV and -56.10 ± 0.71 mV, respectively. The atomic force microscopy (AFM) results showed lamellar structures characteristic of NE. The minimum inhibitory concentration (MIC) values of 3'hidroxychalcone (3'chalc) range from 0.97- 7.8 µg/mL and NE3'chalc from 0.24 - 3.9 µg/mL improved the antibacterial activity when compared with 3'chalc-free, no cytotoxicity. Antibiofilm assays proved the efficacy of 3'chalc-free incorporation in NE. These findings contribute to a greater understanding of the formation of M. tuberculosis and P. brasiliensis in the mixed biofilm. In addition, the findings present a new possible NE3'chalc treatment alternative for the mixed biofilms of these microorganisms, with a high degree of relevance due to the lack of other treatments for these comorbidities.

Dynamics of Mono- and Dual-Species Biofilm Formation and Interactions Between Paracoccidioides brasiliensis and Candida albicans.

The oral cavity is a highly diverse microbial environment in which microorganisms interact with each other, growing as biofilms on biotic and abiotic surfaces. Understanding the interaction among oral microbiota counterparts is pivotal for clarifying the pathogenesis of oral diseases. Candida spp. is one of the most abundant fungi in the oral mycobiome with the ability to cause severe soft tissue lesions under certain conditions. Paracoccidioides spp., the causative agent of paracoccidioidomycosis, may also colonize the oral cavity leading to soft tissue damage. It was hypothesized that both fungi can interact with each other, increasing the growth of the biofilm and its virulence, which in turn can lead to a more aggressive infectivity. Therefore, this study aimed to evaluate the dynamics of mono- and dual-species biofilm growth of Paracoccidioides brasiliensis and Candida albicans and their infectivity using the Galleria mellonella model. Biomass and fungi metabolic activity were determined by the crystal violet and the tetrazolium salt reduction tests (XTT), respectively, and the colony-forming unit (CFU) was obtained by plating. Biofilm structure was characterized by both scanning electronic- and confocal laser scanning- microscopy techniques. Survival analysis of G. mellonella was evaluated to assess infectivity. Our results showed that dual-species biofilm with P. brasiliensis plus C. albicans presented a higher biomass, higher metabolic activity and CFU than their mono-species biofilms. Furthermore, G. mellonella larvae infected with P. brasiliensis plus C. albicans presented a decrease in the survival rate compared to those infected with P. brasiliensis or C. albicans, mainly in the form of biofilms. Our data indicate that P. brasiliensis and C. albicans co-existence is likely to occur on oral mucosal biofilms, as per in vitro and in vivo analysis. These data further widen the knowledge associated with the dynamics of fungal biofilm growth that can potentially lead to the discovery of new therapeutic strategies for these infections.