RESUMO

Candidemia is an opportunistic mycosis with high morbidity and mortality rates. Even though Candida albicans is the main causative agent, other Candida species, such as Candida tropicalis, are relevant etiological agents of candidiasis and candidemia. Compared with C. albicans, there is currently limited information about C. tropicalis' biological aspects, including those related to the cell wall and the interaction with the host. Currently, it is known that its cell wall contains O-linked mannans, and the contribution of these structures to cell fitness has previously been addressed using cells subjected to chemical treatments or in mutants where O-linked mannans and other wall components are affected. Here, we generated a C. tropicalis pmt2∆ null mutant, which was affected in the first step of the O-linked mannosylation pathway. The null mutant was viable, contrasting with C. albicans where this gene is essential. The phenotypical characterization showed that O-linked mannans were required for filamentation; proper cell wall integrity and organization; biofilm formation; protein secretion; and adhesion to extracellular matrix components, in particular to fibronectin; and type I and type II collagen. When interacting with human innate immune cells, it was found that this cell wall structure is dispensable for cytokine production, but mutant cells were more phagocytosed by monocyte-derived macrophages. Furthermore, the null mutant cells showed virulence attenuation in Galleria mellonella larvae. Thus, O-linked mannans are minor components of the cell wall that are involved in different aspects of C. tropicalis' biology.

RESUMO

Candida glabrata (Nakaseomyces glabrata) is an emergent and opportunistic fungal pathogen that colonizes and persists in different niches within its human host. In this work, we studied five clinical isolates from one patient (P7), that have a clonal origin, and all of which come from blood cultures except one, P7-3, obtained from a urine culture. We found phenotypic variation such as sensitivity to high temperature, oxidative stress, susceptibility to two classes of antifungal agents, and cell wall porosity. Only isolate P7-3 is highly resistant to the echinocandin caspofungin while the other four isolates from P7 are sensitive. However, this same isolate P7-3, is the only one that displays susceptibility to fluconazole (FLC), while the rest of the isolates are resistant to this antifungal. We sequenced the PDR1 gene which encodes a transcription factor required to induce the expression of several genes involved in the resistance to FLC and found that all the isolates encode for the same Pdr1 amino acid sequence except for the last isolate P7-5, which contains a single amino acid change, G1099C in the putative Pdr1 transactivation domain. Consistent with the resistance to FLC, we found that the CDR1 gene, encoding the main drug efflux pump in C. glabrata, is highly overexpressed in the FLC-resistant isolates, but not in the FLC-sensitive P7-3. In addition, the resistance to FLC observed in these isolates is dependent on the PDR1 gene. Additionally, we found that all P7 isolates have a different proportion of cell wall carbohydrates compared to our standard strains CBS138 and BG14. In P7 isolates, mannan is the most abundant cell wall component, whereas ß-glucan is the most abundant component in our standard strains. Consistently, all P7 isolates have a relatively low cell wall porosity compared to our standard strains. These data show phenotypic and genotypic variability between clonal isolates from different niches within a single host, suggesting microevolution of C. glabrata during an infection.

Assuntos

Antifúngicos , Candida glabrata , Farmacorresistência Fúngica , Proteínas Fúngicas , Testes de Sensibilidade Microbiana , Candida glabrata/genética , Candida glabrata/efeitos dos fármacos , Antifúngicos/farmacologia , Humanos , Farmacorresistência Fúngica/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fluconazol/farmacologia , Parede Celular/genética , Parede Celular/efeitos dos fármacos , Candidíase/microbiologia , Caspofungina/farmacologia , Evolução Molecular , Estresse Oxidativo/genética , Equinocandinas/farmacologia , Fatores de Transcrição/genética

RESUMO

Mannans are components of the fungal wall attached to proteins via N- or O-linkages. In Candida albicans, Och1 is an α1,6-mannosyltransferase that adds the first mannose unit to the N-linked mannan outer chain; whereas Pmr1 is an ion pump that imports Mn2+ into the Golgi lumen. This cation is the cofactor of Golgi-resident mannosyltransferases, and thus Pmr1 is involved in the synthesis of both N- and O-linked mannans. Since we currently have limited information about the genetic network behind the Candida tropicalis protein mannosylation machinery, we disrupted OCH1 and PMR1 in this organism. The C. tropicalis pmr1Δ and och1Δ mutants showed increased doubling times, aberrant colony and cellular morphology, reduction in the wall mannan content, and increased susceptibility to wall perturbing agents. These changes were accompanied by increased exposure of both ß1,3-glucan and chitin at the wall surface of both mutant strains. Our results showed that O-linked mannans are dispensable for cytokine production by human mononuclear cells, but N-linked mannans and ß1,3-glucan are key ligands to trigger cytokine production in a co-stimulatory pathway involving dectin-1 and mannose receptor. Moreover, we found that the N-linked mannan core found on the surface of C. tropicalis och1Δ null mutant was capable of inducing cytokine production; and that a mannan-independent pathway for IL-10 production is present in the C. tropicalis-mononuclear cell interaction. Both mutant strains showed virulence attenuation in the Galleria mellonella and the mouse model of systemic candidiasis. Therefore, mannans are relevant for cell wall composition and organization, and for the C. tropicalis-host interaction.

RESUMO

Phosphomannosylation is a modification of cell wall proteins that occurs in some species of yeast-like organisms, including the human pathogen Candida albicans. These modified mannans confer a negative charge to the wall, which is important for the interactions with phagocytic cells of the immune systems and cationic antimicrobial peptides. In Saccharomyces cerevisiae, the synthesis of phosphomannan relies on two enzymes, the phosphomannosyltransferase Ktr6 and its positive regulator Mnn4. However, in C. albicans, at least three phosphomannosyltransferases, Mnn4, Mnt3 and Mnt5, participate in the addition of phosphomannan. In addition to MNN4, C. albicans has a MNN4-like gene family composed of seven other homologous members that have no known function. Here, using the classical mini-Ura-blaster approach and the new gene knockout CRISPR-Cas9 system for gene disruption, we generated mutants lacking single and multiple genes of the MNN4 family; and demonstrate that, although Mnn4 has a major impact on the phosphomannan content, MNN42 was also required for full protein phosphomannosylation. The reintroduction of MNN41, MNN42, MNN46, or MNN47 in a genetic background lacking MNN4 partially restored the phenotype associated with the mnn4Δ null mutant, suggesting that there is partial redundancy of function between some family members and that the dominant effect of MNN4 over other genes could be due to its relative abundance within the cell. We observed that additional copies of alleles number of any of the other family members, with the exception of MNN46, restored the phosphomannan content in cells lacking both MNT3 and MNT5. We, therefore, suggest that phosphomannosylation is achieved by three groups of proteins: [i] enzymes solely activated by Mnn4, [ii] enzymes activated by the dual action of Mnn4 and any of the products of other MNN4-like genes, with exception of MNN46, and [iii] activation of Mnt3 and Mnt5 by Mnn4 and Mnn46. Therefore, although the MNN4-like genes have the potential to functionally redundant with Mnn4, they apparently do not play a major role in cell wall mannosylation under most in vitro growth conditions. In addition, our phenotypic analyses indicate that several members of this gene family influence the ability of macrophages to phagocytose C. albicans cells.