Invasive candidiasis is among the most life-threatening infections in patients in intensive care units. Although Candida albicans is the leading cause of candidaemia, the incidence of Candida ...parapsilosis infections is also rising, particularly among the neonates. Due to differences in their biology, these species employ different antifungal resistance and virulence mechanisms and also induce dissimilar immune responses. Previously, it has been suggested that core virulence effecting transcription regulators could be attractive ligands for future antifungal drugs. Although the virulence regulatory mechanisms of C. albicans are well studied, less is known about similar mechanisms in C. parapsilosis. In order to search for potential targets for future antifungal drugs against this species, we analyzed the fungal transcriptome during host-pathogen interaction using an in vitro infection model. Selected genes with high expression levels were further examined through their respective null mutant strains, under conditions that mimic the host environment or influence pathogenicity. As a result, we identified several mutants with relevant pathogenicity affecting phenotypes. During the study we highlight three potentially tractable signaling regulators that influence C. parapsilosis pathogenicity in distinct mechanisms. During infection, CPAR2_100540 is responsible for nutrient acquisition, CPAR2_200390 for cell wall assembly and morphology switching and CPAR2_303700 for fungal viability.
•CRISPR/Cas9 can be used to successfully edit Candida orthopsilosis ALS genes.•CoALS4210 is involved in C. orthopsilosis adhesion to human buccal epithelial cells.•CoALS4210 does not play a ...significant role in disseminated candidiasis.•CoALS4210 inactivation alters transcript levels of the remaining ALS paralogues.
Candida orthopsilosis is a human fungal pathogen belonging to the Candida parapsilosis sensu lato species complex. C. orthopsilosis annotated genome harbors 3 putative agglutinin-like sequence (ALS) genes named CORT0B00800, CORT0C04210 and CORT0C04220. The aim of this study was to investigate the role played by CORT0C04210 (CoALS4210) in the virulence and pathogenicity of this opportunistic yeast. Heterozygous and null mutant strains lacking one or both copies of CoALS4210 were obtained using the SAT1-flipper cassette strategy and were characterized in in vitro, ex vivo and in vivo models. While no differences between the mutant and the wild-type strains were observed in in vitro growth or in the ability to undergo morphogenesis, the CoALS4210 null mutant showed an impaired adhesion to human buccal epithelial cells compared to heterozygous and wild type strains. When the pathogenicity of CoALS4210 mutant and wild type strains was evaluated in a murine model of systemic candidiasis, no statistically significant differences were observed in fungal burden of target organs. Since gene disruption could alter chromatin structure and influence transcriptional regulation of other genes, two independent CRISPR/Cas9 edited mutant strains were generated in the same genetic background used to create the deleted strains. CoALS4210-edited strains were tested for their in vitro growing ability, and compared with the deleted strain for adhesion ability to human buccal epithelial cells. The results obtained confirmed a reduction in the adhesion ability of C. orthopsilosis edited strains to buccal cells. These findings provide the first evidence that CRISPR/Cas9 can be successfully used in C. orthopsilosis and demonstrate that CoALS4210 plays a direct role in the adhesion of C. orthopsilosis to human buccal cells but is not primarily involved in the onset of disseminated candidiasis.
Abstract Aims Exogenously administered biglycan (core protein with high-molecular weight glycosaminoglycan chains) has been shown to protect neonatal cardiomyocytes against simulated ...ischemia/reperfusion injury (SI/R), however, the mechanism of action is not clear. In this study we aimed to investigate, which structural component of biglycan is responsible for its cardiocytoprotective effect and to further explore the molecular mechanisms involved in the cytoprotection. Methods and results A pilot study was conducted to demonstrate that both native (glycanated) and deglycanated biglycan can attenuate cell death induced by SI/R in a dose-dependent manner in primary neonatal cardiomyocytes isolated from Wistar rats. In separate experiments, we have shown that similarly to glycanated biglycan, recombinant human biglycan core protein (rhBGNc) protects cardiomyocytes against SI/R injury. In contrast, the glycosaminoglycan component dermatan sulfate had no significant effect on cell viability, while chondroitin sulfate further enhanced cell death induced by SI/R. Treatment of cardiomyocytes with rhBGNc reverses the effect of SI/R upon markers of necrosis, apoptosis, mitochondrial membrane potential, and autophagy. We have also shown that pharmacological blockade of Toll-like receptor 4 (TLR4) signaling or its downstream mediators (IRAK1/4, ERK, JNK and p38 MAP kinases) abolished the cytoprotective effect of rhBGNc against SI/R injury. Pretreatment of cardiomyocytes with rhBGNc for 20 h resulted in increased Akt phosphorylation and NO production without having significant effect on phosphorylation of ERK1/2, STAT3, and on the production of superoxide. Treatment over 10 min and 1 h with rhBGNc increased ERK1 phosphorylation, while the SI/R-induced increase in superoxide production was attenuated by rhBGNc. Blockade of NO synthesis also prevented the cardiocytoprotective effect of rhBGNc. Conclusions The core protein of exogenous biglycan protects myocardial cells from SI/R injury via TLR4-mediated mechanisms involving activation of ERK, JNK and p38 MAP kinases and increased NO production. The cytoprotective effect of rhBGNc is due to modulation of SI/R-induced changes in necrosis, apoptosis and autophagy.
species are a major cause of life-threatening bloodstream infections worldwide. Although
is responsible for the vast majority of infections, the clinical relevance of other
species has also emerged ...over the last twenty years. This shift might be due in part to changes in clinical guidelines, as echinocandins became the first line of therapeutics for the treatment.
is an emerging non-
species that exhibits lower susceptibility levels to these drugs.
species frequently display resistance to echinocandins, and the mechanism for this is well-known in
and
, where it is mediated by amino acid substitutions at defined locations of the β-1,3-glucan synthase, Fks1p. In
isolates, Fks1p harbors an intrinsic amino acid change at position 660 of the hot spot 1 (HS1) region, which is thought to be responsible for the high MIC values. Less is known about acquired substitutions in this species. In this study, we used directed evolution experiments to generate
strains with acquired resistance to caspofungin, anidulafungin, and micafungin. We showed that cross-resistance was dependent on the type of echinocandin used to generate the evolved strains. During their characterization, all mutant strains showed attenuated virulence
and also displayed alterations in the exposure of inner cell wall components. The evolved strains harbored 251 amino acid changes, including three in the HS1, HS2, and HS3 regions of Fks1p. Altogether, our results demonstrate a direct connection between acquired antifungal resistance and virulence of
is an opportunistic fungal pathogen with the ability to cause infections in immunocompromised patients. Echinocandins are the currently recommended first line of treatment for all
species. Resistance of
to this drug type is well characterized.
strains have the lowest
susceptibility to echinocandins; however, patients with such infections typically respond well to echinocandin therapy. There is little knowledge of acquired resistance in
and its consequences on other characteristics such as virulence properties. In this study, we aimed to dissect how acquired echinocandin resistance influences the pathogenicity of
and to develop explanations for why echinocandins are clinically effective in the setting of acquired resistance.
Candida parapsilosis is an important, emerging opportunistic fungal pathogen. Highly mannosylated fungal cell wall proteins are initial contact points with host immune systems. In Candida albicans, ...Och1 is a Golgi α1,6-mannosyltransferase that plays a key role in the elaboration of the N-linked mannan outer chain. Here, we disrupted C. parapsilosis OCH1 to gain insights into the contribution of N-linked mannosylation to cell fitness and to interactions with immune cells. Loss of Och1 in C. parapsilosis resulted in cellular aggregation, failure of morphogenesis, enhanced susceptibility to cell wall perturbing agents and defects in wall composition. We removed the cell wall O-linked mannans by β-elimination, and assessed the relevance of mannans during interaction with human monocytes. Results indicated that O-linked mannans are important for IL-1β stimulation in a dectin-1 and TLR4-dependent pathway; whereas both, N- and O-linked mannans are equally important ligands for TNFα and IL-6 stimulation, but neither is involved in IL-10 production. Furthermore, mice infected with C. parapsilosis och1Δ null mutant cells had significantly lower fungal burdens compared to wild-type (WT)-challenged counterparts. Therefore, our data are the first to demonstrate that C. parapsilosis N- and O-linked mannans have different roles in host interactions than those reported for C. albicans.
The production of lipases can affect microbial fitness and virulence. We examined the role of the lipase 8 (LIP8) gene in the virulence of Candida albicans by constructing Δlip8 strains by the ...URA-blaster disruption method. Reverse transcription-PCR experiments demonstrated the absence of LIP8 expression in the homozygous knockout mutants. Reconstituted strains and overexpression mutants were generated by introducing a LIP8 open reading frame under control of a constitutive actin promoter. Knockout mutants produced more mycelium, particularly at higher temperatures and pH >=7. Diminished LIP8 expression resulted in reduced growth in lipid-containing media. Mutants deficient in the LIP8 gene were significantly less virulent in a murine intravenous infection model. The results clearly indicate that Lip8p is an important virulence factor of C. albicans.
Methamphetamine (Meth) is abused by over 35 million people worldwide. Chronic Meth abuse may be particularly devastating in individuals who engage in unprotected sex with multiple partners because it ...is associated with a 2-fold higher risk for obtaining HIV and associated secondary infections. We report the first specific evidence that Meth at pharmacological concentrations exerts a direct immunosuppressive effect on dendritic cells and macrophages. As a weak base, Meth collapses the pH gradient across acidic organelles, including lysosomes and associated autophagic organelles. This in turn inhibits receptor-mediated phagocytosis of antibody-coated particles, MHC class II antigen processing by the endosomal-lysosomal pathway, and antigen presentation to splenic T cells by dendritic cells. More importantly Meth facilitates intracellular replication and inhibits intracellular killing of Candida albicans and Cryptococcus neoformans, two major AIDS-related pathogens. Meth exerts previously unreported direct immunosuppressive effects that contribute to increased risk of infection and exacerbate AIDS pathology.
Summary
Seven HMG‐box proteins of Aspergillus nidulans have been identified in the genomic databases. Three of these have the characteristics of non‐specific DNA‐binding proteins. One of these, ...AN1267 (HmbB), comprises one canonical HMG‐box in its C‐terminus and upstream of the canonical box two structurally related boxes, to be called Shadow‐HMG‐boxes. This protein defines, together with the Podospora anserina mtHMG1, a clade of proteins present in the Pezizomycotina, with orthologues in some of the Taphrinomycotina. HmbB localizes primarily to the mitochondria but occasionally in nuclei. The deletion of the cognate gene results in a number of pleiotropic effects, including those on hyphal morphology, sensitivity to oxidative stress, absence of sterigmatocystin production and changes in the profile of conidial metabolites. The most striking phenotype of deletion strains is a dramatic decrease in conidial and ascospore viability. We show that this is most likely due to the protein being essential to maintain mitochondrial DNA in spores.
The spleen tyrosine kinase (Syk) and the downstream adaptor protein CARD9 are crucial signaling molecules in antimicrobial immunity. Candida parapsilosis is an emerging fungal pathogen with a high ...incidence in neonates, while Candida albicans is the most common agent of candidiasis. While signaling through Syk/CARD9 promotes protective host mechanisms in response to C. albicans, its function in immunity against C. parapsilosis remains unclear. Here, we generated Syk
and CARD9
bone marrow chimeric mice to study the role of Syk/CARD9 signaling in immune responses to C. parapsilosis compared to C. albicans. We demonstrate various functions of this pathway (e.g., phagocytosis, phagosome acidification, and killing) in
-challenged, bone marrow-derived macrophages with differential involvement of Syk and CARD9 along with species-specific differences in cytokine production. We report that Syk
or CARD9
chimeras rapidly display high susceptibility to C. albicans, while C. parapsilosis infection exacerbates over a prolonged period in these animals. Thus, our results establish that Syk and CARD9 contribute to systemic resistance to C. parapsilosis and C. albicans differently. Additionally, we confirm prior studies but also detail new insights into the fundamental roles of both proteins in immunity against C. albicans. Our data further suggest that Syk has a more prominent influence on anti-
immunity than CARD9. Therefore, this study reinforces the Syk/CARD9 pathway as a potential target for anti-
immune therapy.
While C. albicans remains the most clinically significant
species, C. parapsilosis is an emerging pathogen with increased affinity to neonates. Syk/CARD9 signaling is crucial in immunity to C. albicans, but its role in
responses to other pathogenic
species is largely unexplored. We used mice with hematopoietic systems deficient in Syk or CARD9 to comparatively study the function of these proteins in anti-
immunity. We demonstrate that Syk/CARD9 signaling has a protective role against C. parapsilosis differently than against C. albicans. Thus, this study is the first to reveal that Syk can exert immune responses during systemic
infections species specifically. Additionally, Syk-dependent immunity to a nonalbicans
species in an
murine model has not been reported previously. We highlight that the contribution of Syk and CARD9 to fungal infections are not identical and underline this pathway as a promising immune-therapeutic target to fight
infections.
Candida albicans and C. parapsilosis are human pathogens causing severe infections. The NLRP3 inflammasome plays a crucial role in host defence against C. albicans, but it has been previously unknown ...whether C. parapsilosis activates this complex. Here we show that C. parapsilosis induces caspase-1 activation and interleukin-1β (IL-1β) secretion in THP-1, as well as primary, human macrophages. IL-1β secretion was dependent on NLRP3, K
-efflux, TLR4, IRAK, Syk, caspase-1, caspase-8 and NADPH-oxidase. Importantly, while C. albicans induced robust IL-1β release after 4 h, C. parapsilosis was not able to stimulate the production of IL-1β after this short incubation period. We also found that C. parapsilosis was phagocytosed to a lesser extent, and induced significantly lower ROS production and lysosomal cathepsin B release compared to C. albicans, suggesting that the low extent of inflammasome activation by C. parapsilosis may result from a delay in the so-called "signal 2". In conclusion, this is the first study to examine the molecular pathways responsible for the IL-1β production in response to a non-albicans Candida species, and these results enhance our understanding about the immune response against C. parapsilosis.
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