Fungal Cell Wall Components Research Articles

Variation in Bombyx mori immune response against fungal pathogen Beauveria bassiana with variability in cell wall β-1,3-glucan.

Entomopathogenic fungi are protected by a cell wall with dynamic structure for adapting to various environmental conditions. β-1,3-Glucan recognition proteins activate the innate immune system of insects by recognizing surface molecules of fungi. However, the associations between pathogenicity and the different components of entomopathogenic fungal cell walls remain unclear. Three Beauveria bassiana strains were selected that have significantly differing virulence against Bombyx mori. The molecular mechanisms underlying the immune response in B. mori were investigated using RNA sequencing, which revealed differences in the immune response to different B. bassiana strains at 12h post-infection. Immunofluorescence assays revealed that β-1,3-glucan content had an opposite trend to that of fungal virulence. β-1,3-Glucan injection upregulated BmβGRP4 expression and significantly reduced the virulence of the high-virulence strain but not that of the medium-virulence or low-virulence strains. BmβGRP4 silencing in B. mori with RNA interference resulted in the opposite virulence pattern, indicating that the virulence of B. bassiana was affected by the cell walls' content of β-1,3-glucan, which could be recognized by BmβGRP4. Furthermore, interference with the gene CnA (calcineurin catalytic A subunit) involved in β-1,3-glucan synthesis eliminated differences in virulence between B. bassiana strains. These results indicate that strains of a single species of pathogenic fungi that have differing cell wall components are recognized differently by the innate immune system of B. mori.

Transgenic tobacco plants expressing a chimeric antimicrobial protein show disease resistance to plant diseases

Plant-pathogen interactions are universal in nature and jeopardise plant health and ultimately global food security. Plant innate immunity can be enhanced by expressing antimicrobial proteins that interfere with the life cycle of plant pathogens. To this end, a recombinant protein comprising a chitinase chitin-binding domain (CBD) from rice and the Phyllomedusa bicolor Dermaseptin B1 (DrsB1) antimicrobial peptide was designed to target fungal cell wall component, chitin. Agrobacterium-mediated transformation of tobacco plants led to the transgenic plants exhibiting significantly resistance to fungal and bacterial plant pathogens. Fungal colonization as well as appearance of fungal disease symptoms delayed for one-week, taking a crucial opportunity from pathogens to establish infections at appropriate time and opportune environmental condition. Furthermore, results of in vitro analysis showed that total protein from transgenic plants significantly hindered growth and development of selected plant pathogens. Predictably, the CBD-DrsB1 recombinant protein had a stronger antimicrobial effect against fungal than bacterial and oomycete pathogens, suggesting that the CBD interferes with fungal cell wall component, chitin oligomers. Scanning electron micrograph (SEM) revealed that CBD-DrsB1 indeed interferes with fungal pathogens by damaging the overall cell wall integrity. Inoculating in vitro transgenic lines with Fusarium oxysporum, Alternaria solani and Pythium sp showed that plants remained green and vigorous and survived up to 51, 56 and 16 days post inoculation (dpi), respectively, whereas non-transgenic plants collapsed completely a few days post inoculation. The results of this study demonstrate that CBD-DrsB1 over-expression limits fungal and bacterial infections, suggesting both in vivo antibacterial activity and possible biotechnological application to engineer crop plants resistant to destructive plant pathogens.

Silencing a Chitinase Gene, PstChia1, Reduces Virulence of Puccinia striiformis f. sp. tritici.

Chitin is the main component of fungal cell walls, which can be recognized by pattern recognition receptors (PRRs) as pathogen-associated molecular patterns (PAMP). Chitinase in filamentous fungi has been reported to degrade immunogenic chitin oligomers, thereby preventing chitin-induced immune activation. In this study, we identified the chitinase families in 10 fungal genomes. A total of 131 chitinase genes were identified. Among the chitinase families, 16 chitinase genes from Puccinia striiformis f. sp. tritici (Pst) were identified, and the expression of PstChia1 was the highest during Pst infection. Further studies indicated that PstChia1 is highly induced during the early stages of the interaction of wheat and Pst and has chitinase enzyme activity. The silencing of PstChia1 revealed that PstChia1 limited the growth and reduced the virulence of Pst. The expression level of TaPR1 and TaPR2 was induced in PstChia1 knockdown plants, suggesting that PstChia1 is involved in regulating wheat resistance to Pst. Our data suggest that PstChia1 contributes to pathogenicity by interfering with plant immunity and regulating the growth of Pst.

Tomato LysM receptor kinase 4 mediates chitin-elicited fungal resistance in both leaves and fruit.

Fungal infection is a major cause of crop and fruit losses. Recognition of chitin, a component of fungal cell walls, endows plants with enhanced fungal resistance. Here, we found that mutation of tomato LysM receptor kinase 4 (SlLYK4) and chitin elicitor receptor kinase 1 (SlCERK1) impaired chitin-induced immune responses in tomato leaves. Compared with the wild type, sllyk4 and slcerk1 mutant leaves were more susceptible to Botrytis cinerea (gray mold). SlLYK4 extracellular domain showed strong binding affinity to chitin, and the binding of SlLYK4 induced SlLYK4-SlCERK1 association. Remarkably, qRT-PCR analysis indicated that SlLYK4 was highly expressed in tomato fruit, and β-GLUCURONIDASE (GUS) expression driven by the SlLYK4 promoter was observed in tomato fruit. Furthermore, SlLYK4 overexpression enhanced disease resistance not only in leaves but also in fruit. Our study suggests that chitin-mediated immunity plays a role in fruit, providing a possible way to reduce fungal infection-related fruit losses by enhancing the chitin-induced immune responses.

Regulation of Surfactant Protein Gene Expression by Aspergillus fumigatus in NCl-H441 Cells.

Aspergillus fumigatus is an opportunistic fungal pathogen that causes serious lung diseases in immunocompromised patients. The lung surfactant produced by alveolar type II and Clara cells in the lungs is an important line of defense against A. fumigatus. The surfactant consists of phospholipids and surfactant proteins (SP-A, SP-B, SP-C and SP-D). The binding to SP-A and SP-D proteins leads to the agglutination and neutralization of lung pathogens as well as the modulation of immune responses. SP-B and SP-C proteins are essential for surfactant metabolism and can modulate the local immune response; however, the molecular mechanisms remain unclear. We investigated changes in the SP gene expression in human lung NCI-H441 cells infected with conidia or treated with culture filtrates obtained from A. fumigatus. To further identify fungal cell wall components that may affect the expression of SP genes, we examined the effect of different A. fumigatus mutant strains, including dihydroxynaphthalene (DHN)-melanin-deficient ΔpksP, galactomannan (GM)-deficient Δugm1 and galactosaminogalactan (GAG)-deficient Δgt4bc strains. Our results show that the tested strains alter the mRNA expression of SP, with the most prominent and consistent downregulation of the lung-specific SP-C. Our findings also suggest that secondary metabolites rather than the membrane composition of conidia/hyphae inhibit SP-C mRNA expression in NCI-H441 cells.

Updates in Laboratory Identification of Invasive Fungal Infection in Neonates.

Invasive fungal infection (IFI) in immunocompromised neonates is significantly associated with high morbidity and mortality and has become the third most common infection in Neonatal Intensive Care Units. The early diagnosis of IFI for neonatal patients is difficult because of the lack of specific symptoms. The traditional blood culture remains the gold standard in clinical diagnosis for neonatal patients but it requires a long duration, which delays treatment initiation. Detections of fungal cell-wall components are developed for early diagnosis but the diagnostic accuracy in neonates needs to be improved. PCR-based laboratory methods, such as real-time PCR, droplet digital PCR, and the cationic conjugated polymer fluorescence resonance energy transfer (CCP-FRET) system, distinguish the infected fungal species by their specific nucleic acids and show a high sensitivity and specificity. Particularly, the CCP-FRET system, which contains a cationic conjugated polymer (CCP) fluorescent probe and pathogen-specific DNA labeled with fluorescent dyes, could identify multiple infections simultaneously. In the CCP-FRET system, the CCP and fungal DNA fragments can self-assemble into a complex with an electrostatic interaction and the CCP triggers the FRET effect under ultraviolet light to make the infection visible. Here, we summarize the recent laboratory methods for neonatal IFI identification and provide a new perspective for early clinical fungal diagnosis.

Maize Antifungal Protein AFP1 Elevates Fungal Chitin Levels by Targeting Chitin Deacetylases and Other Glycoproteins.

Pathogenic fungi convert chitin to chitosan to evade plant perception and disarm chitin-triggered immune responses. Whether plants have evolved factors to counteract this evasion mechanism remains obscure. Here, we decipher the mechanism underlying the antifungal activity of maize secretory mannose-binding cysteine-rich receptor-like secreted protein (CRRSP), antifungal protein 1 (AFP1). AFP1 binds to multiple sites on the surface of sporidial cells, filaments, and germinated spores of the biotrophic fungus Ustilago maydis. It inhibits cell growth and budding, as well as spore germination. AFP1 promiscuously interacts with most chitin deacetylases (CDAs) by recognizing the conserved NodB domain to interfere with the enzyme activity. Deletion of O-mannosyltransferase 4 decreases protein mannosylation, which correlates with reduced AFP1 binding and antifungal activity, suggesting that AFP1 interacts with mannosylated proteins to exhibit an inhibitory effect. AFP1 also has extended inhibitory activity against Saccharomyces cerevisiae; however, AFP1 did not reduce binding to the double ΔΔcda1,2 mutant, suggesting the targets of AFP1 have expanded to other cell surface glycoproteins, probably facilitated by its mannose-binding property. Increasing chitin levels by modulating the activity of cell surface glycoproteins is a universal feature of AFP1 interacting with a broad spectrum of fungi to inhibit their growth. IMPORTANCE Plants alert immune systems by recognizing the fungal pathogen cell wall component chitin via pattern recognition cell surface receptors. Successful fungal pathogens escape the perception by deacetylating chitin to chitosan, which is also necessary for fungal cell development and virulence. Targeting glycoproteins that are associated with regulating chitin metabolism and maintaining cell wall morphogenesis presents an effective strategy to combat fungal pathogens by simultaneously altering cell wall plasticity, activating chitin-triggered immunity, and impairing fungal viability. Our study provides molecular insights into a plant DUF26 domain-containing secretory protein in warding off a broad range of fungal pathogens by acting on more than one glycoprotein target.

Understanding the impact of main cell wall polysaccharides on the decomposition of ectomycorrhizal fungal necromass

AbstractThe extramatrical mycelium of ectomycorrhizal fungi (EMF) is an important source of soil carbon and nitrogen. While the importance of recalcitrant compounds in the fungal cell wall has been explored earlier, the contribution of highly abundant but labile components, like glucans, and the role of their temporal dynamics during decomposition remains unknown. For the first time, we examined how the concentration of three main fungal cell wall components (chitin, melanin, glucans) in EMF necromass are related to necromass decomposition, over a period of 6 weeks. Although the initial concentrations of the three components were not good predictors of necromass loss, we found species–specific trends of chitin and glucans loss over time. The chitin concentration during decomposition was tightly linked to the weekly necromass degradation, with trends of chitin loss being dissimilar across fungal species. Chitin concentration was positively correlated with the mass loss in the first week, but in the remaining 5 weeks, it was found to be weakly negatively correlated with mass loss. The similarity in susceptibility to the decomposition of glucans and chitin likely compensates for the impacts of interspecific differences in their initial concentration, leading to overall similar decomposition patterns. Alternatively, other, non‐measured, components (e.g., glycoproteins, N content) may contribute to explaining similar decomposition patterns. Our results indicate that ectomycorrhizal necromass decomposition processes differ from those of plant litter decomposition with, unlike in plants, differences in initial concentrations of major structural carbohydrates (e.g., glucans) being unrelated to differences in decomposition rates. These findings indicate that the decomposition of fungal material cannot be inferred from assumptions based on data provided by studies of plant decomposition.Highlights Necromass of different ectomycorrhizal species decomposes similarly Main cell wall components show different decomposition patterns The relationship between cell wall compounds and mass loss changes over time Mycorrhizal necromass decomposition differs substantially from that of plant litter.

The grapevine LysM receptor-like kinase VvLYK5-1 recognizes chitin oligomers through its association with VvLYK1-1.

The establishment of defense reactions to protect plants against pathogens requires the recognition of invasion patterns (IPs), mainly detected by plasma membrane-bound pattern recognition receptors (PRRs). Some IPs, also termed elicitors, are used in several biocontrol products that are gradually being developed to reduce the use of chemicals in agriculture. Chitin, the major component of fungal cell walls, as well as its deacetylated derivative, chitosan, are two elicitors known to activate plant defense responses. However, recognition of chitooligosaccharides (COS) in Vitis vinifera is still poorly understood, hampering the improvement and generalization of protection tools for this important crop. In contrast, COS perception in the model plant Arabidopsis thaliana is well described and mainly relies on a tripartite complex formed by the cell surface lysin motif receptor-like kinases (LysM-RLKs) AtLYK1/CERK1, AtLYK4 and AtLYK5, the latter having the strongest affinity for COS. In grapevine, COS perception has for the moment only been demonstrated to rely on two PRRs VvLYK1-1 and VvLYK1-2. Here, we investigated additional players by overexpressing in Arabidopsis the two putative AtLYK5 orthologs from grapevine, VvLYK5-1 and VvLYK5-2. Expression of VvLYK5-1 in the atlyk4/5 double mutant background restored COS sensitivity, such as chitin-induced MAPK activation, defense gene expression, callose deposition and conferred non-host resistance to grapevine downy mildew (Erysiphe necator). Protein-protein interaction studies conducted in planta revealed a chitin oligomer-triggered interaction between VvLYK5-1 and VvLYK1-1. Interestingly, our results also indicate that VvLYK5-1 mediates the perception of chitin but not chitosan oligomers showing a part of its specificity.

Multiple enzymatic approaches to hydrolysis of fungal β-glucans by the soil bacterium Chitinophaga pinensis.

The genome of the soil Bacteroidota Chitinophaga pinensis encodes a large number of glycoside hydrolases (GHs) with noteworthy features and potentially novel functions. Several are predicted to be active on polysaccharide components of fungal and oomycete cell walls, such as chitin, β-1,3-glucan and β-1,6-glucan. While several fungal β-1,6-glucanase enzymes are known, relatively few bacterial examples have been characterised to date. We have previously demonstrated that C.pinensis shows strong growth using β-1,6-glucan as the sole carbon source, with the efficient release of oligosaccharides from the polymer. We here characterise the capacity of the C.pinensis secretome to hydrolyse the β-1,6-glucan pustulan and describe three distinct enzymes encoded by its genome, all of which show different levels of β-1,6-glucanase activity and which are classified into different GH families. Our data show that C.pinensis has multiple tools to deconstruct pustulan, allowing the species' broad utility of this substrate, with potential implications for bacterial biocontrol of pathogens via cell wall disruption. Oligosaccharides derived from fungal β-1,6-glucans are valuable in biomedical research and drug synthesis, and these enzymes could be useful tools for releasing such molecules from microbial biomass, an underexploited source of complex carbohydrates.

Antifungal Activity of Spent Coffee Ground Extracts

Coffee is one of the most popular and consumed products in the world, generating tons of solid waste known as spent coffee grounds (SCG), containing several bioactive compounds. Here, the antifungal activity of ethanolic SCG extract from caffeinated and decaffeinated coffee capsules was evaluated against yeasts and filamentous fungi. These extracts had antifungal activity against Candida krusei, Candida parapsilosis, Trichophyton mentagrophytes, and Trichophyton rubrum, all skin fungal agents. Moreover, SCG had fungicidal activity against T. mentagrophytes and T. rubrum. To understand the underlying mechanisms of the antifungal activity, fungal cell membrane and cell wall components were quantified. SCG caused a significant reduction of the ergosterol, chitin, and β-(1,3)-glucan content of C. parapsilosis, revealing the synthesis of this membrane component and cell wall components as possible targets of these extracts. These extracts were cytotoxic for the tumoral cell lines tested but not for the non-tumoral PLP2 cell line. The analysis of the phenolic compounds of these extracts revealed the presence of caffeoylquinic acid, feruloylquinic acid, and caffeoylshikimic acid derivatives. Overall, this confirmed the antifungal activity of spent coffee grounds, presenting a potential increase in the sustainability of the life cycle of coffee grounds, as a source for the development of novel antifungal formulations, especially for skin or mucosal fungal infections.

Dysbiotic but nonpathogenic shift in the fecal mycobiota of patients with rheumatoid arthritis

ABSTRACT Rheumatoid arthritis (RA) is closely associated with the oral and gut microbiomes. Fungal cell wall components initiate inflammatory arthritis in mouse models. However, little is known regarding the role of the fungal community in the pathogenesis of RA. To evaluate the association between RA and the gut microbiome, investigations of bacterial and fungal communities in patients with RA are necessary. Therefore, we investigated the compositions and associations of fecal bacterial and fungal communities in 30 healthy controls and 99 patients with RA. The relative abundances of Bifidobacterium and Blautia decreased, whereas the relative abundance of Streptococcus increased, in patients with RA. The relative abundance of Candida in the fecal fungal community was higher in patients with RA than in healthy controls, while the relative abundance of Aspergillus was higher in healthy controls than in patients with RA. Candida species-specific gene amplification showed that C. albicans was the most abundant species of Candida. Ordination analysis and random forest classification models supported the findings of structural changes in bacterial and fungal communities. Aspergillus was the core fecal fungal genus in healthy controls, although Saccharomyces spp. are typically predominant in Western cohorts. In addition, bacterial–fungal association analyses showed that the hub node had shifted from fungi to bacteria in patients with RA. The finding of fungal dysbiosis in patients with RA suggests that fungi play critical roles in the fecal microbial communities and pathogenesis of RA.

Quantitative enzymatic-mass spectrometric analysis of the chitinous polymers in fungal cell walls

Chitin is an essential structural component of complex and dynamic fungal cell walls. It may be converted by partial or full deacetylation to yield chitosan. Here, we describe a method to quantify N-acetyl d-glucosamine (GlcNAc, A) and d-glucosamine (GlcN, D) units and, thus, total amount and average fraction of acetylation (x̅ FA) of the chitinous polymers by complete enzyme hydrolysis of the polymers followed by mass spectrometric analyses of the monomers. First, the native polymers were isotopically N-acetylated, then enzymatically hydrolyzed to A and R (2H3N-acetyl-d-glucosamine – former D) monomers. Relative abundances of A and R units were used to calculate x̅ FA, and a double-isotopically labeled internal standard R* ([13C2,2H3] N-acetyl-d-glucosamine) monomer was used to calculate the absolute amounts of GlcNAc and GlcN units present in the fungal samples. The method was validated using known chitosan polymers and is suitable for both purified cell walls and whole mycelia.

Chitosan triggers actin remodelling and activation of defence genes that is repressed by calcium influx in grapevine cells

Defence to pathogens must be specific. In the past, we have dissected early signalling deployed by bacterial elicitors in a grapevine cell system. In the current work, we asked, how defence of fungi differs. Fungal diseases of grapevine pose great challenges for global viticulture and require massive plant protection measures. Plant cells are able to sense chitin, a central component of fungal cell walls and respond by activation of basal defence. We, therefore mapped early defence responses evoked by chitosan, a chitin fragment able to bind to chitin receptors. We found an activation of calcium influx, monitored by extracellular alkalinisation due to a co-transport of protons, remodelling of actin (but not of microtubules), and the activation of transcripts for phytoalexin synthesis, jasmonate-signalling, salicylate signalling, and chitinase. Interestingly, Gadolinium, an inhibitor of calcium influx, can inhibit extracellular alkalinisation in response to chitosan, while the induction of the phytoalexin synthesis transcripts was specifically promoted. In contrast, both DMSO and benzyl alcohol, compounds known to modulate membrane fluidity, partially inhibited the transcript responses to chitosan. We discuss these data with a model, where chitosan deploys signalling culminating in activation of defence related transcripts, but at the same time activates calcium influx that negatively feeds back on the same signal chain, which might be a mechanism to achieve a temporal signature that is rapid, but transient.

The protective role and mechanism of melanin for Aspergillus niger and Aspergillus flavus against chlorine-based disinfectants

Melanin is a critical component of fungal cell wall which protect fungi from adverse environmental tress. However, the role of melanin for fungi during the disinfection with chlorine-based disinfectants has not been elucidated. The results showed that the inactivation rate constants of Aspergillus niger with chlorine and chlorine dioxide decreased from 0.08 to 2.10 min−1 to 0 after addition of 0.32 mg/L melanin. The results indicated addition of extracted fungal melanin inhibited the inactivation efficiency of chlorine and chlorine dioxide. In contrast, the k of Aspergillus niger after inactivation with monochloramine ranged from 1.50 to 1.78 min−1 after addition of melanin which indicated effect of melanin on the inactivation efficiency of monochloramine was negligible. In addition, the extracted fungal melanin exhibited high reactivity with chlorine and chlorine dioxide but very low reactivity with monochloramine. The different inactivation mechanisms of chlorine-based disinfectants and different reactivity of melanin with chlorine-based disinfectants led to the different protective mechanism of melanin for A. niger and A. flavus spores against disinfection with chlorine-based disinfectants. The chlorine and chlorine dioxide appeared to react with functional groups of melanin in cell wall of spores, so sacrificial reactions between melanin and disinfectants decreased the available disinfectants and limited the diffusion of disinfectants to the reactive site on cell membrane, which led to the decrease of the disinfection efficiency for chlorine and chlorine dioxide. The monochloramine could penetrate into cell and damage DNA without the effect of melanin due to its strong penetration and low reactivity with melanin. Our results systematically demonstrate the protective roles of melanin on the fungal spores against chlorine-based disinfectants and the underlying mechanisms in resisting the environmental stress caused by chlorine-based disinfectants, which provides important implications for the control of fungi, especially for fungi producing melanin.

Fungal dual-domain LysM effectors undergo chitin-induced intermolecular, and not intramolecular, dimerization.

Chitin is a homopolymer of β-(1,4)-linked N-acetyl-D-glucosamine (GlcNAc) and a major structural component of fungal cell walls. In plants, chitin acts as a microbe-associated molecular pattern (MAMP) that is recognized by lysin motif (LysM)-containing plant cell surface-localized pattern recognition receptors (PRRs) that activate a plethora of downstream immune responses. To deregulate chitin-induced plant immunity and successfully establish infection, many fungal pathogens secrete LysM domain-containing effector proteins during host colonization. The LysM effector Ecp6 from the tomato (Solanum lycopersicum) leaf mold fungus Cladosporium fulvum can outcompete plant PRRs for chitin binding because two of its three LysM domains cooperate to form a composite groove with ultra-high (pM) chitin-binding affinity. However, most functionally characterized LysM effectors contain only two LysMs, including Magnaporthe oryzae MoSlp1, Verticillium dahliae Vd2LysM, and Colletotrichum higginsianum ChElp1 and ChElp2. Here, we performed modeling, structural, and functional analyses to investigate whether such dual-domain LysM effectors can also form ultra-high chitin-binding affinity grooves through intramolecular LysM dimerization. However, our study suggests that intramolecular LysM dimerization does not occur. Rather, our data support the occurrence of intermolecular LysM dimerization for these effectors, associated with a substantially lower chitin binding affinity than monitored for Ecp6. Interestingly, the intermolecular LysM dimerization allows for the formation of polymeric complexes in the presence of chitin. Possibly, such polymers may precipitate at infection sites to eliminate chitin oligomers, and thus suppress the activation of chitin-induced plant immunity.

Chitin Synthase Genes Are Differentially Required for Growth, Stress Response, and Virulence in Verticillium dahliae.

Crop wilt disease caused by Verticillium dahliae usually leads to serious yield loss. Chitin, an important component of most fungal cell walls, functions to maintain the rigidity of cell walls and septa. Chitin synthesis mainly relies on the activity of chitin synthase (CHS). Eight CHS genes have been predicted in V. dahliae. In this study, we characterized the functions of these genes in terms of growth, stress responses, penetration, and virulence. Results showed that VdCHS5 is important for conidia germination and resistance to hyperosmotic stress. Conidial production is significantly decreased in Vdchs1, Vdchs4, and Vdchs8 mutants. VdCHS1, VdCHS2, VdCHS4, VdCHS6, VdCHS7, and VdCHS8 genes are important for cell wall integrity, while all mutants are important for cell membrane integrity. All of the VdCHS genes, except for VdCHS3, are required for the full pathogenicity of V. dahliae to Arabidopsis thaliana and cotton plants. The in vitro and in vivo penetration of Vdchs1, Vdchs4, Vdchs6, and Vdchs7 mutants was impaired, while that of the other mutants was normal. Overall, our results indicate that the VdCHS genes exert diverse functions to regulate the growth and development, conidial germination, conidial production, stress response, penetration, and virulence in V. dahliae.

Efficacy of Candida dubliniensis and Fungal β-Glucans in Inducing Trained Innate Immune Protection Against Inducers of Sepsis

Fungal-bacterial intra-abdominal infections (IAI) can lead to sepsis with significant morbidity and mortality. We have established a murine model of Candida albicans (Ca) and Staphylococcus aureus (Sa) IAI that results in acute lethal sepsis. Prior intraperitoneal or intravenous inoculation with low virulence Candida dubliniensis (Cd) confers high level protection against lethal Ca/Sa IAI and sepsis. Protection via Cd immunization is associated with decreased pro-inflammatory cytokines and mediated by Gr-1+ putative myeloid-derived suppressor cells (MDSCs) representing a novel form of trained innate immunity (TII). The objective of these studies was to determine the extent of Cd-mediated TII against sepsis of broad origin and explore the potential of fungal cell wall components as abiotic immunogen alternatives to induce TII, including zymosan depleted of TLR2 activity (d-zymosan), or purified preparations of β-glucan. Immunized mice were challenged 14 days post-immunization with a lethal array of live or abiotic inducers of sepsis, including Ca/Sa, Ca/Escherichia coli (Ca/Ec), LPS or untreated zymosan. Results showed that live Cd immunization was protective against sepsis induced by Ca/Ec and zymosan, but not LPS. Similar to protection against Ca/Sa, survival was dependent on Gr-1+ cells with no role for macrophages. Among the fungal cell wall compounds as immunogens, immunization with d-zymosan and an alkali-treated form of β-glucan also resulted in significant protection against sepsis induced by Ca/Sa or Ca/Ec, but not LPS sepsis. Again, there was a strong dependence on Gr-1+ cells for protection with one exception, an added role for macrophages in the case of protection induced by alkali-treated β-glucan. Overall, these results demonstrate that immunization with Cd as well as abiotic fungal cell components are capable of Gr-1+ cell-mediated trained innate immune protection against sepsis of broad microbial origin. In addition, abiotic β-glucans represent potential alternatives to live Cd for protection against lethal polymicrobial sepsis.

Different effectiveness of fungal pathogen-associated molecular patterns (PAMPs) in activating rat peritoneal mast cells

Mast cells (MCs) are the first immune cell type that can contact with the external environment, where they may rapidly sense the presence of pathogens. These cells are directly involved in innate defense through their ability to pathogen destruction by several mechanisms and the pattern recognition receptors (PRRs) they express. Several studies have focused on the aspects of MC responses to bacterial and viral pathogens or their specific components and the role of those cells in antibacterial or antiviral defense mechanisms. However, to date, the knowledge of the influence of various fungi-derived molecules on MC activity is primarily based on limited data. Thus, this study aims to compare the effect of the major fungi cell wall-associated antigens, i.e., two β-(1,3)-glucans: zymosan - β-(1,3)-glucan containing mannan and chitin, and curdlan - purified linear model β-(1,3)-glucan as well as mannan on peritoneal MC activity. In particular, the potency of various fungal cell wall components to induce MC migration, degranulation, and generation and/or release of de novo-synthesized mediators/cytokines/chemokines was analyzed. The most striking result to emerge from the data is that MC activation differs depending on the fungal stimuli. Our study outlines that components of the inner layer of the fungi cell wall - β-glucans, i.e., zymosan and curdlan, are more potent stimulators of MC activity compared to mannan. On this note, the data described here may provide a foundation for further studying the role of MC in antifungal immunity and be helpful for a better understanding of host-pathogenic fungi interactions.

Predictors and outcomes of peritoneal dialysis-related infections due to filamentous molds (MycoPDICS).

We sought to evaluate the predictors and outcomes of mold peritonitis in patients with peritoneal dialysis (PD). This cohort study included PD patients from the MycoPDICS database who had fungal peritonitis between July 2015-June 2020. Patient outcomes were analyzed by Kaplan Meier curves and the Log-rank test. Multivariable Cox proportional hazards model regression was used to estimating associations between fungal types and patients' outcomes. The study included 304 fungal peritonitis episodes (yeasts n = 129, hyaline molds n = 122, non-hyaline molds n = 44, and mixed fungi n = 9) in 303 patients. Fungal infections were common during the wet season (p <0.001). Mold peritonitis was significantly more frequent in patients with higher hemoglobin levels, presentations with catheter problems, and positive galactomannan (a fungal cell wall component) tests. Patient survival rates were lowest for non-hyaline mold peritonitis. A higher hazard of death was significantly associated with leaving the catheter in-situ (adjusted hazard ratio [HR] = 6.15, 95%confidence interval [CI]: 2.86-13.23) or delaying catheter removal after the diagnosis of fungal peritonitis (HR = 1.56, 95%CI: 1.00-2.44), as well as not receiving antifungal treatment (HR = 2.23, 95%CI: 1.25-4.01) or receiving it for less than 2 weeks (HR = 2.13, 95%CI: 1.33-3.43). Each additional day of antifungal therapy beyond the minimum 14-day duration was associated with a 2% lower risk of death (HR = 0.98, 95%CI: 0.95-0.999). Non-hyaline-mold peritonitis had worse survival. Longer duration and higher daily dosage of antifungal treatment were associated with better survival. Deviations from the 2016 ISPD Peritonitis Guideline recommendations concerning treatment duration and catheter removal timing were independently associated with higher mortality.