Cellular and Molecular Mechanisms of Fungal β-(1→6)-Glucan in Macrophages

Over the last 40-yr, the majority of research on glucans has focused on β-(1→3)-glucans. Recent studies indicate that β-(1→6)-glucans may be even more potent immune modulators than β-(1→3)-glucans. Mechanisms by which β-(1→6)-glucans are recognized and modulate immunity are unknown. In this study, we examined the interaction of purified water-soluble β-(1→6)-glucans with macrophage cell lines and primary peritoneal macrophages and the cellular and molecular consequences of this interaction. Our results indicate the existence of a specific β-(1→6)-glucan receptor that internalizes the glucan ligand via a clathrin-dependent mechanism. We show that the known β-(1→3)-glucans receptors are not responsible for β-(1→6)-glucan recognition and interaction. The receptor-ligand uptake/interaction has an apparent dissociation constant (KD) of ∼4-μM, and was associated with phosphorylation of ERK and JNK but not Iκ-α or p38. Our results indicate that macrophage interaction with β-(1→6)-glucans may lead to modulation of genes associated with anti-fungal immunity and recruitment/activation of neutrophils. In summary, we show that macrophages specifically bind and internalize β-(1→6)-glucans followed by activation of intracellular signaling and modulation of anti-fungal immune response-related gene regulation. Thus, we conclude that the interaction between innate immunity and β-(1→6)-glucans may play an important role in shaping the anti-fungal immune response

New Insights into the Structure of (1→3,1→6)-β-D-Glucan Side Chains in the Candida glabrata Cell Wall

β-glucan is a (1→3)-β-linked glucose polymer with (1→6)-β-linked side chains and a major component of fungal cell walls. β-glucans provide structural integrity to the fungal cell wall. The nature of the (1–6)-β-linked side chain structure of fungal (1→3,1→6)-β-D-glucans has been very difficult to elucidate. Herein, we report the first detailed structural characterization of the (1→6)-β-linked side chains of Candida glabrata using high-field NMR. The (1→6)-β-linked side chains have an average length of 4 to 5 repeat units spaced every 21 repeat units along the (1→3)-linked polymer backbone. Computer modeling suggests that the side chains have a bent curve structure that allows for a flexible interconnection with parallel (1→3)-β-D-glucan polymers, and/or as a point of attachment for proteins. Based on these observations we propose new approaches to how (1→6)-β-linked side chains interconnect with neighboring glucan polymers in a manner that maximizes fungal cell wall strength, while also allowing for flexibility, or plasticity

Binding of Elementary Bodies by the Opportunistic Fungal Pathogen Candida albicansor Soluble β-Glucan, Laminarin, Inhibits Chlamydia Trachomatisinfectivity

Microbial interactions represent an understudied facet of human health and disease. In this study, the interactions that occur between Chlamydia trachomatis and the opportunistic fungal pathogen, Candida albicans were investigated. Candida albicans is a common component of the oral and vaginal microbiota responsible for thrush and vaginal yeast infections. Normally, Candida exist in the body as yeast. However, disruptions to the microbiota create conditions that allow expanded growth of Candida, conversion to the hyphal form, and tissue invasion. Previous studies have shown that a myriad of outcomes can occur when Candida albicans interacts with pathogenic bacteria. To determine if C. trachomatis physically interacts with C. albicans, we incubated chlamydial elementary bodies (EB) in medium alone or with C. albicans yeast or hyphal forms for 1 h. Following incubation, the samples were formaldehyde-fixed and processed for immunofluorescence assays using anti-chlamydial MOMP or anti- chlamydial LPS antibodies. Replicate samples were replenished with culture medium and incubated at 35°C for 0-120 h prior to fixation for immunofluorescence analysis or collection for EB infectivity assays. Data from this study indicates that both C. trachomatis serovar E and C. muridarum EB bind to C. albicans yeast and hyphal forms. This interaction was not blocked by pre-incubation of EB with the Candida cell wall components, mannan or β-glucans, suggesting that EB interact with a Candida cell wall protein or other structure. Bound EB remained attached to C. albicans for a minimum of 5 days (120 h). Infectivity assays demonstrated that EB bound to C. albicans are infectious immediately following binding (0h). However, once bound to C. albicans, EB infectivity decreased at a faster rate than EB in medium alone. At 6h post binding, 40% of EB incubated in medium alone remained infectious compared to only 16% of EB bound to C. albicans. Likewise, pre-incubation of EB with laminarin, a soluble preparation of β-glucan, alone or in combination with other fungal cell wall components significantly decreases chlamydial infectivity in HeLa cells. These data indicate that interactions between EB and C. albicans inhibit chlamydial infectivity, possibly by physically blocking EB interactions with host cell receptors

Mannan Molecular Substructures Control Nanoscale Glucan Exposure in Candida

Cell wall mannans of Candida albicans mask β-(1,3)-glucan from recognition by Dectin-1, contributing to innate immune evasion. Glucan exposures are predominantly single receptor-ligand interaction sites of nanoscale dimensions. Candida species vary in basal glucan exposure and molecular complexity of mannans. We used super-resolution fluorescence imaging and a series of protein mannosylation mutants in C. albicans and C. glabrata to investigate the role of specific N-mannan features in regulating the nanoscale geometry of glucan exposure. Decreasing acid labile mannan abundance and α-(1,6)-mannan backbone length correlated most strongly with increased density and nanoscopic size of glucan exposures in C. albicans and C. glabrata, respectively. Additionally, a C. albicans clinical isolate with high glucan exposure produced similarly perturbed N-mannan structures and elevated glucan exposure geometry. Thus, acid labile mannan structure influences the nanoscale features of glucan exposure, impacting the nature of the pathogenic surface that triggers immunoreceptor engagement, aggregation, and signaling. Graus et al. find that N-mannan structural features regulated by Candida mannosyltransfersases control glucan exposure. Loss of mannan increased the frequency and size of glucan exposures and changed multivalent receptor engagement. Changes to mannan structure in a bloodstream isolate are associated with elevated glucan exposure at the nanoscale

Binding of Elementary Bodies by the Opportunistic Fungal Pathogen Candida albicans or Soluble β-Glucan, Laminarin, Inhibits Chlamydia trachomatis Infectivity

Microbial interactions represent an understudied facet of human health and disease. In this study, the interactions that occur between Chlamydia trachomatis and the opportunistic fungal pathogen, Candida albicans were investigated. Candida albicans is a common component of the oral and vaginal microbiota responsible for thrush and vaginal yeast infections. Normally, Candida exist in the body as yeast. However, disruptions to the microbiota create conditions that allow expanded growth of Candida, conversion to the hyphal form, and tissue invasion. Previous studies have shown that a myriad of outcomes can occur when Candida albicans interacts with pathogenic bacteria. To determine if C. trachomatis physically interacts with C. albicans, we incubated chlamydial elementary bodies (EB) in medium alone or with C. albicans yeast or hyphal forms for 1 h. Following incubation, the samples were formaldehyde-fixed and processed for immunofluorescence assays using anti-chlamydial MOMP or anti- chlamydial LPS antibodies. Replicate samples were replenished with culture medium and incubated at 35°C for 0–120 h prior to fixation for immunofluorescence analysis or collection for EB infectivity assays. Data from this study indicates that both C. trachomatis serovar E and C. muridarum EB bind to C. albicans yeast and hyphal forms. This interaction was not blocked by pre-incubation of EB with the Candida cell wall components, mannan or β-glucans, suggesting that EB interact with a Candida cell wall protein or other structure. Bound EB remained attached to C. albicans for a minimum of 5 days (120 h). Infectivity assays demonstrated that EB bound to C. albicans are infectious immediately following binding (0h). However, once bound to C. albicans, EB infectivity decreased at a faster rate than EB in medium alone. At 6h post binding, 40% of EB incubated in medium alone remained infectious compared to only 16% of EB bound to C. albicans. Likewise, pre-incubation of EB with laminarin, a soluble preparation of β-glucan, alone or in combination with other fungal cell wall components significantly decreases chlamydial infectivity in HeLa cells. These data indicate that interactions between EB and C. albicans inhibit chlamydial infectivity, possibly by physically blocking EB interactions with host cell receptors

Discrimination of Methionine Sulfoxide and Sulfone by Human Neutrophil Elastase

Human neutrophil elastase (HNE) is a uniquely destructive serine protease with the ability to unleash a wave of proteolytic activity by destroying the inhibitors of other proteases. Although this phenomenon forms an important part of the innate immune response to invading pathogens, it is responsible for the collateral host tissue damage observed in chronic conditions such as chronic obstructive pulmonary disease (COPD), and in more acute disorders such as the lung injuries associated with COVID-19 infection. Previously, a combinatorially selected activity-based probe revealed an unexpected substrate preference for oxidised methionine, which suggests a link to oxida-tive pathogen clearance by neutrophils. Here we use oxidised model substrates and inhibitors to confirm this observation and to show that neutrophil elastase is specifically selective for the di-oxygenated methionine sulfone rather than the mono-oxygenated methionine sulfoxide. We also posit a critical role for ordered solvent in the mechanism of HNE discrimination between the two oxidised forms methionine residue. Preference for the sulfone form of oxidised methionine is especially significant. While both host and pathogens have the ability to reduce methionine sulfoxide back to methionine, a biological pathway to reduce methionine sulfone is not known. Taken to-gether, these data suggest that the oxidative activity of neutrophils may create rapidly cleaved elas-tase “super substrates” that directly damage tissue, while initiating a cycle of neutrophil oxidation that increases elastase tissue damage and further neutrophil recruitment

Chemical crosslinking and mass spectrometry studies of the structure and dynamics of membrane proteins and receptors.

Membrane proteins make up a diverse and important subset of proteins for which structural information is limited. In this study, chemical cross-linking and mass spectrometry were used to explore the structure of the G-protein-coupled photoreceptor bovine rhodopsin in the dark-state conformation. All experiments were performed in rod outer segment membranes using amino acid 'handles' in the native protein sequence and thus minimizing perturbations to the native protein structure. Cysteine and lysine residues were covalently cross-linked using commercially available reagents with a range of linker arm lengths. Following chemical digestion of cross-linked protein, cross-linked peptides were identified by accurate mass measurement using liquid chromatography-fourier transform mass spectrometry and an automated data analysis pipeline. Assignments were confirmed and, if necessary, resolved, by tandem MS. The relative reactivity of lysine residues participating in cross-links was evaluated by labeling with NHS-esters. A distinct pattern of cross-link formation within the C-terminal domain, and between loop I and the C-terminal domain, emerged. Theoretical distances based on cross-linking were compared to inter-atomic distances determined from the energy-minimized X-ray crystal structure and Monte Carlo conformational search procedures. In general, the observed cross-links can be explained by re-positioning participating side-chains without significantly altering backbone structure. One exception, between C3 16 and K325, requires backbone motion to bring the reactive atoms into sufficient proximity for cross-linking. Evidence from other studies suggests that residues around K325 for a region of high backbone mobility. These findings show that cross-linking studies can provide insight into the structural dynamics of membrane proteins in their native environment

The Mnn2 Mannosyltransferase Family Modulates Mannoprotein Fibril Length, Immune Recognition and Virulence of Candida albicans

The fungal cell wall is the first point of interaction between an invading fungal pathogen and the host immune system. The outer layer of the cell wall is comprised of GPI anchored proteins, which are post-translationally modified by both N- and O-linked glycans. These glycans are important pathogen associated molecular patterns (PAMPs) recognised by the innate immune system. Glycan synthesis is mediated by a series of glycosyl transferases, located in the endoplasmic reticulum and Golgi apparatus. Mnn2 is responsible for the addition of the initial α1,2-mannose residue onto the α1,6-mannose backbone, forming the N-mannan outer chain branches. In Candida albicans, the MNN2 gene family is comprised of six members (MNN2, MNN21, MNN22, MNN23, MNN24 and MNN26). Using a series of single, double, triple, quintuple and sextuple mutants, we show, for the first time, that addition of α1,2-mannose is required for stabilisation of the α1,6-mannose backbone and hence regulates mannan fibril length. Sequential deletion of members of the MNN2 gene family resulted in the synthesis of lower molecular weight, less complex and more uniform N-glycans, with the sextuple mutant displaying only un-substituted α1,6-mannose. TEM images confirmed that the sextuple mutant was completely devoid of the outer mannan fibril layer, while deletion of two MNN2 orthologues resulted in short mannan fibrils. These changes in cell wall architecture correlated with decreased proinflammatory cytokine induction from monocytes and a decrease in fungal virulence in two animal models. Therefore, α1,2-mannose of N-mannan is important for both immune recognition and virulence of C. albicans

Large-scale genome-wide analysis identifies genetic variants associated with cardiac structure and function

BACKGROUND: Understanding the genetic architecture of cardiac structure and function may help to prevent and treat heart disease. This investigation sought to identify common genetic variations associated with inter-individual variability in cardiac structure and function. METHODS: A GWAS meta-analysis of echocardiographic traits was performed, including 46,533 individuals from 30 studies (EchoGen consortium). The analysis included 16 traits of left ventricular (LV) structure, and systolic and diastolic function. RESULTS: The discovery analysis included 21 cohorts for structural and systolic function traits (n = 32,212) and 17 cohorts for diastolic function traits (n = 21,852). Replication was performed in 5 cohorts (n = 14,321) and 6 cohorts (n = 16,308), respectively. Besides 5 previously reported loci, the combined meta-analysis identified 10 additional genome-wide significant SNPs: rs12541595 near MTSS1 and rs10774625 in ATXN2 for LV end-diastolic internal dimension; rs806322 near KCNRG, rs4765663 in CACNA1C, rs6702619 near PALMD, rs7127129 in TMEM16A, rs11207426 near FGGY, rs17608766 in GOSR2, and rs17696696 in CFDP1 for aortic root diameter; and rs12440869 in IQCH for Doppler transmitral A-wave peak velocity. Findings were in part validated in other cohorts and in GWAS of related disease traits. The genetic loci showed associations with putative signaling pathways, and with gene expression in whole blood, monocytes, and myocardial tissue. CONCLUSION: The additional genetic loci identified in this large meta-analysis of cardiac structure and function provide insights into the underlying genetic architecture of cardiac structure and warrant follow-up in future functional studies. FUNDING: For detailed information per study, see Acknowledgments.This work was supported by a grant from the US National Heart, Lung, and Blood Institute (N01-HL-25195; R01HL 093328 to RSV), a MAIFOR grant from the University Medical Center Mainz, Germany (to PSW), the Center for Translational Vascular Biology (CTVB) of the Johannes Gutenberg-University of Mainz, and the Federal Ministry of Research and Education, Germany (BMBF 01EO1003 to PSW). This work was also supported by the research project Greifswald Approach to Individualized Medicine (GANI_MED). GANI_MED was funded by the Federal Ministry of Education and Research and the Ministry of Cultural Affairs of the Federal State of Mecklenburg, West Pomerania (contract 03IS2061A). We thank all study participants, and the colleagues and coworkers from all cohorts and sites who were involved in the generation of data or in the analysis. We especially thank Andrew Johnson (FHS) for generation of the gene annotation database used for analysis. We thank the German Center for Cardiovascular Research (DZHK e.V.) for supporting the analysis and publication of this project. RSV is a member of the Scientific Advisory Board of the DZHK. Data on CAD and MI were contributed by CARDIoGRAMplusC4D investigators. See Supplemental Acknowledgments for consortium details. PSW, JFF, AS, AT, TZ, RSV, and MD had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis

A C. albicans two component pathway regulates the CDR4 and SSU1 transport genes involved in quorum sensing and response to bacterial signaling molecules.

Polymicrobial communities of bacterial and fungal species are present on the skin and mucosal surfaces of the body. Invasive infections caused by Candida species are commonly seen in immunocompromised individuals (HIV, transplants, cancer) and ranks as the third leading cause of infection in hospitalized patients. C. albicans is a polymorphic opportunistic fungus that infects critically ill patients and has the ability to change its morphology from yeast to hyphal form.The morphogenesis of C. albicans is a major aspect of its virulence and is regulated by quorum sensing (QS) molecules they produce, as well as the presence of neighboring microbes.In this study, we examined two transporter mutants, cdr4 and ssu1, for their ability to form biofilms in the presence of cyclic-di-GMP and 3-oxo-12-homoserine lactone. To quantify biomass, wild type and mutant cells were grown overnight at 30˚C in YPD. The cells were washed, counted and diluted to a desired density of 106 cells/ml in medium 199, pH7.5. Cells were added to 96-well plates pre-incubated with 5% fetal bovine serum at densities of 105, 104, and 103 cells/well and allowed to adhere at 37˚C for one hour. The wells were then covered with fresh M199 media containing the QS molecule and monitored for 48 hours at 37˚C. After this time, the media and planktonic cells were removed. The biofilms were fixed with methanol, dried, then stained with 0.05% crystal violet. Bulk biomass was assessed by spectrophotometry. We did observe a difference in biofilm density when incubated in the presence of cyclic-diGMP. We noted that for the wild type and ssu1 strain their biofilms biomass increased by as much as 10% at 104 and 103 cell densities when compared with the control. While the cdr4 strain had a slight reduction in biofilm density when cyclic-diGMP was present. This result also indicates a potentially positive role in which cyclic-diGMP can help C. albicans develop denser biofilms, potentially in the presence of bacteria like P. aeruginsa, which secrete cyclic di-GMP, but kill hyphal forms of C. albicans. Additionally, it has been shown that C. albicans mutants lacking the hybrid histidine kinase, Chk1p, are refractory to the effects of farnesol, a QS molecule that inhibits morphogenesis.Given that mutations in CDR4 and SSU1 impact the QS response in C. albicans, we investigated whether these genes were regulated through two-component signaling by Chk1. To assess CDR4 and SSU1 expression, wild type and mutant strains were grown overnight in YPD media at 30˚C. Cells were then harvested and RNA was obtained by acid phenol extraction. Using RT-PCR, we determined both CDR4 and SSU1 expression is reduced or highly repressed in the chk1, ypd1, and skn7 null strains. These results suggest the two genes are downstream targets in a pathway regulated by Chk1p. The finding that QS proceeds through a two-component pathway can be exploited in antifungal drug development. Given that two-component signaling is absent in mammalian cells, development of novel compounds that interfere with this pathway may be a useful alternative for treating patients with candidiasis