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1.
Molecules ; 26(17)2021 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-34500777

RESUMO

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 oxidative 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 together, these data suggest that the oxidative activity of neutrophils may create rapidly cleaved elastase "super substrates" that directly damage tissue, while initiating a cycle of neutrophil oxidation that increases elastase tissue damage and further neutrophil recruitment.


Assuntos
Imunidade Inata , Elastase de Leucócito/metabolismo , Metionina/análogos & derivados , Neutrófilos/imunologia , Biocatálise , COVID-19/imunologia , COVID-19/patologia , COVID-19/virologia , Domínio Catalítico/genética , Ensaios Enzimáticos , Interações Hospedeiro-Patógeno/imunologia , Humanos , Elastase de Leucócito/antagonistas & inibidores , Elastase de Leucócito/genética , Pulmão/imunologia , Pulmão/patologia , Pulmão/virologia , Metionina/metabolismo , Simulação de Dinâmica Molecular , Infiltração de Neutrófilos , Neutrófilos/enzimologia , Oxirredução/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Doença Pulmonar Obstrutiva Crônica/imunologia , Doença Pulmonar Obstrutiva Crônica/patologia , SARS-CoV-2/imunologia , Especificidade por Substrato/imunologia
2.
mSphere ; 6(3): e0040621, 2021 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-34160238

RESUMO

Candida auris, a recently emergent fungal pathogen, has caused invasive infections in health care settings worldwide. Mortality rates approach 60% and hospital spread poses a public health threat. Compared to other Candida spp., C. auris avoids triggering the antifungal activity of neutrophils, innate immune cells that are critical for responding to many invasive fungal infections, including candidiasis. However, the mechanism underpinning this immune evasion has been largely unknown. Here, we show that C. auris cell wall mannosylation contributes to the evasion of neutrophils ex vivo and in a zebrafish infection model. Genetic disruption of mannosylation pathways (PMR1 and VAN1) diminishes the outer cell wall mannan, unmasks immunostimulatory components, and promotes neutrophil engagement, phagocytosis, and killing. Upon examination of these pathways in other Candida spp. (Candida albicans and Candida glabrata), we did not find an impact on neutrophil interactions. These studies show how C. auris mannosylation contributes to neutrophil evasion though pathways distinct from other common Candida spp. The findings shed light on innate immune evasion for this emerging pathogen. IMPORTANCE The emerging fungal pathogen Candida auris presents a global public health threat. Therapeutic options are often limited for this frequently drug-resistant pathogen, and mortality rates for invasive disease are high. Previous study has demonstrated that neutrophils, leukocytes critical for the antifungal host defense, do not efficiently recognize and kill C. auris. Here, we show how the outer cell wall of C. auris promotes immune evasion. Disruption of this mannan polysaccharide layer renders C. auris susceptible to neutrophil killing ex vivo and in a zebrafish model of invasive candidiasis. The role of these mannosylation pathways for neutrophil evasion appears divergent from other common Candida species.

3.
Nat Microbiol ; 5(12): 1516-1531, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32839538

RESUMO

Candida auris is among the most important emerging fungal pathogens, yet mechanistic insights into its immune recognition and control are lacking. Here, we integrate transcriptional and functional immune-cell profiling to uncover innate defence mechanisms against C. auris. C. auris induces a specific transcriptome in human mononuclear cells, a stronger cytokine response compared with Candida albicans, but a lower macrophage lysis capacity. C. auris-induced innate immune activation is mediated through the recognition of C-type lectin receptors, mainly elicited by structurally unique C. auris mannoproteins. In in vivo experimental models of disseminated candidiasis, C. auris was less virulent than C. albicans. Collectively, these results demonstrate that C. auris is a strong inducer of innate host defence, and identify possible targets for adjuvant immunotherapy.


Assuntos
Candida/fisiologia , Candidíase/genética , Candidíase/microbiologia , Animais , Candida/genética , Candida/patogenicidade , Candidíase/imunologia , Citocinas/genética , Citocinas/imunologia , Humanos , Imunidade , Lectinas Tipo C/genética , Lectinas Tipo C/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transcrição Genética , Virulência
4.
Cell Rep ; 24(9): 2432-2442.e5, 2018 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-30157435

RESUMO

Cell wall mannans of Candida albicans mask ß-(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.


Assuntos
Candida/metabolismo , Glucanos/metabolismo , Mananas/metabolismo , Humanos
5.
Front Microbiol ; 9: 3270, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30692972

RESUMO

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 ß-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 ß-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.

6.
Innate Immun ; 21(7): 759-69, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26209532

RESUMO

Over the last 40 yr, the majority of research on glucans has focused on ß-(1→3)-glucans. Recent studies indicate that ß-(1→6)-glucans may be even more potent immune modulators than ß-(1→3)-glucans. Mechanisms by which ß-(1→6)-glucans are recognized and modulate immunity are unknown. In this study, we examined the interaction of purified water-soluble ß-(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 ß-(1→6)-glucan receptor that internalizes the glucan ligand via a clathrin-dependent mechanism. We show that the known ß-(1→3)-glucans receptors are not responsible for ß-(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 IκB-α or p38. Our results indicate that macrophage interaction with ß-(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 ß-(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 ß-(1→6)-glucans may play an important role in shaping the anti-fungal immune response.


Assuntos
Macrófagos/fisiologia , Malassezia/imunologia , Saccharomyces cerevisiae/imunologia , beta-Glucanas/metabolismo , Animais , Linhagem Celular , Regulação da Expressão Gênica , Humanos , Imunidade Inata , Sistema de Sinalização das MAP Quinases , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ativação de Neutrófilo , Receptores de Reconhecimento de Padrão/metabolismo
7.
J Biol Chem ; 289(6): 3432-43, 2014 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-24344127

RESUMO

The innate immune system differentially recognizes Candida albicans yeast and hyphae. It is not clear how the innate immune system effectively discriminates between yeast and hyphal forms of C. albicans. Glucans are major components of the fungal cell wall and key fungal pathogen-associated molecular patterns. C. albicans yeast glucan has been characterized; however, little is known about glucan structure in C. albicans hyphae. Using an extraction procedure that minimizes degradation of the native structure, we extracted glucans from C. albicans hyphal cell walls. (1)H NMR data analysis revealed that, when compared with reference (1→3,1→6) ß-linked glucans and C. albicans yeast glucan, hyphal glucan has a unique cyclical or "closed chain" structure that is not found in yeast glucan. GC/MS analyses showed a high abundance of 3- and 6-linked glucose units when compared with yeast ß-glucan. In addition to the expected (1→3), (1→6), and 3,6 linkages, we also identified a 2,3 linkage that has not been reported previously in C. albicans. Hyphal glucan induced robust immune responses in human peripheral blood mononuclear cells and macrophages via a Dectin-1-dependent mechanism. In contrast, C. albicans yeast glucan was a much less potent stimulus. We also demonstrated the capacity of C. albicans hyphal glucan, but not yeast glucan, to induce IL-1ß processing and secretion. This finding provides important evidence for understanding the immune discrimination between colonization and invasion at the mucosal level. When taken together, these data provide a structural basis for differential innate immune recognition of C. albicans yeast versus hyphae.


Assuntos
Candida albicans/imunologia , Polissacarídeos Fúngicos/imunologia , Hifas/metabolismo , Imunidade Inata , Macrófagos/imunologia , Candida albicans/química , Configuração de Carboidratos , Feminino , Polissacarídeos Fúngicos/química , Humanos , Hifas/química , Interleucina-1beta/imunologia , Macrófagos/citologia , Espectroscopia de Ressonância Magnética , Masculino
8.
PLoS One ; 8(8): e71939, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23951271

RESUMO

In the human body, fungi and bacteria share many niches where the close contact of these organisms maintains a balance among the microbial population. However, when this microbial balance is disrupted, as with antibiotic treatment, other bacteria or fungi can grow uninhibited. C. albicans is the most common opportunistic fungal pathogen affecting humans and can uniquely control its morphogenesis between yeast, pseudohyphal, and hyphal forms. Numerous studies have shown that C. albicans interactions with bacteria can impact its ability to undergo morphogenesis; however, the genetics that govern this morphological control via these bacterial interactions are still relatively unknown. To aid in the understanding of the cross-kingdom interactions of C. albicans with bacteria and the impact on morphology we utilized a haploinsufficiency based C. albicans mutant screen to test for the ability of C. albicans to produce hyphae in the presence of three bacterial species (Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus). Of the 18,144 mutant strains tested, 295 mutants produced hyphae in the presence of all three bacterial species. The 295 mutants identified 132 points of insertion, which included identified/predicted genes, major repeat sequences, and a number of non-coding/unannotated transcripts. One gene, CDR4, displayed increased expression when co-cultured with S. aureus, but not E. coli or P. aeruginosa. Our data demonstrates the ability to use a large scale library screen to identify genes involved in Candida-bacterial interactions and provides the foundation for comprehending the genetic pathways relating to bacterial control of C. albicans morphogenesis.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Candida albicans/crescimento & desenvolvimento , Proteínas Fúngicas/metabolismo , Hifas/crescimento & desenvolvimento , Staphylococcus aureus/fisiologia , Transportadores de Cassetes de Ligação de ATP/genética , Candida albicans/genética , Candida albicans/metabolismo , Escherichia coli/fisiologia , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Haploinsuficiência , Hifas/genética , Hifas/metabolismo , Interações Microbianas , Morfogênese , Mutação , Pseudomonas aeruginosa/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa
9.
PLoS Pathog ; 9(4): e1003276, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23633946

RESUMO

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.


Assuntos
Candida albicans/imunologia , Candida albicans/patogenicidade , Mananas/imunologia , Manose/metabolismo , Manosiltransferases/metabolismo , Glicoproteínas de Membrana/imunologia , Animais , Candida albicans/enzimologia , Candidíase/imunologia , Parede Celular/química , Parede Celular/imunologia , Feminino , Proteínas Fúngicas/genética , Proteínas Fúngicas/imunologia , Proteínas Fúngicas/metabolismo , Humanos , Mananas/química , Manose/química , Manosiltransferases/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Polissacarídeos/metabolismo , Receptores de Reconhecimento de Padrão/imunologia , Receptores de Reconhecimento de Padrão/metabolismo , Alinhamento de Sequência , Deleção de Sequência
10.
PLoS One ; 6(11): e27614, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22096604

RESUMO

ß-Glucan is a (1→3)-ß-linked glucose polymer with (1→6)-ß-linked side chains and a major component of fungal cell walls. ß-Glucans provide structural integrity to the fungal cell wall. The nature of the (1-6)-ß-linked side chain structure of fungal (1→3,1→6)-ß-D-glucans has been very difficult to elucidate. Herein, we report the first detailed structural characterization of the (1→6)-ß-linked side chains of Candida glabrata using high-field NMR. The (1→6)-ß-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)-ß-D-glucan polymers, and/or as a point of attachment for proteins. Based on these observations we propose new approaches to how (1→6)-ß-linked side chains interconnect with neighboring glucan polymers in a manner that maximizes fungal cell wall strength, while also allowing for flexibility, or plasticity.


Assuntos
Candida glabrata/química , Parede Celular/química , beta-Glucanas/química , Espectroscopia de Ressonância Magnética
11.
Carbohydr Res ; 346(17): 2752-9, 2011 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-22030461

RESUMO

The Candida albicans cell wall provides an architecture that allows for the organism to survive environmental stress as well as interaction with host tissues. Previous work has focused on growing C. albicans on media such as Sabouraud or YPD at 30°C. Because C. albicans normally colonizes a host, we hypothesized that cultivation on blood or serum at 37°C would result in structural changes in cell wall mannan. C. albicans SC5314 was inoculated onto YPD, 5% blood, or 5% serum agar media three successive times at 30°C and 37°C, then cultivated overnight at 30°C in YPD. The mannan was extracted and characterized using 1D and 2D (1)H NMR techniques. At 30°C cells grown in blood and serum contain less acid-stable terminal ß-(1→2)-linked d-mannose and α-(1→2)-linked d-mannose-containing side chains, while the acid-labile side chains of mannan grown in blood and serum contain fewer ß-Man-(1→2)-α-Man-(1→ side chains. The decrement in acid-stable mannan side chains is greater at 37°C than at 30°C. Cells grown on blood at 37°C show fewer →6)-α-Man-(1→ structural motifs in the acid-stable polymer backbone. The data indicate that C. albicans, grown on media containing host-derived components, produces less complex mannan. This is accentuated when the cells are cultured at 37°C. This study demonstrates that the C. albicans cell wall is a dynamic and adaptive organelle, which alters its structural phenotype in response to growth in host-derived media at physiological temperature.


Assuntos
Candida albicans/metabolismo , Parede Celular/metabolismo , Mananas/metabolismo , Animais , Sangue , Candida albicans/crescimento & desenvolvimento , Configuração de Carboidratos , Sequência de Carboidratos , Parede Celular/química , Meios de Cultura , Espectroscopia de Ressonância Magnética , Mananas/química , Dados de Sequência Molecular , Fenótipo , Ovinos , Temperatura
12.
Carbohydr Res ; 344(18): 2474-9, 2009 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-19853245

RESUMO

Members of the genus Malassezia are commensal fungi found on the skin of both human and domestic animals and are associated with skin diseases including dandruff/seborrheic dermatitis, pityriasis versicolor, and atopic eczema (AE) in humans. In this study we have characterized the cell-wall carbohydrates of Malassezia sympodialis, one of the species most frequently isolated from both AE patients and healthy individuals. Cells were grown in liquid Dixon media at 32 degrees C, harvested, and processed using a standard Fehling's precipitation methodology for the isolation of mannan and a standard base/acid extraction for (1-->3)-beta-D-glucans. Using these classic extraction methods we were unable to isolate precipitable mannan or insoluble (1-->3)-beta-D-glucan. However, acidification and addition of methanol to the remaining Fehling's-treated sample resulted in a very clean precipitate. This material was characterized by GPC-MALLS, 1D and 2D NMR, and GC-MS for monomer-type and linkage-type composition. We determined that trace amounts of both mannan and branched (1-->3, 1-->6)-beta-D-glucan were present in the recovered precipitate, but not linear (1-->3)-beta-D-glucan. Surprisingly, NMR analysis indicated that (1-->6)-beta-D-glucan was the major carbohydrate component isolated from M. sympodialis cell wall. GC-MS linkage analysis confirmed the (1-->6)-beta-D-glucan structure. Based on these studies we have determined that the M. sympodialis cell wall contains (1-->6)-beta-D-glucan as the major carbohydrate component along with trace amounts of mannan and (1-->3, 1-->6)-beta-d-glucan. In addition, these data indicate that modification of the classic mannan isolation methodology may be useful in the simultaneous isolation of both mannan and (1-->6)-beta-D-glucan from other fungi.


Assuntos
Parede Celular/química , Malassezia/ultraestrutura , Polissacarídeos/química , beta-Glucanas/isolamento & purificação , Configuração de Carboidratos , Espectroscopia de Ressonância Magnética , Malassezia/química , Mananas/isolamento & purificação
13.
Methods Mol Biol ; 499: 77-83, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19152041

RESUMO

A general procedure is described for the analysis of gene expression of Candida albicans cultured in a mouse infection model. This technique involves first infecting mice with Candida and subsequently harvesting blood and other tissue at specific time points during infection. The tissues are homogenized and the infecting Candida isolated. Finally, RNA is extracted from recovered Candida cells and subjected to microarray analysis.


Assuntos
Candida albicans/genética , Candidíase/genética , Perfilação da Expressão Gênica/métodos , Animais , Candida albicans/metabolismo , Candida albicans/patogenicidade , Candidíase/metabolismo , Candidíase/microbiologia , Modelos Animais de Doenças , Humanos , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos
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