Emerging Fungal PathogenCandida aurisEvades Neutrophil Attack

ABSTRACTCandida aurishas recently emerged as the first fungal pathogen to cause a global public health threat. The reason this species is causing hospital-associated outbreaks of invasive candidiasis with high mortality is unknown. In this study, we examine the interaction ofC. auriswith neutrophils, leukocytes critical for control of invasive fungal infections. We show that human neutrophils do not effectively killC. auris. Compared toCandida albicans, neutrophils poorly recruited toC. aurisand failed to form neutrophil extracellular traps (NETs), which are structures of DNA, histones, and proteins with antimicrobial activity. In mixed cultures, neutrophils preferentially engaged and killedC. albicansoverC. auris. Imaging of neutrophils in a zebrafish larval model of invasive candidiasis revealed the recruitment of approximately 50% fewer neutrophils in response toC. auriscompared toC. albicans. Upon encounter withC. albicansin the zebrafish hindbrain, neutrophils produced clouds of histones, suggesting the formation of NETs. These structures were not observed inC. aurisinfection. Evasion of neutrophil attack and innate immunity offers an explanation for the virulence of this pathogen.IMPORTANCEThe emerging fungal pathogenCandida aurishas produced numerous outbreaks of invasive disease in hospitals worldwide. Why this species causes deadly disease is unknown. Our findings reveal a failure of neutrophils to killC. auriscompared to the most commonly encounteredCandidaspecies,C. albicans. While neutrophils produce neutrophil extracellular traps (NETs) upon encounter withC. albicans, these antimicrobial structures are not formed in response toC. auris. Using human neutrophils and a zebrafish model of invasive candidiasis, we show thatC. aurispoorly recruits neutrophils and evades immune attack. Identification of this impaired innate immune response toC. aurissheds light on the dismal outcomes for patients with invasive disease.

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Extracellular Nucleic Acids Present in the Candida albicans Biofilm Trigger the Release of Neutrophil Extracellular Traps

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2021.681030 ◽

2021 ◽

Vol 11 ◽

Author(s):

Magdalena Smolarz ◽

Marcin Zawrotniak ◽

Dorota Satala ◽

Maria Rapala-Kozik

Keyword(s):

Candida Albicans ◽

Nucleic Acids ◽

Fungal Infection ◽

Fungal Infections ◽

Neutrophil Extracellular Traps ◽

Human Neutrophils ◽

High Concentration ◽

First Line ◽

Extracellular Traps ◽

Biofilm Structure

Neutrophils, the first line of the host’s defense, use a variety of antimicrobial mechanisms to fight invading pathogens. One of the most crucial is the production of neutrophil extracellular traps (NETs) in the process called NETosis. The unique structure of NETs effectively inhibits the spread of pathogens and ensures their exposure to a high concentration of NET-embedded antimicrobial compounds. NETosis strategy is often used by the host to defend against fungal infection caused by Candida albicans. In immunocompromised patients, this microorganism is responsible for developing systemic fungal infections (candidiasis). This is correlated with the use of a vast array of virulence factors, leading to the acquisition of specific resistance to host defense factors and available drug therapies. One of the most important features favoring the development of drug resistance is a C. albicans ability to form biofilms that protect fungal cells mainly through the production of an extracellular matrix (ECM). Among the main ECM-building macromolecules extracellular nucleic acids have been identified and their role is probably associated with the stbilization of the biofilm structure. The complex interactions of immune cells with the thick ECM layer, comprising the first line of contact between these cells and the biofilm structure, are still poorly understood. Therefore, the current studies aimed to assess the release of extracellular nucleic acids by C. albicans strains at different stages of biofilm formation, and to determine the role of these molecules in triggering the NETosis. We showed for the first time that fungal nucleic acids, purified directly from mature C. albicans biofilm structure or obtained from the whole fungal cells, have the potential to induce NET release in vitro. In this study, we considered the involvement of TLR8 and TLR9 in NETosis activation. We showed that DNA and RNA molecules initiated the production of reactive oxygen species (ROS) by activation of the NADPH oxidase complex, essential for ROS-dependent NETosis. Furthermore, analysis of the cell migration showed that the nucleic acids located in the extracellular space surrounding the biofilm may be also effective chemotactic factors, driving the dynamic migration of human neutrophils to the site of ongoing fungal infection.

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3′-Nucleotidase/Nuclease Activity Allows Leishmania Parasites To Escape Killing by Neutrophil Extracellular Traps

Infection and Immunity ◽

10.1128/iai.01232-13 ◽

2014 ◽

Vol 82 (4) ◽

pp. 1732-1740 ◽

Cited By ~ 66

Author(s):

Anderson B. Guimarães-Costa ◽

Thiago S. DeSouza-Vieira ◽

Rafael Paletta-Silva ◽

Anita Leocádio Freitas-Mesquita ◽

José Roberto Meyer-Fernandes ◽

...

Keyword(s):

Leishmania Infantum ◽

Nuclease Activity ◽

Neutrophil Extracellular Traps ◽

Human Neutrophils ◽

Dependent Manner ◽

Content Type ◽

Extracellular Traps ◽

Leishmania Parasites ◽

High Phosphate

ABSTRACTLeishmaniasis is a widespread neglected tropical disease caused by parasites of theLeishmaniagenus. These parasites express the enzyme 3′-nucleotidase/nuclease (3′NT/NU), which has been described to be involved in parasite nutrition and infection. Bacteria that express nucleases escape the toxic effects of neutrophil extracellular traps (NETs). Hence, we investigated the role of 3′NT/NU inLeishmaniasurvival of NET-mediated killing. Promastigotes ofLeishmania infantumwere cultured in high-phosphate (HP) or low-phosphate (LP) medium to modulate nuclease activity. We compared the survival of the two different groups ofLeishmaniaduring interaction with human neutrophils, assessing the role of neutrophil extracellular traps. As previously reported, we detected higher nuclease activity in parasites cultured in LP medium. Both LP and HP promastigotes were capable of inducing the release of neutrophil extracellular traps from human neutrophils in a dose- and time-dependent manner. LP parasites had 2.4 times more survival than HP promastigotes. NET disruption was prevented by the treatment of the parasites with ammonium tetrathiomolybdate (TTM), a 3′NT/NU inhibitor. Inhibition of 3′NT/NU by 3′-AMP, 5′-GMP, or TTM decreased promastigote survival upon interaction with neutrophils. Our results show thatLeishmania infantuminduces NET release and that promastigotes can escape NET-mediated killing by 3′-nucleotidase/nuclease activity, thus ascribing a new function to this enzyme.

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970. Emerging Pathogen Candida auris Evades Neutrophil Attack

Open Forum Infectious Diseases ◽

10.1093/ofid/ofy209.086 ◽

2018 ◽

Vol 5 (suppl_1) ◽

pp. S37-S37

Author(s):

Chad Johnson ◽

J Muse Davis ◽

Anna Huttenlocher ◽

John Kernien ◽

Jeniel Nett

Keyword(s):

Invasive Candidiasis ◽

Fluorescent Microscopy ◽

Human Neutrophils ◽

Infection Model ◽

Candida Auris ◽

Zebrafish Model ◽

Signaling Mechanism ◽

Sytox Green ◽

Neutrophil Response

Abstract Background Candida auris, an emerging fungal pathogen, causes hospital-associated outbreaks of invasive candidiasis with mortality near 60%. Little is known about the pathogenesis of this species that has newly arisen in the last 10 years, and it is unclear why this species is rapidly spreading worldwide. Neutrophils, critical for control of invasive candidiasis, kill fungi through phagocytosis or the release of neutrophil extracellular traps (NETs), which are structures of DNA, histones, and proteins with antimicrobial activity. The objective of this study was to delineate the neutrophil response to C. auris. Methods We examined interactions of human neutrophils with C. auris and included C. albicans for comparison. Neutrophil–Candida interactions were visualized by time-lapse fluorescent microscopy and scanning electron microscopy (SEM). We utilized oxidative stress indicator CM-H2DCFDA to measure the generation of reactive oxygen species (ROS) in neutrophils. NET formation was quantified by Sytox Green staining and assessed by SEM and immunofluorescent labeling of NET-associated proteins. Fungal viability was evaluated using microbiological counts and viability stains. We utilized a zebrafish larvae infection model to evaluate neutrophil–Candida interactions in vivo. Results Imaging revealed the phagocytosis of C. albicans by human neutrophils followed by the formation of NETs. In contrast, neutrophils encountering C. auris rarely engaged in phagocytosis or produced NETs. By Sytox Green staining, C. auris triggered negligible NET release by human neutrophils, with levels 7-fold lower when compared with C. albicans (Figure A). C. auris did not induce neutrophils to generate ROS, a key signaling mechanism for NET formation. The ineffective neutrophil response to C. auris correlated with diminished fungal killing (Figure B). Imaging of neutrophils in a zebrafish model of invasive candidiasis revealed the recruitment of approximately 50% fewer neutrophils in response to C. auris when compared with C. albicans (Figure C). Conclusion C. auris evades neutrophils by altering multiple aspects of their usual anti-candidal responses. We propose that this diminished innate immune response may contribute to the unexpected virulence of C. auris. Disclosures All authors: No reported disclosures.

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Comparative Pathogenicity of United Kingdom Isolates of the Emerging Pathogen Candida auris and Other Key Pathogenic Candida Species

mSphere ◽

10.1128/msphere.00189-16 ◽

2016 ◽

Vol 1 (4) ◽

Cited By ~ 116

Author(s):

Andrew M. Borman ◽

Adrien Szekely ◽

Elizabeth M. Johnson

Keyword(s):

Invasive Candidiasis ◽

Fungal Pathogen ◽

Galleria Mellonella ◽

Systemic Infection ◽

Mortality Rates ◽

Infection Model ◽

Candida Auris ◽

Deep Tissue ◽

Content Type

ABSTRACT The incidence of invasive candidiasis, which includes candidemia and deep tissue infections, continues to rise and is associated with considerable mortality rates. Candida albicans remains the most common cause of invasive candidiasis, although the prevalence of non-albicans species has increased over recent years. Since its first description in 2009, Candida auris has emerged as a serious nosocomial health risk, with widespread outbreaks in numerous hospitals worldwide. However, despite receiving considerable attention, little is known concerning the pathogenicity of this emerging fungal pathogen. Here, using the Galleria mellonella insect systemic infection model, we show strain-specific differences in the virulence of C. auris, with the most virulent isolates exhibiting pathogenicity comparable to that of C. albicans, which is currently accepted as the most pathogenic member of the genus. Candida auris, first described in 2009, has since emerged as an important, multidrug-resistant, nosocomial agent of candidemia, with large outbreaks reported worldwide and high mortality rates associated with therapeutic failure. The current study employed C. auris isolates from a variety of centers in the United Kingdom to evaluate the pathogenicity of this emerging pathogen compared to that of other common pathogenic yeast species in the invertebrate Galleria mellonella infection model. We showed that C. auris isolates differ in their growth characteristics in vitro, with a proportion of isolates failing to release daughter cells after budding, resulting in the formation of large aggregates of cells that cannot be physically disrupted. Our results also demonstrate strain-specific differences in the behavior of C. auris in G. mellonella, with the aggregate-forming isolates exhibiting significantly less pathogenicity than their nonaggregating counterparts. Importantly, the nonaggregating isolates exhibited pathogenicity comparable to that of C. albicans, which is currently accepted as the most pathogenic member of the genus, despite the fact that C. auris isolates do not produce hyphae and produce only rudimentary pseudohyphae either in vitro or in G. mellonella. IMPORTANCE The incidence of invasive candidiasis, which includes candidemia and deep tissue infections, continues to rise and is associated with considerable mortality rates. Candida albicans remains the most common cause of invasive candidiasis, although the prevalence of non-albicans species has increased over recent years. Since its first description in 2009, Candida auris has emerged as a serious nosocomial health risk, with widespread outbreaks in numerous hospitals worldwide. However, despite receiving considerable attention, little is known concerning the pathogenicity of this emerging fungal pathogen. Here, using the Galleria mellonella insect systemic infection model, we show strain-specific differences in the virulence of C. auris, with the most virulent isolates exhibiting pathogenicity comparable to that of C. albicans, which is currently accepted as the most pathogenic member of the genus.

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Natural Variation in Clinical Isolates of Candida albicans Modulates Neutrophil Responses

mSphere ◽

10.1128/msphere.00501-20 ◽

2020 ◽

Vol 5 (4) ◽

Cited By ~ 1

Author(s):

Madhu Shankar ◽

Tricia L. Lo ◽

Ana Traven

Keyword(s):

Reactive Oxygen Species ◽

Candida Albicans ◽

Reactive Oxygen ◽

Neutrophil Extracellular Traps ◽

Prototype Strain ◽

Human Neutrophils ◽

Oxygen Species ◽

Content Type ◽

Extracellular Traps ◽

Strain Sc5314

ABSTRACT Neutropenia predisposes patients to life-threatening infection with Candida albicans, a commensal and opportunistic fungal pathogen. How phenotypic variation in C. albicans isolates dictates neutrophil responses is poorly understood. By using a panel of clinical C. albicans strains, here we report that the prototype strain SC5314 induces the most potent accumulation of reactive oxygen species (ROS) and neutrophil extracellular traps (NETs) by human neutrophils of all tested isolates. ROS and NET accumulation positively correlated with the degree of hyphal formation by the isolates, the hypha being the fungal morphotype that promotes pathogenesis. However, there was no correlation of ROS and NET accumulation with fungal killing by neutrophils. Fungal killing was also not correlated with phagocytosis levels or oxidative stress susceptibility of the isolates. The bloodstream isolate P94015 cannot make hyphae and was previously shown to be hyperfit in the murine gut commensalism model. Our results show that P94015 displays poor phagocytosis by neutrophils, the least ROS and NET accumulation of all tested isolates, and resistance to neutrophil-mediated killing. Our data suggest that reduced susceptibility to neutrophils is likely to be independent from a previously described genetic mutation in P94015 that promotes commensalism. Reduced clearance by neutrophils could benefit commensal fitness of C. albicans and could also have promoted the virulence of P94015 in the human patient in the absence of hyphal morphogenesis. Collectively, our study provides new insights into neutrophil interactions with C. albicans and suggests that studying diverse isolates informs knowledge of the relevant aspects of this key immune interaction. IMPORTANCE Neutrophils are the key immune cell type for host defenses against infections with Candida albicans. C. albicans strains isolated from patients display large phenotypic diversity, but how this diversity impacts host-pathogen interactions with neutrophils is incompletely defined. Here, we show that important neutrophil responses, such as accumulation of reactive oxygen species and neutrophil extracellular traps, as well as the levels of phagocytosis and killing of the pathogen, differ when comparing diverse C. albicans isolates. A bloodstream patient isolate previously described as more suited to commensalism than pathogenesis in animal models is relatively “silent” to neutrophils and resistant to killing. Our findings illuminate the relationships between fungal morphogenesis, neutrophil responses, and C. albicans survival. Our findings suggest that host phenotypes of a commensally adapted strain could be driven by resistance to immune clearance and indicate that we should extend our studies beyond the “prototype” strain SC5314 for deeper understanding of Candida-neutrophil interactions.

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Candida auris Cell Wall Mannosylation Contributes to Neutrophil Evasion through Pathways Divergent from Candida albicans and Candida glabrata

mSphere ◽

10.1128/msphere.00406-21 ◽

2021 ◽

Author(s):

Mark V. Horton ◽

Chad J. Johnson ◽

Robert Zarnowski ◽

Brody D. Andes ◽

Taylor J. Schoen ◽

...

Keyword(s):

Public Health ◽

Fungal Pathogen ◽

Candida Glabrata ◽

Mortality Rates ◽

Invasive Disease ◽

Drug Resistant ◽

Global Public Health ◽

Candida Auris ◽

Content Type ◽

Public Health Threat

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.

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Silver Nanoparticles Induce Neutrophil Extracellular Traps Via Activation of PAD and Neutrophil Elastase

Biomolecules ◽

10.3390/biom11020317 ◽

2021 ◽

Vol 11 (2) ◽

pp. 317

Author(s):

HanGoo Kang ◽

Jinwon Seo ◽

Eun-Jeong Yang ◽

In-Hong Choi

Keyword(s):

Silver Nanoparticles ◽

Neutrophil Elastase ◽

Mammalian Cells ◽

Antimicrobial Properties ◽

Neutrophil Extracellular Traps ◽

Arginine Deiminase ◽

Human Neutrophils ◽

Extracellular Traps ◽

Peptidyl Arginine Deiminase ◽

Key Factor

Silver nanoparticles (AgNPs) are widely used in various fields because of their antimicrobial properties. However, many studies have reported that AgNPs can be harmful to both microorganisms and humans. Reactive oxygen species (ROS) are a key factor of cytotoxicity of AgNPs in mammalian cells and an important factor in the immune reaction of neutrophils. The immune reactions of neutrophils include the expulsion of webs of DNA surrounded by histones and granular proteins. These webs of DNA are termed neutrophil extracellular traps (NETs). NETs allow neutrophils to catch and destroy pathogens in extracellular spaces. In this study, we investigated how AgNPs stimulate neutrophils, specifically focusing on NETs. Freshly isolated human neutrophils were treated with 5 or 100 nm AgNPs. The 5 nm AgNPs induced NET formation, but the 100 nm AgNPs did not. Subsequently, we investigated the mechanism of AgNP-induced NETs using known inhibitors related to NET formation. AgNP-induced NETs were dependent on ROS, peptidyl arginine deiminase, and neutrophil elastase. The result in this study indicates that treatment of 5 nm AgNPs induce NET formation through histone citrullination by peptidyl arginine deiminase and histone cleavage by neutrophil elastase.

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Neutrophil Extracellular Traps Exhibit Antibacterial Activity against Burkholderia pseudomallei and Are Influenced by Bacterial and Host Factors

Infection and Immunity ◽

10.1128/iai.00806-12 ◽

2012 ◽

Vol 80 (11) ◽

pp. 3921-3929 ◽

Cited By ~ 51

Author(s):

Donporn Riyapa ◽

Surachat Buddhisa ◽

Sunee Korbsrisate ◽

Jon Cuccui ◽

Brendan W. Wren ◽

...

Keyword(s):

Burkholderia Pseudomallei ◽

Capsular Polysaccharide ◽

Neutrophil Extracellular Traps ◽

Polymorphonuclear Neutrophils ◽

Dependent Manner ◽

Predisposing Factor ◽

Bacterial Killing ◽

Content Type ◽

Extracellular Traps ◽

Type Iii Protein Secretion

ABSTRACTBurkholderia pseudomalleiis the causative pathogen of melioidosis, of which a major predisposing factor is diabetes mellitus. Polymorphonuclear neutrophils (PMNs) kill microbes extracellularly by the release of neutrophil extracellular traps (NETs). PMNs play a key role in the control of melioidosis, but the involvement of NETs in killing ofB. pseudomalleiremains obscure. Here, we showed that bactericidal NETs were released from human PMNs in response toB. pseudomalleiin a dose- and time-dependent manner.B. pseudomallei-induced NET formation required NADPH oxidase activation but not phosphatidylinositol-3 kinase, mitogen-activated protein kinases, or Src family kinase signaling pathways.B. pseudomalleimutants defective in the virulence-associated Bsa type III protein secretion system (T3SS) or capsular polysaccharide I (CPS-I) induced elevated levels of NETs. NET induction by such mutants was associated with increased bacterial killing, phagocytosis, and oxidative burst by PMNs. Taken together the data imply that T3SS and the capsule may play a role in evading the induction of NETs. Importantly, PMNs from diabetic subjects released NETs at a lower level than PMNs from healthy subjects. Modulation of NET formation may therefore be associated with the pathogenesis and control of melioidosis.

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Capsular polysaccharides from Cryptococcus neoformans modulate production of neutrophil extracellular traps (NETs) by human neutrophils

Scientific Reports ◽

10.1038/srep08008 ◽

2015 ◽

Vol 5 (1) ◽

Cited By ~ 51

Author(s):

Juliana D. B. Rocha ◽

Michelle T. C. Nascimento ◽

Debora Decote-Ricardo ◽

Suzana Côrte-Real ◽

Alexandre Morrot ◽

...

Keyword(s):

Cryptococcus Neoformans ◽

Neutrophil Extracellular Traps ◽

Human Neutrophils ◽

Capsular Polysaccharides ◽

Extracellular Traps

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Control of Mucosal Candidiasis in the Zebrafish Swim Bladder Depends on Neutrophils That Block Filament Invasion and Drive Extracellular-Trap Production

Infection and Immunity ◽

10.1128/iai.00276-17 ◽

2017 ◽

Vol 85 (9) ◽

Cited By ~ 17

Author(s):

Remi L. Gratacap ◽

Allison K. Scherer ◽

Brittany G. Seman ◽

Robert T. Wheeler

Keyword(s):

Invasive Disease ◽

Extracellular Dna ◽

Epithelial Barrier ◽

Infection Model ◽

Swim Bladder ◽

Spatiotemporal Resolution ◽

Zebrafish Model ◽

Content Type ◽

Mucosal Candidiasis ◽

Disease Pressure

ABSTRACT Candida albicans is a ubiquitous mucosal commensal that is normally prevented from causing acute or chronic invasive disease. Neutrophils contribute to protection in oral infection but exacerbate vulvovaginal candidiasis. To dissect the role of neutrophils during mucosal candidiasis, we took advantage of a new, transparent zebrafish swim bladder infection model. Intravital microscopic tracking of individual animals revealed that the blocking of neutrophil recruitment leads to rapid mortality in this model through faster disease progression. Conversely, artificial recruitment of neutrophils during early infection reduces disease pressure. Noninvasive longitudinal tracking showed that mortality is a consequence of C. albicans breaching the epithelial barrier and invading surrounding tissues. Accordingly, we found that a hyperfilamentous C. albicans strain breaches the epithelial barrier more frequently and causes mortality in immunocompetent zebrafish. A lack of neutrophils at the infection site is associated with less fungus-associated extracellular DNA and less damage to fungal filaments, suggesting that neutrophil extracellular traps help to protect the epithelial barrier from C. albicans breach. We propose a homeostatic model where C. albicans disease pressure is balanced by neutrophil-mediated damage of fungi, maintaining this organism as a commensal while minimizing the risk of damage to host tissue. The unequaled ability to dissect infection dynamics at a high spatiotemporal resolution makes this zebrafish model a unique tool for understanding mucosal host-pathogen interactions.

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