Candida albicans Hypha Formation and Mannan Masking of β-Glucan Inhibit Macrophage Phagosome Maturation

ABSTRACTCandida albicansis a major life-threatening human fungal pathogen in the immunocompromised host. Host defense against systemicCandidainfection relies heavily on the capacity of professional phagocytes of the innate immune system to ingest and destroy fungal cells. A number of pathogens, includingC. albicans, have evolved mechanisms that attenuate the efficiency of phagosome-mediated inactivation, promoting their survival and replication within the host. Here we visualize host-pathogen interactions using live-cell imaging and show that viable, but not heat- or UV-killedC. albicanscells profoundly delay phagosome maturation in macrophage cell lines and primary macrophages. The ability ofC. albicansto delay phagosome maturation is dependent on cell wall composition and fungal morphology. Loss of cell wallO-mannan is associated with enhanced acquisition of phagosome maturation markers, distinct changes in Rab GTPase acquisition by the maturing phagosome, impaired hyphal growth within macrophage phagosomes, profound changes in macrophage actin dynamics, and ultimately a reduced ability of fungal cells to escape from macrophage phagosomes. The loss of cell wallO-mannan leads to exposure of β-glucan in the inner cell wall, facilitating recognition by Dectin-1, which is associated with enhanced phagosome maturation.IMPORTANCEInnate cells engulf and destroy invading organisms by phagocytosis, which is essential for the elimination of fungal cells to protect against systemic life-threatening infections. Yet comparatively little is known about what controls the maturation of phagosomes following ingestion of fungal cells. We used live-cell microscopy and fluorescent protein reporter macrophages to understand howC. albicansviability, filamentous growth, and cell wall composition affect phagosome maturation and the survival of the pathogen within host macrophages. We have demonstrated that cell wall glycosylation and yeast-hypha morphogenesis are required for disruption of host processes that function to inactivate pathogens, leading to survival and escape of this fungal pathogen from within host phagocytes. The methods employed here are applicable to study interactions of other pathogens with phagocytic cells to dissect how specific microbial features impact different stages of phagosome maturation and the survival of the pathogen or host.

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Rab14 Regulates Maturation of Macrophage Phagosomes Containing the Fungal Pathogen Candida albicans and Outcome of the Host-Pathogen Interaction

Infection and Immunity ◽

10.1128/iai.02917-14 ◽

2015 ◽

Vol 83 (4) ◽

pp. 1523-1535 ◽

Cited By ~ 26

Author(s):

Blessing Okai ◽

Natalie Lyall ◽

Neil A. R. Gow ◽

Judith M. Bain ◽

Lars-Peter Erwig

Keyword(s):

Candida Albicans ◽

Fungal Pathogen ◽

Fluorescent Protein ◽

Temporal Dynamics ◽

Immune Defense ◽

Dominant Negative ◽

Phagosome Maturation ◽

Hyphal Length ◽

Content Type ◽

Green Fluorescent

Avoidance of innate immune defense is an important mechanism contributing to the pathogenicity of microorganisms. The fungal pathogenCandida albicansundergoes morphogenetic switching from the yeast to the filamentous hyphal form following phagocytosis by macrophages, facilitating its escape from the phagosome, which can result in host cell lysis. We show that the intracellular host trafficking GTPase Rab14 plays an important role in protecting macrophages from lysis mediated byC. albicanshyphae. Live-cell imaging of macrophages expressing green fluorescent protein (GFP)-tagged Rab14 or dominant negative Rab14, or with small interfering RNA (siRNA)-mediated knockdown of Rab14, revealed the temporal dynamics of this protein and its influence on the maturation of macrophage phagosomes following the engulfment ofC. albicanscells. Phagosomes containing liveC. albicanscells became transiently Rab14 positive within 2 min following engulfment. The duration of Rab14 retention on phagosomes was prolonged for hyphal cargo and was directly proportional to hyphal length. Interference with endogenous Rab14 did not affect the migration of macrophages towardC. albicanscells, the rate of engulfment, the overall uptake of fungal cells, or early phagosome processing. However, Rab14 depletion delayed the acquisition of the late phagosome maturation markers LAMP1 and lysosomal cathepsin, indicating delayed formation of a fully bioactive lysosome. This was associated with a significant increase in the level of macrophage killing byC. albicans. Therefore, Rab14 activity promotes phagosome maturation duringC. albicansinfection but is dysregulated on the phagosome in the presence of the invasive hyphal form, which favors fungal survival and escape.

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Melanin Externalization in Candida albicans Depends on Cell Wall Chitin Structures

Eukaryotic Cell ◽

10.1128/ec.00051-10 ◽

2010 ◽

Vol 9 (9) ◽

pp. 1329-1342 ◽

Cited By ~ 55

Author(s):

Claire A. Walker ◽

Beatriz L. Gómez ◽

Héctor M. Mora-Montes ◽

Kevin S. Mackenzie ◽

Carol A. Munro ◽

...

Keyword(s):

Cell Wall ◽

Candida Albicans ◽

Fungal Pathogen ◽

Cell Walls ◽

Chitin Synthesis ◽

Melanin Production ◽

Content Type ◽

Encoding Gene ◽

Chitinase Genes

ABSTRACT The fungal pathogen Candida albicans produces dark-pigmented melanin after 3 to 4 days of incubation in medium containing l-3,4-dihydroxyphenylalanine (l-DOPA) as a substrate. Expression profiling of C. albicans revealed very few genes significantly up- or downregulated by growth in l-DOPA. We were unable to determine a possible role for melanin in the virulence of C. albicans. However, we showed that melanin was externalized from the fungal cells in the form of electron-dense melanosomes that were free or often loosely bound to the cell wall exterior. Melanin production was boosted by the addition of N-acetylglucosamine to the medium, indicating a possible association between melanin production and chitin synthesis. Melanin externalization was blocked in a mutant specifically disrupted in the chitin synthase-encoding gene CHS2. Melanosomes remained within the outermost cell wall layers in chs3Δ and chs2Δ chs3Δ mutants but were fully externalized in chs8Δ and chs2Δ chs8Δ mutants. All the CHS mutants synthesized dark pigment at equivalent rates from mixed membrane fractions in vitro, suggesting it was the form of chitin structure produced by the enzymes, not the enzymes themselves, that was involved in the melanin externalization process. Mutants with single and double disruptions of the chitinase genes CHT2 and CHT3 and the chitin pathway regulator ECM33 also showed impaired melanin externalization. We hypothesize that the chitin product of Chs3 forms a scaffold essential for normal externalization of melanosomes, while the Chs8 chitin product, probably produced in cell walls in greater quantity in the absence of CHS2, impedes externalization.

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In Vivo Indicators of Cytoplasmic, Vacuolar, and Extracellular pH Using pHluorin2 in Candida albicans

mSphere ◽

10.1128/msphere.00276-17 ◽

2017 ◽

Vol 2 (4) ◽

Cited By ~ 9

Author(s):

Hélène Tournu ◽

Arturo Luna-Tapia ◽

Brian M. Peters ◽

Glen E. Palmer

Keyword(s):

Candida Albicans ◽

Intracellular Ph ◽

Fungal Pathogen ◽

Fluorescent Protein ◽

Extracellular Ph ◽

Adaptive Responses ◽

Ph Homeostasis ◽

Content Type ◽

A Cell

ABSTRACT Candida albicans is an opportunistic fungal pathogen that colonizes the reproductive and gastrointestinal tracts of its human host. It can also invade the bloodstream and deeper organs of immunosuppressed individuals, and thus it encounters enormous variations in external pH in vivo. Accordingly, survival within such diverse niches necessitates robust adaptive responses to regulate intracellular pH. However, the impact of antifungal drugs upon these adaptive responses, and on intracellular pH in general, is not well characterized. Furthermore, the tools and methods currently available to directly monitor intracellular pH in C. albicans, as well as other fungal pathogens, have significant limitations. To address these issues, we developed a new and improved set of pH sensors based on the pH-responsive fluorescent protein pHluorin. This includes a cytoplasmic sensor, a probe that localizes inside the fungal vacuole (an acidified compartment that plays a central role in intracellular pH homeostasis), and a cell surface probe that can detect changes in extracellular pH. These tools can be used to monitor pH within single C. albicans cells or in cell populations in real time through convenient and high-throughput assays. Environmental or chemically induced stresses often trigger physiological responses that regulate intracellular pH. As such, the capacity to detect pH changes in real time and within live cells is of fundamental importance to essentially all aspects of biology. In this respect, pHluorin, a pH-sensitive variant of green fluorescent protein, has provided an invaluable tool to detect such responses. Here, we report the adaptation of pHluorin2 (PHL2), a substantially brighter variant of pHluorin, for use with the human fungal pathogen Candida albicans. As well as a cytoplasmic PHL2 indicator, we describe a version that specifically localizes within the fungal vacuole, an acidified subcellular compartment with important functions in nutrient storage and pH homeostasis. In addition, by means of a glycophosphatidylinositol-anchored PHL2-fusion protein, we generated a cell surface pH sensor. We demonstrated the utility of these tools in several applications, including accurate intracellular and extracellular pH measurements in individual cells via flow cytometry and in cell populations via a convenient plate reader-based protocol. The PHL2 tools can also be used for endpoint as well as time course experiments and to conduct chemical screens to identify drugs that alter normal pH homeostasis. These tools enable observation of the highly dynamic intracellular pH shifts that occur throughout the fungal growth cycle, as well as in response to various chemical treatments. IMPORTANCE Candida albicans is an opportunistic fungal pathogen that colonizes the reproductive and gastrointestinal tracts of its human host. It can also invade the bloodstream and deeper organs of immunosuppressed individuals, and thus it encounters enormous variations in external pH in vivo. Accordingly, survival within such diverse niches necessitates robust adaptive responses to regulate intracellular pH. However, the impact of antifungal drugs upon these adaptive responses, and on intracellular pH in general, is not well characterized. Furthermore, the tools and methods currently available to directly monitor intracellular pH in C. albicans, as well as other fungal pathogens, have significant limitations. To address these issues, we developed a new and improved set of pH sensors based on the pH-responsive fluorescent protein pHluorin. This includes a cytoplasmic sensor, a probe that localizes inside the fungal vacuole (an acidified compartment that plays a central role in intracellular pH homeostasis), and a cell surface probe that can detect changes in extracellular pH. These tools can be used to monitor pH within single C. albicans cells or in cell populations in real time through convenient and high-throughput assays.

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F-Actin Dynamics in Neurospora crassa

Eukaryotic Cell ◽

10.1128/ec.00253-09 ◽

2010 ◽

Vol 9 (4) ◽

pp. 547-557 ◽

Cited By ~ 107

Author(s):

Adokiye Berepiki ◽

Alexander Lichius ◽

Jun-Ya Shoji ◽

Jens Tilsner ◽

Nick D. Read

Keyword(s):

Neurospora Crassa ◽

Live Cell Imaging ◽

Cell Imaging ◽

Fluorescent Protein ◽

Live Cell ◽

Actin Dynamics ◽

Actin Binding ◽

Content Type ◽

Actin Cables ◽

Germ Tubes

ABSTRACT This study demonstrates the utility of Lifeact for the investigation of actin dynamics in Neurospora crassa and also represents the first report of simultaneous live-cell imaging of the actin and microtubule cytoskeletons in filamentous fungi. Lifeact is a 17-amino-acid peptide derived from the nonessential Saccharomyces cerevisiae actin-binding protein Abp140p. Fused to green fluorescent protein (GFP) or red fluorescent protein (TagRFP), Lifeact allowed live-cell imaging of actin patches, cables, and rings in N. crassa without interfering with cellular functions. Actin cables and patches localized to sites of active growth during the establishment and maintenance of cell polarity in germ tubes and conidial anastomosis tubes (CATs). Recurrent phases of formation and retrograde movement of complex arrays of actin cables were observed at growing tips of germ tubes and CATs. Two populations of actin patches exhibiting slow and fast movement were distinguished, and rapid (1.2 μm/s) saltatory transport of patches along cables was observed. Actin cables accumulated and subsequently condensed into actin rings associated with septum formation. F-actin organization was markedly different in the tip regions of mature hyphae and in germ tubes. Only mature hyphae displayed a subapical collar of actin patches and a concentration of F-actin within the core of the Spitzenkörper. Coexpression of Lifeact-TagRFP and β-tubulin–GFP revealed distinct but interrelated localization patterns of F-actin and microtubules during the initiation and maintenance of tip growth.

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Pathways That Synthesize Phosphatidylethanolamine Impact Candida albicans Hyphal Length and Cell Wall Composition through Transcriptional and Posttranscriptional Mechanisms

Infection and Immunity ◽

10.1128/iai.00480-19 ◽

2019 ◽

Vol 88 (3) ◽

Author(s):

Robert N. Tams ◽

Andrew S. Wagner ◽

Joseph W. Jackson ◽

Eric R. Gann ◽

Timothy E. Sparer ◽

...

Keyword(s):

Cell Wall ◽

Candida Albicans ◽

Bloodstream Infections ◽

Cell Wall Composition ◽

Wild Type ◽

Hyphal Length ◽

Content Type ◽

Kennedy Pathway ◽

Overexpression Strain ◽

Cytidine Diphosphate

ABSTRACT Candida albicans is a leading cause of systemic bloodstream infections, and synthesis of the phospholipid phosphatidylethanolamine (PE) is required for virulence. The psd1Δ/Δ psd2Δ/Δ mutant, which cannot synthesize PE by the cytidine diphosphate diacylglycerol (CDP-DAG) pathway, is avirulent in the mouse model of systemic candidiasis. Similarly, an ept1Δ/Δ mutant, which cannot produce PE by the Kennedy pathway, exhibits decreased kidney fungal burden in systemically infected mice. Conversely, overexpression of EPT1 results in a hypervirulent phenotype in this model. Thus, mutations that increase PE synthesis increase virulence, and mutations that decrease PE synthesis decrease virulence. However, the mechanism by which virulence is regulated by PE synthesis is only partially understood. RNA sequencing was performed on strains with deficient or excessive PE biosynthesis to elucidate the mechanism. Decreased PE synthesis from loss of EPT1 or PSD1 and PSD2 leads to downregulation of genes that impact mitochondrial function. Losses of PSD1 and PSD2, but not EPT1, cause significant increases in transcription of glycosylation genes, which may reflect the substantial cell wall defects in the psd1Δ/Δ psd2Δ/Δ mutant. These accumulated defects could contribute to the decreased virulence observed for mutants with deficient PE synthesis. In contrast to mutants with decreased PE synthesis, there were no transcriptional differences between the EPT1 overexpression strain and the wild type, indicating that the hypervirulent phenotype is a consequence of posttranscriptional changes. It was found that overexpression of EPT1 causes increased chitin content and increased hyphal length. These phenotypes may help to explain the previously observed hypervirulence in the EPT1 overexpressor.

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Epitope Shaving Promotes Fungal Immune Evasion

mBio ◽

10.1128/mbio.00984-20 ◽

2020 ◽

Vol 11 (4) ◽

Cited By ~ 4

Author(s):

Delma S. Childers ◽

Gabriela Mol Avelar ◽

Judith M. Bain ◽

Arnab Pradhan ◽

Daniel E. Larcombe ◽

...

Keyword(s):

Cell Wall ◽

Candida Albicans ◽

Cell Surface ◽

Immune Evasion ◽

Fungal Pathogen ◽

Fungal Pathogens ◽

Specific Cell ◽

Immune Recognition ◽

Content Type ◽

Evasion Strategy

ABSTRACT The cell wall provides a major physical interface between fungal pathogens and their mammalian host. This extracellular armor is critical for fungal cell homeostasis and survival. Fungus-specific cell wall moieties, such as β-1,3-glucan, are recognized as pathogen-associated molecular patterns (PAMPs) that activate immune-mediated clearance mechanisms. We have reported that the opportunistic human fungal pathogen Candida albicans masks β-1,3-glucan following exposure to lactate, hypoxia, or iron depletion. However, the precise mechanism(s) by which C. albicans masks β-1,3-glucan has remained obscure. Here, we identify a secreted exoglucanase, Xog1, that is induced in response to lactate or hypoxia. Xog1 functions downstream of the lactate-induced β-glucan “masking” pathway to promote β-1,3-glucan “shaving.” Inactivation of XOG1 blocks most but not all β-1,3-glucan masking in response to lactate, suggesting that other activities contribute to this phenomenon. Nevertheless, XOG1 deletion attenuates the lactate-induced reductions in phagocytosis and cytokine stimulation normally observed for wild-type cells. We also demonstrate that the pharmacological inhibition of exoglucanases undermines β-glucan shaving, enhances the immune visibility of the fungus, and attenuates its virulence. Our study establishes a new mechanism underlying environmentally induced PAMP remodeling that can be manipulated pharmacologically to influence immune recognition and infection outcomes. IMPORTANCE The immune system plays a critical role in protecting us against potentially fatal fungal infections. However, some fungal pathogens have evolved evasion strategies that reduce the efficacy of our immune defenses. Previously, we reported that the fungal pathogen Candida albicans exploits specific host-derived signals (such as lactate and hypoxia) to trigger an immune evasion strategy that involves reducing the exposure of β-glucan at its cell surface. Here, we show that this phenomenon is mediated by the induction of a major secreted exoglucanase (Xog1) by the fungus in response to these host signals. Inactivating XOG1-mediated “shaving” of cell surface-exposed β-glucan enhances immune responses against the fungus. Furthermore, inhibiting exoglucanase activity pharmacologically attenuates C. albicans virulence. In addition to revealing the mechanism underlying a key immune evasion strategy in a major fungal pathogen of humans, our work highlights the potential therapeutic value of drugs that block fungal immune evasion.

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Hypoxia Promotes Immune Evasion by Triggering β-Glucan Masking on theCandida albicansCell Surface via Mitochondrial and cAMP-Protein Kinase A Signaling

mBio ◽

10.1128/mbio.01318-18 ◽

2018 ◽

Vol 9 (6) ◽

Cited By ~ 38

Author(s):

Arnab Pradhan ◽

Gabriela M. Avelar ◽

Judith M. Bain ◽

Delma S. Childers ◽

Daniel E. Larcombe ◽

...

Keyword(s):

Cell Wall ◽

Candida Albicans ◽

Protein Kinase ◽

Immune Evasion ◽

Fungal Pathogen ◽

Wall Structure ◽

Adaptation To Hypoxia ◽

Low Oxygen ◽

Content Type ◽

Kinase A

ABSTRACTOrganisms must adapt to changes in oxygen tension if they are to exploit the energetic benefits of reducing oxygen while minimizing the potentially damaging effects of oxidation. Consequently, organisms in all eukaryotic kingdoms display robust adaptation to hypoxia (low oxygen levels). This is particularly important for fungal pathogens that colonize hypoxic niches in the host. We show that adaptation to hypoxia in the major fungal pathogen of humansCandida albicansincludes changes in cell wall structure and reduced exposure, at the cell surface, of β-glucan, a key pathogen-associated molecular pattern (PAMP). This leads to reduced phagocytosis by murine bone marrow-derived macrophages and decreased production of IL-10, RANTES, and TNF-α by peripheral blood mononuclear cells, suggesting that hypoxia-induced β-glucan masking has a significant effect uponC. albicans-host interactions. We show that hypoxia-induced β-glucan masking is dependent upon both mitochondrial and cAMP-protein kinase A (PKA) signaling. The decrease in β-glucan exposure is blocked by mutations that affect mitochondrial functionality (goa1Δ andupc2Δ) or that decrease production of hydrogen peroxide in the inner membrane space (sod1Δ). Furthermore, β-glucan masking is enhanced by mutations that elevate mitochondrial reactive oxygen species (aox1Δ). The β-glucan masking defects displayed bygoa1Δ andupc2Δ cells are suppressed by exogenous dibutyryl-cAMP. Also, mutations that inactivate cAMP synthesis (cyr1Δ) or PKA (tpk1Δtpk2Δ) block the masking phenotype. Our data suggest thatC. albicansresponds to hypoxic niches by inducing β-glucan masking via a mitochondrial cAMP-PKA signaling pathway, thereby modulating local immune responses and promoting fungal colonization.IMPORTANCEAnimal, plant, and fungal cells occupy environments that impose changes in oxygen tension. Consequently, many species have evolved mechanisms that permit robust adaptation to these changes. The fungal pathogenCandida albicanscan colonize hypoxic (low oxygen) niches in its human host, such as the lower gastrointestinal tract and inflamed tissues, but to colonize its host, the fungus must also evade local immune defenses. We reveal, for the first time, a defined link between hypoxic adaptation and immune evasion inC. albicans. As this pathogen adapts to hypoxia, it undergoes changes in cell wall structure that include masking of β-glucan at its cell surface, and it becomes better able to evade phagocytosis by innate immune cells. We also define the signaling mechanisms that mediate hypoxia-induced β-glucan masking, showing that they are dependent on mitochondrial signaling and the cAMP-protein kinase pathway. Therefore, hypoxia appears to trigger immune evasion in this fungal pathogen.

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SpRY Cas9 Can Utilize a Variety of Protospacer Adjacent Motif Site Sequences To Edit the Candida albicans Genome

mSphere ◽

10.1128/msphere.00303-21 ◽

2021 ◽

Vol 6 (3) ◽

Author(s):

Ben A. Evans ◽

Douglas A. Bernstein

Keyword(s):

Candida Albicans ◽

Genome Editing ◽

Fungal Pathogen ◽

Wild Type ◽

Content Type ◽

Human Fungal Pathogen ◽

Protospacer Adjacent Motif ◽

Motif Site ◽

Life Threatening ◽

Genomic Locations

ABSTRACT Candida albicans is a human fungal pathogen capable of causing life-threatening infections. The ability to edit the C. albicans genome using CRISPR/Cas9 is an important tool investigators can leverage in their search for novel therapeutic targets. However, wild-type Cas9 requires an NGG protospacer adjacent motif (PAM), leaving many AT-rich regions of DNA inaccessible. A recently described near-PAMless CRISPR system that utilizes the SpRY Cas9 variant can target non-NGG PAM sequences. Using this system as a model, we developed C. albicans CRISPR/SpRY. We tested our system by mutating C. albicans ADE2 and show that CRISPR/SpRY can utilize non-NGG PAM sequences in C. albicans. Our CRISPR/SpRY system will allow researchers to efficiently modify C. albicans DNA that lacks NGG PAM sequences. IMPORTANCE Genetic modification of the human fungal pathogen Candida albicans allows us to better understand how fungi differ from humans at the molecular level and play essential roles in the development of therapeutics. CRISPR/Cas9-mediated genome editing systems can be used to introduce site-specific mutations to C. albicans. However, wild-type Cas9 is limited by the requirement of an NGG PAM site. CRISPR/SpRY targets a variety of different PAM sequences. We modified the C. albicans CRISPR/Cas9 system using the CRISPR/SpRY as a guide. We tested CRISPR/SpRY on C. albicans ADE2 and show that our SpRY system can facilitate genome editing independent of an NGG PAM sequence, thus allowing the investigator to target AT-rich sequences. Our system will potentially enable mutation of the 125 C. albicans genes which have been previously untargetable with CRISPR/Cas9. Additionally, our system will allow for precise targeting of many genomic locations that lack NGG PAM sites.

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Candida albicans ENT2 Contributes to Efficient Endocytosis, Cell Wall Integrity, Filamentation, and Virulence

mSphere ◽

10.1128/msphere.00707-21 ◽

2021 ◽

Author(s):

Christiane Rollenhagen ◽

Harrison Agyeman ◽

Susan Eszterhas ◽

Samuel A. Lee

Keyword(s):

Cell Wall ◽

Candida Albicans ◽

Fungal Pathogen ◽

Clinical Importance ◽

Hospitalized Patients ◽

Cell Wall Integrity ◽

Content Type ◽

Invasive Infections ◽

Opportunistic Fungal Pathogen ◽

Considerable Morbidity

The opportunistic fungal pathogen Candida albicans is an important cause of invasive infections in hospitalized patients and a source of considerable morbidity and mortality. Despite its clinical importance, we still need to improve our ability to diagnose and treat this common pathogen.

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The MARVEL Domain Protein Nce102 Regulates Actin Organization and Invasive Growth of Candida albicans

mBio ◽

10.1128/mbio.00723-13 ◽

2013 ◽

Vol 4 (6) ◽

Cited By ~ 19

Author(s):

Lois M. Douglas ◽

Hong X. Wang ◽

James B. Konopka

Keyword(s):

Cell Wall ◽

Candida Albicans ◽

Plasma Membrane ◽

Virulence Factors ◽

Fungal Pathogen ◽

Invasive Growth ◽

Cell Wall Synthesis ◽

Content Type ◽

Actin Organization ◽

Domain Protein

ABSTRACTInvasive growth of the fungal pathogenCandida albicansinto tissues promotes disseminated infections in humans. The plasma membrane is essential for pathogenesis because this important barrier mediates morphogenesis and invasive growth, as well as secretion of virulence factors, cell wall synthesis, nutrient import, and other processes. Previous studies showed that the Sur7 tetraspan protein that localizes to MCC (membrane compartment occupied by Can1)/eisosome subdomains of the plasma membrane regulates a broad range of key functions, including cell wall synthesis, morphogenesis, and resistance to copper. Therefore, a distinct tetraspan protein found in MCC/eisosomes, Nce102, was investigated. Nce102 belongs to the MARVEL domain protein family, which is implicated in regulating membrane structure and function. Deletion ofNCE102did not cause the broad defects seen insur7Δcells. Instead, thence102Δmutant displayed a unique phenotype in that it was defective in forming hyphae and invading low concentrations of agar but could invade well in higher agar concentrations. This phenotype was likely due to a defect in actin organization that was observed by phalloidin staining. In support of this, the invasive growth defect of abni1Δmutant that mislocalizes actin due to lack of the Bni1 formin was also reversed at high agar concentrations. This suggests that a denser matrix provides a signal that compensates for the actin defects. Thence102Δmutant displayed decreased virulence and formed abnormal hyphae in mice. These studies identify novel ways that Nce102 and the physical environment surroundingC. albicansregulate morphogenesis and pathogenesis.IMPORTANCEThe plasma membrane promotes virulence of the human fungal pathogenCandida albicansby acting as a protective barrier around the cell and mediating dynamic activities, such as morphogenesis, cell wall synthesis, secretion of virulence factors, and nutrient uptake. To better understand how the plasma membrane contributes to virulence, we analyzed a set of eight genes encoding MARVEL family proteins that are predicted to function in membrane organization. Interestingly, deletion of one gene,NCE102, caused a strong defect in formation of invasive hyphal growthin vitroand decreased virulence in mice. Thence102Δmutant cells showed defects in actin organization that underlie the morphogenesis defect, since mutation of a known regulator of actin organization caused a similar defect. These studies identify a novel way in which the plasma membrane regulates the actin cytoskeleton and contributes to pathogenesis.

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