Heterogeneous Expression of the Virulence-Related Adhesin Epa1 between Individual Cells and Strains of the Pathogen Candida glabrata

ABSTRACTWe investigated the relevance of gene expression heterogeneity to virulence properties of a major fungal pathogen,Candida glabrata. The organism's key virulence-associated factors include glycosylphosphatidylinositol-anchored adhesins, encoded subtelomerically by theEPAgene family. Individual-cell analyses of expression revealed very striking heterogeneity for Epa1, an adhesin that mediates ∼95% of adherence to epithelial cellsin vitro. The heterogeneity in Epa1 was markedly greater than that known for other yeast genes. Sorted cells expressing high or low levels of Epa1 exhibited high and low adherence to epithelial cells, indicating a link between gene expression noise and potential virulence. The phenotypes of sorted subpopulations reverted to mixed phenotypes within a few generations. Variation in single-cell Epa1 protein and mRNA levels was correlated, consistent with transcriptional regulation of heterogeneity. Sir-dependent transcriptional silencing was the primary mechanism driving heterogeneous Epa1 expression inC. glabrataBG2, but not in CBS138 (ATCC 2001). Inefficient silencing in the latter strain was not due to a difference inEPA1sequence or (sub)telomere length and was overcome by ectopicSIR3expression. Moreover, differences between strains in the silencing dependence ofEPA1expression were evident across a range of clinical isolates, with heterogeneity being the greatest in strains whereEPA1was subject to silencing. The study shows how heterogeneity can impact the virulence-related properties ofC. glabratacell populations, with potential implications for microbial pathogenesis more broadly.

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Upregulation of the Adhesin Gene EPA1 Mediated by PDR1 in Candida glabrata Leads to Enhanced Host Colonization

mSphere ◽

10.1128/msphere.00065-15 ◽

2016 ◽

Vol 1 (2) ◽

Cited By ~ 19

Author(s):

Luis A. Vale-Silva ◽

Beat Moeckli ◽

Riccardo Torelli ◽

Brunella Posteraro ◽

Maurizio Sanguinetti ◽

...

Keyword(s):

Drug Resistance ◽

Epithelial Cells ◽

Candida Glabrata ◽

Resistance Mechanism ◽

Cell Types ◽

Host Cells ◽

Regulation Mechanism ◽

Content Type

ABSTRACT Candida glabrata is an important fungal pathogen in human diseases and is also rapidly acquiring drug resistance. Drug resistance can be mediated by the transcriptional activator PDR1, and this results in the upregulation of multidrug transporters. Intriguingly, this resistance mechanism is associated in C. glabrata with increased virulence in animal models and also with increased adherence to specific host cell types. The C. glabrata adhesin gene EPA1 is a major contributor of virulence and adherence to host cells. Here, we show that EPA1 expression is controlled by PDR1 independently of subtelomeric silencing, a known EPA1 regulation mechanism. Thus, a relationship exists between PDR1, EPA1 expression, and adherence to host cells, which is critical for efficient virulence. Our results demonstrate that acquisition of drug resistance is beneficial for C. glabrata in fungus-host relationships. These findings further highlight the challenges of the therapeutic management of C. glabrata infections in human patients. Candida glabrata is the second most common Candida species causing disseminated infection, after C. albicans. C. glabrata is intrinsically less susceptible to the widely used azole antifungal drugs and quickly develops secondary resistance. Resistance typically relies on drug efflux with transporters regulated by the transcription factor Pdr1. Gain-of-function (GOF) mutations in PDR1 lead to a hyperactive state and thus efflux transporter upregulation. Our laboratory has characterized a collection of C. glabrata clinical isolates in which azole resistance was found to correlate with increased virulence in vivo. Contributing phenotypes were the evasion of adhesion and phagocytosis by macrophages and an increased adhesion to epithelial cells. These phenotypes were found to be dependent on PDR1 GOF mutation and/or C. glabrata strain background. In the search for the molecular effectors, we found that PDR1 hyperactivity leads to overexpression of specific cell wall adhesins of C. glabrata. Further study revealed that EPA1 regulation, in particular, explained the increase in adherence to epithelial cells. Deleting EPA1 eliminates the increase in adherence in an in vitro model of interaction with epithelial cells. In a murine model of urinary tract infection, PDR1 hyperactivity conferred increased ability to colonize the bladder and kidneys in an EPA1-dependent way. In conclusion, this study establishes a relationship between PDR1 and the regulation of cell wall adhesins, an important virulence attribute of C. glabrata. Furthermore, our data show that PDR1 hyperactivity mediates increased adherence to host epithelial tissues both in vitro and in vivo through upregulation of the adhesin gene EPA1. IMPORTANCE Candida glabrata is an important fungal pathogen in human diseases and is also rapidly acquiring drug resistance. Drug resistance can be mediated by the transcriptional activator PDR1, and this results in the upregulation of multidrug transporters. Intriguingly, this resistance mechanism is associated in C. glabrata with increased virulence in animal models and also with increased adherence to specific host cell types. The C. glabrata adhesin gene EPA1 is a major contributor of virulence and adherence to host cells. Here, we show that EPA1 expression is controlled by PDR1 independently of subtelomeric silencing, a known EPA1 regulation mechanism. Thus, a relationship exists between PDR1, EPA1 expression, and adherence to host cells, which is critical for efficient virulence. Our results demonstrate that acquisition of drug resistance is beneficial for C. glabrata in fungus-host relationships. These findings further highlight the challenges of the therapeutic management of C. glabrata infections in human patients.

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The Ferredoxin-Like Protein FerR Regulates PrbP Activity inLiberibacter asiaticus

Applied and Environmental Microbiology ◽

10.1128/aem.02605-18 ◽

2018 ◽

Vol 85 (4) ◽

Cited By ~ 1

Author(s):

Lei Pan ◽

Danilo da Silva ◽

Fernando A. Pagliai ◽

Natalie A. Harrison ◽

Claudio F. Gonzalez ◽

...

Keyword(s):

Gene Expression ◽

Rna Polymerase ◽

Regulation Of Gene Expression ◽

Accessory Protein ◽

Mrna Levels ◽

Plant Host ◽

Content Type ◽

Environmental Signals ◽

Liberibacter Asiaticus

ABSTRACTInLiberibacter asiaticus, PrbP is an important transcriptional accessory protein that regulates gene expression through interactions with the RNA polymerase β-subunit and a specific sequence on the promoter region. The constitutive expression ofprbPobserved upon chemical inactivation of PrbP-DNA interactionsin vivoindicated that the expression ofprbPwas not autoregulated at the level of transcription. This observation suggested that a modulatory mechanism via protein-protein interactions may be involved.In silicogenome association analysis identified FerR (CLIBASIA_01505), a putative ferredoxin-like protein, as a PrbP-interacting protein. Using a bacterial two-hybrid system and immunoprecipitation assays, interactions between PrbP and FerR were confirmed.In vitrotranscription assays were used to show that FerR can increase the activity of PrbP by 16-fold when present in the PrbP-RNA polymerase reaction mixture. The FerR protein-protein interaction surface was predicted by structural modeling and followed by site-directed mutagenesis. Amino acids V20, V23, and C40 were identified as the most important residues in FerR involved in the modulation of PrbP activityin vitro. The regulatory mechanism of FerR abundance was examined at the transcription level. In contrast toprbPofL. asiaticus(prbPLas), mRNA levels offerRofL. asiaticus(ferRLas) are induced by an increase in osmotic pressure. The results of this study revealed that the activity of the transcriptional activator PrbPLasis modulated via interactions with FerRLas. The induction offerRLasexpression by osmolarity provides insight into the mechanisms of adjusting gene expression in response to host environmental signals inL. asiaticus.IMPORTANCEThe rapid spread and aggressive progression of huanglongbing (HLB) in the major citrus-producing areas have raised global recognition of and vigilance to this disease. As a result, the causative agent,Liberibacter asiaticus, has been investigated from various perspectives. However, gene expression regulatory mechanisms that are important for the survival and persistence of this intracellular pathogen remain largely unexplored. PrbP is a transcriptional accessory protein important forL. asiaticussurvival in the plant host. In this study, we investigated the interactions between PrbP inL. asiaticus(PrbPLas) and a ferredoxin-like protein (FerR) inL. asiaticus, FerRLas. We show that the presence of FerR stabilizes and augments the activity of PrbPLas. In addition, we demonstrate that the expression offerRis induced by increases in osmolarity inLiberibacter crescens. Altogether, these results suggest that FerRLasand PrbPLasmay play important roles in the regulation of gene expression in response to changing environmental signals duringL. asiaticusinfection in the citrus host.

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In Vitro Activity of Ibrexafungerp, a Novel Glucan Synthase Inhibitor against Candida glabrata Isolates with FKS Mutations

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01692-19 ◽

2019 ◽

Vol 63 (11) ◽

Cited By ~ 8

Author(s):

Natalie S. Nunnally ◽

Kizee A. Etienne ◽

David Angulo ◽

Shawn R. Lockhart ◽

Elizabeth L. Berkow

Keyword(s):

Candida Glabrata ◽

Vitro Activity ◽

Good Activity ◽

In Vitro Activity ◽

Broth Microdilution ◽

Glucan Synthase ◽

Content Type ◽

Synthase Inhibitor

ABSTRACT Ibrexafungerp is a first-in-class glucan synthase inhibitor. In vitro activity was determined for 89 Candida glabrata isolates with molecularly identified FKS1 or FKS2 mutations conferring resistance to the echinocandins. All isolates were resistant to at least one echinocandin (i.e., anidulafungin, caspofungin, or micafungin) by broth microdilution. Results for ibrexafungerp were compared with those for each echinocandin. Ibrexafungerp had good activity against all echinocandin-resistant C. glabrata isolates.

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Discovery of a Novel Class of Orally Active Antifungal β-1,3-d-Glucan Synthase Inhibitors

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00432-11 ◽

2011 ◽

Vol 55 (11) ◽

pp. 5099-5106 ◽

Cited By ~ 38

Author(s):

Scott S. Walker ◽

Yiming Xu ◽

Ilias Triantafyllou ◽

Michelle F. Waldman ◽

Cara Mendrick ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Candida Glabrata ◽

Pharmacokinetic Data ◽

Morphological Response ◽

Glucan Synthase ◽

Content Type ◽

Safety Issues ◽

Spontaneous Mutants ◽

Orally Active

ABSTRACTThe echinocandins are a class of semisynthetic natural products that target β-1,3-glucan synthase (GS). Their proven clinical efficacy combined with minimal safety issues has made the echinocandins an important asset in the management of fungal infection in a variety of patient populations. However, the echinocandins are delivered only parenterally. A screen for antifungal bioactivities combined with mechanism-of-action studies identified a class of piperazinyl-pyridazinones that target GS. The compounds exhibitedin vitroactivity comparable, and in some cases superior, to that of the echinocandins. The compounds inhibit GSin vitro, and there was a strong correlation between enzyme inhibition andin vitroantifungal activity. In addition, like the echinocandins, the compounds caused a leakage of cytoplasmic contents from yeast and produced a morphological response in molds characteristic of GS inhibitors. Spontaneous mutants ofSaccharomyces cerevisiaewith reduced susceptibility to the piperazinyl-pyridazinones had substitutions inFKS1. The sites of these substitutions were distinct from those conferring resistance to echinocandins; likewise, echinocandin-resistant isolates remained susceptible to the test compounds. Finally, we present efficacy and pharmacokinetic data on an example of the piperazinyl-pyridazinone compounds that demonstrated efficacy in a murine model ofCandida glabratainfection.

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Nitric oxide increases IL-8 gene transcription and mRNA stability to enhance IL-8 gene expression in lung epithelial cells

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00165.2004 ◽

2004 ◽

Vol 287 (4) ◽

pp. L764-L773 ◽

Cited By ~ 34

Author(s):

Loretta Sparkman ◽

Vijayakumar Boggaram

Keyword(s):

Gene Expression ◽

Nitric Oxide ◽

Protein Kinase ◽

Epithelial Cells ◽

Gene Transcription ◽

Mrna Stability ◽

Inflammatory Responses ◽

Lung Epithelial Cells ◽

Mrna Levels ◽

Lung Epithelial

Interleukin (IL)-8, a C-X-C chemokine, is a potent chemoattractant and an activator for neutrophils, T cells, and other immune cells. The airway and respiratory epithelia play important roles in the initiation and modulation of inflammatory responses via production of cytokines and surfactant. The association between elevated levels of nitric oxide (NO) and IL-8 in acute lung injury associated with sepsis, acute respiratory distress syndrome, respiratory syncytial virus infection in infants, and other inflammatory diseases suggested that NO may play important roles in the control of IL-8 gene expression in the lung. We investigated the role of NO in the control of IL-8 gene expression in H441 lung epithelial cells. We found that a variety of NO donors significantly induced IL-8 mRNA levels, and the increase in IL-8 mRNA was associated with an increase in IL-8 protein. NO induction of IL-8 mRNA was due to increases in IL-8 gene transcription and mRNA stability. NO induction of IL-8 mRNA levels was not inhibited by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one and KT-5823, inhibitors of soluble guanylate cyclase and protein kinase G, respectively, and 8-bromo-cGMP did not increase IL-8 mRNA levels. This indicated that NO induces IL-8 mRNA levels independently of changes in the intracellular cGMP levels. NO induction of IL-8 mRNA was significantly reduced by inhibitors of extracellular regulated kinase and protein kinase C. IL-8 induction by NO was also reduced by hydroxyl radical scavengers such as dimethyl sulfoxide and dimethylthiourea, indicating the involvement of hydroxyl radicals in the induction process. NO induction of IL-8 gene expression could be a significant contributing factor in the initiation and induction of inflammatory response in the respiratory epithelium.

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Differential Effects of Gonadotropin-Releasing Hormone (GnRH) Pulse Frequency on Gonadotropin Subunit and GnRH Receptor Messenger Ribonucleic Acid Levels in Vitro*

Endocrinology ◽

10.1210/endo.138.3.4968 ◽

1997 ◽

Vol 138 (3) ◽

pp. 1224-1231 ◽

Cited By ~ 112

Author(s):

Ursula B. Kaiser ◽

Andrzej Jakubowiak ◽

Anna Steinberger ◽

William W. Chin

Keyword(s):

Gene Expression ◽

Messenger Rna ◽

Pulse Frequency ◽

Gnrh Receptor ◽

Pituitary Cells ◽

Mrna Levels ◽

Subunit Gene ◽

Differential Effects ◽

Messenger Ribonucleic Acid

Abstract The hypothalamic hormone, GnRH, is released and transported to the anterior pituitary in a pulsatile manner, where it binds to specific high-affinity receptors and regulates gonadotropin biosynthesis and secretion. The frequency of GnRH pulses changes under various physiological conditions, and varying GnRH pulse frequencies have been shown to regulate differentially the secretion of LH and FSH and the expression of the gonadotropin α, LHβ, and FSHβ subunit genes in vivo. We demonstrate differential effects of varying GnRH pulse frequency in vitro in superfused primary monolayer cultures of rat pituitary cells. Cells were treated with 10 nm GnRH pulses for 24 h at a frequency of every 0.5, 1, 2, or 4 h. α, LHβ, and FSHβ messenger RNA (mRNA) levels were increased by GnRH at all pulse frequencies. α and LHβ mRNA levels and LH secretion were stimulated to the greatest extent at a GnRH pulse frequency of every 30 min, whereas FSHβ mRNA levels and FSH secretion were stimulated maximally at a lower GnRH pulse frequency, every 2 h. GnRH receptor (GnRHR) mRNA levels also were increased by GnRH at all pulse frequencies and were stimulated maximally at a GnRH pulse frequency of every 30 min. Similar results were obtained when the dose of each pulse of GnRH was adjusted to maintain a constant total cumulative dose of GnRH over 24 h. These data show that gonadotropin subunit gene expression is regulated differentially by varying GnRH pulse frequencies in vitro, suggesting that the differential effects of varying GnRH pulse frequencies on gonadotropin subunit gene expression occur directly at the level of the pituitary. The pattern of regulation of GnRHR mRNA levels correlated with that of α and LHβ but was different from that of FSHβ. This suggests that α and LHβ mRNA levels are maximally stimulated when GnRHR levels are relatively high, whereas FSHβ mRNA levels are maximally stimulated at lower levels of GnRHR expression, and that the mechanism for differential regulation of the gonadotropins by varying pulse frequencies of GnRH may involve levels of GnRHR. Furthermore, these data suggest that the mechanisms whereby varying GnRH pulse frequencies stimulate α, LHβ, and GnRHR gene expression are similar, whereas the stimulation of FSHβ mRNA levels may be different.

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A Dual Role for Oncostatin M Signaling in the Differentiation and Death of Mammary Epithelial Cells in Vivo

Molecular Endocrinology ◽

10.1210/me.2008-0097 ◽

2008 ◽

Vol 22 (12) ◽

pp. 2677-2688 ◽

Cited By ~ 45

Author(s):

Paul G. Tiffen ◽

Nader Omidvar ◽

Nuria Marquez-Almuina ◽

Dawn Croston ◽

Christine J. Watson ◽

...

Keyword(s):

Gene Expression ◽

Epithelial Cell ◽

Epithelial Cells ◽

Mammary Epithelial Cells ◽

Mammary Epithelial ◽

Oncostatin M ◽

Specific Gene ◽

Mammary Involution

Abstract Recent studies in breast cancer cell lines have shown that oncostatin M (OSM) not only inhibits proliferation but also promotes cell detachment and enhances cell motility. In this study, we have looked at the role of OSM signaling in nontransformed mouse mammary epithelial cells in vitro using the KIM-2 mammary epithelial cell line and in vivo using OSM receptor (OSMR)-deficient mice. OSM and its receptor were up-regulated approximately 2 d after the onset of postlactational mammary regression, in response to leukemia inhibitory factor (LIF)-induced signal transducer and activator of transcription-3 (STAT3). This resulted in sustained STAT3 activity, increased epithelial apoptosis, and enhanced clearance of epithelial structures during the remodeling phase of mammary involution. Concurrently, OSM signaling precipitated the dephosphorylation of STAT5 and repressed expression of the milk protein genes β-casein and whey acidic protein (WAP). Similarly, during pregnancy, OSM signaling suppressed β-casein and WAP gene expression. In vitro, OSM but not LIF persistently down-regulated phosphorylated (p)-STAT5, even in the continued presence of prolactin. OSM also promoted the expression of metalloproteinases MMP3, MMP12, and MMP14, which, in vitro, were responsible for OSM-specific apoptosis. Thus, the sequential activation of IL-6-related cytokines during mammary involution culminates in an OSM-dependent repression of epithelial-specific gene expression and the potentiation of epithelial cell extinction mediated, at least in part, by the reciprocal regulation of p-STAT5 and p-STAT3.

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Downregulation of the Central Noradrenergic System by Toxoplasma gondii Infection

Infection and Immunity ◽

10.1128/iai.00789-18 ◽

2018 ◽

Vol 87 (2) ◽

Cited By ~ 7

Author(s):

Isra Alsaady ◽

Ellen Tedford ◽

Mohammad Alsaad ◽

Greg Bristow ◽

Shivali Kohli ◽

...

Keyword(s):

Toxoplasma Gondii ◽

Infection Intensity ◽

Noradrenergic System ◽

Neural Cells ◽

Mrna Levels ◽

Female Rat ◽

Motor Impairments ◽

Content Type ◽

General Response

ABSTRACT Toxoplasma gondii is associated with physiological effects in the host. Dysregulation of catecholamines in the central nervous system has previously been observed in chronically infected animals. In the study described here, the noradrenergic system was found to be suppressed with decreased levels of norepinephrine (NE) in brains of infected animals and in infected human and rat neural cells in vitro. The mechanism responsible for the NE suppression was found to be downregulation of dopamine β-hydroxylase (DBH) gene expression, encoding the enzyme that synthesizes norepinephrine from dopamine, with downregulation observed in vitro and in infected brain tissue, particularly in the dorsal locus coeruleus/pons region. The downregulation was sex specific, with males expressing reduced DBH mRNA levels whereas females were unchanged. Rather, DBH expression correlated with estrogen receptor in the female rat brains for this estrogen-regulated gene. DBH silencing was not a general response of neurons to infection, as human cytomegalovirus did not downregulate DBH expression. The noradrenergic-linked behaviors of sociability and arousal were altered in chronically infected animals, with a high correlation between DBH expression and infection intensity. A decrease in DBH expression in noradrenergic neurons can elevate dopamine levels, which provides a possible explanation for mixed observations of changes in this neurotransmitter with infection. Decreased NE is consistent with the loss of coordination and motor impairments associated with toxoplasmosis. Further, the altered norepinephrine synthesis observed here may, in part, explain behavioral effects of infection and associations with mental illness.

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Antifungal Activity of SCY-078 and Standard Antifungal Agents against 178 Clinical Isolates of Resistant and Susceptible Candida Species

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01102-17 ◽

2017 ◽

Vol 61 (11) ◽

Cited By ~ 20

Author(s):

Wiley A. Schell ◽

A. M. Jones ◽

Katyna Borroto-Esoda ◽

Barbara D. Alexander

Keyword(s):

Candida Glabrata ◽

Antifungal Agents ◽

Candida Parapsilosis ◽

Candida Krusei ◽

Wild Type ◽

Content Type ◽

Clinical Resistance ◽

Excellent Activity ◽

Candida Lusitaniae

ABSTRACT SCY-078 in vitro activity was determined for 178 isolates of resistant or susceptible Candida albicans, Candida dubliniensis, Candida glabrata, Candida krusei, Candida lusitaniae, and Candida parapsilosis, including 44 Candida isolates with known genotypic (FKS1 or FKS2 mutations), phenotypic, or clinical resistance to echinocandins. Results were compared to those for anidulafungin, caspofungin, micafungin, fluconazole, and voriconazole. SCY-078 was shown to have excellent activity against both wild-type isolates and echinocandin- and azole-resistant isolates of Candida species.

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Eosinophil Deficiency Compromises Lung Defense against Aspergillus fumigatus

Infection and Immunity ◽

10.1128/iai.01172-13 ◽

2013 ◽

Vol 82 (3) ◽

pp. 1315-1325 ◽

Cited By ~ 45

Author(s):

Lauren M. Lilly ◽

Michaella Scopel ◽

Michael P. Nelson ◽

Ashley R. Burg ◽

Chad W. Dunaway ◽

...

Keyword(s):

Aspergillus Fumigatus ◽

Allergic Bronchopulmonary Aspergillosis ◽

Cell Contact ◽

Mrna Levels ◽

Colony Stimulating Factor ◽

Content Type ◽

Eosinophil Peroxidase ◽

Stimulating Factor ◽

Deficient Mice

ABSTRACTExposure to the moldAspergillus fumigatusmay result in allergic bronchopulmonary aspergillosis, chronic necrotizing pulmonary aspergillosis, or invasive aspergillosis (IA), depending on the host's immune status. Neutrophil deficiency is the predominant risk factor for the development of IA, the most life-threatening condition associated withA. fumigatusexposure. Here we demonstrate that in addition to neutrophils, eosinophils are an important contributor to the clearance ofA. fumigatusfrom the lung. AcuteA. fumigatuschallenge in normal mice induced the recruitment of CD11b+Siglec F+Ly-6GloLy-6CnegCCR3+eosinophils to the lungs, which was accompanied by an increase in lungEpx(eosinophil peroxidase) mRNA levels. Mice deficient in the transcription factor dblGATA1, which exhibit a selective deficiency in eosinophils, demonstrated impairedA. fumigatusclearance and evidence of germinating organisms in the lung. Higher burden correlated with lower mRNA expression ofEpx(eosinophil peroxidase) andPrg2(major basic protein) as well as lower interleukin 1β (IL-1β), IL-6, IL-17A, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and CXCL1 levels. However, examination of lung inflammatory cell populations failed to demonstrate defects in monocyte/macrophage, dendritic cell, or neutrophil recruitment in dblGATA1-deficient mice, suggesting that the absence of eosinophils in dlbGATA1-deficient mice was the sole cause of impaired lung clearance. We show that eosinophils generated from bone marrow have potent killing activity againstA. fumigtausin vitro, which does not require cell contact and can be recapitulated by eosinophil whole-cell lysates. Collectively, our data support a role for eosinophils in the lung response afterA. fumigatusexposure.

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