Glycosylation and Immunoreactivity of the Histoplasma capsulatum Cfp4 Yeast-Phase Exoantigen

Holbrook, Eric D.; Kemski, Megan M.; Richer, Sarah M.; Wheat, L. Joseph; Rappleye, Chad A.

doi:10.1128/iai.01893-14

Cited by 13 publications

(13 citation statements)

References 43 publications

(50 reference statements)

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“…We previously observed that many known virulence factors are Ryp-associated [2]; consistent with this observation, the 16 Ryp-associated yeast-specific genes include 2 virulence factors, CBP1 [21–24] and CTR3 [25], as well as two genes required for efficient lysis of macrophages, LDF1 and a gene previously designated as UA35-G3 [26]. Several others are putative secreted factors of unknown function, including the paralogs Yps21 [27] and GH17/Cfp4 [28, 29] as well as ucsf.hc_01.G217B.05476 which, along with UA35-G3, is predicted to be a secreted knottin (cystine knot proteins that show yeast-specific expression and may play a role in virulence in H. capsulatum ) [4]. Notably, these yeast-associated genes, which are normally expressed by wild-type yeast cells at 37°C, are expressed in the yeast-locked msb2 mutant even at room temperature, suggesting that their expression can be unlinked to temperature but remains linked to cell morphology.…”

Section: Resultsmentioning

confidence: 90%

Opposing signaling pathways regulate morphology in response to temperature in the fungal pathogenHistoplasma capsulatum

Rodriguez

Voorhies

Gilmore

et al. 2019

Preprint

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19 20 21 22 23 2 24 25 ABSTRACT26 Phenotypic switching between two opposing cellular states is a fundamental aspect of biology, 27 and fungi provide facile systems to analyze the interactions between regulons that control this 28 type of switch. A long-standing mystery in fungal pathogens of humans is how thermally 29 dimorphic fungi switch their developmental form in response to temperature. These fungi, 30 including the subject of this study, Histoplasma capsulatum, are temperature-responsive 31 organisms that utilize unknown regulatory pathways to couple their cell shape and associated 32 attributes to the temperature of their environment. H. capsulatum grows as a multicellular hypha 33 in the soil that switches to a pathogenic yeast form in response to the temperature of a 34 mammalian host. These states can be triggered in the laboratory simply by growing the fungus 35 either at room temperature (where it grows as hyphae) or at 37ºC (where it grows as yeast). Prior 36 worked revealed that 15-20% of transcripts are differentially expressed in response to 37 temperature, but it is unclear which transcripts are linked to specific phenotypic changes such as 38 cell morphology or virulence. To elucidate temperature-responsive regulons, we previously 39 identified four transcription factors (Ryp1-4) that are required for yeast-phase growth at 37ºC; in 40 each ryp mutant, the fungus grows constitutively as hyphae regardless of temperature and the 41 cells fail to express genes that are normally induced in response to growth at 37ºC. Here we 42 perform the first genetic screen to identify genes required for hyphal growth of H. capsulatum at 43 room temperature and find that disruption of the signaling mucin MSB2 results in a yeast-locked 44 phenotype. RNAseq experiments reveal that MSB2 is not required for the majority of gene 45 expression changes that occur when cells are shifted to room temperature. However, a small 46 subset of temperature-responsive genes is dependent on MSB2 for its expression, thereby 3 47 implicating these genes in the process of filamentation. Disruption or knockdown of an Msb2-48 dependent MAP kinase (HOG2) and an APSES transcription factor (STU1) prevents hyphal 49 growth at room temperature, validating that the Msb2 regulon contains genes that control 50 filamentation. Notably, the Msb2 regulon shows conserved hyphal-specific expression in other 51 dimorphic fungi, suggesting that this work defines a small set of genes that are likely to be 52 conserved regulators and effectors of filamentation in multiple fungi. In contrast, a few yeast-53 specific transcripts, including virulence factors that are normally expressed only at 37ºC, are 54 inappropriately expressed at room temperature in the msb2 mutant, suggesting that expression of 55 these genes is coupled to growth in the yeast form rather than to temperature. Finally, we find 56 that the yeast-promoting transcription factor Ryp3 associates with the MSB2 promoter and 57 inhibits MSB2 transcript expression at 37ºC, whereas Msb2 inhibits...

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Section: Resultsmentioning

confidence: 90%

Opposing signaling pathways regulate morphology in response to temperature in the fungal pathogenHistoplasma capsulatum

Rodriguez

Voorhies

Gilmore

et al. 2019

Preprint

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“…To confirm the function of Histoplasma Pmt proteins in protein glycosylation, secreted proteins were examined for the loss of protein glycosylation by reduction of protein molecular mass. The glycoprotein Cfp4 ( 8 , 21 ) and β-glucanases Eng1 and Exg8 ( 8 ) contain mucin-like domains in the primary amino acid sequence, suggestive of regions potentially modified by O-linked mannosylation ( 22 ). Consistent with O-mannosylation of Cfp4, the electrophoretic mobility of the Cfp4 protein is reduced by approximately 2 kDa when produced by Pmt2-deficient yeast cells, and the higher molecular mass is restored by PMT2 complementation ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Culture filtrate proteins from wild-type ( PMT2 ) or mutant ( pmt2 ) yeast cells were treated with PNGase F (New England Biolabs) to remove N-linked glycans and then separated under reducing conditions by 10% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Proteins were detected with monoclonal antibodies to Cfp4 (clone 2D20) ( 21 ) and Sod3 (clone 3J23) and visualized with horseradish peroxidase (HRP)-conjugated anti-mouse antibody and HRP chemiluminescent substrate (Millipore).…”

Section: Methodsmentioning

confidence: 99%

O-Mannosylation of Proteins Enables Histoplasma Yeast Survival at Mammalian Body Temperatures

Garfoot

Goughenour

Wüthrich

et al. 2018

mBio

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The ability to grow at mammalian body temperatures is critical for pathogen infection of humans. For the thermally dimorphic fungal pathogen Histoplasma capsulatum, elevated temperature is required for differentiation of mycelia or conidia into yeast cells, a step critical for invasion and replication within phagocytic immune cells. Posttranslational glycosylation of extracellular proteins characterizes factors produced by the pathogenic yeast cells but not those of avirulent mycelia, correlating glycosylation with infection. Histoplasma yeast cells lacking the Pmt1 and Pmt2 protein mannosyltransferases, which catalyze O-linked mannosylation of proteins, are severely attenuated during infection of mammalian hosts. Cells lacking Pmt2 have altered surface characteristics that increase recognition of yeast cells by the macrophage mannose receptor and reduce recognition by the ␤-glucan receptor Dectin-1. Despite these changes, yeast cells lacking these factors still associate with and survive within phagocytes. Depletion of macrophages or neutrophils in vivo does not recover the virulence of the mutant yeast cells. We show that yeast cells lacking Pmt functions are more sensitive to thermal stress in vitro and consequently are unable to productively infect mice, even in the absence of fever. Treatment of mice with cyclophosphamide reduces the normal core body temperature of mice, and this decrease is sufficient to restore the infectivity of O-mannosylation-deficient yeast cells. These findings demonstrate that O-mannosylation of proteins increases the thermotolerance of Histoplasma yeast cells, which facilitates infection of mammalian hosts.IMPORTANCE For dimorphic fungal pathogens, mammalian body temperature can have contrasting roles. Mammalian body temperature induces differentiation of the fungal pathogen Histoplasma capsulatum into a pathogenic state characterized by infection of host phagocytes. On the other hand, elevated temperatures represent a significant barrier to infection by many microbes. By functionally characterizing cells lacking O-linked mannosylation enzymes, we show that protein mannosylation confers thermotolerance on H. capsulatum, enabling infection of mammalian hosts.

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“…The majority of infected persons could have either no symptoms or a very less mild sickness, which is hard to recognize as the cause of histoplasmosis [5]. The clinical symptoms generally occur only in a small number (around 1%) of the population when exposed with H. capsulatum spores [6]. Persons who are usually immuno-compromised and are unable to develop effective cell-mediated response are likely to develop symptomatic disease during the period of acute dissemination in body [5], which includes infant child, patients with HIV/AIDS, organ transplant recipients, and those with hematologic deficiencies, and also those patients, who are on corticosteroids drugs treatment [7].…”

Section: Introductionmentioning

confidence: 99%

Infectious sources of Histoplasmosis and molecular techniques for its identification

Bhandari

Rayamajhee²,

Dhungel

et al. 2019

Nepal J Biotechnol

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Histoplasmosis, a fungal infection caused by Histoplasma capsulatum (H. capsulatum), acquired from contaminated soil with droppings of chicken or birds and found to be distributed in many parts of the world. The prevalence of histoplasmosis has not well studied in Nepal. The common symptoms of acute and epidemic histoplasmosis include high fever, cough, and asthenia and weight loss. Most of the infections associated with histoplasmosis are asymptomatic. People with compromised immune systems such as HIV/AIDS (PLWHA), cancer, and organ transplant recipients are at risk of developing this disease. In this review, we have summarised the current status of histoplasmosis in Nepal and molecular techniques available for its identification. To date, the significant outbreak is not reported in Nepal, but the risk of infection for the vulnerable population cannot be undermined. Appropriate preventive measures and treatment on time can reduce the burden of this fungal disease. Further, this review is also focused on molecular identification of H. capsulatum. Hence, careful considerations by concerned stakeholders for national surveillance programs and the treatment of patients on time after proper diagnosis is highly recommended.

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Glycosylation and Immunoreactivity of the Histoplasma capsulatum Cfp4 Yeast-Phase Exoantigen

Cited by 13 publications

References 43 publications

Opposing signaling pathways regulate morphology in response to temperature in the fungal pathogenHistoplasma capsulatum

Opposing signaling pathways regulate morphology in response to temperature in the fungal pathogenHistoplasma capsulatum

O-Mannosylation of Proteins Enables Histoplasma Yeast Survival at Mammalian Body Temperatures

Infectious sources of Histoplasmosis and molecular techniques for its identification

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