scholarly journals Additional C-type lectin receptors mediate interactions with Pneumocystis organisms and major surface glycoprotein

2021 ◽  
Vol 70 (12) ◽  
Author(s):  
Theodore J. Kottom ◽  
Eva M. Carmona ◽  
Kyle Schaefbauer ◽  
Andrew H. Limper
Keyword(s):  
Mrna Expression ◽  
Immune Cell ◽  
Expression Profiles ◽  
Pneumocystis Carinii ◽  
Pneumocystis Pneumonia ◽  
Immunofluorescence Assay ◽  
Surface Glycoprotein ◽  
Lectin Receptors ◽  
Major Surface ◽  
Surface Glycoproteins

Introduction. Pathogen-associated molecular patterns’ (PAMPs) are microbial signatures that are recognized by host myeloid C-type lectin receptors (CLRs). These CLRs interact with micro-organisms via their carbohydrate recognition domains (CRDs) and engage signalling pathways within the cell resulting in pro-inflammatory and microbicidal responses. Gap statement. In this article, we extend our laboratory study of additional CLRs that recognize fungal ligands against Pneumocystis murina and Pneumocystis carinii and their purified major surface glycoproteins (Msgs). Aim. To study the potential of newly synthesized hFc-CLR fusions on binding to Pneumocystis and its Msg. Methods. A library of new synthesized hFc-CLR fusions was screened against Pneumocystis murina and Pneumocystis carinii organisms and their purified major surface glycoproteins (Msgs) found on the respective fungi via modified ELISA. Immunofluorescence assay (IFA) was implemented and quantified to verify results. mRNA expression analysis by quantitative PCR (q-PCR) was employed to detect respective CLRs found to bind fungal organisms in the ELISA and determine their expression levels in the mouse immunosuppressed Pneumocystis pneumonia (PCP) model. Results. We detected a number of the CLR hFc-fusions displayed significant binding with P. murina and P. carinii organisms, and similarly to their respective Msgs. Significant organism and Msg binding was observed for CLR members C-type lectin domain family 12 member A (CLEC12A), Langerin, macrophage galactose-type lectin-1 (MGL-1), and specific intracellular adhesion molecule-3 grabbing non-integrin homologue-related 3 (SIGNR3). Immunofluorescence assay (IFA) with the respective CLR hFc-fusions against whole P. murina life forms corroborated these findings. Lastly, we surveyed the mRNA expression profiles of the respective CLRs tested above in the mouse immunosuppressed Pneumocystis pneumonia (PCP) model and determined that macrophage galactose type C-type lectin (Mgl-1), implicated in recognizing terminal N-acetylgalactosamine (GalNAc) found in the glycoproteins of microbial pathogens was significantly up-regulated during infection. Conclusion. The data herein add to the growing list of CLRs recognizing Pneumocystis and provide insights for further study of organism/host immune cell interactions.

scholarly journals Pneumocystis carinii Major Surface Glycoprotein Dampens Macrophage Inflammatory Responses to Fungal β-Glucan

2020 ◽  
Vol 222 (7) ◽  
pp. 1213-1221
Author(s):  
Theodore J Kottom ◽  
Deanne M Hebrink ◽  
Eva M Carmona ◽  
Andrew H Limper
Keyword(s):  
Protein Complex ◽  
Inflammatory Responses ◽  
Pneumocystis Carinii ◽  
Surface Protein ◽  
Surface Glycoprotein ◽  
Immune Recognition ◽  
Lectin Receptors ◽  
Major Surface Glycoprotein ◽  
Tnf Α ◽  
Major Surface

Abstract Background Pneumocystis major surface glycoprotein (Msg) is a 120-kD surface protein complex on the organism with importance in adhesion and immune recognition. In this study, we show that Msg significantly impairs tumor necrosis factor (TNF)-α secretion by macrophages induced by Saccharomyces cerevisiae and Pneumocystis carinii (Pc) β-glucans. Methods Major surface glycoprotein was shown to greatly reduce β-glucan-induced Dectin-1 immunoreceptor tyrosine-based activating motif (ITAM) phosphorylation. Major surface glycoprotein also down regulated Dectin-1 receptor messenger ribonucleic acid (mRNA) expression in the macrophages. It is interesting that Msg incubation with macrophages resulted in significant mRNA upregulation of both C-type lectin receptors (CLR) Mincle and MCL in Msg protein presence alone but to even greater amounts in the presence of Pc β-glucan. Results The silencing of MCL and Mincle resulted in TNF-α secretions similar to that of macrophages treated with Pneumocystis β-glucan alone, which is suggestive of an inhibitory role for these 2 CLRs in Msg-suppressive effects on host cell immune response. Conclusions Taken together, these data indicate that the Pneumocystis Msg surface protein complex can act to suppress host macrophage inflammatory responses to the proinflammatory β -glucan components of the organisms.

scholarly journals Effect of Bronchoalveolar Lavage Fluid from Pneumocystis carinii- Infected Hosts on Phagocytic Activity of Alveolar Macrophages

2004 ◽  
Vol 72 (4) ◽  
pp. 2140-2147 ◽  
Author(s):  
Mark E. Lasbury ◽  
Peimao Lin ◽  
Dennis Tschang ◽  
Pamela J. Durant ◽  
Chao-Hung Lee
Keyword(s):  
Bronchoalveolar Lavage ◽  
Concanavalin A ◽  
Alveolar Macrophages ◽  
Phagocytic Activity ◽  
Pneumocystis Carinii ◽  
Surface Glycoprotein ◽  
Suppressive Activity ◽  
Agarose Beads ◽  
Major Surface Glycoprotein ◽  
Major Surface

ABSTRACT Alveolar macrophages from Pneumocystis carinii-infected rats are defective in phagocytosis. To investigate whether this defect is due to a certain factor present in P. carinii-infected lungs, alveolar macrophages from uninfected rats were incubated with bronchoalveolar lavage (BAL) fluid samples from P. carinii-infected rats. Alveolar macrophages treated with these BAL fluid samples became defective in phagocytosis but remained normal when treated with BAL fluid samples from noninfected or Toxoplasma gondii-infected rats. The suppressive activity of the BAL fluid samples from P. carinii-infected rats on phagocytosis was retained when the BAL fluid samples were passed through a filter with a pore size of 0.45 μm but was lost when the BAL fluid samples were digested with proteases such as trypsin, pepsin, papain, or endopeptidase Gly-C. Lipid fractions of these BAL fluid samples had no suppressive activity on phagocytosis. The suppressive activity of these BAL fluid samples was also lost when they were incubated with concanavalin A-agarose beads, suggesting that the inhibitor is a glycoprotein. The inhibitor was estimated to be larger than 100,000 Da by exclusion filtration. After binding to the concanavalin A-agarose beads, the inhibitor in BAL fluid samples and P. carinii lysate could be eluted with 200 mM methylmannose. Treatment of both the crude BAL fluid samples and P. carinii lysate and the 200 mM methylmannose eluate with antibody against the major surface glycoprotein of P. carinii eliminated their suppressive activity. These results suggest that the factor capable of suppressing the phagocytic activity of alveolar macrophages is P. carinii major surface glycoprotein or one or more of its derivatives.

scholarly journals Characterization of Major Surface Glycoprotein Genes of Human Pneumocystis carinii and High-Level Expression of a Conserved Region

1998 ◽  
Vol 66 (9) ◽  
pp. 4268-4273 ◽  
Author(s):  
Qin Mei ◽  
Ross E. Turner ◽  
Vivian Sorial ◽  
Diane Klivington ◽  
C. William Angus ◽  
...  
Keyword(s):  
Genomic Library ◽  
Pneumocystis Carinii ◽  
Surface Glycoprotein ◽  
High Level Expression ◽  
Serum Samples ◽  
Major Surface Glycoprotein ◽  
Adult Humans ◽  
Major Surface ◽  
High Level

ABSTRACT To facilitate studies of Pneumocystis cariniiinfection in humans, we undertook to better characterize and to express the major surface glycoprotein (MSG) of human P. carinii, an important protein in host-pathogen interactions. Seven MSG genes were cloned from a single isolate by PCR or genomic library screening and were sequenced. The predicted proteins, like rat MSGs, were closely related but unique variants, with a high level of conservation among cysteine residues. A conserved immunodominant region (of approximately 100 amino acids) near the carboxy terminus was expressed at high levels in Escherichia coli and used in Western blot studies. All 49 of the serum samples, which were taken from healthy controls as well as from patients with and withoutP. carinii pneumonia, were reactive with this peptide by Western blotting, supporting the hypothesis that most adult humans have been infected with P. carinii at some point. This recombinant MSG fragment, which is the first human P. carinii antigen available in large quantities, may be a useful reagent for investigating the epidemiology of P. cariniiinfection in humans.

scholarly journals Myeloid C-type lectin receptors that recognize fungal mannans interact with Pneumocystis organisms and major surface glycoprotein

2019 ◽  
Vol 68 (11) ◽  
pp. 1649-1654 ◽  
Author(s):  
Theodore J. Kottom ◽  
Deanne M. Hebrink ◽  
Joao T. Monteiro ◽  
Bernd Lepenies ◽  
Eva M. Carmona ◽  
...  
Keyword(s):  
Surface Glycoprotein ◽  
Lectin Receptors ◽  
Major Surface Glycoprotein ◽  
Major Surface

scholarly journals Identification of a Putative Precursor to the Major Surface Glycoprotein of Pneumocystis carinii

1998 ◽  
Vol 66 (2) ◽  
pp. 741-746 ◽  
Author(s):  
Susan M. Sunkin ◽  
Michael J. Linke ◽  
Francis X. McCormack ◽  
Peter D. Walzer ◽  
James R. Stringer
Keyword(s):  
Insect Cells ◽  
Pneumocystis Carinii ◽  
Recombinant Baculovirus ◽  
Polyclonal Antiserum ◽  
Surface Glycoprotein ◽  
Messenger Rnas ◽  
Conserved Sequence ◽  
Amino Acid Peptide ◽  
Major Surface Glycoprotein ◽  
Major Surface

ABSTRACT The major surface glycoprotein (MSG) of Pneumocystis cariniif. sp. carinii is a family of proteins encoded by a family of heterogeneous genes. Messenger RNAs encoding different MSGs each begin with the same 365-bp sequence, called the Upstream Conserved Sequence (UCS), which is in frame with the contiguous MSG sequence. The UCS contains several potential start sites for translation. To determine if translation of MSG mRNAs begins in the UCS, polyclonal antiserum was raised against the 123-amino-acid peptide encoded by the UCS. The anti-UCS serum reacted with a P. carinii protein that migrated at 170 kDa; however, it did not react with the mature MSG protein, which migrates at 116 kDa. A 170-kDa protein was immunoprecipitated with anti-UCS serum and shown to react with a monoclonal antibody against a conserved MSG epitope. To explore the functional role of the UCS in the trafficking of MSG, the nucleotide sequence encoding the UCS peptide was ligated to the 5′ end of an MSG gene and incorporated into a recombinant baculovirus. Insect cells infected with the UCS-MSG hybrid gene expressed a 160-kDa protein which was N-glycosylated. By contrast, insect cells infected with a baculovirus carrying an MSG gene lacking the UCS expressed a nonglycosylated 130-kDa protein. These data suggest that in P. carinii, translation begins in the UCS to produce a pre-MSG protein, which is subsequently directed to the endoplasmic reticulum and processed to the mature form by proteolytic cleavage.

Characterization of Major Surface Glycoprotein Genes of Human Pneumocystis carinii and High-Level Expression of a Conserved Region

1998 ◽  
Vol 66 (9) ◽  
pp. 4268-4273
Author(s):  
Qin Mei ◽  
Ross E. Turner ◽  
Vivian Sorial ◽  
Diane Klivington ◽  
C. William Angus ◽  
...  
Keyword(s):  
Pneumocystis Carinii ◽  
Surface Glycoprotein ◽  
High Level Expression ◽  
Major Surface Glycoprotein ◽  
Conserved Region ◽  
Major Surface ◽  
High Level

scholarly journals The Fungal PCR Initiative's evaluation of in-house and commercial Pneumocystis jirovecii qPCR assays: Toward a standard for a diagnostics assay

2019 ◽  
Vol 58 (6) ◽  
pp. 779-788 ◽  
Author(s):  
Maud Gits-Muselli ◽  
P Lewis White ◽  
Carlo Mengoli ◽  
Sharon Chen ◽  
Brendan Crowley ◽  
...  
Keyword(s):  
Target Genes ◽  
Clinical Care ◽  
Large Subunit ◽  
Reference Method ◽  
Pneumocystis Pneumonia ◽  
Surface Glycoprotein ◽  
Pneumocystis Jirovecii ◽  
Tubulin Genes ◽  
Major Surface Glycoprotein ◽  
Major Surface

Abstract Quantitative real-time PCR (qPCR) is increasingly used to detect Pneumocystis jirovecii for the diagnosis of Pneumocystis pneumonia (PCP), but there are differences in the nucleic acids targeted, DNA only versus whole nucleic acid (WNA), and also the target genes for amplification. Through the Fungal PCR Initiative, a working group of the International Society for Human and Animal Mycology, a multicenter and monocenter evaluation of PCP qPCR assays was performed. For the multicenter study, 16 reference laboratories from eight different countries, performing 20 assays analyzed a panel consisting of two negative and three PCP positive samples. Aliquots were prepared by pooling residual material from 20 negative or positive- P. jirovecii bronchoalveolar lavage fluids (BALFs). The positive pool was diluted to obtain three concentrations (pure 1:1; 1:100; and 1:1000 to mimic high, medium, and low fungal loads, respectively). The monocenter study compared five in-house and five commercial qPCR assays testing 19 individual BALFs on the same amplification platform. Across both evaluations and for all fungal loads, targeting WNA and the mitochondrial small sub-unit (mtSSU) provided the earliest Cq values, compared to only targeting DNA and the mitochondrial large subunit, the major surface glycoprotein or the beta-tubulin genes. Thus, reverse transcriptase-qPCR targeting the mtSSU gene could serve as a basis for standardizing the P. jirovecii load, which is essential if qPCR is to be incorporated into clinical care pathways as the reference method, accepting that additional parameters such as amplification platforms still need evaluation.

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