scholarly journals Ubiquitylation and Developmental Regulation of Invariant Surface Protein Expression in Trypanosomes

2011 ◽  
Vol 10 (7) ◽  
pp. 916-931 ◽  
Author(s):  
Ka Fai Leung ◽  
Fay S. Riley ◽  
Mark Carrington ◽  
Mark C. Field
Keyword(s):  
Cell Surface ◽  
Protein Turnover ◽  
Full Length ◽  
Surface Proteins ◽  
Surface Glycoprotein ◽  
General Mechanism ◽  
Membrane Domain ◽  
Reporter Protein ◽  
Invariant Surface ◽  
Content Type

ABSTRACTThe cell surface ofTrypanosoma bruceiis dominated by the glycosylphosphatidylinositol-anchored variant surface glycoprotein (VSG), which is essential for immune evasion. VSG biosynthesis, trafficking, and turnover are well documented, buttrans-membrane domain (TMD) proteins, including the invariant surface glycoproteins (ISGs), are less well characterized. Internalization and degradation of ISG65 depend on ubiquitylation of conserved cytoplasmic lysines. Using epitope-tagged ISG75 and reporter chimeric proteins bearing the cytoplasmic andtrans-membrane regions of ISG75, together with multiple mutants with lysine-to-arginine mutations, we demonstrate that the cytoplasmic tail of ISG75 is both sufficient and necessary for endosomal targeting and degradation. The ISG75 chimeric reporter protein localized to endocytic organelles, while lysine-null versions were significantly stabilized at the cell surface. Importantly, ISG75 cytoplasmic lysines are modified by extensive oligoubiquitin chains and ubiquitylation is abolished in the lysine-null version. Furthermore, we find evidence for differential modes of turnover of ISG65 and ISG75. Full-length lysine-null ISG65 localization and protein turnover are significantly perturbed, but ISG75 localization and protein turnover are not, while ubiquitin conjugates can be detected for full-length lysine-null ISG75 but not ISG65. We find that the ISG75 ectodomain has a predicted coiled-coil, suggesting that ISG75 could be part of a complex, while ISG65 behaves independently. We also demonstrate a developmental stage-specific mechanism for exclusion of surface ISG expression in insect-stage cells by a ubiquitin-independent mechanism. We suggest that ubiquitylation may be a general mechanism for regulatingtrans-membrane domain surface proteins in trypanosomes.

scholarly journals The endosomal TbTpr86/TbUsp7/SkpZ (TUS) complex controls surface protein abundance in trypanosomes

2020 ◽  
Author(s):  
Kayo Yamada ◽  
Farzana K. Yaqub ◽  
Martin Zoltner ◽  
Mark C. Field
Keyword(s):  
Cell Cycle Progression ◽  
Surface Protein ◽  
Surface Proteins ◽  
Surface Glycoprotein ◽  
Tetratricopeptide Repeat ◽  
Protein Abundance ◽  
Membrane Domain ◽  
Cycle Progression ◽  
Invariant Surface ◽  
Tetratricopeptide Repeat Protein

AbstractIn trypanosomes the orthologs of human USP7 and VDU1 control abundance of a cohort of surface proteins, including invariant surface glycoproteins (ISGs) by functioning as deubiquitinases (DUBs) Silencing TbUsp7 partially inhibits endocytosis and invariant surface glycoprotein turnover. As a component of cullin E3 ubiquitin ligases, S-phase kinase-associated protein 1 (Skp1) has crucial roles in cell cycle progression, transcriptional regulation, signal transduction and other processes in animals and fungi. Unexpectedly, trypanosomes possess multiple Skp1 paralogs, including a divergent paralog designated SkpZ. SkpZ is implicated in suramin-sensitivity and endocytosis and decreases in abundance following TbUsp7 knockdown and physically interacts with TbUsp7 and TbTpr86. The latter is a tetratricopeptide-repeat protein also implicated in suramin sensitivity and located close to the flagellar pocket/endosomes, consistent with a role in endocytosis. Further, silencing SkpZ reduced abundance of TbUsp7 and TbTpr86 and many trans-membrane domain surface proteins. Our data indicate that TbTpr86, TbUsp7 and SkpZ form the ‘TUS’ complex that regulates abundance of a significant cohort of trypanosome surface proteins.

scholarly journals Visualizing the dynamics of exported bacterial proteins with the chemogenetic fluorescent reporter FAST

2020 ◽  
Author(s):  
Yankel Chekli ◽  
Caroline Peron-Cane ◽  
Dario Dell’Arciprete ◽  
Jean-François Allemand ◽  
Chenge Li ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Listeria Monocytogenes ◽  
Cell Surface ◽  
Bacterial Cell ◽  
Surface Proteins ◽  
Cellular Functions ◽  
Reporter Protein ◽  
Cell Surface Proteins ◽  
Fluorescent Reporter ◽  
Bacterial Proteins

AbstractBacterial proteins exported to the cell surface play key cellular functions. However, despite the interest to study the localization of surface proteins such as adhesins, transporters or hydrolases, monitoring their dynamics in live imaging remains challenging, due to the limited availability of fluorescent probes remaining functional after secretion. In this work, we used the Escherichia coli intimin and the Listeria monocytogenes InlB invasin as surface exposed scaffolds fused with the recently developed chemogenetic fluorescent reporter protein FAST. Using both membrane permeant (HBR-3,5DM) and non-permeant (HBRAA-3E) fluorogens that fluoresce upon binding to FAST, we demonstrated that fully functional FAST can be exposed at the cell surface and specifically tagged on the external side of the bacterial envelop in both diderm and monoderm bacteria. Our work opens new avenues to study of the organization and dynamics of the bacterial cell surface proteins.

scholarly journals Molecular characterization and expression of a novel human leukocyte cell-surface marker homologous to mouse Ly-9

Blood ◽  
2001 ◽  
Vol 97 (11) ◽  
pp. 3513-3520 ◽  
Author(s):  
Miguel Angel de la Fuente ◽  
Victoria Tovar ◽  
Neus Villamor ◽  
Nuria Zapater ◽  
Pilar Pizcueta ◽  
...  
Keyword(s):  
Cell Surface ◽  
B Cell ◽  
Cell Line ◽  
B Lymphocytes ◽  
Full Length ◽  
Surface Glycoprotein ◽  
T And B Lymphocytes ◽  
Cdna Clones ◽  
Cell Surface Glycoprotein ◽  
Full Length Cdna

Ly-9 is a mouse cell-surface glycoprotein that is selectively expressed on thymocytes and on mature T and B lymphocytes. Ly-9 belongs to the CD2 subset of the immunoglobulin superfamily, an emerging family of cell signaling receptors. Recently, a partial human Ly-9 complementary DNA (cDNA) sequence has been described. Full-length cDNA clones were isolated that included the initiation codon, the sequence encoding the full signal peptide, and 14 amino acids more in the cytoplasmic domain than in the previously reported clone. The predicted extracellular domain of human Ly-9 contains 4 immunoglobulinlike domains, similar to those in mouse Ly-9. Northern blot analysis revealed that the human Ly-9 messenger RNA (2.6 kb) is expressed predominantly in lymph node, spleen, thymus, and peripheral blood leukocytes. Four monoclonal antibodies (mAbs) were raised against human Ly-9 by immunizing mice with the pre-B-cell line 300.19 stably transfected with human Ly-9 full-length cDNA. These mAbs strongly stained the surfaces of cells transfected with human Ly-9 cDNA but not of untransfected cells. Human Ly-9 expression was restricted to T and B lymphocytes and thymocytes, with the highest levels of expression on CD4+CD8− and CD4−CD8+ thymocytes. Monocytes, granulocytes, platelets, and red blood cells were uniformly negative for Ly-9. These mAbs immunoprecipitated major polypeptides of 120 kd from the transfected cells and 120 kd and 100 kd from B-cell line Daudi, probably because of the cell-surface–expressed isoforms. These data demonstrate that human Ly-9 is a new marker for the study of normal and malignant leukocytes.

scholarly journals The Distinct Immune-Stimulatory Capacities of Porphyromonas gingivalis Strains 381 and ATCC 33277 Are Determined by the fimB Allele and Gingipain Activity

2019 ◽  
Vol 87 (12) ◽  
Author(s):  
Stephen R. Coats ◽  
Nutthapong Kantrong ◽  
Thao T. To ◽  
Sumita Jain ◽  
Caroline A. Genco ◽  
...  
Keyword(s):  
Cell Surface ◽  
Immune Responses ◽  
Host Cell ◽  
Porphyromonas Gingivalis ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Innate Immune ◽  
Surface Proteins ◽  
Innate Immune Responses ◽  
Content Type

ABSTRACT The Porphyromonas gingivalis strain ATCC 33277 (33277) and 381 genomes are nearly identical. However, strain 33277 displays a significantly diminished capacity to stimulate host cell Toll-like receptor 2 (TLR2)-dependent signaling and interleukin-1β (IL-1β) production relative to 381, suggesting that there are strain-specific differences in one or more bacterial immune-modulatory factors. Genomic sequencing identified a single nucleotide polymorphism in the 33277 fimB allele (A→T), creating a premature stop codon in the 33277 fimB open reading frame relative to the 381 fimB allele. Gene exchange experiments established that the 33277 fimB allele reduces the immune-stimulatory capacity of this strain. Transcriptome comparisons revealed that multiple genes related to carboxy-terminal domain (CTD) family proteins, including the gingipains, were upregulated in 33277 relative to 381. A gingipain substrate degradation assay demonstrated that cell surface gingipain activity is higher in 33277, and an isogenic mutant strain deficient for the gingipains exhibited an increased ability to induce TLR2 signaling and IL-1β production. Furthermore, 33277 and 381 mutant strains lacking CTD cell surface proteins were more immune-stimulatory than the parental wild-type strains, consistent with an immune-suppressive role for the gingipains. Our data show that the combination of an intact fimB allele and limited cell surface gingipain activity in P. gingivalis 381 renders this strain more immune-stimulatory. Conversely, a defective fimB allele and high-level cell surface gingipain activity reduce the capacity of P. gingivalis 33277 to stimulate host cell innate immune responses. In summary, genomic and transcriptomic comparisons identified key virulence characteristics that confer divergent host cell innate immune responses to these highly related P. gingivalis strains.

scholarly journals Staphylococcus aureus Fibronectin-Binding Protein A Mediates Cell-Cell Adhesion through Low-Affinity Homophilic Bonds

mBio ◽  
2015 ◽  
Vol 6 (3) ◽  
Author(s):  
Philippe Herman-Bausier ◽  
Sofiane El-Kirat-Chatel ◽  
Timothy J. Foster ◽  
Joan A. Geoghegan ◽  
Yves F. Dufrêne
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Surface ◽  
Biofilm Formation ◽  
Protein A ◽  
Intercellular Adhesion ◽  
Surface Proteins ◽  
Content Type ◽  
Fibronectin Binding ◽  
A Domains ◽  
Cell Cell

ABSTRACT Staphylococcus aureus is an important opportunistic pathogen which is a leading cause of biofilm-associated infections on indwelling medical devices. The cell surface-located fibronectin-binding protein A (FnBPA) plays an important role in the accumulation phase of biofilm formation by methicillin-resistant S. aureus (MRSA), but the underlying molecular interactions are not yet established. Here, we use single-cell and single-molecule atomic force microscopy to unravel the mechanism by which FnBPA mediates intercellular adhesion. We show that FnBPA is responsible for specific cell-cell interactions that involve the FnBPA A domain and cause microscale cell aggregation. We demonstrate that the strength of FnBPA-mediated adhesion originates from multiple low-affinity homophilic interactions between FnBPA A domains on neighboring cells. Low-affinity binding by means of FnBPA may be important for biofilm dynamics. These results provide a molecular basis for the ability of FnBPA to promote cell accumulation during S. aureus biofilm formation. We speculate that homophilic interactions may represent a generic strategy among staphylococcal cell surface proteins for guiding intercellular adhesion. As biofilm formation by MRSA strains depends on proteins rather than polysaccharides, our approach offers exciting prospects for the design of drugs or vaccines to inhibit protein-dependent intercellular interactions in MRSA biofilms. IMPORTANCE Staphylococcus aureus is a human pathogen that forms biofilms on indwelling medical devices, such as central venous catheters and prosthetic joints. This leads to biofilm infections that are difficult to treat with antibiotics because many cells within the biofilm matrix are dormant. The fibronectin-binding proteins (FnBPs) FnBPA and FnBPB promote biofilm formation by clinically relevant methicillin-resistant S. aureus (MRSA) strains, but the molecular mechanisms involved remain poorly understood. We used atomic force microscopy techniques to demonstrate that FnBPA mediates cell-cell adhesion via multiple, low-affinity homophilic bonds between FnBPA A domains on adjacent cells. Therefore, FnBP-mediated homophilic interactions represent an interesting target to prevent MRSA biofilms. We propose that such homophilic mechanisms may be widespread among staphylococcal cell surface proteins, providing a means to guide intercellular adhesion and biofilm accumulation.

scholarly journals Periplasmic Cytophaga hutchinsonii Endoglucanases Are Required for Use of Crystalline Cellulose as the Sole Source of Carbon and Energy

2016 ◽  
Vol 82 (15) ◽  
pp. 4835-4845 ◽  
Author(s):  
Yongtao Zhu ◽  
Lanlan Han ◽  
Kathleen L. Hefferon ◽  
Nicholas R. Silvaggi ◽  
David B. Wilson ◽  
...  
Keyword(s):  
Cell Surface ◽  
Outer Membrane ◽  
Soluble Sugars ◽  
Surface Proteins ◽  
Cellulolytic Enzymes ◽  
Crystalline Cellulose ◽  
Cell Fractionation ◽  
Unknown Mechanism ◽  
Content Type ◽  
Cytophaga Hutchinsonii

ABSTRACTThe soil bacteriumCytophaga hutchinsoniiactively digests crystalline cellulose by a poorly understood mechanism. Genome analyses identified nine genes predicted to encode endoglucanases with roles in this process. No predicted cellobiohydrolases, which are usually involved in the utilization of crystalline cellulose, were identified. Chromosomal deletions were performed in eight of the endoglucanase-encoding genes:cel5A,cel5B,cel5C,cel9A,cel9B,cel9C,cel9E, andcel9F. Each mutant retained the ability to digest crystalline cellulose, although the deletion ofcel9Ccaused a modest decrease in cellulose utilization. Strains with multiple deletions were constructed to identify the critical cellulases. Cells of a mutant lacking bothcel5Bandcel9Cwere completely deficient in growth on cellulose. Cell fractionation and biochemical analyses indicate that Cel5B and Cel9C are periplasmic nonprocessive endoglucanases. The requirement of periplasmic endoglucanases for cellulose utilization suggests that cellodextrins are transported across the outer membrane during this process. Bioinformatic analyses predict that Cel5A, Cel9A, Cel9B, Cel9D, and Cel9E are secreted across the outer membrane by the type IX secretion system, which has been linked to cellulose utilization. These secreted endoglucanases may perform the initial digestion within amorphous regions on the cellulose fibers, releasing oligomers that are transported into the periplasm for further digestion by Cel5B and Cel9C. The results suggest that both cell surface and periplasmic endoglucanases are required for the growth ofC. hutchinsoniion cellulose and that novel cell surface proteins may solubilize and transport cellodextrins across the outer membrane.IMPORTANCEThe bacteriumCytophaga hutchinsoniidigests crystalline cellulose by an unknown mechanism. It lacks processive cellobiohydrolases that are often involved in cellulose digestion. Critical cellulolytic enzymes were identified by genetic analyses. Intracellular (periplasmic) nonprocessive endoglucanases performed an important role in cellulose utilization. The results suggest a model involving partial digestion at the cell surface, solubilization and uptake of cellodextrins across the outer membrane by an unknown mechanism, and further digestion within the periplasm. The ability to sequester cellodextrins and digest them intracellularly may limit losses of soluble cellobiose to other organisms.C. hutchinsoniiuses an unusual approach to digest cellulose and is a potential source of novel proteins to increase the efficiency of conversion of cellulose into soluble sugars and biofuels.

scholarly journals The GPI anchor of cell-surface proteins is synthesized on the cytoplasmic face of the endoplasmic reticulum.

1994 ◽  
Vol 127 (2) ◽  
pp. 333-341 ◽  
Author(s):  
J Vidugiriene ◽  
A K Menon
Keyword(s):  
Cell Surface ◽  
Surface Proteins ◽  
Surface Glycoprotein ◽  
Proteinase K ◽  
Con A ◽  
Membrane Bilayer ◽  
Gpi Anchor ◽  
Assembly Pathway ◽  
Cytoplasmic Face ◽  
External Face

Glycosylphosphatidylinositol (GPI) membrane protein anchors are synthesized from sugar nucleotides and phospholipids in the ER and transferred to newly synthesized proteins destined for the cell surface. The topology of GPI synthesis in the ER was investigated using sealed trypanosome microsomes and the membrane-impermeant probes phosphatidylinositol-specific phospholipase C, Con A, and proteinase K. All the GPI biosynthetic intermediates examined were found to be located on the external face of the microsomal vesicles suggesting that the principal steps of GPI assembly occur in the cytoplasmic leaflet of the ER. Protease protection experiments showed that newly GPI-modified trypanosome variant surface glycoprotein was primarily oriented towards the ER lumen, consistent with eventual expression at the cell surface. The unusual topographical arrangement of the GPI assembly pathway suggests that a biosynthetic intermediate, possibly the phosphoethanolamine-containing anchor precursor, must be translocated across the ER membrane bilayer in the process of constructing a GPI anchor.

scholarly journals Functional Cell Surface Display and Controlled Secretion of Diverse Agarolytic Enzymes by Escherichia coli with a Novel Ligation-Independent Cloning Vector Based on the Autotransporter YfaL

2012 ◽  
Vol 78 (9) ◽  
pp. 3051-3058 ◽  
Author(s):  
Hyeok-Jin Ko ◽  
Eunhye Park ◽  
Joseph Song ◽  
Taek Ho Yang ◽  
Hee Jong Lee ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Surface ◽  
Fluorescent Protein ◽  
Surface Display ◽  
Cell Surface Display ◽  
Heterologous Proteins ◽  
Display System ◽  
Reporter Protein ◽  
Content Type ◽  
Ligation Independent Cloning

ABSTRACTAutotransporters have been employed as the anchoring scaffold for cell surface display by replacing their passenger domains with heterologous proteins to be displayed. We adopted an autotransporter (YfaL) ofEscherichia colifor the cell surface display system. The critical regions in YfaL for surface display were identified for the construction of a ligation-independent cloning (LIC)-based display system. The designed system showed no detrimental effect on either the growth of the host cell or overexpressing heterologous proteins on the cell surface. We functionally displayed monomeric red fluorescent protein (mRFP1) as a reporter protein and diverse agarolytic enzymes fromSaccharophagus degradans2-40, including Aga86C and Aga86E, which previously had failed to be functional expressed. The system could display different sizes of proteins ranging from 25.3 to 143 kDa. We also attempted controlled release of the displayed proteins by incorporating a tobacco etch virus protease cleavage site into the C termini of the displayed proteins. The maximum level of the displayed protein was 6.1 × 104molecules per a single cell, which corresponds to 5.6% of the entire cell surface of actively growingE. coli.

scholarly journals Lipid Anchoring of Archaeosortase Substrates and Midcell Growth in Haloarchaea

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
Author(s):  
Mohd Farid Abdul-Halim ◽  
Stefan Schulze ◽  
Anthony DiLucido ◽  
Friedhelm Pfeiffer ◽  
Alexandre Wilson Bisson Filho ◽  
...  
Keyword(s):  
Cell Growth ◽  
Cell Surface ◽  
Cell Biology ◽  
Fluorescent Protein ◽  
De Novo ◽  
Hot Spot ◽  
Control Cell ◽  
Haloferax Volcanii ◽  
Surface Proteins ◽  
Content Type

ABSTRACT The archaeal cytoplasmic membrane provides an anchor for many surface proteins. Recently, a novel membrane anchoring mechanism involving a peptidase, archaeosortase A (ArtA), and C-terminal lipid attachment of surface proteins was identified in the model archaeon Haloferax volcanii. ArtA is required for optimal cell growth and morphogenesis, and the S-layer glycoprotein (SLG), the sole component of the H. volcanii cell wall, is one of the targets for this anchoring mechanism. However, how exactly ArtA function and regulation control cell growth and morphogenesis is still elusive. Here, we report that archaeal homologs to the bacterial phosphatidylserine synthase (PssA) and phosphatidylserine decarboxylase (PssD) are involved in ArtA-dependent protein maturation. Haloferax volcanii strains lacking either HvPssA or HvPssD exhibited motility, growth, and morphological phenotypes similar to those of an ΔartA mutant. Moreover, we showed a loss of covalent lipid attachment to SLG in the ΔhvpssA mutant and that proteolytic cleavage of the ArtA substrate HVO_0405 was blocked in the ΔhvpssA and ΔhvpssD mutant strains. Strikingly, ArtA, HvPssA, and HvPssD green fluorescent protein (GFP) fusions colocalized to the midcell position of H. volcanii cells, strongly supporting that they are involved in the same pathway. Finally, we have shown that the SLG is also recruited to the midcell before being secreted and lipid anchored at the cell outer surface. Collectively, our data suggest that haloarchaea use the midcell as the main surface processing hot spot for cell elongation, division, and shape determination. IMPORTANCE The subcellular organization of biochemical processes in space and time is still one of the most mysterious topics in archaeal cell biology. Despite the fact that haloarchaea largely rely on covalent lipid anchoring to coat the cell envelope, little is known about how cells coordinate de novo synthesis and about the insertion of this proteinaceous layer throughout the cell cycle. Here, we report the identification of two novel contributors to ArtA-dependent lipid-mediated protein anchoring to the cell surface, HvPssA and HvPssD. ArtA, HvPssA, and HvPssD, as well as SLG, showed midcell localization during growth and cytokinesis, indicating that haloarchaeal cells confine phospholipid processing in order to promote midcell elongation. Our findings have important implications for the biogenesis of the cell surface.

scholarly journals The Identification and Functional Characterization of WxL Proteins from Enterococcus faecium Reveal Surface Proteins Involved in Extracellular Matrix Interactions

2014 ◽  
Vol 197 (5) ◽  
pp. 882-892 ◽  
Author(s):  
Jessica R. Galloway-Peña ◽  
Xiaowen Liang ◽  
Kavindra V. Singh ◽  
Puja Yadav ◽  
Chungyu Chang ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Cell Surface ◽  
Bile Salt ◽  
Enterococcus Faecium ◽  
Bacterial Species ◽  
Functional Characterization ◽  
Surface Proteins ◽  
Type I ◽  
Gram Positive ◽  
Content Type

The WxL domain recently has been identified as a novel cell wall binding domain found in numerous predicted proteins within multiple Gram-positive bacterial species. However, little is known about the function of proteins containing this novel domain. Here, we identify and characterize 6Enterococcus faeciumproteins containing the WxL domain which, by reverse transcription-PCR (RT-PCR) and genomic analyses, are located in three similarly organized operons, deemed WxL loci A, B, and C. Western blotting, electron microscopy, and enzyme-linked immunosorbent assays (ELISAs) determined that genes of WxL loci A and C encode antigenic, cell surface proteins exposed at higher levels in clinical isolates than in commensal isolates. Secondary structural analyses of locus A recombinant WxL domain-containing proteins found they are rich in β-sheet structure and disordered segments. Using Biacore analyses, we discovered that recombinant WxL proteins from locus A bind human extracellular matrix proteins, specifically type I collagen and fibronectin. Proteins encoded by locus A also were found to bind to each other, suggesting a novel cell surface complex. Furthermore, bile salt survival assays and animal models using a mutant from which all three WxL loci were deleted revealed the involvement of WxL operons in bile salt stress and endocarditis pathogenesis. In summary, these studies extend our understanding of proteins containing the WxL domain and their potential impact on colonization and virulence inE. faeciumand possibly other Gram-positive bacterial species.

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