Molecular Dissection of the secA2 Locus of Group B Streptococcus Reveals that Glycosylation of the Srr1 LPXTG Protein Is Required for Full Virulence

ABSTRACT In streptococci, the secA2 locus includes genes encoding the following: (i) the accessory Sec components (SecA2, SecY2, and at least three accessory secretion proteins), (ii) two essential glycosyltranferases (GTs) (GtfA and GtfB), (iii) a variable number of dispensable additional GTs, and (iv) a secreted serine-rich LPXTG protein which is glycosylated in the cytoplasm and transported to the cell surface by this accessory Sec system. The secA2 locus of Streptococcus agalactiae strain NEM316 is structurally related to those found in other streptococci and encodes the serine-rich surface protein Srr1. We demonstrated that expression of Srr1 but not that of the SecA2 components and the associated GTs is regulated by the standalone transcriptional regulator Rga. Srr1 is synthesized as a glycosylated precursor, secreted by the SecA2 system, and anchored to the cell wall by the housekeeping sortase A. Srr1 was localized preferentially at the old poles. GtfA and/or GtfB, but not the six additional GTs, is essential for the production of Srr1. These GTs are involved in the attachment of GlcNac and sialic acid to Srr1. Full glycosylation of Srr1 is associated with the cell surface display of a protein that is more resistant to proteolytic attack. Srr1 contributes to bacterial adherence to human epithelial cell lines and virulence in a neonatal rat model. The extent of Srr1 glycosylation by GtfC to -H modulates bacterial adherence and virulence.

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Protein rib: a novel group B streptococcal cell surface protein that confers protective immunity and is expressed by most strains causing invasive infections.

Journal of Experimental Medicine ◽

10.1084/jem.177.6.1593 ◽

1993 ◽

Vol 177 (6) ◽

pp. 1593-1603 ◽

Cited By ~ 143

Author(s):

M Stålhammar-Carlemalm ◽

L Stenberg ◽

G Lindahl

Keyword(s):

Cell Surface ◽

Protective Immunity ◽

Group B Streptococcus ◽

Rabbit Antiserum ◽

Surface Protein ◽

Amino Acid Sequences ◽

Cell Surface Protein ◽

Type Iii ◽

Invasive Infections ◽

Group B

The group B Streptococcus, an important cause of invasive infections in the neonate, is classified into four major serotypes (Ia, Ib, II, and III) based on the structure of the polysaccharide capsule. Since the capsule is a known virulence factor, it has been extensively studied, in particular in type III strains, which cause the majority of invasive infections. Two cell surface proteins, alpha and beta, have also been studied in detail since they confer protective immunity, but these proteins are usually not expressed by type III strains. We describe here a cell surface protein, designated protein Rib (resistance to proteases, immunity, group B), that confers protective immunity and is expressed by most strains of type III. Protein Rib was first identified as a distinct 95-kD protein in extracts of a type III strain, and was purified to homogeneity from that strain. Rabbit antiserum to protein Rib was used to demonstrate that it is expressed on the cell surface of 31 out of 33 type III strains, but only on 1 out of 25 strains representing the other three serotypes. Mouse protection tests showed that antiserum to protein Rib protects against lethal infection with three different strains expressing this antigen, including a strain representing a recently identified high virulence type III clone. Protein Rib is immunologically unrelated to the alpha and beta proteins, but shares several features with the alpha protein. Most importantly, the NH2-terminal amino acid sequences of the Rib and alpha proteins are identical at 6 out of 12 positions. In addition, both protein Rib and the alpha protein are relatively resistant to trypsin (and Rib is also resistant to pepsin) and both proteins vary greatly in size between different clinical isolates. Finally, both protein Rib and the alpha protein exhibit a regular ladderlike pattern in immunoblotting experiments, which may reflect a repetitive structure. Taken together, these data suggest that the Rib and alpha proteins are members of a family of proteins with related structure and function. Since protein Rib confers protective immunity, it may be valuable for the development of a protein vaccine against the group B Streptococcus, an encapsulated bacterium.

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Anaplasma marginale major surface protein 1a directs cell surface display of tick BM95 immunogenic peptides on Escherichia coli

Journal of Biotechnology ◽

10.1016/j.jbiotec.2008.05.006 ◽

2008 ◽

Vol 135 (4) ◽

pp. 326-332 ◽

Cited By ~ 12

Author(s):

Mario Canales ◽

Consuelo Almazán ◽

José M. Pérez de la Lastra ◽

José de la Fuente

Keyword(s):

Escherichia Coli ◽

Cell Surface ◽

Surface Display ◽

Surface Protein ◽

Anaplasma Marginale ◽

Cell Surface Display ◽

Major Surface Protein ◽

Immunogenic Peptides ◽

Major Surface

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Construction of a Yeast Cell-Surface Display System and Expression of Trametes sp. laccase

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.347-353.3635 ◽

2011 ◽

Vol 347-353 ◽

pp. 3635-3640 ◽

Cited By ~ 1

Author(s):

Jian Zhang Lu ◽

Qin Guo ◽

Mei Lin Cui ◽

Lu Yang ◽

Shan Shan Du ◽

...

Keyword(s):

Cell Surface ◽

Surface Display ◽

Cell Surface Display ◽

Heterologous Proteins ◽

Secretion Signal ◽

Yeast Cell Surface ◽

Gal1 Promoter ◽

Genes Encoding ◽

Display Technology ◽

A Cell

Laccases (1.10.3.2, p-diphenol: dioxygen oxidoreductases) is a family of blue copper-containing oxidases that are commonly found in bacteria, fungi and plants. It is able to oxidize and degrade a variety of aromatic compounds and other organic compounds. Due to this ability, laccases can serve environmental bioremediation processes and industrial purposes. Cell-surface display of enzymes is one of the most attractive applications in yeast. It is a effective utilization to construct the whole cell biocatalyst. The cDNA sequence of Trametes sp. C30 LAC3 was optimized and synthesized according to the codon bias of Saccharomyces Italic textcerevisiae, because codon optimization has been proved to be effective to maximize production of heterologous proteins in yeast. The genes encoding galactokinase (GAL1) promoter, α-mating factor 1 (MFα1) pre-pro secretion signal, fully codon-optimized LAC3, the 320 amino acids of C terminal of α-agglutinin, alcohol dehydrogenase (ADH1) terminator and kanMX cassette were amplified and cloned into YEplac181 to construct a cell-surface display vector called pGMAAK-lac3 with α-agglutinin as an anchor. Then pGMAAK-lac3 was transformed into S. cerevisiae. The results show LAC3 was immobilized and actively expressed on S. cerevisiae. However, the substrate specifity and activity were obviously changed. The displayed LAC3 lost the activity to phenolic substrate (guaiacol) and its activity to non-phenolic substrate (ABTS) was greatly reduced. To our knowledge, this was the first attempt to construct and express laccase through cell-surface display technology.

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Genetic characterization and diversity of Streptococcus agalactiae isolates with macrolide resistance

Journal of Medical Microbiology ◽

10.1099/jmm.0.018176-0 ◽

2010 ◽

Vol 59 (7) ◽

pp. 780-786 ◽

Cited By ~ 13

Author(s):

Monika Brzychczy-Włoch ◽

Tomasz Gosiewski ◽

Małgorzata Bodaszewska ◽

Wojciech Pabian ◽

Małgorzata Bulanda ◽

...

Keyword(s):

Streptococcus Agalactiae ◽

Group B Streptococcus ◽

Surface Protein ◽

Macrolide Resistance ◽

Surface Proteins ◽

Erythromycin Resistance ◽

High Genetic Diversity ◽

Genes Encoding ◽

Resistant Strains ◽

Group B

Macrolide resistance in 169 Streptococcus agalactiae [group B streptococcus (GBS)] isolates originating from pregnant carriers was investigated. Using multiplex PCR the presence of genes encoding erythromycin resistance and capsular polysaccharides, as well as surface proteins, was determined. Random amplification of polymorphic DNA (RAPD) and PFGE were used to characterize specific clones among the isolates. In the examined population of women, erythromycin-resistant strains were found in 4.5 % of patients, whereas clindamycin-resistant strains were found in 3 % of patients, which was 16 % of strains resistant to erythromycin and 10 % of strains resistant to clindamycin among GBS isolates, respectively. Among the isolates, the largest percentage was represented by the constitutive macrolide–lincosamide–streptogramin B (cMLSB) phenotype (63 %), then the inductive macrolide–lincosamide–streptogramin B (iMLSB) phenotype (26 %) and the macrolide resistance (M) phenotype (11 %). The ermB gene was indicated in all isolates with the cMLSB phenotype and V serotype, whereas mefA/mefE genes were found in isolates with the M phenotype and Ia serotype. Among resistance isolates, serotype V was predominant (67 %), followed by serotypes II (15 %), Ia (11 %) and III (7 %). The most common surface protein encoding genes were alp3 (70 %), then rib (11 %), epsilon (7.5 %), bca (7.5 %) and alp2 (4 %). A statistically significant relationship between macrolide resistance, serotype V and the alp3 gene was demonstrated. PFGE, in comparison to the RAPD method, gave better genetic discrimination of GBS isolates. A relatively high genetic diversity among investigated strains was shown. In addition, the largest genetic homogeneity was found in serotype V.

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Research on Construction of a Whole-Cell Biocatalyst for Xylan Degradation

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.347-353.2599 ◽

2011 ◽

Vol 347-353 ◽

pp. 2599-2603

Author(s):

Jian Zhang Lu ◽

Mei Lin Cui ◽

Shan Shan Du ◽

Lu Yang ◽

Qin Guo ◽

...

Keyword(s):

Cell Surface ◽

Surface Display ◽

Cell Surface Display ◽

Whole Cell ◽

Secretion Signal ◽

Gal1 Promoter ◽

Dry Cell Weight ◽

Whole Cell Biocatalyst ◽

Genes Encoding ◽

Secretion Expression

Endo-1,4-β-xylanase (E.C.3.2.1.8) is a family of glycoside hydrolase. It is capable of hydrolyzing the backbone of substituted xylan polymers into fragments of random size. Due to this ability, xylanase can serve the degradation of lignocellulose, and facilitate the application of xylan. Cell-surface display of enzymes is one of the most attractive applications in yeast. It is a promising utilization in constructing the whole-cell biocatalyst of xylanase. For this purpose, a cDNA sequence of endo-1,4-β-xylanase B (XylB) from Aspergillus niger BCC14405 was optimized and synthesized according to the codon bias of Saccharomyces cerevisiae. The genes encoding galactokinase (GAL1) promoter, α-mating factor 1 (MFα1) pre-pro secretion signal, fully codon-optimized XylB, the 320 amino acids of C terminal of α-agglutinin, alcohol dehydrogenase (ADH1) terminator and kanMX cassette were amplified and cloned into YEplac181 to construct a cell-surface display vector called pGMAAK-XylB with α-agglutinin as an anchor. Then pGMAAK-XylB was transformed into S. cerevisiae. The results show XylB was immobilized and actively expressed on S. cerevisiae. Meanwhile, a secretion expression plasmid was also constructed using the above elements except α-agglutinin as a control strain in the study of characteristic of XylB. After an induction of 48 h by 2% galactose, the activity of displayed XylB reached 63 U/g dry-cell weight. The optimal pH of displayed XylB has changed from 5 to 6 and the optimal temperature has changed from 50 °C to 60 °C, comparing to the recombinant secretion XylB.

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Recent Advances in Cell Surface Display Technologies for Directed Protein Evolution

Protein Engineering ◽

10.1002/9783527815128.ch4 ◽

2021 ◽

pp. 81-103

Author(s):

Maryam Raeeszadeh‐Sarmazdeh ◽

Wilfred Chen

Keyword(s):

Cell Surface ◽

Protein Evolution ◽

Surface Display ◽

Cell Surface Display ◽

Recent Advances

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Ligand fishing based on cell surface display of enzymes for inhibitor screening

Analytica Chimica Acta ◽

10.1016/j.aca.2021.338359 ◽

2021 ◽

pp. 338359

Author(s):

Yun-Cong Yuan ◽

Xiao-Lin Bai ◽

Yi-Ming Liu ◽

Xiao-Yue Tang ◽

Hao Yuan ◽

...

Keyword(s):

Cell Surface ◽

Surface Display ◽

Cell Surface Display ◽

Inhibitor Screening ◽

Ligand Fishing

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Cell Surface Display of the Chlamydial Glycolipid Exoantigen (GLXA) Demonstrated by Antibody-Dependent Complement-Mediated Cytotoxicity

Current Microbiology ◽

10.1007/s00284-003-4181-7 ◽

2004 ◽

Vol 49 (1) ◽

Cited By ~ 2

Author(s):

Wilmore C. Webley ◽

Gary J. Vora ◽

Elizabeth S. Stuart

Keyword(s):

Cell Surface ◽

Surface Display ◽

Cell Surface Display ◽

Glycolipid Exoantigen

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Abstract 028: CCN1 Enables Fas Ligand-Induced Apoptosis by Elevating Fas Expression in Primary Cardiomyocytes or by Increasing Cytoplasmic Smac in Cardiomyoblast H9c2 Cells

Circulation Research ◽

10.1161/res.113.suppl_1.a028 ◽

2013 ◽

Vol 113 (suppl_1) ◽

Author(s):

Bor-Chyuan Su ◽

Fan-E Mo

Keyword(s):

Cell Surface ◽

Fas Ligand ◽

Ischemia Reperfusion ◽

Surface Display ◽

Critical Role ◽

Mitogen Activated Protein Kinase ◽

Neonatal Rat ◽

Ros Generation ◽

H9c2 Cells ◽

Induced Apoptosis

Fas/Fas ligand (FasL) is implicated in cardiac ischemia/reperfusion injury. However, cardiomyocytes in culture are resistant to FasL-induced apoptosis, suggesting that additional factor(s) are required for FasL-induced apoptosis. Matricellular protein CCN1 has been demonstrated to promote cytotoxicity of FasL in human skin fibroblasts. CCN1 is induced in a variety of cardiac pathologies. We assessed the hypothesis that CCN1 may be involved in the regulation of FasL-induced apoptosis in cardiomyocytes. We found that either FasL or CCN1 did not induce cell death in neonatal rat ventricular cardiomyocytes (NRVM). Interestingly, the combination of FasL+CCN1 generated 2-fold induction of apoptosis (vs. control p<0.001). An integrin-α 6 β 1 -binding defective mutant CCN1, CCN1-DM failed to exert synergy with FasL to induce apoptosis, indicating a critical role of α 6 β 1 . The engagement between CCN1 and α 6 β 1 instigated the elevation of cellular reactive oxygen species (ROS), the activation of mitogen activated protein kinase p38, and followed by the induction of cell surface display of Fas, thereby sensitizing NRVM to FasL-induced apoptosis. Pretreatment of the p38 inhibitor SB202190 abolished the CCN1-induced cell-surface Fas expression and the apoptosis induced by FasL+CCN1. In addition, we tested the interaction between CCN1 and FasL on the cardiomyoblast H9c2 cells. We found that FasL or CCN1 alone did not cause apoptosis in H9c2, and required the combination of FasL+CCN1 to induced apoptosis (vs. control p<0.001) in H9c2 cells, reminiscent of the observation in NRVM. Mechanistically, CCN1 acted through binding to integrin α 6 β 1 , ROS generation, and p38 activation, however, did not increase the expression of cell surface Fas for its synergy with FasL in H9c2 cells. Instead, CCN1 induced Bax translocation to mitochondria, which in turn led to the release of Smac from mitochondria to cytosol. The cytosolic Smac functions to neutralize XIAP. Smac is critical for CCN1 action, because the knockdown of Smac blunted the apoptotic activities of CCN1. In conclusion, CCN1 may play a detrimental role in a stressed heart to both the differentiated cardiomyocytes and the proliferative cardioblasts through distinct signaling mechanisms.

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