Multiplex PCR Assay for Direct Identification of Group B Streptococcal Alpha-Protein-Like Protein Genes

Creti, Roberta; Fabretti, Francesca; Orefici, Graziella; Hunolstein, Christina von

doi:10.1128/jcm.42.3.1326-1329.2004

Cited by 111 publications

(121 citation statements)

References 18 publications

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“…In FnIII10, this loop contains a canonical Arg-Gly-Asp (RGD) integrin-binding motif, in which the Arg (23), Rib (NCBI accession number AAC44468) (39), R28 (NCBI accession number AAD39085) (17), Alp1 (NCBI accession number AAR08144, L. C. Madoff, unpublished data), Alp2 and Alp3 (NCBI accession numbers AAG01392 and AAG02097, respectively) (37), and Alp4 (NCBI accession number CAD32934) (68) are aligned. Highly conserved residues are shaded in black and gray.…”

Section: Resultsmentioning

confidence: 99%

Crystal Structure of the N-terminal Domain of the Group B Streptococcus Alpha C Protein

Auperin

Bolduc

Baron

et al. 2005

Journal of Biological Chemistry

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Group B Streptococcus (GBS) is the leading cause of bacterial pneumonia, sepsis, and meningitis among neonates and an important cause of morbidity among pregnant women and immunocompromised adults. Invasive diseases due to GBS are attributed to the ability of the pathogen to translocate across human epithelial surfaces. The alpha C protein (ACP) has been identified as an invasin that plays a role in internalization and translocation of GBS across epithelial cells. The soluble N-terminal domain of ACP (NtACP) blocks the internalization of GBS. We determined the 1.86-Å resolution crystal structure of NtACP comprising residues Ser 52 through Leu 225 of the full-length ACP. NtACP has two domains, an N-terminal ␤-sandwich and a C-terminal three-helix bundle. Structural and topological alignments reveal that the ␤-sandwich shares structural elements with the type III fibronectin fold (FnIII), but includes structural elaborations that make it unique. We have identified a potential integrin-binding motif consisting of Lys-Thr-Asp 146 , Arg 110 , and Asp 118 . A similar arrangement of charged residues has been described in other invasins. ACP shows a heparin binding activity that requires NtACP. We propose a possible heparin-binding site, including one surface of the three-helix bundle, and nearby portions of the sandwich and repeat domains. We have validated this prediction using assays of the heparin binding and cell-adhesion properties of engineered fragments of ACP. This is the first crystal structure of a member of the highly conserved Gram-positive surface alpha-like protein family, and it will enable the internalization mechanism of GBS to be dissected at the atomic level.Group B Streptococcus (GBS) 1 (Streptococcus agalactiae) remains the leading cause of invasive bacterial diseases in neonates, despite its decline in prevalence during the last decade because of intrapartum chemoprophylatic therapy (1, 2). It is also an important cause of morbidity in pregnant women and non-pregnant adults with underlying medical conditions (3-5). GBS colonizes the human gastrointestinal and genitourinary tracts and may cause chorioamnionitis and urinary tract infection in pregnant women and a range of invasive infections in elderly and immunocompromised adults (1, 6 -8). During labor and delivery, GBS may be transmitted to neonates, causing pneumonia, sepsis, or meningitis. Four to six percent of all neonatal GBS infections result in death (6, 9). In vitro, GBS adheres to (10, 11), internalizes within (12-14), and translocates across (15) intact human epithelial and endothelial cells. Little is known about the bacterial components that allow this pathogen to adhere to and penetrate cellular membranes. Previous studies suggest that surface proteins are significantly involved in the process (11, 16). The surface-expressed GBS alpha C protein (ACP) has been shown to act as an invasin (15). ACP is the prototype of a family of surface-expressed proteins containing long tandem repeats (alpha-like proteins (Alp)). Members of this famil...

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Section: Resultsmentioning

confidence: 99%

Crystal Structure of the N-terminal Domain of the Group B Streptococcus Alpha C Protein

Auperin

Bolduc

Baron

et al. 2005

Journal of Biological Chemistry

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“…Since our previous study, we have designed primers targeting newly described C␣-like protein genes, namely, Alp1, Alp5, epsilon (alp1, alp5, and epsilon genes), and Alp4 (alp4) (4). Analysis of sequence data in GenBank showed that the alp1, alp5, and epsilon gene sequences are very similar and indistinguishable by use of our primers.…”

Section: Methodsmentioning

confidence: 99%

Comparison of a 3-Set Genotyping System with Multilocus Sequence Typing for Streptococcus agalactiae (Group B Streptococcus)

et al. 2005

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Group B streptococcus (GBS; Streptococcus agalactiae) is the most common cause of neonatal and obstetric sepsis and is an increasingly important cause of septicemia in elderly individuals and immunocompromised patients. Epidemiological studies of GBS infections require comprehensive typing systems that provide information about variable characteristics, such as antigenic type, virulence, or antibiotic resistance, as well as the "backbone" structure or the genetic lineage of isolates. We have previously described a 3-set genotyping system that identifies the molecular serotype ( ) were examined by using the 3-set genotyping system. Genotypes were assigned to five isolates that were nontypeable by conventional serotyping. There were 27 "3-set" genotypes, 24 multilocus sequence types (STs), and 35 unique combinations (or strains), of which the 4 most common, msst III-2 (ST-17), msst III-1 (ST-19), Ia-1 (ST-23), and V-1 (ST-1), accounted for more than 60% of isolates. The 83 isolates were grouped into seven clusters, with a good correlation between the multilocus STs and the genotypes. The combination of 3-set genotyping and MLST adds discriminatory power to strain typing of GBS, which will be useful for future studies of the epidemiology and pathogenesis of GBS disease.

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“…Amino acid sequence alignment of Alps indicates that KTD is highly conserved (Auperin et al, 2005;Creti et al, 2004), suggesting biological importance. The RGD motif present on FnIII10 interacts directly with integrin and is the canonical integrin-binding motif.…”

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

“…Nearly all strains of serotype Ia, II and most of Ib express ACP on their surface (Madoff et al, 1991). In addition, some human serotype III, IV and NT strains and bovine V, VII and non-typable strains contain the ACP gene (bca) (Creti et al, 2004).The biological function(s) of the Alp family of proteins remains unclear (Lindahl et al, 2005 , 1992). The DNA sequence encoding the ACP (pCL1), NtACP (pDEK14), repeat region RR9 (pET24RR9) and D2R has been cloned previously (Auperin et al, 2005; Bolduc et al, 2002;Gravekamp et al, 1996;Kling et al, 1997).…”

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confidence: 99%

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The group B streptococcal alpha C protein binds α 1 β 1-integrin through a novel KTD motif that promotes internalization of GBS within human epithelial cells

Bolduc

Madoff

2007

Microbiology

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Group B Streptococcus (GBS) is the leading cause of bacterial pneumonia, sepsis and meningitis among neonates and a cause of morbidity among pregnant women and immunocompromised adults. GBS epithelial cell invasion is associated with expression of alpha C protein (ACP). Loss of ACP expression results in a decrease in GBS internalization and translocation across human cervical epithelial cells (ME180). Soluble ACP and its 170 amino acid N-terminal region (NtACP), but not the repeat protein RR9, bind to ME180 cells and reduce internalization of wild-type GBS to levels obtained with an ACP-deficient isogenic mutant. In the current study, ACP colocalized with a 1 b 1 -integrin, resulting in integrin clustering as determined by laser scanning confocal microscopy. NtACP contains two structural domains, D1 and D2. D1 is structurally similar to fibronectin's integrin-binding region (FnIII10). D1's (KT)D146 motif is structurally similar to the FnIII10 (RG)D1495 integrin-binding motif, suggesting that ACP binds a 1 b 1 -integrin via the D1 domain. The (KT)D146A mutation within soluble NtACP reduced its ability to bind a 1 b 1 -integrin and inhibit GBS internalization within ME180 cells. Thus ACP binding to human epithelial cell integrins appears to contribute to GBS internalization within epithelial cells. INTRODUCTIONGroup B Streptococcus (GBS; Streptococcus agalactiae) is the leading cause of neonatal bacterial invasive diseases (Arisoy et al., 2003;Manning et al., 2004). The organism colonizes the vagina, rectum and urethra of 10-35 % of adults without causing disease. Neonates acquire GBS either in utero or during birth, aspirating GBS-containing fluid. GBS enters the lungs, binds to and invades the alveolar epithelial cells, and enters the blood, allowing the organisms to infect multiple organs. Invasive disease occurs within hours of birth, with 4-6 % mortality (Baker & Edwards, 1995;Schrag et al., 2000). In a survey in the USA, the incidence of early-onset disease (within the first week of life) declined by 65 % (from 1.7 per 1000 live births to 0.6 per 1000) from 1993 to 1998, due to intrapartum antimicrobial prophylaxis (Schrag et al., 2000) and stabilized at 0.35 per 1000 in 2004 . The incidence of late-onset disease (.1 week to 3 months of age) remained stable, with an average of 0.35 per 1000 live births during the same time period (CDC, 1997(CDC, , 2005.GBS was more recently found to be pathogenic in the elderly, and in adults with underlying medical conditions, including diabetes (Dahl et al., 2003;Jackson et al., 1995;Tyrrell et al., 2000). Adult invasive GBS diseases result in bacteraemia, meningitis, skin and soft-tissue infection, and bone and joint infections. The rate of invasive diseases is significantly higher in neonates than in adults. However, the case fatality rates are greater in adults (Schuchat, 1999), with the incidence of GBS disease increasing with advanced age (Farley et al., 1993). Despite the clinical importance of these diseases, little is known about the events that lead up to GBS inv...

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Multiplex PCR Assay for Direct Identification of Group B Streptococcal Alpha-Protein-Like Protein Genes

Cited by 111 publications

References 18 publications

Crystal Structure of the N-terminal Domain of the Group B Streptococcus Alpha C Protein

Crystal Structure of the N-terminal Domain of the Group B Streptococcus Alpha C Protein

Comparison of a 3-Set Genotyping System with Multilocus Sequence Typing for Streptococcus agalactiae (Group B Streptococcus)

The group B streptococcal alpha C protein binds α 1 β 1-integrin through a novel KTD motif that promotes internalization of GBS within human epithelial cells

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