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Role of pilus proteins in adherence and invasion of Streptococcus agalactiae to the lung and cervical epithelial cells.

ABSTRACT: Streptococcus agalactiae, or group B Streptococcus (GBS), is an important opportunistic pathogen that causes pneumonia, sepsis, and meningitis in neonates and severe diseases in immunocompromised adults. We have performed comparative genomics of prevalent GBS serotypes of Indian origin (i.e. Ia, III, V, and VII). Pilus-proteins were commonly found up-regulated, and their expression was studied by using antiserum for GBS80 (backbone protein of pilus island-I), GBS67 (ancillary protein of PI-2a), and SAN1518 (backbone protein of PI-2b) by whole cell and Western blot analysis. To check the role of pilus proteins in adherence and invasion, an inhibition assay was performed. Comparative immunoblotting experiments revealed that expression of pili proteins does not differ in geographically different selected serotypes, Ia and V, of India and the United States. In the case of A549 cells, we found that GBS VII invasion and adherence was inhibited by pilus protein-specific antiserum SAN1518 significantly (p < 0.001) by 88.5 and 91%, respectively. We found that mutant strains, deficient in the pilus proteins (?gbs80 and ?san1518) exhibit a significant decrease in adherence in the case of type Ia, III, and VII. In the case of type VII, we have found a 95% reduction in invasion when ?san1518 was used with A549 cells. Because the pilus proteins were identified previously as vaccine candidates against GBS serotypes of developed countries, we also found their role in the attachment and invasion of GBS of Indian origin. Thus, the present work supports the idea of making a more effective pilus protein-based vaccine that can be used universally.

SUBMITTER: Sharma P

PROVIDER: S-EPMC3567654 | biostudies-literature | 2013 Feb

REPOSITORIES: biostudies-literature

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Role of pilus proteins in adherence and invasion of Streptococcus agalactiae to the lung and cervical epithelial cells.

Sharma Puja P Lata Hem H Arya Deepak Kumar DK Kashyap Arun Kumar AK Kumar Hemant H Kumar Hemant H Dua Meenakshi M Ali Arif A Johri Atul Kumar AK

The Journal of biological chemistry 20121203 6

Streptococcus agalactiae, or group B Streptococcus (GBS), is an important opportunistic pathogen that causes pneumonia, sepsis, and meningitis in neonates and severe diseases in immunocompromised adults. We have performed comparative genomics of prevalent GBS serotypes of Indian origin (i.e. Ia, III, V, and VII). Pilus-proteins were commonly found up-regulated, and their expression was studied by using antiserum for GBS80 (backbone protein of pilus island-I), GBS67 (ancillary protein of PI-2a), ...[more]

PMID: 23209289

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Project description:Streptococcus agalactiae is a common human commensal and a major life-threatening pathogen in neonates. Adherence to host epithelial cells is the first critical step of the infectious process. Pili have been observed on the surface of several gram-positive bacteria including S. agalactiae. We previously characterized the pilus-encoding operon gbs1479-1474 in strain NEM316. This pilus is composed of three structural subunit proteins: Gbs1478 (PilA), Gbs1477 (PilB), and Gbs1474 (PilC), and its assembly involves two class C sortases (SrtC3 and SrtC4). PilB, the bona fide pilin, is the major component; PilA, the pilus associated adhesin, and PilC, are both accessory proteins incorporated into the pilus backbone. We first addressed the role of the housekeeping sortase A in pilus biogenesis and showed that it is essential for the covalent anchoring of the pilus fiber to the peptidoglycan. We next aimed at understanding the role of the pilus fiber in bacterial adherence and at resolving the paradox of an adhesive but dispensable pilus. Combining immunoblotting and electron microscopy analyses, we showed that the PilB fiber is essential for efficient PilA display on the surface of the capsulated strain NEM316. We then demonstrated that pilus integrity becomes critical for adherence to respiratory epithelial cells under flow-conditions mimicking an in vivo situation and revealing the limitations of the commonly used static adherence model. Interestingly, PilA exhibits a von Willebrand adhesion domain (VWA) found in many extracellular eucaryotic proteins. We show here that the VWA domain of PilA is essential for its adhesive function, demonstrating for the first time the functionality of a prokaryotic VWA homolog. Furthermore, the auto aggregative phenotype of NEM316 observed in standing liquid culture was strongly reduced in all three individual pilus mutants. S. agalactiae strain NEM316 was able to form biofilm in microtiter plate and, strikingly, the PilA and PilB mutants were strongly impaired in biofilm formation. Surprisingly, the VWA domain involved in adherence to epithelial cells was not required for biofilm formation.

Project description:The widely spread Streptococcus agalactiae (also known as Group B Streptococcus, GBS) "hypervirulent" ST17 clone is strongly associated with neonatal meningitis. The PI-2b locus is mainly found in ST17 strains but is also present in a few non ST17 human isolates such as the ST-7 prototype strain A909. Here, we analysed the expression of the PI-2b pilus in the ST17 strain BM110 as compared to the non ST17 A909. Comparative genome analyses revealed the presence of a 43-base pair (bp) hairpin-like structure in the upstream region of PI-2b operon in all 26 ST17 genomes, which was absent in the 8 non-ST17 strains carrying the PI-2b locus. Deletion of this 43-bp sequence in strain BM110 resulted in a 3- to 5-fold increased transcription of PI-2b. Characterization of PI-2b promoter region in A909 and BM110 strains was carried out by RNAseq, primer extension, qRT-PCR and transcriptional fusions with gfp as reporter gene. Our results indicate the presence of a single promoter (Ppi2b) with a transcriptional start site (TSS) mapped 37 bases upstream of the start codon of the first PI-2b gene. The large operon of 16 genes located upstream of PI-2b codes for the group B carbohydrate (also known as antigen B), a major constituent of the bacterial cell wall. We showed that the hairpin sequence located between antigen B and PI-2b operons is a transcriptional terminator. In A909, increased expression of PI-2b probably results from read-through transcription from antigen B operon. In addition, we showed that an extended 5' promoter region is required for maximal transcription of gfp as a reporter gene in S. agalactiae from Ppi2b promoter. Gene reporter assays performed in Lactococcus lactis strain NZ9000, a related non-pathogenic Gram-positive species, revealed that GBS-specific regulatory factors are required to drive PI-2b transcription. PI-2b expression is up-regulated in the BM110?covR mutant as compared to the parental BM110 strain, but this effect is probably indirect. Collectively, our results indicate that PI-2b expression is regulated in GBS ST17 strains, which may confer a selective advantage in the human host either by reducing host immune responses and/or increasing their dissemination potential.

Dataset Information

Role of pilus proteins in adherence and invasion of Streptococcus agalactiae to the lung and cervical epithelial cells.

Publications

Role of pilus proteins in adherence and invasion of Streptococcus agalactiae to the lung and cervical epithelial cells.

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