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Involvement of lipid microdomains in human endothelial cells infected by Streptococcus agalactiae type III belonging to the hypervirulent ST-17.

ABSTRACT: BACKGROUND:Streptococcus agalactiae capsular type III strains are a leading cause of invasive neonatal infections. Many pathogens have developed mechanisms to escape from host defense response using the host membrane microdomain machinery. Lipid rafts play an important role in a variety of cellular functions and the benefit provided by interaction with lipid rafts can vary from one pathogen to another. OBJECTIVES:This study aims to evaluate the involvement of membrane microdomains during infection of human endothelial cell by S. agalactiae. METHODS:The effects of cholesterol depletion and PI3K/AKT signaling pathway activation during S. agalactiae-human umbilical vein endothelial cells (HUVEC) interaction were analysed by pre-treatment with methyl-?-cyclodextrin (M?CD) or LY294002 inhibitors, immunofluorescence and immunoblot analysis. The involvement of lipid rafts was analysed by colocalisation of bacteria with flotillin-1 and caveolin-1 using fluorescence confocal microscopy. FINDINGS:In this work, we demonstrated the importance of the integrity of lipid rafts microdomains and activation of PI3K/Akt pathway during invasion of S. agalactiae strain to HUVEC cells. Our results suggest the involvement of flotillin-1 and caveolin-1 during the invasion of S. agalactiae strain in HUVEC cells. CONCLUSIONS:The collection of our results suggests that lipid microdomain affects the interaction of S. agalactiae type III belonging to the hypervirulent ST-17 with HUVEC cells through PI3K/Akt signaling pathway.

SUBMITTER: Ferreira BJ

PROVIDER: S-EPMC7066991 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Involvement of lipid microdomains in human endothelial cells infected by Streptococcus agalactiae type III belonging to the hypervirulent ST-17.

Ferreira Beatriz Jandre BJ Lannes-Costa Pamella Silva PS Santos Gabriela da Silva GDS Mermelstein Cláudia C Einicker-Lamas Marcelo M Nagao Prescilla Emy PE

Memorias do Instituto Oswaldo Cruz 20200316

<h4>Background</h4>Streptococcus agalactiae capsular type III strains are a leading cause of invasive neonatal infections. Many pathogens have developed mechanisms to escape from host defense response using the host membrane microdomain machinery. Lipid rafts play an important role in a variety of cellular functions and the benefit provided by interaction with lipid rafts can vary from one pathogen to another.<h4>Objectives</h4>This study aims to evaluate the involvement of membrane microdomains ...[more]

PMID: 32187326

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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.

Project description:Group B Streptococcus (GBS) is a commensal of the gastrointestinal and genitourinary tracts, while a prevailing cause of neonatal disease worldwide. Of the various clonal complexes (CCs), CC17 is overrepresented in GBS-infected newborns for reasons that are still largely unknown. Here, we report a comprehensive genomic analysis of 626 CC17 isolates collected worldwide, identifying the genetic traits behind their successful adaptation to humans and the underlying differences between carriage and clinical strains. Comparative analysis with 923 GBS genomes belonging to CC1, CC19, and CC23 revealed that the evolution of CC17 is distinct from that of other human-adapted lineages and recurrently targets functions related to nucleotide and amino acid metabolism, cell adhesion, regulation, and immune evasion. We show that the most distinctive features of disease-specific CC17 isolates were frequent mutations in the virulence-associated CovS and Stk1 kinases, underscoring the crucial role of the entire CovRS regulatory pathway in modulating the pathogenicity of GBS. Importantly, parallel and convergent evolution of major components of the bacterial cell envelope, such as the capsule biosynthesis operon, the pilus, and Rib, reflects adaptation to host immune pressures and should be taken into account in the ongoing development of a GBS vaccine. The presence of recurrent targets of evolution not previously implicated in virulence also opens the way for uncovering new functions involved in host colonization and GBS pathogenesis. IMPORTANCE The incidence of group B Streptococcus (GBS) neonatal disease continues to be a significant cause of concern worldwide. Strains belonging to clonal complex 17 (CC17) are the most frequently responsible for GBS infections in neonates, especially among late-onset disease cases. Therefore, we undertook the largest genomic study of GBS CC17 strains to date to decipher the genetic bases of their remarkable colonization and infection ability. We show that crucial functions involved in different steps of the colonization or infection process of GBS are distinctly mutated during the adaptation of CC17 to the human host. In particular, our results implicate the CovRS two-component regulator of virulence in the differentiation between carriage- and disease-associated isolates. Not only does this work raise important implications for the ongoing development of a vaccine against GBS but might also drive the discovery of key functions for GBS adaptation and pathogenesis that have been overlooked until now. Author Video: An author video summary of this article is available.

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Dataset Information

Involvement of lipid microdomains in human endothelial cells infected by Streptococcus agalactiae type III belonging to the hypervirulent ST-17.

Publications

Involvement of lipid microdomains in human endothelial cells infected by Streptococcus agalactiae type III belonging to the hypervirulent ST-17.

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