Image_1_Engagement of α3β1 and α2β1 integrins by hypervirulent Streptococcus agalactiae in invasion of polarized enterocytes.TIF

The gut represents an important site of colonization of the commensal bacterium Streptococcus agalactiae (group B Streptococcus or GBS), which can also behave as a deadly pathogen in neonates and adults. Invasion of the intestinal epithelial barrier is likely a crucial step in the pathogenesis of neonatal infections caused by GBS belonging to clonal complex 17 (CC17). We have previously shown that the prototypical CC17 BM110 strain invades polarized enterocyte-like cells through their lateral surfaces using an endocytic pathway. By analyzing the cellular distribution of putative GBS receptors in human enterocyte-like Caco-2 cells, we find here that the alpha 3 (α3) and alpha 2 (α2) integrin subunits are selectively expressed on lateral enterocyte surfaces at equatorial and parabasal levels along the vertical axis of polarized cells, in an area corresponding to GBS entry sites. The α3β1 and α2β1 integrins were not readily accessible in fully differentiated Caco-2 monolayers but could be exposed to specific antibodies after weakening of intercellular junctions in calcium-free media. Under these conditions, anti-α3β1 and anti-α2β1 antibodies significantly reduced GBS adhesion to and invasion of enterocytes. After endocytosis, α3β1 and α2β1 integrins localized to areas of actin remodeling around GBS containing vacuoles. Taken together, these data indicate that GBS can invade enterocytes by binding to α3β1 and α2β1 integrins on the lateral membrane of polarized enterocytes, resulting in cytoskeletal remodeling and bacterial internalization. Blocking integrins might represent a viable strategy to prevent GBS invasion of gut epithelial tissues.</p

Image_3_Engagement of α3β1 and α2β1 integrins by hypervirulent Streptococcus agalactiae in invasion of polarized enterocytes.TIF

The gut represents an important site of colonization of the commensal bacterium Streptococcus agalactiae (group B Streptococcus or GBS), which can also behave as a deadly pathogen in neonates and adults. Invasion of the intestinal epithelial barrier is likely a crucial step in the pathogenesis of neonatal infections caused by GBS belonging to clonal complex 17 (CC17). We have previously shown that the prototypical CC17 BM110 strain invades polarized enterocyte-like cells through their lateral surfaces using an endocytic pathway. By analyzing the cellular distribution of putative GBS receptors in human enterocyte-like Caco-2 cells, we find here that the alpha 3 (α3) and alpha 2 (α2) integrin subunits are selectively expressed on lateral enterocyte surfaces at equatorial and parabasal levels along the vertical axis of polarized cells, in an area corresponding to GBS entry sites. The α3β1 and α2β1 integrins were not readily accessible in fully differentiated Caco-2 monolayers but could be exposed to specific antibodies after weakening of intercellular junctions in calcium-free media. Under these conditions, anti-α3β1 and anti-α2β1 antibodies significantly reduced GBS adhesion to and invasion of enterocytes. After endocytosis, α3β1 and α2β1 integrins localized to areas of actin remodeling around GBS containing vacuoles. Taken together, these data indicate that GBS can invade enterocytes by binding to α3β1 and α2β1 integrins on the lateral membrane of polarized enterocytes, resulting in cytoskeletal remodeling and bacterial internalization. Blocking integrins might represent a viable strategy to prevent GBS invasion of gut epithelial tissues.</p

Image_2_Engagement of α3β1 and α2β1 integrins by hypervirulent Streptococcus agalactiae in invasion of polarized enterocytes.TIF

The gut represents an important site of colonization of the commensal bacterium Streptococcus agalactiae (group B Streptococcus or GBS), which can also behave as a deadly pathogen in neonates and adults. Invasion of the intestinal epithelial barrier is likely a crucial step in the pathogenesis of neonatal infections caused by GBS belonging to clonal complex 17 (CC17). We have previously shown that the prototypical CC17 BM110 strain invades polarized enterocyte-like cells through their lateral surfaces using an endocytic pathway. By analyzing the cellular distribution of putative GBS receptors in human enterocyte-like Caco-2 cells, we find here that the alpha 3 (α3) and alpha 2 (α2) integrin subunits are selectively expressed on lateral enterocyte surfaces at equatorial and parabasal levels along the vertical axis of polarized cells, in an area corresponding to GBS entry sites. The α3β1 and α2β1 integrins were not readily accessible in fully differentiated Caco-2 monolayers but could be exposed to specific antibodies after weakening of intercellular junctions in calcium-free media. Under these conditions, anti-α3β1 and anti-α2β1 antibodies significantly reduced GBS adhesion to and invasion of enterocytes. After endocytosis, α3β1 and α2β1 integrins localized to areas of actin remodeling around GBS containing vacuoles. Taken together, these data indicate that GBS can invade enterocytes by binding to α3β1 and α2β1 integrins on the lateral membrane of polarized enterocytes, resulting in cytoskeletal remodeling and bacterial internalization. Blocking integrins might represent a viable strategy to prevent GBS invasion of gut epithelial tissues.</p

Image_5_Engagement of α3β1 and α2β1 integrins by hypervirulent Streptococcus agalactiae in invasion of polarized enterocytes.TIF

The gut represents an important site of colonization of the commensal bacterium Streptococcus agalactiae (group B Streptococcus or GBS), which can also behave as a deadly pathogen in neonates and adults. Invasion of the intestinal epithelial barrier is likely a crucial step in the pathogenesis of neonatal infections caused by GBS belonging to clonal complex 17 (CC17). We have previously shown that the prototypical CC17 BM110 strain invades polarized enterocyte-like cells through their lateral surfaces using an endocytic pathway. By analyzing the cellular distribution of putative GBS receptors in human enterocyte-like Caco-2 cells, we find here that the alpha 3 (α3) and alpha 2 (α2) integrin subunits are selectively expressed on lateral enterocyte surfaces at equatorial and parabasal levels along the vertical axis of polarized cells, in an area corresponding to GBS entry sites. The α3β1 and α2β1 integrins were not readily accessible in fully differentiated Caco-2 monolayers but could be exposed to specific antibodies after weakening of intercellular junctions in calcium-free media. Under these conditions, anti-α3β1 and anti-α2β1 antibodies significantly reduced GBS adhesion to and invasion of enterocytes. After endocytosis, α3β1 and α2β1 integrins localized to areas of actin remodeling around GBS containing vacuoles. Taken together, these data indicate that GBS can invade enterocytes by binding to α3β1 and α2β1 integrins on the lateral membrane of polarized enterocytes, resulting in cytoskeletal remodeling and bacterial internalization. Blocking integrins might represent a viable strategy to prevent GBS invasion of gut epithelial tissues.</p

Image_4_Engagement of α3β1 and α2β1 integrins by hypervirulent Streptococcus agalactiae in invasion of polarized enterocytes.TIF

The gut represents an important site of colonization of the commensal bacterium Streptococcus agalactiae (group B Streptococcus or GBS), which can also behave as a deadly pathogen in neonates and adults. Invasion of the intestinal epithelial barrier is likely a crucial step in the pathogenesis of neonatal infections caused by GBS belonging to clonal complex 17 (CC17). We have previously shown that the prototypical CC17 BM110 strain invades polarized enterocyte-like cells through their lateral surfaces using an endocytic pathway. By analyzing the cellular distribution of putative GBS receptors in human enterocyte-like Caco-2 cells, we find here that the alpha 3 (α3) and alpha 2 (α2) integrin subunits are selectively expressed on lateral enterocyte surfaces at equatorial and parabasal levels along the vertical axis of polarized cells, in an area corresponding to GBS entry sites. The α3β1 and α2β1 integrins were not readily accessible in fully differentiated Caco-2 monolayers but could be exposed to specific antibodies after weakening of intercellular junctions in calcium-free media. Under these conditions, anti-α3β1 and anti-α2β1 antibodies significantly reduced GBS adhesion to and invasion of enterocytes. After endocytosis, α3β1 and α2β1 integrins localized to areas of actin remodeling around GBS containing vacuoles. Taken together, these data indicate that GBS can invade enterocytes by binding to α3β1 and α2β1 integrins on the lateral membrane of polarized enterocytes, resulting in cytoskeletal remodeling and bacterial internalization. Blocking integrins might represent a viable strategy to prevent GBS invasion of gut epithelial tissues.</p

Neutrophils Directly Recognize Group B Streptococci and Contribute to Interleukin-1β Production during Infection

<div><p>Previous studies have shown that the pro-inflammatory cytokine IL-1β has a crucial role in host defenses against group B streptococcus (GBS), a frequent human pathogen, by recruiting neutrophils to infection sites. We examined here the cell types and mechanisms involved in IL-1β production during infection. Using a GBS-induced peritonitis model in mice, we first found that a large proportion of exudate cells contain intracellular IL-1β by immunofluorescence. Of the IL-1β positive cells, 82 and 7% were neutrophils and macrophages, respectively, suggesting that the former cell type might significantly contribute to IL-1β production. Accordingly, depletion of neutrophils with anti-Ly6G antibodies resulted in a significant reduction in the levels of IL-1β, but not of TNF-α or IL-6. We next found that neutrophils are capable of releasing mature IL-1β and TNF-α directly in response to <i>in vitro</i> stimulation with GBS. The production of pro-IL-1β and TNF-α in these cells required the Toll-like receptor (TLR) adaptor MyD88 and the chaperone protein UNC93B1, which is involved in mobilization of a subfamily of TLRs to the endosomes. Moreover, pro-IL-1β processing and IL-1β release was triggered by GBS hemolysin and required components of the canonical inflammasome, including caspase-1, ASC and NLRP3. Collectively our findings indicate that neutrophils make a significant contribution to IL-1β production during GBS infection, thereby amplifying their own recruitment. These cells directly recognize GBS by means of endosomal TLRs and cytosolic sensors, leading to activation of the caspase-1 inflammasome.</p></div

IL-1β processing by neutrophils is mediated by caspase-1.

<p>Mouse neutrophils (5 x 10⁵/well) were incubated for 1 h in the presence of Z-VAD, YVAD-CHO, IETD, AEBSF, NE (all at a concentration of 10μM), or CGi (0.5μM). Live GBS (MOI 20) were added and secreted IL-1β (<b>A, C</b>) and TNF-α (<b>B, D</b>) were measured in culture supernatants after 24 h of incubation. Data are expressed as means + SD of three independent observations, each conducted with cells from a different animal. *, <i>p</i><0.05 versus untreated cells, as determined by one-way analysis of variance and the Student's–Keuls–Newman test.</p

Neutrophils are the predominant cell type expressing IL-1β in response to challenge with GBS.

<p>Flow cytometry analysis showing cells positive for intracellular IL-1β staining in peritoneal lavage fluid samples from WT C57BL/6 mice challenged i.p. with HK-GBS. Neutrophils and macrophages were identified based on expression of Ly6G and F4/80, respectively. Data are from one representative experiment of three producing similar results.</p

The caspase 1 inflammasome is involved in pro-IL-1β processing and IL-1β release in GBS-infected neutrophils.

<p>Concentrations of IL-1β (<b>A</b> and <b>C</b>) or TNF-α (<b>B</b> and <b>D</b>) in culture supernatants of neutrophils lacking the indicated TLRs or inflammasome components. Supernatants were collected at 24 h after infection with live bacteria (MOI 20). Positive controls consisted of cells treated with LPS (0.1μg/ml) and then pulsed with ATP (5mM) for 30 min before collecting the supernatants. Data are expressed as means + SD of three independent observations, each conducted with cells from a different animal. *, <i>p</i><0.05 versus WT mice, as determined by one-way analysis of variance and the Student's-Keuls-Newman test. <b>E</b>, Western blot analysis, using anti-IL-1β antibodies, of lysates from neutrophils lacking the indicated inflammasome components. Neutrophils were infected with GBS (MOI 20) for 4h.</p

Effects of neutrophil depletion on cytokine production in response to GBS.

<p>WT C57BL/6 mice were pretreated with rat anti-Ly6G monoclonal antibody or isotype control Ig before i.p. challenge with HK-GBS. <b>A-C,</b> numbers of peritoneal cells positive for Ly6G (granulocytes), F4/80 (macrophages) and CD11c (dendritic cells) at the indicated times after HK-GBS challenge. <b>D-F</b>, Cytokine concentrations in peritoneal lavage fluid samples at the indicated times after HK-GBS challenge. Data are expressed as means±SD of three independent observations, each conducted on a different animal. *, <i>p</i><0.05, relative to isotype control-pretreated mice by one-way analysis of variance and the Student’s-Keuls-Newman test.</p