Streptococcus pneumoniae Interacts with pIgR Expressed by the Brain Microvascular Endothelium but Does Not Co-Localize with PAF Receptor

Streptococcus pneumoniae is thought to adhere to the blood-brain barrier (BBB) endothelium prior to causing meningitis. The platelet activating factor receptor (PAFR) has been implicated in this adhesion but there is a paucity of data demonstrating direct binding of the bacteria to PAFR. Additionally, studies that inhibit PAFR strongly suggest that alternative receptors for pneumococci are present on the endothelium. Therefore, we studied the roles of PAFR and pIgR, an established epithelial pneumococcal receptor, in pneumococcal adhesion to brain endothelial cells in vivo. Mice were intravenously infected with pneumococci and sacrificed at various time points before meningitis onset. Co-localization of bacteria with PAFR and pIgR was investigated using immunofluorescent analysis of the brain tissue. In vitro blocking with antibodies and incubation of pneumococci with endothelial cell lysates were used to further probe bacteria-receptor interaction. In vivo as well as in vitro pneumococci did not co-localize with PAFR. On the other hand the majority of S. pneumoniae co-localized with endothelial pIgR and pIgR blocking reduced pneumococcal adhesion to endothelial cells. Pneumococci physically interacted with pIgR in endothelial cell lysates. In conclusion, bacteria did not associate with PAFR, indicating an indirect role of PAFR in pneumococcal adhesion to endothelial cells. In contrast, pIgR on the BBB endothelium may represent a novel pneumococcal adhesion receptor

BAGEL:a web-based bacteriocin genome mining tool

A common problem in the annotation of open reading frames (ORFs) is the identification of genes that are functionally similar but have limited or no sequence homology. This is particularly the case for bacteriocins, a very diverse group of antimicrobial peptides produced by bacteria and usually encoded by small, poorly conserved ORFs. ORFs surrounding bacteriocin genes are often biosynthetic genes. This information can be used to locate putative structural bacteriocin genes. Here, we describe BAGEL, a web server that identifies putative bacteriocin ORFs in a DNA sequence using novel, knowledge-based bacteriocin databases and motif databases. Many bacteriocins are encoded by small genes that are often omitted in the annotation process of bacterial genomes. Thus, we have implemented ORF detection using a number of published ORF prediction tools. In addition, BAGEL takes into account the genomic context, i.e. for each potential bacteriocin-encoding ORF, the sequence of the surrounding region on the genome is analyzed for genes that might encode proteins involved in biosynthesis, transport, regulation and/or immunity. These innovations make BAGEL unique in its ability to detect putative bacteriocin gene clusters in (new) bacterial genomes. BAGEL is freely accessible at:

Pneumococcal Gene Complex Involved in Resistance to Extracellular Oxidative Stress

Streptococcus pneumoniae is a Gram-positive bacterium which is a member of the normal human nasopharyngeal flora but can also cause serious disease such as pneumonia, bacteremia, and meningitis. Throughout its life cycle, S. pneumoniae is exposed to significant oxidative stress derived from endogenously produced hydrogen peroxide (H2O2) and from the host through the oxidative burst. How S. pneumoniae, an aerotolerant anaerobic bacterium that lacks catalase, protects itself against hydrogen peroxide stress is still unclear. Bioinformatic analysis of its genome identified a hypothetical open reading frame belonging to the thiol-specific antioxidant (TlpA/TSA) family, located in an operon consisting of three open reading frames. For all four strains tested, deletion of the gene resulted in an approximately 10-fold reduction in survival when strains were exposed to external peroxide stress. However, no role for this gene in survival of internal superoxide stress was observed. Mutagenesis and complementation analysis demonstrated that all three genes are necessary and sufficient for protection against oxidative stress. Interestingly, in a competitive index mouse pneumonia model, deletion of the operon had no impact shortly after infection but was detrimental during the later stages of disease. Thus, we have identified a gene complex involved in the protection of S. pneumoniae against external oxidative stress, which plays an important role during invasive disease.

Platelet Endothelial Cell Adhesion Molecule-1, a Putative Receptor for the Adhesion of Streptococcus pneumoniae to the Vascular Endothelium of the Blood-Brain Barrier

The Gram-positive bacterium Streptococcus pneumoniae is the main causative agent of bacterial meningitis. S. pneumoniae is thought to invade the central nervous system via the bloodstream by crossing the vascular endothelium of the blood-brain barrier. The exact mechanism by which pneumococci cross endothelial cell barriers before meningitis develops is unknown. Here, we investigated the role of PECAM-1/CD31, one of the major endothelial cell adhesion molecules, in S. pneumoniae adhesion to vascular endothelium of the blood-brain barrier. Mice were intravenously infected with pneumococci and sacrificed at various time points to represent stages preceding meningitis. Immunofluorescent analysis of brain tissue of infected mice showed that pneumococci colocalized with PECAM-1. In human brain microvascular endothelial cells (HBMEC) incubated with S. pneumoniae, we observed a clear colocalization between PECAM-1 and pneumococci. Blocking of PECAM-1 reduced the adhesion of S. pneumoniae to endothelial cells in vitro, implying that PECAM-1 is involved in pneumococcal adhesion to the cells. Furthermore, using endothelial cell protein lysates, we demonstrated that S. pneumoniae physically binds to PECAM-1. Moreover, both in vitro and in vivo PECAM-1 colocalizes with the S. pneumoniae adhesion receptor pIgR. Lastly, immunoprecipitation experiments revealed that PECAM-1 can physically interact with pIgR. In summary, we show for the first time that blood-borne S. pneumoniae colocalizes with PECAM-1 expressed by brain microvascular endothelium and that, in addition, they colocalize with pIgR. We hypothesize that this interaction plays a role in pneumococcal binding to the blood-brain barrier vasculature prior to invasion into the brain

Generic and Specific Adaptive Responses of Streptococcus pneumoniae to Challenge with Three Distinct Antimicrobial Peptides, Bacitracin, LL-37, and Nisin▿ †

To investigate the response of Streptococcus pneumoniae to three distinct antimicrobial peptides (AMPs), bacitracin, nisin, and LL-37, transcriptome analysis of challenged bacteria was performed. Only a limited number of genes were found to be up- or downregulated in all cases. Several of these common highly induced genes were chosen for further analysis, i.e., SP0385-SP0387 (SP0385-0387 herein), SP0912-0913, SP0785-0787, SP1714-1715, and the blp gene cluster. Deletion of these genes in combination with MIC determinations showed that several putative transporters, i.e., SP0785-0787 and SP0912-0913, were indeed involved in resistance to lincomycin and LL-37 and to bacitracin, nisin, and lincomycin, respectively. Mutation of the blp bacteriocin immunity genes resulted in an increased sensitivity to LL-37. Interestingly, a putative ABC transporter (SP1715) protected against bacitracin and Hoechst 33342 but conferred sensitivity to LL-37. A GntR-like regulator, SP1714, was identified as a negative regulator of itself and two of the putative transporters. In conclusion, we show that resistance to three different AMPs in S. pneumoniae is mediated by several putative ABC transporters, some of which have not been associated with antimicrobial resistance in this organism before. In addition, a GntR-like regulator that regulates two of these transporters was identified. Our findings extend the understanding of defense mechanisms of this important human pathogen against antimicrobial compounds and point toward novel proteins, i.e., putative ABC transporters, which can be used as targets for the development of new antimicrobials

Confocal example of intracellular <i>S. pneumoniae</i> colocalized with CD63 in endothelial cells.

<p>A: Green <i>S. pneumoniae</i> (Alexa-488), B: Phasecontrast, C: Red CD63 (Alexa-594). D: A merge of green and red with a circle around the intracellular <i>S. pneumoniae</i> (see results for how this was determined), and the sites of colocalization in white, as identified with the ImageJ Colocalization function. The ImageJ orthogonal view function was used to generate the XZ plane (E) and YZ plane (F) and the site of colocalization between CD36 and intracellular <i>S. pneumoniae</i> is seen in yellowish (red and green combined) as highlighted with the white circle. The combined results from image D, E, and F clearly shows that the internalized bacteria are co-localized with the lysosomal marker CD63.</p

<i>S. pneumoniae</i> does not co-localize with PAFR on brain endothelium during the course of infection.

<p>Brain slides of mice intravenously challenged with <i>S. pneumoniae</i> were stained for vasculature endothelium (red), pneumococci (green), and PAFR (blue). Total magnification 630X. For each time point, the panel “Co-localization analysis <i>S. pneumoniae</i>-PAFR” shows bacteria co-localizing with PAFR in white as determined with ImageJ, and bacteria not co-localizing with PAFR in green; white arrows indicate the only pneumococci co-localized with PAFR. For each time point of infection, brains from 3 mice were analyzed, and of each mouse 3 brain sections were used for the immunofluorescent detection. The images are representative of the situation in each brain compartment during the entire time course of infection and in each mouse that was analyzed.</p

PIgR is involved in <i>S. pneumoniae</i> adhesion to human endothelial cells and pneumococci co-localize with pIgR.

<p>A. Immunofluorescent detection of pIgR (green), adherent <i>S. pneumoniae</i> (red) and cellular nuclei (blue) in HBMEC cells after 1 hour incubation with pneumococci. After 1 hour incubation with pneumococci, HBMEC cells were washed with PBS to remove the non-adherent bacteria and fixed. Total magnification 400X. B. Co-localization between pneumococci and pIgR detected in panel A. White pixels represent the areas of bacterial signal co-localized with pIgR, while red pixels indicate the area of bacterial signal not co-localized with pIgR. C. Immunofluorescent detection of pIgR (green), adherent <i>S. pneumoniae</i> (red) and cellular nuclei (blue) in HUVEC cells after 1 hour incubation with pneumococci. After 1 hour incubation with pneumococci, HUVEC cells were washed with PBS to remove the non-adherent bacteria, subsequently immune fluorescent staining was performed. Total magnification 400X. D. Co-localization between pneumococci and pIgR detected in panel C. White pixels represent the areas of bacterial signal co-localized with pIgR, while red pixels indicate the area of bacterial signal not co-localized with pIgR. E-G. Blocking of pIgR (white column) in Detroit cells (E), HBMEC cells (F) and HUVEC cells (G) leads to a significant reduction of pneumococcal adhesion in comparison with Detroit/HBMEC/HUVEC treated with isotype control IgG (hatched column) or with Detroit/HBMEC/HUVEC cells incubated without a blocking antibody (black column). * P value <0.05.</p

Heterogeneous expression of pIgR by brain endothelium in mock-infected mice.

<p>A. Immunofluorescent detection of pIgR (green) and endothelial cells (red) in the brain in absence of bacterial infection shows heterogeneous expression of pIgR on brain endothelium <i>in vivo</i>. Total magnification 630X. B. Confocal microscopy images visualizing pIgR (green) on the brain vascular endothelium (red). Overlaps in the pIgR signal and endothelial cell staining resulted in a yellow color, suggestive of pIgR expression by endothelial cells. The white scale bar in each image represents 20 µm. Brains analyzed were from 2 mock treated mice, of each mouse 3 brain sections were used for immunofluorescent detection. These images are representative of the situation in i) each brain compartment and ii) each mouse that was analyzed.</p

Relative translocation of internalized <i>S. pneumoniae</i> when the lysosomal function is inhibited with NH₄Cl.

<p>Translocation in the presence of the vehicle of the inhibitor is set at 100%, and translocation in experimental conditions is related to this percentage. O designates the relative invasion in one individual experiment, ― is the average of all experiments. (n = 6), p.<0.05. Translocation was defined as the cumulative number of bacteria obtained in the lower compartment over 5 hours divided by the number of bacteria obtained at initial invasion point.</p