s pneumoniae  (ATCC)

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: The Role of Minor Pilins in Assembly and Function of the Competence Pilus of Streptococcus pneumoniae

doi: 10.3389/fcimb.2021.808601

Figure Lengend Snippet: S. pneumoniae strains used in this study.

Article Snippet: For this, PCR fragments flanking the upstream and downstream regions of the target genes were amplified from the S. pneumoniae R6 genomic DNA using Fusion Flash polymerase (Thermo Fisher Scientific).

Techniques:

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: The Role of Minor Pilins in Assembly and Function of the Competence Pilus of Streptococcus pneumoniae

doi: 10.3389/fcimb.2021.808601

Figure Lengend Snippet: Competence pilus assembly and transformability depends on the pneumococcal minor pilins. (A) Schematic representation of the comG operon in S. pneumoniae R6 strain encoding comGA and comGB (white), the major pilin gene comGC (black) and the minor pilin genes comGD, comGE, comGF and comGG (grey). The invariant Glu5 residue, present in all pneumococcal pilins except ComGG, is highlighted (E5). (B) ComGC was detected by immunoblotting in bacterial whole cell lysates and sheared pili of mutants lacking Δ comGC , Δ comGG , Δ comGFG , Δ comGEFG . The WT strain was included as a positive control and GAPDH as loading control. (C) Transformation frequency of WT , comG mutants and R6Δ comGDEFG complemented strains. The detection limit (dl) of the assay was 2.39e -9 and the error bars represent the standard deviation of a minimum of three independent experiments (n=3). (D) Western blotting analysis of ComGC in bacterial whole cell lysates and sheared pili of Δ comGDEFG strains expressing individual minor pilins, comGDEF or comGDEFG at the ectopic bgaA locus. The WT strain was included as a positive control and GAPDH as loading control. (E) Immunofluorescence microscopy (IF) of competence-induced pili in WT R6 and R6Δ comGDEFG complemented with comGG, comGDEF or comGDEFG. Bacteria were visualized by bright field (BF) microscopy and competence pili were labelled with antisera specific for ComGC (red). Scale bars represent 2 µm.

Article Snippet: For this, PCR fragments flanking the upstream and downstream regions of the target genes were amplified from the S. pneumoniae R6 genomic DNA using Fusion Flash polymerase (Thermo Fisher Scientific).

Techniques: Residue, Western Blot, Positive Control, Control, Transformation Assay, Standard Deviation, Expressing, Immunofluorescence, Microscopy, Bacteria

Journal: Immunotherapy Advances

Article Title: Neuraminidase A controls pneumococcal recognition and fate: deficiency enhances immune sensing and intracellular survival, while treatment promotes phagocytic clearance.

doi: 10.1093/immadv/ltag002

Figure Lengend Snippet: Adhesion and intracellular survival capabilities of S. pneumoniae with and without NanA in microglia. Lack of released NanA caused increased adhesion of the T4 strain to BV-2 cells (a), while the membrane-bound form of NanA had no effect on adhesion rates for the pneumococcus in the D39 strains (b). (c) Intracellular survival of wild-type T4 and its isogenic mutant strains 5 h post-infection (2 h post-antibiotic treatment). Values of biological replicates represent the percentage of strain-specific adherent (a, b) or intracellular (c) CFUs relative to total recovered CFUs, normalized to wild-type T4 (a, c) or D39 (b). (a-c) Statistical analysis was performed using a one-way ANOVA, followed by Dunn’s multiple comparisons test, comparing each mutant strain to T4. Columns represent mean values; error bars indicate standard deviations (SD), n = 3–4. (d) Uptake of S. pneumoniae T4 and its isogenic Δ nanA mutant in HMC3 human microglia; values of biological replicates represent the percentage of strain-specific intracellular CFUs relative to total recovered CFUs, normalized to wild-type T4. Statistical analysis was performed using an unpaired two-tailed t -test to compare wild-type T4 and the Δ nanA mutant; columns represent mean values; error bars indicate standard deviations (SD), n = 3.

Article Snippet: Coding fragments of soluble NanA (Q54-N800) and soluble RrgA (E39-G868) were PCR-amplified from S. pneumoniae T4 genomic DNA and subcloned into the pNIC28-Bsa4 plasmid (Addgene #26103).

Techniques: Membrane, Mutagenesis, Infection, Two Tailed Test

Journal: Immunotherapy Advances

Article Title: Neuraminidase A controls pneumococcal recognition and fate: deficiency enhances immune sensing and intracellular survival, while treatment promotes phagocytic clearance.

doi: 10.1093/immadv/ltag002

Figure Lengend Snippet: Microglia infected with S. pneumoniae depicted an increased bacterial signal when infected with pneumococci lacking NanA. Confocal microscopy analysis (63 × magnification) was performed to detect BV2 microglia (CD45, magenta) with intracellular pneumococci (T4 capsule, cyan). (a) Invasion of S. pneumoniae wild-type T4 and deletion mutants after antibiotic treatment (assessment of bacterial invasion), and respective semi-quantification analysis of bacterial signal (b). (c) Intracellular survival of S. pneumoniae mutants at 1 h timepoint after antibiotic treatment (assessment of bacterial intracellular survival), semi-quantification analysis of bacterial signal (d). In a and c, scale bar 50 µm, images shown are representative of three independent experiments. In b and d, values of biological replicates represent the percentage of strain-specific intracellular CFUs relative to total recovered CFUs, normalized to wild-type T4. Statistical analysis was performed using one-way ANOVA, followed by Dunn’s multiple comparisons test, comparing each experimental group to T4. Columns represent mean values; error bars indicate SD, n = 3.

Article Snippet: Coding fragments of soluble NanA (Q54-N800) and soluble RrgA (E39-G868) were PCR-amplified from S. pneumoniae T4 genomic DNA and subcloned into the pNIC28-Bsa4 plasmid (Addgene #26103).

Techniques: Infection, Confocal Microscopy

Journal: Immunotherapy Advances

Article Title: Neuraminidase A controls pneumococcal recognition and fate: deficiency enhances immune sensing and intracellular survival, while treatment promotes phagocytic clearance.

doi: 10.1093/immadv/ltag002

Figure Lengend Snippet: Intracellular presence of bacteria is due to the immune cell phagocytosis process, and the viability of pneumococci lacking NanA inside microglia is associated with increased phagocytic activity. (a) Invasion assay of BV-2 infected cells with S. pneumoniae T4 and its isogenic mutant strains after treatment with cytochalasin D shows decreased invasion of the T4 strains, indicating that bacterial presence inside microglia is the result of an active phagocytic process of microglia towards bacteria. Values of biological replicates represent the percentage of strain-specific intracellular CFUs relative to total recovered CFUs. (b) Confocal microscopy analysis of BV-2 microglial cells infected with S. pneumoniae T4 or Δ nanA mutant strain showed an increase of phagocytic activity after infection with Δ nanA ; 40 × magnification objective was used to detect phagocytic activity of BV2 microglia (CD68, red), microglial cells (CD45, green) and the nuclei (DAPI, blue), scale bar 25 µm; each image is representative of three images (fields of view) taken per each fluorescence channel per each experimental group. (c) Quantification of the mean intensity of CD68 normalized to T4 after infection with wild-type T4 and Δ nanA or uninfected. Statistical analysis was performed using two-way ANOVA with Bonferroni correction for multiple comparisons, n = 3 (a), and one-way ANOVA followed by Dunn’s multiple comparisons test, comparing each experimental group to T4, n = 4 (c). Columns represent mean values; error bars indicate SD.

Article Snippet: Coding fragments of soluble NanA (Q54-N800) and soluble RrgA (E39-G868) were PCR-amplified from S. pneumoniae T4 genomic DNA and subcloned into the pNIC28-Bsa4 plasmid (Addgene #26103).

Techniques: Bacteria, Activity Assay, Invasion Assay, Infection, Mutagenesis, Confocal Microscopy, Fluorescence

Journal: Immunotherapy Advances

Article Title: Neuraminidase A controls pneumococcal recognition and fate: deficiency enhances immune sensing and intracellular survival, while treatment promotes phagocytic clearance.

doi: 10.1093/immadv/ltag002

Figure Lengend Snippet: Increased intracellular presence of pneumococci lacking NanA is not associated with enhanced degrading capability of microglia. (a) Confocal microscopy analysis of BV-2 microglial cells infected with S. pneumoniae T4 or mutant strains after antibiotic treatment; 40 × magnification objective was used to detect lysosomal proteolytic activity of BV-2 cells (Cathepsin B, green), microglial cells (Iba1, red) and the nuclei (DAPI, blue), scale bar 20 µm; each image is representative of three images (fields of view) taken per each fluorescence channel per each experimental group. (b) Quantification of the mean intensity of Cathepsin B normalized to T4 after infection with wild-type T4 and Δ nanA or non-infected. Statistical analysis was performed using one-way ANOVA followed by Dunn’s multiple comparisons test, comparing each experimental group to T4-infected cells, n = 3. Columns represent mean values; error bars indicate SD.

Article Snippet: Coding fragments of soluble NanA (Q54-N800) and soluble RrgA (E39-G868) were PCR-amplified from S. pneumoniae T4 genomic DNA and subcloned into the pNIC28-Bsa4 plasmid (Addgene #26103).

Techniques: Confocal Microscopy, Infection, Mutagenesis, Activity Assay, Fluorescence

Journal: Immunotherapy Advances

Article Title: Neuraminidase A controls pneumococcal recognition and fate: deficiency enhances immune sensing and intracellular survival, while treatment promotes phagocytic clearance.

doi: 10.1093/immadv/ltag002

Figure Lengend Snippet: Recombinant and endogenous NanA enhance microglial and macrophage clearance of S. pneumoniae. (a) Uptake of S. pneumoniae T4, its isogenic Δ nanA mutant, and Δ nanA infections in the presence of recombinant NanA, active or heat-inactivated (HI), or RrgA. Values of biological replicates represent the percentage of experimental group intracellular CFUs relative to the total recovered CFUs, normalized to wild-type T4. Statistical analysis was performed using one-way ANOVA followed by Dunn’s multiple comparisons test. Columns represent mean values; error bars indicate standard deviations (SD), n = 3–6. (b) Uptake of S. pneumoniae T4 and its isogenic mutant strains in RAW 264.7 macrophages. Values of biological replicates represent the percentage of experimental group intracellular CFUs relative to the total recovered CFUs, normalized to wild-type T4. Statistical analysis was performed using one-way ANOVA, with a mixed-effect model for (a), followed by Dunn’s multiple comparisons test. Columns represent mean values; error bars indicate SD, n = 3–6.

Article Snippet: Coding fragments of soluble NanA (Q54-N800) and soluble RrgA (E39-G868) were PCR-amplified from S. pneumoniae T4 genomic DNA and subcloned into the pNIC28-Bsa4 plasmid (Addgene #26103).

Techniques: Recombinant, Mutagenesis