Difference in Resistance to Streptococcus pneumoniae Infection in Mice

Streptococcus pneumoniae is a major pathogen that causes various diseases, including pneumonia and sepsis, as millions of people suffer from S. pneumoniae infection worldwide. To better understand the immune and inflammatory responses to S. pneumoniae, we produced murine models. To investigate the differences between intranasal and intratracheal infection, BALB/c mice were infected with S. pneumoniae D39 intranasally or intratracheally. Mice showed no significant differences in survival rates, body weight changes, and bacterial loads. To investigate resistance and susceptibility among mouse strains, BALB/c, C57BL/6J, tumor necrosis factor-α (TNF-α) knockout, and interleukin-10 (IL-10) knockout mice were infected with S. pneumoniae D39 via intranasal or intravenous routes. In this study, BALB/c and C57BL/6J mice were resistant, IL-10 knockout mice were intermediate, and TNF-α knokout mice were susceptible to S. pneumoniae infection. These data show that intranasal and intratracheal infection induced similar results after S. pneumoniae infection, and the genetic background of mice must be considered when studying S. pneumoniae infection in vivo

Impact of Capsular Switch on Invasive Pneumococcal Disease Incidence in a Vaccinated Population

BACKGROUND: Despite the dramatic decline in the incidence of invasive pneumococcal disease (IPD) observed since the introduction of conjugate vaccination, it is feared that several factors may undermine the future effectiveness of the vaccines. In particular, pathogenic pneumococci may switch their capsular types and evade vaccine-conferred immunity. METHODOLOGY/PRINCIPAL FINDINGS: Here, we first review the literature and summarize the available epidemiological data on capsular switch for S. pneumoniae. We estimate the weekly probability that a persistently carried strain may switch its capsule from four studies, totalling 516 children and 6 years of follow-up, at 1.5x10(-3)/week [4.6x10(-5)-4.8x10(-3)/week]. There is not enough power to assess an increase in this frequency in vaccinated individuals. Then, we use a mathematical model of pneumococcal transmission to quantify the impact of capsular switch on the incidence of IPD in a vaccinated population. In this model, we investigate a wide range of values for the frequency of vaccine-selected capsular switch. Predictions show that, with vaccine-independent switching only, IPD incidence in children should be down by 48% 5 years after the introduction of the vaccine with high coverage. Introducing vaccine-selected capsular switch at a frequency up to 0.01/week shows little effect on this decrease; yearly, at most 3 excess cases of IPD per 10(6) children might occur due to switched pneumococcal strains. CONCLUSIONS: Based on all available data and model predictions, the existence of capsular switch by itself should not impact significantly the efficacy of pneumococcal conjugate vaccination on IPD incidence. This optimistic result should be tempered by the fact that the selective pressure induced by the vaccine is currently increasing along with vaccine coverage worldwide; continued surveillance of pneumococcal populations remains of the utmost importance, in particular during clinical trials of the new conjugate vaccines

The Natural Cytotoxicity Receptor 1 Contribution to Early Clearance of Streptococcus pneumoniae and to Natural Killer-Macrophage Cross Talk

Natural killer (NK) cells serve as a crucial first line of defense against tumors, viral and bacterial infections. We studied the involvement of a principal activating natural killer cell receptor, natural cytotoxicity receptor 1 (NCR1), in the innate immune response to S. pneumoniae infection. Our results demonstrate that the presence of the NCR1 receptor is imperative for the early clearance of S. pneumoniae. We tied the ends in vivo by showing that deficiency in NCR1 resulted in reduced lung NK cell activation and lung IFNγ production at the early stages of S. pneumoniae infection. NCR1 did not mediate direct recognition of S. pneumoniae. Therefore, we studied the involvement of lung macrophages and dendritic cells (DC) as the mediators of NK-expressed NCR1 involvement in response to S. pneumoniae. In vitro, wild type BM-derived macrophages and DC expressed ligands to NCR1 and co-incubation of S. pneumoniae-infected macrophages/DC with NCR1-deficient NK cells resulted in significantly lesser IFNγ levels compared to NCR1-expressing NK cells. In vivo, ablation of lung macrophages and DC was detrimental to the early clearance of S. pneumoniae. NCR1-expressing mice had more potent alveolar macrophages as compared to NCR1-deficient mice. This result correlated with the higher fraction of NCR1-ligandhigh lung macrophages, in NCR1-expressing mice, that had better phagocytic activity compared to NCR1-liganddull macrophages. Overall, our results point to the essential contribution of NK-expressed NCR1 in early response to S. pneumoniae infection and to NCR1-mediated interaction of NK and S. pneumoniae infected-macrophages and -DC

<i>In vitro</i> regulation of the capsule by SpxR and CpsR through the <i>37-CE</i> and <i>in vivo</i> imaging studies.

(A) Luciferase reporter assay in Δ37-CE, and strains where only SpxR (SpxR-only), CpsR (CpsR-only), or neither SpxR nor CpsR can bind (Neither) the 37-CE sequence. Individual data points, the mean, and SEM are plotted from three biological replicates. RLU; relative light units. (B) Western blot analysis of CpsA protein. A representative blot of three independent experiments is shown with quantification below. (C) Quantification of capsule content using dextran exclusion assay. Individual cells from five biological replicates, the mean, and SEM are plotted. (D) Representative images of C showing a fluorescence and phase contrast microscopy overlay. The dark area around the cell represents the capsule. (E) (Left) In vivo imaging quantification (total luminescence over lung normalized to CFU/mg of lung tissue) of Pcps::CBRluc reporters during a pneumonia model of infection. (Right) Example images of one set of mice. Statistical differences in A and C were determined using a one-way ANOVA with Tukey’s multiple comparisons test. Statistical differences in B were determined using a one sample t test and Wilcoxon test. ns = not significant, *** p≤0.001, **** p≤0.0001.</p

pPP3 <i>CBRluc</i> luciferase reporter vector.

(A) Schematic diagram of pPP3 luciferase reporter plasmid. Multiple Cloning Site (MCS) DNA sequence is shown above with unique restriction sites, save EcoRI which has two cut sites. (B) Click beetle luciferese (CBRluc) expression and enzymatic activity driven by the D39 capsule promoter (Pcps) does not affect pneumococcal growth. Growth of Pcps::CBRluc reporter induced (+) and uninduced (-) with luciferin (inset: luciferase activity during log phase growth). Plasmid map was generated using SnapGene software (Insightful Science, San Diego, CA). (JPG)</p

Analysis of the <i>37-CE</i> / <i>21-CE</i> sequence in different pneumococcal serotypes.

(A) Sequences were obtained from the EMBL database (33) and aligned using Clustal Omega (53). The four proposed SpxR ACTA sequences within the 37-CE are highlighted in grey. Proposed CpsR sequences are highlighted in green. D39 (serotype 2) and TIGR4 (serotype 4) are labelled in bold and found at the top of the alignment. (B) Meta-MEME (54) generated a motif using the sequences in A displayed in Hidden Markov Model (HMM) Logo format. (C) Phylogenetic tree generated using Clustal Omega from the 37-CE alignment shown in A displayed using iTOL (55). 37-CE clades are highlighted in different colors. Asterisks indicate serotype 2 (D39) and serotype 4 (TIGR4).</p

Quaternary structure of SpxR.

(A) SEC-MALS analysis (left) and numeric mass data (right) of SpxR in high salt (500 mM NaCl). A major tetramer peak and a minor octamer peak were observed. (B) EM negative stain 2D class averages of SpxR with DNA in both high and low salt. (C) Low resolution 3D models for SpxR with 37-CE DNA in high salt (tetramer) and low salt (trimer “wheel”). The diagram shows three views of the complexes obtained from the EM negative stain data. The long axis of the tetramer complex (high salt) is approximately 200 Å long, whereas the diameter of the trimeric (low salt) wheel is approximately 135 Å. In the low salt complex there is a visible opening formed by the domains, which could be where the double stranded DNA resides. For reference, the three Cartesian axes are shown (X red, Y green, Z blue) on each picture. (D) Fluorescence polarization of SpxR titrated into either 37-CE DNA or (E) 21-CE DNA, in either low or high salt buffer (Tris pH 7.0 with 50 mM NaCl or 500 mM NaCl, respectively). Polarization is expressed in millipolarization units (mPs). The mean of three independent experiments is plotted. Error bars represent standard deviation and are indicated by dashed lines.</p

Additional SpxR_DBD and CpsR EMSAs.

(A) SpxRDBD EMSAs and (B) CpsR EMSAs. For reference, the cps promoter from Fig 2A is shown (C). The precise 37-CE/21-CE double stranded DNA oligos used in experiments are shown above the gel shifts. To define the minimal sequences required for SpxR and CpsR interaction within the 37/21-CE a series of 37-CE truncations were tested for SpxR and CpsR interaction using EMSA. It was first hypothesized that the interactions occurred at either the 10 bp inverted repeats highlighted in light blue, or within the 17 bp spacer region highlighted in pink. Neither SpxR nor CpsR interacted with these regions alone, indicating that they must occur around the junctions of these sequences. Therefore, we tested interactions with oligos consisting of the 10 bp inverted repeat regions (light blue) that had been extended by either 5 or 10 nucleotides (37 CE 5’ + 5/10 and 37 CE 3’ + 5/10). For SpxRDBD, interaction was strongest with the 37 CE 5’ + 10 oligo, followed by the 37 CE 3’ + 10 oligo. Closer examination of these two sequences identified two similar inverted repeat sequences, one that is imperfect on the 5’ half of the 37-CE and another that is perfect on the 3’ half. These are underlined in red for the 37 CE 5’ IR/3’ IR oligos. To achieve robust interaction, the 37 CE 5’ IR and 3’ IR oligos had to be extended (37 CE 5/3’ IR +3/4 each end). We have defined the spxR1 and spxR2 sites as these two inverted repeats within the 37-CE (see C). Unlike SpxR, CpsR was found to only interact with the 5’ end of the 37-CE (see shifts with 37 CE 5’ + 10 and 37-CE 3’ + 10). We hypothesize that CpsR interacts with the direct repeat underlined in black in the 21 CE oligo shift (also depicted in C). Making the spxR1 site a perfect inverted repeat (21 CE perfect oligo) dramatically reduces the affinity of interaction between CpsR and the 21 CE. Gels are representative of 3 independent experiments. (D) Fluorescence polarization with CpsR. We were unable to obtain sigmoidal binding curves with CpsR, likely due to its small size in solution (dimer, 65.72 kDa) relative to the 21-CE oligo. Experiments were carried out as per Fig 2 using protein concentrations between 0 and 5 μM. (JPG)</p

Schematic representation of the proposed mechanism of <i>cps</i> regulation during pneumococcal infection.

SpxR and CpsR respond to unknown ligands in the airways whose presence or absence could influence their interaction with the 37-CE. SpxR binds as a trimer and CpsR as a dimer. Interaction is associated with the nucleoid-like protein HlpA, which assists in SpxR and/or CpsR repression by bending the DNA to occlude RNAP promoter access. After lung to blood transition, we hypothesize that changes in ligand concentrations result in the de-repression of cps operon transcription by alleviating transcription factor binding and allowing for RNAP recruitment. The capsule machinery (cps operon) is then increased substantially during sepsis to avoid phagocytosis. The importance of SpxR and CpsR in regulating cps control might be serotype-specific depending on the precise composition of the 37-CE sequence.</p