Streptococcus pneumoniae translocates into the myocardium and forms unique microlesions that disrupt cardiac function

Abstract

Hospitalization of the elderly for invasive pneumococcal disease is frequently accompanied by the occurrence of an adverse cardiac event; these are primarily new or worsened heart failure and cardiac arrhythmia. Herein, we describe previously unrecognized microscopic lesions (microlesions) formed within the myocardium of mice, rhesus macaques, and humans during bacteremic Streptococcus pneumoniae infection. In mice, invasive pneumococcal disease (IPD) severity correlated with levels of serum troponin, a marker for cardiac damage, the development of aberrant cardiac electrophysiology, and the number and size of cardiac microlesions. Microlesions were prominent in the ventricles, vacuolar in appearance with extracellular pneumococci, and remarkable due to the absence of infiltrating immune cells. The pore-forming toxin pneumolysin was required for microlesion formation but Interleukin-1β was not detected at the microlesion site ruling out pneumolysin-mediated pyroptosis as a cause of cell death. Antibiotic treatment resulted in maturing of the lesions over one week with robust immune cell infiltration and collagen deposition suggestive of long-term cardiac scarring. Bacterial translocation into the heart tissue required the pneumococcal adhesin CbpA and the host ligands Laminin receptor (LR) and Platelet-activating factor receptor. Immunization of mice with a fusion construct of CbpA or the LR binding domain of CbpA with the pneumolysin toxoid L460D protected against microlesion formation. We conclude that microlesion formation may contribute to the acute and long-term adverse cardiac events seen in humans with IPD.

Figure 1. IPD is associated with alterations in cardiac electrophysiology and heart damage.

A) Bacterial titers in blood of mice at 12 (n = 24), 24 (n = 17), and 30 (n = 11) h following intraperitoneal challenge with 10³ CFU of S. pneumoniae, strain TIGR4. *P<0.05 by two-tailed Student's t-test. B) Regression analysis of blood bacterial titers and cardiac troponin-I concentrations at various time points following intraperitoneal challenge with TIGR4 (n = 16). Statistical analysis was done using a Pearson correlation coefficient calculator. C) Limb-lead electrocardiogram (ECG) tracings from a single mouse prior to and following intraperitoneal infection at 0, 12, 24 and 30 h. Letters at 0 h identify the corresponding ECG waves. D) ECG tracings obtained from 3 representative mice (Mouse [M] 2–4) 24–30 h post infection highlighting the variety of arrhythmias observed among the infected mice (n = 8 for 0, 12, and 24 h; n = 6 for 30 h). The ECGs of control saline treated mice showed no electrical disturbances despite repeated anesthesia and ECG measurement (n = 2; see Fig. S1). Note in panels C and D the pronounced bifurcated P-wave (blue dot), the early compensatory increase and then reduced R wave at late time points (purple dot), the presence of a J-wave (orange dot), the elongated intervals for contraction (red dot), PQ wave (black dot) and fibrillation (green dot). ECG tracings were acquired at 200 kHz using the 100B electrocardiogram data acquisition system (iWorx) with mice under isoflurane anesthesia. Fig. S1 shows an extended ECG rhythm strip for these infected mice.

Figure 2. Cardiac lesions form as the result of IPD.

H&E stained cross section of a heart obtained from a BALB/c mouse 30 h post-intraperitoneal challenge with TIGR4. A) Cardiac microlesions were randomly distributed throughout the mouse myocardium. The circled regions demarcate lesion areas. B) Pericarditis was observed in rare mice at 30 h post infection. C) Cardiac microlesions were often observed to be adjacent to blood vessels. D&E) Representative images of cardiac microlesions seen at 24 h (n = 8) and 30 h (n = 11) post infection, respectively. F) Higher powered magnification of the 30 h cardiac microlesion shows S. pneumoniae bacterial aggregates within the microlesion. G) As a point of contrast, in mice infected i.p. with Staphylococcus aureus (Sa) abscesses were large and characterized by a robust neutrophil response (white arrow). Tissue sample a gift from Dr. Eric Skaar, Nashville, TN. H) Transmission electron microscopy image of cardiac lesion indicates that the bacteria within have diplococcal morphology. I) Immunofluorescent detection of the bacterial capsule (serotype 4) confirmed that the granular bodies are S. pneumoniae. J) Representative cardiac lesion seen in the heart of 3 SIV-infected macaques that had succumbed to experimental pneumococcal challenge despite antimicrobial therapy. Similar lesions were absent in the hearts of macaques that cleared the infection (n = 2). K) Cardiac lesion detected in heart of a human adult that had succumbed to IPD. Lesions were observed in 2 of 9 human heart samples. L) Cardiac microlesion from a mouse with IPD that had been treated with ampicillin beginning at 30 h post-infection. Cardiac section was collected 12 hours after initiating antimicrobial therapy (n = 4).

Figure 3. Lesion formation is dependent on the host protein PAFR and the bacterial adhesin CbpA.

A) Total counts, size of lesions, and bacterial burden in BALB/c mice infected with TIGR4 (24 h n = 8; 30 h n = 12), T4 ΔcbpA (24 h n = 8; 30 h n = 13), and T4 Δpln (24 h n = 8; 30 h n = 15) post-infection. B) Counts of cardiac lesions and bacterial burden found in sections from TIGR4 infected wild-type C57BL/6 (n = 6, n = 13, respectively) and PAFR^−/− (n = 9, n = 6, respectively) mice. C) Cardiac lesions and bacterial titers in the blood in TIGR4 infected BALB/c mice following passive immunization with monoclonal antibodies against LR (anti-LR n = 8) or with an isotype control (n = 8). D) Immunofluorescence microscopy of a cardiac section treated with FITC conjugated anti-PAFr or anti-LR antibodies in addition to tomato lectin that is selective for vascular endothelial cells. DAPI was used to stain nuclei. Top left image was taken under bright field with the rectangle indicating the location of cardiac blood vessels. Note that on overlaid images of the same tissue section PAFR and LR were found primarily on the vascular endothelial cells and not on cardiomyocytes. E) Comparison of pneumococcal invasion rates into rat HL-1 cardiomyocytes, human type II pneumocytes (A549) and rat brain endothelial (RBCEC6) cell lines. The graph represents the ratio of invasive over adherent CFUs (n = 3, each with 4 replicates). Statistical analysis on panels A–C and E was performed using a non-parametric Mann-Whitney rank sum test; *P<0.05.

Figure 4. Effect of pneumolysin on cardiomyocyte viability.

A) Immunofluorescent TUNEL (red) staining of cardiac microlesions from BALB/c mice 30 h following intraperitoneal infection. Pneumococci were detected using antibodies against serotype 4 capsular polysaccharide (green) and cardiomyocyte nuclei stained with DAPI. B) Detection of pneumolysin (red) in a microlesion using anti-pneumolysin monoclonal antibody. C) Detection of pneumococcal cell wall (red) in microlesions by immunohistochemistry using TEPC-15 an IgA monoclonal antibody against cell wall. For panels A–C) fluorescent microscopy using the corresponding control antibody is shown immediately below. Dashed line demarcates the site of the lesion. Vybrant MTT Cell Proliferation Assay was used to determine cell viability of HL-1, A549, and RBCEC6 cells following their exposure to D) recombinant pneumolysin (rPLY) or E) purified pneumococcal cell wall. Experiments were done 3 times each with 4 replicates. Shown are the results from single, representative experiments.

Figure 5. YLN immunized mice are protected against lesion formation.

A) Top left: Schematic representation of the anti-parallel helices of R domains of CbpA. Square: binding site for polymeric immunoglobulin receptor showing amino acids at the turn (i.e. YPT); Circle: binding site for LR showing amino acids at the turn (i.e. NEEK). Top right: Schematic representation of various fusion protein derivatives of CbpA and the pneumolysin toxoid L460D used for vaccination. YLN is identified as composed of YPT-L460D-NEEK in our studies. B) Cardiac lesion number per individual mouse (circle) at 30 h post infection obtained from immunized mice. Experimental cohort size: Alum = 20; CbpA-R12 = 19; L460D = 10; YPT-L460D = 10; L460D-NEEK = 10; YLN = 20. Asterisks denote a statistical significant difference versus the alum control. Statistical analysis was done using Kruskall-Wallis a One-way ANOVA on Ranks.

Figure 6. Immune cell infiltration and collagen deposition following antimicrobial therapy.

BALB/c mice were infected with S. pneumoniae and beginning at 30 h treated with ampicillin for rescue. A) Representative H&E stained cross sections of hearts from BALB/c mouse at time when ampicillin treatment was initiated as well as 3 and 7 days post-infection. Note that former microlesion sites are now characterized by robust immune cell infiltration. B) Heart sections were also stained with Picrosirius Red to visualize collagen deposition.