Streptococcus pneumoniae in the heart subvert the host response through biofilm-mediated resident macrophage killing

Abstract

For over 130 years, invasive pneumococcal disease has been associated with the presence of extracellular planktonic pneumococci, i.e. diplococci or short chains in affected tissues. Herein, we show that Streptococcus pneumoniae that invade the myocardium instead replicate within cellular vesicles and transition into non-purulent biofilms. Pneumococci within mature cardiac microlesions exhibited salient biofilm features including intrinsic resistance to antibiotic killing and the presence of an extracellular matrix. Dual RNA-seq and subsequent principal component analyses of heart- and blood-isolated pneumococci confirmed the biofilm phenotype in vivo and revealed stark anatomical site-specific differences in virulence gene expression; the latter having major implications on future vaccine antigen selection. Our RNA-seq approach also identified three genomic islands as exclusively expressed in vivo. Deletion of one such island, Region of Diversity 12, resulted in a biofilm-deficient and highly inflammogenic phenotype within the heart; indicating a possible link between the biofilm phenotype and a dampened host-response. We subsequently determined that biofilm pneumococci released greater amounts of the toxin pneumolysin than did planktonic or RD12 deficient pneumococci. This allowed heart-invaded wildtype pneumococci to kill resident cardiac macrophages and subsequently subvert cytokine/chemokine production and neutrophil infiltration into the myocardium. This is the first report for pneumococcal biofilm formation in an invasive disease setting. We show that biofilm pneumococci actively suppress the host response through pneumolysin-mediated immune cell killing. As such, our findings contradict the emerging notion that biofilm pneumococci are passively immunoquiescent.

Emerging concepts in the pathogenesis of theStreptococcus pneumoniae: From nasopharyngeal colonizer to intracellular pathogen

“…However, the intriguing question is, How does the pneumococcus establish itself within the myocardium without eliciting immune responses? Shenoy et al () showed by transmission electron microscopy that pneumococci replicate within cardiac lesions in unique intracellular vesicles of sizes 4–8 μm. Further, they showed that pneumococci formed biofilms within the heart and possessed a distinct transcriptomic profile when compared with blood‐isolated pneumococci.…”

“…However, the intriguing question is, How does the pneumococcus establish itself within the myocardium without eliciting immune responses? Shenoy et al (2017) showed by transmission electron microscopy that pneumococci repli- van de Beek, de Gans, Tunkel, & Wijdicks, 2006;Weisfelt et al, 2006). To cause meningitis, pneumococci in the bloodstream need to pass the blood-brain barrier (BBB), which is composed of brain microvascular endothelial cells.…”

Pathogenesis and prevention of risk of cardiovascular events in patients with pneumococcal community‐acquired pneumonia

“…Invasion of the myocardium by the pneumococcus has been reported to result in the formation of early microlesions, enabling the pathogen to undergo intracellular invasion, survival and replication in small vesicles, which was dependent on bacterial adhesins, as well as the cytotoxins, PLY and pneumococcus‐derived hydrogen peroxide, acting in concert . This was followed by development of mature microlesions, facilitated by PLY‐mediated neutralization of resident, sentinel macrophages, in which the pneumococcus transitioned to a more persistent intracellular, biofilm‐forming phenotype associated with high‐level production of PLY . In this setting, myocardial damage and dysfunction appeared to result from the sublethal and lethal effects of the toxin, also acting in concert with hydrogen peroxide, on cardiomyocytes .…”

“… —Immunogenicity of the capsular serotype of the pneumococcus, magnitude of bacteraemia and impact on rate of clearance of the pathogen —Propensity to produce pneumolysin and intramyocardial biofilm, enabling persistence in cardiomyocytes, cardiac fibroblasts and macrophages ; —Propensity to survive intracellularly in CD169 + splenic macrophages, M2‐polarized macrophages and dendritic cells ; —Persistence of pneumococcal antigens in pathogen‐derived extracellular vesicles ; —Persistence due to capsular switching . …”

“…development of mature microlesions, facilitated by PLY-mediated neutralization of resident, sentinel macrophages, in which the pneumococcus transitioned to a more persistent intracellular, biofilm-forming phenotype associated with highlevel production of PLY [59,69]. In this setting, myocardial damage and dysfunction appeared to result from the sublethal and lethal effects of the toxin, also acting in concert with hydrogen peroxide, on cardiomyocytes [57,58,68].…”