Pneumococcal Invasion Research Articles

A 3’UTR-derived small RNA represses pneumolysin synthesis and facilitates pneumococcal brain invasion

Pneumolysin (Ply) of Streptococcus pneumoniae (pneumococcus) at relatively high and low levels facilitates pneumococcal invasion into the lung and brain, respectively; however, the regulatory mechanisms of Ply expression are poorly understood. Here, we find that a small RNA plyT, processed from the 3’UTR of the ply operon, is expressed higher in anaerobically- than in statically-cultured pneumococcus D39. Using bioinformatic, biochemical and genetic approaches, we reveal that PlyT inhibits Ply synthesis and hemolytic activities by pairing with an RBS-embedded intergenic region of the ply operon. The RNA-binding protein SPD_1558 facilitates the pairing. Importantly, PlyT inhibition of Ply synthesis is stronger in anaerobic culture and leads to lower Ply abundance. Deletion of plyT decreases the number of pneumococci in the infected mouse brain and reduces the virulence, demonstrating that PlyT-regulated lower Ply in oxygen-void microenvironments, such as the blood, is important for pneumococcus to cross the blood-brain barrier and invade the brain. PlyT-mediated repression of Ply synthesis at anoxic niches is also verified in pneumococcal serotype 4 and 14 strains; moreover, the ply operon with a 3’UTR-embedded plyT, and the pairing sequences of IGR and plyT are highly conserved among pneumococcal strains, implying PlyT-regulated Ply synthesis might be widely employed by pneumococcus.

TCS01 Two-Component System Influenced the Virulence of Streptococcus pneumoniae by Regulating PcpA.

Streptococcus pneumoniae relies on two-component systems (TCSs) to regulate the processes of pathogenicity, osmotic pressure, chemotaxis, and energy metabolism. The TCS01 system of S. pneumoniae is composed of HK01 (histidine kinase) and RR01 (response regulator). Previous studies have reported that an rr01 mutant reduced the pneumococcal virulence in rat pneumonia, bacteremia, a nasopharyngeal model, and infective endocarditis. However, the mechanism of TCS01 (HK/RR01) regulating pneumococcal virulence remains unclear. Here, pneumococcal mutant strains Δrr01, Δhk01, and Δrr01&hk01 were constructed, and bacterial adhesion and invasion to A549 cells were compared. RNA sequencing was performed in D39 wild-type and Δrr01 strains, and transcript profile changes were analyzed. Differentially expressed virulence genes in the Δrr01 strain were screened out and identified by quantitative real-time PCR (qRT-PCR). Our results showed that pneumococcal mutant strains exhibited attenuated adhesion and invasion to A549 cells and differential transcript profiles. Results of qRT-PCR identification showed that the differential virulence genes screened out were downregulated. Among those changed virulence genes in the Δrr01 strain, the downregulated expression level of choline binding protein pcpA was the most obvious. Complementation of rr01 and overexpression of pcpA in the Δrr01 strain partially restored both pneumococcal adhesion and invasion, and rr01 complementation made the expression of pcpA upregulated. These findings revealed that rr01 influenced pneumococcal virulence by regulating pcpA.

Streptococcus pneumoniae Rapidly Translocate from the Nasopharynx through the Cribriform Plate to Invade the Outer Meninges.

ABSTRACTThe entry routes and translocation mechanisms of microorganisms or particulate materials into the central nervous system remain obscure We report here that Streptococcus pneumoniae (pneumococcus), or polystyrene microspheres of similar size, appear in the meninges of the dorsal cortex of mice within minutes of inhaled delivery. Recovery of viable bacteria from dissected tissue and fluorescence microscopy show that up to at least 72 h, pneumococci and microspheres were predominantly found in the outer of the two meninges: the pachymeninx. No pneumococci were found in blood or cerebrospinal fluid. Intravital imaging through the skull, aligned with flow cytometry showed recruitment and activation of LysM+ cells in the dorsal pachymeninx at 5 and 10 hours following intranasal infection. Imaging of the cribriform plate suggested that both pneumococci and microspheres entered through the foramina via an inward flow of fluid connecting the nose to the pachymeninx. Our findings bring new insight into the varied mechanisms of pneumococcal invasion of the central nervous system, but they are also pertinent to the delivery of drugs to the brain and the entry of airborne particulate matter into the cranium.

The Role of Neutrophils and Neutrophil Elastase in Pneumococcal Pneumonia.

Streptococcus pneumoniae, also known as pneumococcus, is a Gram-positive diplococcus and a major human pathogen. This bacterium is a leading cause of bacterial pneumonia, otitis media, meningitis, and septicemia, and is a major cause of morbidity and mortality worldwide. To date, studies on S. pneumoniae have mainly focused on the role of its virulence factors including toxins, cell surface proteins, and capsules. However, accumulating evidence indicates that in addition to these studies, knowledge of host factors and host-pathogen interactions is essential for understanding the pathogenesis of pneumococcal diseases. Recent studies have demonstrated that neutrophil accumulation, which is generally considered to play a critical role in host defense during bacterial infections, can significantly contribute to lung injury and immune subversion, leading to pneumococcal invasion of the bloodstream. Here, we review bacterial and host factors, focusing on the role of neutrophils and their elastase, which contribute to the progression of pneumococcal pneumonia.

Pneumococcal Meningitis and Its Sequelae - A Devastating CNS Disease.

IntroductionIn countries where Haemophilus influenzae type B vaccine is used, Streptococcus pneumoniae is the most common cause of bacterial meningitis in young children and notable cause of morbidity/mortality. The authors present material of magnetic resonance imaging (MRI) of patients with pneumococcal meningitis from archive of Department of Diagnostic Imaging of Institute of Mother and Child in Warsaw.Materials and methodsWe performed 27 brain MRI scans and 1 follow-up computed tomography (CT) in 10 children (2 girls and 8 boys) aged from neonate to 5 years at disease onset with proven pneumococcal infection.ResultsFollow-up period range was 0–12 years. Two children underwent only one MRI, one of them died before follow-up and the other was lost from further observation. There was one case of relatively benign disease course with mild changes on MRI. In another seemingly benign case, acute transient hydrocephalus was observed. Six children developed hydrocephalus, and two required ventriculoperitoneal shunting complicated by neuroinfection, shunt malfunction and revisions. Two patients developed epilepsy. In six children, spastic paresis of various severity was diagnosed, up to quadriplegia in one who is under the longest observation (>12 years) and survived in vegetative state. Three other children suffer from delayed psychomotor development to severe intellectual disability.ConclusionsMRI shows perfectly the degree of central nervous system (CNS) damage during and after pneumococcal invasion. Despite appropriate treatment, disease course may be unpredictably serious. Attempts to eliminate the obligation to vaccinate are extremely irresponsible taking into account potential danger of death, vegetative state or another form of severe damage to CNS. Social and financial costs of care of survivors are very high with shunts placements and changes, (neuro)infections, rehabilitation, families breakdown, etc.

Raff-ining our understanding of pneumococcal invasion

Differential raffinose metabolism influences Streptococcus pneumoniae tissue tropism and disease phenotype.

Competence-induced protein Ccs4 facilitates pneumococcal invasion into brain tissue and virulence in meningitis

ABSTRACT Streptococcus pneumoniae is a major pathogen that causes pneumonia, sepsis, and meningitis. The candidate combox site 4 (ccs4) gene has been reported to be a pneumococcal competence-induced gene. Such genes are involved in development of S. pneumoniae competence and virulence, though the functions of ccs4 remain unknown. In the present study, the role of Ccs4 in the pathogenesis of pneumococcal meningitis was examined. We initially constructed a ccs4 deletion mutant and complement strains, then examined their association with and invasion into human brain microvascular endothelial cells. Wild-type and Ccs4-complemented strains exhibited significantly higher rates of association and invasion as compared to the ccs4 mutant strain. Deletion of ccs4 did not change bacterial growth activity or expression of NanA and CbpA, known brain endothelial pneumococcal adhesins. Next, mice were infected either intravenously or intranasally with pneumococcal strains. In the intranasal infection model, survival rates were comparable between wild-type strain-infected and ccs4 mutant strain-infected mice, while the ccs4 mutant strain exhibited a lower level of virulence in the intravenous infection model. In addition, at 24 hours after intravenous infection, the bacterial burden in blood was comparable between the wild-type and ccs4 mutant strain-infected mice, whereas the wild-type strain-infected mice showed a significantly higher bacterial burden in the brain. These results suggest that Ccs4 contributes to pneumococcal invasion of host brain tissues and functions as a virulence factor.

Decline in otitis media associated ambulatory visits in the US

Tapping into the nationally representative databases from the National Center for Health Statistics at the US Centers for Disease Control and Prevention, Kawai et al from Boston queried the number of otitis media associated ambulatory visits in children up to 18 years of age over the years 1997 to 2014. This interval spans years before universal use of pneumococcal conjugate vaccine (PCV) (before 2000), during PCV7 use, and since PCV13 use beginning in late 2010 in children 2 months through 59 months of age. A strength of the study is that the databases include 3 types of ambulatory physician visits: in office practices, hospital-associated outpatient departments, and emergency departments. The results are only broad brush strokes as no attempt was made to assess criteria or providers' clinical acumen to diagnose/code otitis media, whether visits were at the time of diagnosis or for follow up, and did not take in to account changing national recommendations for management that included dropping follow up visits for uncomplicated acute otitis media. The results, however, represent real world incidence and are in line with smaller (less representative samples) that did include more granular data. Almost 25 000 otitis media related visits were identified over the study period. Between the pre-PCV7 and post-PCV13 period, annual physician office visits for those under 2 years of age declined most dramatically (ie, by 51%, CI 42% to 58%; from 826 to 387 visits per 1000 children under 2 years of age). Declines occurred across all ambulatory settings. An equally important finding was decline across demographics of sex, race/ethnicity, age, health insurance status, and across geographic regions. Without debate, PCV7 and PCV13 have all but eliminated the primary “bad actor” pneumococci of previous severe infection that led to the most “problematic” cases as well as protracted middle ear inflammation that facilitated recurrent episodes of acute otitis media. PCVs also have all but eliminated dread pneumococcal invasion of the bloodstream and central nervous system. With these helpful data from Kawai et al in mind, providers should have greatly reduced prescribing of antibiotics in ambulatory settings and parents should expect receiving many fewer prescriptions. Article page 122 ▶ Ambulatory Visits for Otitis Media before and after the Introduction of Pneumococcal Conjugate VaccinationThe Journal of PediatricsVol. 201PreviewTo examine the long-term trend of otitis media (OM)-associated ambulatory visits from 1997 to 2014 and to evaluate the impact of 7-valent and 13-valent pneumococcal conjugate vaccines (PCV7 and PCV13) on OM ambulatory visits. Full-Text PDF

Receptor Blockade: A Novel Approach to Protect the Brain From Pneumococcal Invasion.

Pneumococci are the major cause of bacterial meningitis globally. To cause meningitis pneumococci interact with the 2 endothelial receptors, polymeric immunoglobulin receptor (pIgR) and platelet endothelial cell adhesion molecule (PECAM-1), to penetrate the blood-brain barrier (BBB) and invade the brain. C57BL/6 mice were infected intravenously with bioluminescent pneumococci, and treated with ceftriaxone (1 hour postinfection) and anti-pIgR and PECAM-1 antibodies (1 or 5 hours postinfection), then monitored for 5 and 10 days. Bacterial brain invasion was analyzed using IVIS imaging and bacterial counts. Ceftriaxone, given early after pneumococcal challenge, cleared pneumococci from the blood but not from the brain. After combining ceftriaxone with receptor blockade, using anti-pIgR and PECAM-1 antibodies, we found 100% survival after 5 and 10 days of infection, in contrast to 60% for ceftriaxone alone. Combined antibiotic and antibody treatment resulted in no or few viable bacteria in the brain and no microglia activation. Antibodies remained bound to the receptors during the study period. Receptor blockade did not interfere with antibiotic permeability through the BBB. We suggest that adjunct treatment with pIgR and PECAM-1 antibodies to antibiotics may prevent pneumococcal meningitis development and associated brain damages. However, further evaluations are required.

Rgg-Shp regulators are important for pneumococcal colonization and invasion through their effect on mannose utilization and capsule synthesis

Microbes communicate with each other by using quorum sensing (QS) systems and modulate their collective ‘behavior’ for in-host colonization and virulence, biofilm formation, and environmental adaptation. The recent increase in genome data availability reveals the presence of several putative QS sensing circuits in microbial pathogens, but many of these have not been functionally characterized yet, despite their possible utility as drug targets. To increase the repertoire of functionally characterized QS systems in bacteria, we studied Rgg144/Shp144 and Rgg939/Shp939, two putative QS systems in the important human pathogen Streptococcus pneumoniae. We find that both of these QS circuits are induced by short hydrophobic peptides (Shp) upon sensing sugars found in the respiratory tract, such as galactose and mannose. Microarray analyses using cultures grown on mannose and galactose revealed that the expression of a large number of genes is controlled by these QS systems, especially those encoding for essential physiological functions and virulence-related genes such as the capsular locus. Moreover, the array data revealed evidence for cross-talk between these systems. Finally, these Rgg systems play a key role in colonization and virulence, as deletion mutants of these QS systems are attenuated in the mouse models of colonization and pneumonia.

Mast Cells Are Activated by Streptococcus pneumoniae In Vitro but Dispensable for the Host Defense Against Pneumococcal Central Nervous System Infection In Vivo.

Mast cells reside on and near the cerebral vasculature, the predominant site of pneumococcal entry into the central nervous system (CNS). Although mast cells have been reported to be crucial in protecting from systemic bacterial infections, their role in bacterial infections of the CNS remained elusive. Here, we assessed the role of mast cells in pneumococcal infection in vitro and in vivo. In introductory experiments using mouse bone marrow-derived mast cells (BMMC), we found that (i) BMMC degranulate and release selected cytokines upon exposure to Streptococcus pneumoniae, (ii) the response of BMMC varies between different pneumococcal serotypes and (iii) is dependent on pneumolysin. Intriguingly though, apart from a slight enhancement of cerebrospinal fluid (CSF) pleocytosis, neither two different mast cell-deficient Kit mutant mouse strains (WBB6F1-KitW/Wv and C57BL/6 KitW-sh/W-sh mice) nor pharmacologic mast cell stabilization with cromoglycate had any significant impact on the disease phenotype of experimental pneumococcal meningitis. The incomplete reversal of the enhanced CSF pleocytosis by local mast cell engraftment suggests that this phenomenon is caused by other c-Kit mutation-related mechanisms than mast cell deficiency. In conclusion, our study suggests that mast cells can be activated by S. pneumoniae in vitro. However, mast cells do not play a significant role as sentinels of pneumococcal CSF invasion and initiators of innate immunity in vivo.

Increased Virulence of an Encapsulated Streptococcus pneumoniae Upon Expression of Pneumococcal Surface Protein K

Current Streptococcus pneumoniae vaccines selectively target capsular polysaccharide of specific serotypes, leading to an increase in nonencapsulated S. pneumoniae (NESp). Cocolonization by encapsulated pneumococci and NESp increases the opportunity for intraspecies genetic exchange. Acquisition of NESp genes by encapsulated pneumococci could alter virulence and help vaccine-targeted serotypes persist in the host. Adhesion and invasion assays were performed using immortalized human pharyngeal or lung epithelial cells. In vivo models assessing murine nasopharyngeal colonization and pneumonia, as well as chinchilla otitis media (OM), were also used. Pneumococcal surface protein K (PspK) expression increased encapsulated pneumococcal adhesion and invasion of lung cells and enhanced virulence during pneumonia and OM. Additionally, PspK increased nasopharyngeal colonization, persistence in the lungs, and persistence in the middle ear when expressed in a capsule deletion mutant. Competition experiments demonstrated encapsulated pneumococci expressing PspK also had a selective advantage in both the lungs and nasopharynx. PspK increases pneumococcal virulence during pneumonia and OM. PspK also partially compensates for loss of virulence in the absence of capsule. Additionally, PspK provides a selective advantage in a competitive environment. Therefore, acquisition of PspK increases encapsulated virulence in a condition-dependent manner. Together, these studies demonstrate risks associated with pneumococcal intraspecies genetic exchange.

The influenza of 1918: Evolutionary perspectives in a historical context.

The 1918 influenza pandemic was the deadliest in known human history. It spread globally to the most isolated of human communities, causing clinical disease in a third of the world’s population, and infecting nearly every human alive at the time. Determination of mortality numbers is complicated by weak contemporary surveillance in the developing world, but recent estimates put the death toll at 50 million or even higher. This outbreak is of great interest to modern day epidemiologists, virologists, global health researchers and evolutionary biologists. They ask: Where did it come from? And if it happened once, could it happen again? Understanding how such a virulent epidemic emerged and spread offers hope for prevention and strategies of response. This review uses historical methodology and evolutionary perspectives to revisit the 1918 outbreak. Using the American military experience as a case study, it investigates the emergence of virulence in 1918 by focusing on key susceptibility factors that favored both the influenza virus and the subsequent pneumococcal invasion that took so many lives. This article explores the history of the epidemic and contemporary measures against it, surveys modern research on the virus, and considers what aspects of 1918 human and animal ecology most contributed to the emergence of this pandemic.

A Perfect Storm: Increased Colonization and Failure of Vaccination Leads to Severe Secondary Bacterial Infection in Influenza Virus-Infected Obese Mice.

ABSTRACTObesity is a risk factor for developing severe disease following influenza virus infection; however, the comorbidity of obesity and secondary bacterial infection, a serious complication of influenza virus infections, is unknown. To fill this gap in knowledge, lean and obese C57BL/6 mice were infected with a nonlethal dose of influenza virus followed by a nonlethal dose of Streptococcus pneumoniae. Strikingly, not only did significantly enhanced death occur in obese coinfected mice compared to lean controls, but also high mortality was seen irrespective of influenza virus strain, bacterial strain, or timing of coinfection. This result was unexpected, given that most influenza virus strains, especially seasonal human A and B viruses, are nonlethal in this model. Both viral and bacterial titers were increased in the upper respiratory tract and lungs of obese animals as early as days 1 and 2 post-bacterial infection, leading to a significant decrease in lung function. This increased bacterial load correlated with extensive cellular damage and upregulation of platelet-activating factor receptor, a host receptor central to pneumococcal invasion. Importantly, while vaccination of obese mice against either influenza virus or pneumococcus failed to confer protection, antibiotic treatment was able to resolve secondary bacterial infection-associated mortality. Overall, secondary bacterial pneumonia could be a widespread, unaddressed public health problem in an increasingly obese population.

A Critical Combination of Bacterial Dose and Virus-Induced Alveolar Macrophage Depletion Leads to Pneumococcal Infections During Influenza

Abstract Coinfection with pneumococcus during influenza A virus infection is characterized by rapid, uncontrolled bacterial growth, a rebound in viral titers, and a robust inflammatory response. Several factors contribute to influenza-pneumococcal pathogenicity, including aberrant immune responses, tissue destruction, and pathogen strain and dose. By analyzing pathogen kinetics with a mathematical model, we predicted that bacterial establishment and growth is driven by a defect in clearance by alveolar macrophages (AMs), the first line of defense against pneumococcal invasion. This prediction was tested and confirmed in an experimental study, which suggested that these cells are depleted during influenza. Remarkably, both our model prediction and the follow-up experiments agreed that the AM population is reduced by ~85–90% 7d post-influenza infection (pii). Analyzing our coinfection model in more depth allowed us to quantify an initial dose threshold for successful bacterial infection that depends on the level of AM depletion and predict how the threshold changes throughout an influenza virus infection. To validate this prediction, we infected groups of mice with pneumococcus at 1, 3, 5, 7, 9, or 11d pii and at a dose either higher or lower than the predicted threshold. Within 4h after inoculation, bacterial loads decline for doses below the threshold, increase for doses above the threshold, and are dichotomous for doses close to the threshold. These results give insight into the conditions necessary for a secondary bacterial infection to establish during influenza infection and the probability of a successful secondary bacterial infection occurring with seasonal and pandemic influenza virus strains.

BLT2 receptor protects against lethal acute lung injury induced by a pneumococcal toxin pneumolysin

Pneumonia is one of the major causes of death over the world, and the capital cause of infectious pneumonia is S. pneumoniae (pneumococcus). The key virulence factor of pneumococcus is a pore‐forming toxin pneumolysin (PLY). By its direct cytotoxic effect, PLY is known to cause acute lung injury, leading to pneumococcal invasion and subsequent severe bacteremia. Leukotriene B4 (LTB4) receptor type 2 (BLT2) is a G‐protein coupled receptor that was originally identified as a low‐affinity receptor for LTB4, and recently we elucidated that the high affinity endogenous ligand for BLT2 is 12‐hydroxyheptadecatrienoic acid (12‐HHT), an arachidonic acid metabolite by cyclooxygenase. Previously we reported that BLT2 is expressed in intestinal epithelial cells and skin keratinocytes, and that 12‐HHT/BLT2 signaling exhibits important roles in maintaining mucosal integrity and in accelerating skin wound healing. However, the biological roles of BLT2 in other organs are still uncertified. In this study, we investigated the protective role of BLT2 in mouse lung using PLY‐induced acute lung injury model. Interestingly, BLT2‐deficient mice were more susceptible to PLY administration, and also exhibited increased vascular permeability and potent bronchoconstriction by PLY administration when compared to wild‐type mice. We found BLT2 in mouse lung is expressed in alveolar type II cells and vascular endothelial cells. The molecular mechanisms of how BLT2 exhibits the protective role for PLY‐induced acute lung injury are now under investigation.Support or Funding InformationThis work was supported by Grants‐in‐Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (MEXT) Nos. 22116001, 22116002, 15H05901, 15H05904, and 15H04708.

Infiltrated Macrophages Die of Pneumolysin-Mediated Necroptosis following Pneumococcal Myocardial Invasion.

Streptococcus pneumoniae (the pneumococcus) is capable of invading the heart. Herein we observed that pneumococcal invasion of the myocardium occurred soon after development of bacteremia and was continuous thereafter. Using immunofluorescence microscopy (IFM), we observed that S. pneumoniae replication within the heart preceded visual signs of tissue damage in cardiac tissue sections stained with hematoxylin and eosin. Different S. pneumoniae strains caused distinct cardiac pathologies: strain TIGR4, a serotype 4 isolate, caused discrete pneumococcus-filled microscopic lesions (microlesions), whereas strain D39, a serotype 2 isolate, was, in most instances, detectable only using IFM and was associated with foci of cardiomyocyte hydropic degeneration and immune cell infiltration. Both strains efficiently invaded the myocardium, but cardiac damage was entirely dependent on the pore-forming toxin pneumolysin only for D39. Early microlesions caused by TIGR4 and microlesions formed by a TIGR4 pneumolysin-deficient mutant were infiltrated with CD11b(+) and Ly6G-positive neutrophils and CD11b(+) and F4/80-positive (F4/80(+)) macrophages. We subsequently demonstrated that macrophages in TIGR4-infected hearts died as a result of pneumolysin-induced necroptosis. The effector of necroptosis, phosphorylated mixed-lineage kinase domain-like protein (MLKL), was detected in CD11b(+) and F4/80(+) cells associated with microlesions. Likewise, treatment of infected mice and THP-1 macrophages in vitro with the receptor-interacting protein 1 kinase (RIP1) inhibitor necrostatin-5 promoted the formation of purulent microlesions and blocked cell death, respectively. We conclude that pneumococci that have invaded the myocardium are an important cause of cardiac damage, pneumolysin contributes to cardiac damage in a bacterial strain-specific manner, and pneumolysin kills infiltrated macrophages via necroptosis, which alters the immune response.

Visualization of <em>Streptococcus pneumoniae</em> within Cardiac Microlesions and Subsequent Cardiac Remodeling

During bacteremia Streptococcus pneumoniae can translocate across the vascular endothelium into the myocardium and form discrete bacteria-filled microscopic lesions (microlesions) that are remarkable due to the absence of infiltrating immune cells. Due to their release of cardiotoxic products, S. pneumoniae within microlesions are thought to contribute to the heart failure that is frequently observed during fulminate invasive pneumococcal disease in adults. Herein is demonstrated a protocol for experimental mouse infection that leads to reproducible cardiac microlesion formation within 30 hr. Instruction is provided on microlesion identification in hematoxylin & eosin stained heart sections and the morphological distinctions between early and late microlesions are highlighted. Instruction is provided on a protocol for verification of S. pneumoniae within microlesions using antibodies against pneumococcal capsular polysaccharide and immunofluorescent microscopy. Last, a protocol for antibiotic intervention that rescues infected mice and for the detection and assessment of scar formation in the hearts of convalescent mice is provided. Together, these protocols will facilitate the investigation of the molecular mechanisms underlying pneumococcal cardiac invasion, cardiomyocyte death, cardiac remodeling as a result of exposure to S. pneumoniae, and the immune response to the pneumococci in the heart.

Visualization of Streptococcus pneumoniae within Cardiac Microlesions and Subsequent Cardiac Remodeling.

During bacteremia Streptococcus pneumoniae can translocate across the vascular endothelium into the myocardium and form discrete bacteria-filled microscopic lesions (microlesions) that are remarkable due to the absence of infiltrating immune cells. Due to their release of cardiotoxic products, S. pneumoniae within microlesions are thought to contribute to the heart failure that is frequently observed during fulminate invasive pneumococcal disease in adults. Herein is demonstrated a protocol for experimental mouse infection that leads to reproducible cardiac microlesion formation within 30 hr. Instruction is provided on microlesion identification in hematoxylin & eosin stained heart sections and the morphological distinctions between early and late microlesions are highlighted. Instruction is provided on a protocol for verification of S. pneumoniae within microlesions using antibodies against pneumococcal capsular polysaccharide and immunofluorescent microscopy. Last, a protocol for antibiotic intervention that rescues infected mice and for the detection and assessment of scar formation in the hearts of convalescent mice is provided. Together, these protocols will facilitate the investigation of the molecular mechanisms underlying pneumococcal cardiac invasion, cardiomyocyte death, cardiac remodeling as a result of exposure to S. pneumoniae, and the immune response to the pneumococci in the heart.

Cardiotoxicity during invasive pneumococcal disease.

Streptococcus pneumoniae is the leading cause of community-acquired pneumonia and sepsis, with adult hospitalization linked to approximately 19% incidence of an adverse cardiac event (e.g., heart failure, arrhythmia, infarction). Herein, we review the specific host-pathogen interactions that contribute to cardiac dysfunction during invasive pneumococcal disease: (1) cell wall-mediated inhibition of cardiomyocyte contractility; (2) the new observation that S. pneumoniae is capable of translocation into the myocardium and within the heart, forming discrete, nonpurulent, microscopic lesions that are filled with pneumococci; and (3) the bacterial virulence determinants, pneumolysin and hydrogen peroxide, that are most likely responsible for cardiomyocyte cell death. Pneumococcal invasion of heart tissue is dependent on the bacterial adhesin choline-binding protein A that binds to laminin receptor on vascular endothelial cells and binding of phosphorylcholine residues on pneumococcal cell wall to platelet-activating factor receptor. These are the same interactions responsible for pneumococcal translocation across the blood-brain barrier during the development of meningitis. We discuss these interactions and how their neutralization, either with antibody or therapeutic agents that modulate platelet-activating factor receptor expression, may confer protection against cardiac damage and meningitis. Considerable collagen deposition was observed in hearts of mice that had recovered from invasive pneumococcal disease. We discuss the possibility that cardiac scar formation after severe pneumococcal infection may explain why individuals who are hospitalized for pneumonia are at greater risk for sudden death up to 1 year after infection.