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Intracellular replication of Streptococcus pneumoniae inside splenic macrophages serves as a reservoir for septicaemia.

ABSTRACT: Bacterial septicaemia is a major cause of mortality, but its pathogenesis remains poorly understood. In experimental pneumococcal murine intravenous infection, an initial reduction of bacteria in the blood is followed hours later by a fatal septicaemia. These events represent a population bottleneck driven by efficient clearance of pneumococci by splenic macrophages and neutrophils, but as we show in this study, accompanied by occasional intracellular replication of bacteria that are taken up by a subset of CD169+ splenic macrophages. In this model, proliferation of these sequestered bacteria provides a reservoir for dissemination of pneumococci into the bloodstream, as demonstrated by its prevention using an anti-CD169 monoclonal antibody treatment. Intracellular replication of pneumococci within CD169+ splenic macrophages was also observed in an ex vivo porcine spleen, where the microanatomy is comparable with humans. We also showed that macrolides, which effectively penetrate macrophages, prevented septicaemia, whereas beta-lactams, with inefficient intracellular penetration, failed to prevent dissemination to the blood. Our findings define a shift in our understanding of the pneumococcus from an exclusively extracellular pathogen to one with an intracellular phase. These findings open the door to the development of treatments that target this early, previously unrecognized intracellular phase of bacterial sepsis.

SUBMITTER: Ercoli G

PROVIDER: S-EPMC6207342 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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Intracellular replication of Streptococcus pneumoniae inside splenic macrophages serves as a reservoir for septicaemia.

Ercoli Giuseppe G Fernandes Vitor E VE Chung Wen Y WY Wanford Joseph J JJ Thomson Sarah S Bayliss Christopher D CD Straatman Kornelis K Crocker Paul R PR Dennison Ashley A Martinez-Pomares Luisa L Andrew Peter W PW Moxon E Richard ER Oggioni Marco R MR

Nature microbiology 20180416 5

Bacterial septicaemia is a major cause of mortality, but its pathogenesis remains poorly understood. In experimental pneumococcal murine intravenous infection, an initial reduction of bacteria in the blood is followed hours later by a fatal septicaemia. These events represent a population bottleneck driven by efficient clearance of pneumococci by splenic macrophages and neutrophils, but as we show in this study, accompanied by occasional intracellular replication of bacteria that are taken up by ...[more]

PMID: 29662129

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Project description:Circadian rhythms affect the progression and severity of bacterial infections including those caused by Streptococcus pneumoniae, but the mechanisms responsible for this phenomenon remain largely elusive. Following advances in our understanding of the role of replication of S. pneumoniae within splenic macrophages, we sought to investigate whether events within the spleen correlate with differential outcomes of invasive pneumococcal infection. Utilising murine invasive pneumococcal disease (IPD) models, here we report that infection during the murine active phase (zeitgeber time 15; 15h after start of light cycle, 3h after start of dark cycle) resulted in significantly faster onset of septicaemia compared to rest phase (zeitgeber time 3; 3h after start of light cycle) infection. This correlated with significantly higher pneumococcal burden within the spleen of active phase-infected mice at early time points compared to rest phase-infected mice. Whole-section confocal microscopy analysis of these spleens revealed that the number of pneumococci is significantly higher exclusively within marginal zone metallophilic macrophages (MMMs) known to allow intracellular pneumococcal replication as a prerequisite step to the onset of septicaemia. Pneumococcal clusters within MMMs were more abundant and increased in size over time in active phase-infected mice compared to those in rest phase-infected mice which decreased in size and were present in a lower percentage of MMMs. This phenomenon preceded significantly higher levels of bacteraemia alongside serum IL-6 and TNF-α concentrations in active phase-infected mice following re-seeding of pneumococci into the blood. These data greatly advance our fundamental knowledge of pneumococcal infection by linking susceptibility to invasive pneumococcal infection to variation in the propensity of MMMs to allow persistence and replication of phagocytosed bacteria. These findings also outline a somewhat rare scenario whereby the active phase of an organism's circadian cycle plays a seemingly counterproductive role in the control of invasive infection.

Project description:UnlabelledAs key components of innate immune defense, macrophages are essential in controlling bacterial pathogens, including group A Streptococcus(GAS). Despite this, only a limited number of studies have analyzed the recovery of GAS from within human neutrophils and macrophages. Here, we determined the intracellular fate of GAS in human macrophages by using several quantitative approaches. In both U937 and primary human macrophages, the appearance over time of long GAS chains revealed that despite GAS-mediated cytotoxicity, replication occurred in viable, propidium iodide-negative macrophages. Whereas the major virulence factor M1 did not contribute to bacterial growth, a GAS mutant strain deficient in streptolysin O (SLO) was impaired for intracellular replication. SLO promoted bacterial escape from the GAS-containing vacuole (GCV) into the macrophage cytosol. Up to half of the cytosolic GAS colocalized with ubiquitin and p62, suggesting that the bacteria were targeted by the autophagy machinery. Despite this, live imaging of U937 macrophages revealed proficient replication of GAS after GCV rupture, indicating that escape from the GCV is important for growth of GAS in macrophages. Our results reveal that GAS can replicate within viable human macrophages, with SLO promoting GCV escape and cytosolic growth, despite the recruitment of autophagy receptors to bacteria.ImportanceClassically regarded as an extracellular pathogen, GAS can persist within human epithelial cells, as well as neutrophils and macrophages. Some studies suggest that GAS can modulate its intracellular vacuole to promote survival and perhaps replicate in macrophages. However, an in-depth single-cell analysis of the dynamics of survival and replication is lacking. We used macrophage-like cell lines and primary macrophages to measure the intracellular growth of GAS at both the population and single-cell levels. While CFU counts revealed no increase in overall bacterial growth, quantitative fluorescence microscopy, flow cytometry, and time-lapse imaging revealed bacterial replication in a proportion of infected macrophages. This study emphasizes the importance of single-cell analysis especially when studying the intracellular fate of a pathogen that is cytotoxic and displays heterogeneity in terms of intracellular killing and growth. To our knowledge, this study provides the first direct visualization of GAS replication inside human cells.

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Intracellular replication of Streptococcus pneumoniae inside splenic macrophages serves as a reservoir for septicaemia.

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Intracellular replication of Streptococcus pneumoniae inside splenic macrophages serves as a reservoir for septicaemia.

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