Pneumococcal infections - dependence upon pneumolysin expression

Scientists led by Assoc. Prof. Anirban Banerjee, IIT, Mumbai have made a significant and deep investigation into infection by Streptococcus pneumoniae where tissue-destination and persistence are highly dependent upon the expression level of the major virulence factor pneumolysin (Ply), a pore-forming toxin.  

Even in an isogenic culture Ply was shown to be expressed over a wide range.  Bacterial constructs recapitulating the Ply-low and Ply-high expression gave markedly different capacities for evasion of the bactericidal response mechanisms in human host brain microvascular endothelial cells and for resulting transcytosis across the blood brain barrier, as detected in an animal model.

In one part of the study to explore the response of infected host cells, human host brain microvascular endothelial cells were stably transduced with GFP and mStrawberry to permit real-time tracking of LC3 and Ubiquitin (Ubq), respectively.  To track intracellular wildtype S pneumoniae and their complex interactions with LC3 and Ubq, the bacteria were pre-stained with far-red cell permeant DNA counterstain DRAQ5™.  DRAQ5™ has spectral compatibility with GFP and mStrawberry as shown in the figure below.  We believe this might represent the first example of three-colour, real-time imaging of intracellular bacterial infections.

This publication begins a more complete elucidation of the role Ply has in the varied nature of pneumococcal infections and the complexity of interactions with host cell defences.

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Reference:
Surve MV, Bhutda S, Datey A, Anil A, Rawat S, Pushpakaran A, et al. (2018) Heterogeneity in pneumolysin expression governs the fate of Streptococcus pneumoniae during blood-brain barrier trafficking. PLoS Pathog 14(7): e1007168.

Figure: emission spectrum for eGFP (LC3), mStrawberry (Ubq) and DRAQ5™

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