Structural insights into loss of function of a pore forming toxin and its role in pneumococcal adaptation to an intracellular lifestyle.

Dilip C Badgujar,Anirban Banerjee,Shruti Guha Sarkar,Andrea Mitchell,Ian A Prior,Prachi Kadam More,Alison Beckett,Shilpa Madhavan,Prasenjit Bhaumik,Timothy J Mitchell,Barsa K Godsora,Anjali Anil,Angharad E Green,Sanket B Patil,Prashant S Phale,Rinti Banerjee,Daniel R Neill,Manalee Vishnu Surve

doi:10.1371/journal.ppat.1009016

Abstract

The opportunistic pathogen Streptococcus pneumoniae has dual lifestyles: one of an asymptomatic colonizer in the human nasopharynx and the other of a deadly pathogen invading sterile host compartments. The latter triggers an overwhelming inflammatory response, partly driven via pore forming activity of the cholesterol dependent cytolysin (CDC), pneumolysin. Although pneumolysin-induced inflammation drives person-to-person transmission from nasopharynx, the primary reservoir for pneumococcus, it also contributes to high mortality rates, creating a bottleneck that hampers widespread bacterial dissemination, thus acting as a double-edged sword. Serotype 1 ST306, a widespread pneumococcal clone, harbours a non-hemolytic variant of pneumolysin (Ply-NH). Performing crystal structure analysis of Ply-NH, we identified Y150H and T172I as key substitutions responsible for loss of its pore forming activity. We uncovered a novel inter-molecular cation-π interaction, governing formation of the transmembrane β-hairpins (TMH) in the pore state of Ply, which can be extended to other CDCs. H150 in Ply-NH disrupts this interaction, while I172 provides structural rigidity to domain-3, through hydrophobic interactions, inhibiting TMH formation. Loss of pore forming activity enabled improved cellular invasion and autophagy evasion, promoting an atypical intracellular lifestyle for pneumococcus, a finding that was corroborated in in vivo infection models. Attenuation of inflammatory responses and tissue damage promoted tolerance of Ply-NH-expressing pneumococcus in the lower respiratory tract. Adoption of this altered lifestyle may be necessary for ST306 due to its limited nasopharyngeal carriage, with Ply-NH, aided partly by loss of its pore forming ability, facilitating a benign association of SPN in an alternative, intracellular host niche.

Highlights

Virulence of a microbe is a highly dynamic trait, dependent primarily on the niche it occupies and the availability of an alternate reservoir
We uncovered the structural basis for loss of pore-forming activity of a Ply variant, present in Serotype 1 ST306, and observed that this enabled adoption of an intracellular lifestyle, attenuating inflammatory responses and prolonging host tolerance of pneumococcus in the lower airways
Pneumococcal virulence factors induce cytotoxicity, the host inflammatory response triggered against these factors is the major mediator of pathology and lethality associated with invasive pneumococcal disease (IPD)

Summary

Introduction

Virulence of a microbe is a highly dynamic trait, dependent primarily on the niche it occupies and the availability of an alternate reservoir. Recent studies suggest that pathogens can undergo attenuation of virulence inside the host, as long as transmission remains uncompromised [1] Such traits enable microbial colonization and proliferation, with minimal harm to the host, and form the basis of the “infection tolerance” concept that challenges the paradigm of the arms race in infection biology [2]. Ply induces necroptosis of respiratory epithelial cells and this mode of cell death has been linked to release of IL-1α from host cells [10,11] This IL-1 signaling has been identified to play a key role in inflammatory clearance of SPN from the nasopharynx [12] with Ply-deficient strain exhibiting slower clearance than their wild type counterparts [13]. Ply is a versatile factor, the activities of which can have different (and sometimes opposing)

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Conclusion