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

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.

Intracellular survival of Streptococcus pneumoniae in human alveolar macrophages is augmented with HIV infection

“…The polysaccharide capsule is a major virulence factor of the pneumococci as it inhibits complement and antibody mediated opsonophagocytosis ( 48 – 51 ). Despite Pneumococcal ST3 being biochemically different, our findings are consistent with observations from other serotypes including serotype 1 ( 37 ), serotype 2 ( 43 , 44 ), serotype 6B ( 12 ) and serotype 18, that also survive intracellularly, suggesting intracellular survival may not be capsular specific but is common amongst pneumococcal serotypes.…”

“…The mechanisms for pneumococcal intracellular survival in macrophages are still not fully understood, despite a growing recognition of the ability of pneumococci to survive intracellularly in macrophages ( 12 , 14 ). However, there is some evidence suggesting that pneumolysin, a cholesterol binding and pore-forming toxin, facilitates intracellular lysosomal escape of the bacteria in macrophages and dendritic cells ( 37 , 38 ). Specifically, pneumolysin binds to the mannose receptor (CD206), widely found on AMs, thus facilitating uptake of pneumococci, but also dampening the inflammatory cytokine responses through upregulation of cytokine suppressor-1 (SOCS1) that leads to attenuated intracellular bacterial killing ( 38 ).…”

“…Moreover, the non-hemolytic pneumolysin strains of S . pneumoniae survive better inside both alveolar epithelial cells and THP-1 macrophages than the hemolytic pneumolysin strains ( 37 ). Thus, these findings suggest that this prototypical extracellular bacterium could have an intracellular niche within macrophages and potential other cells for its propagation and maintenance in vivo.…”

Single Point Mutation and Its Role in Specific Pathogenicity to Reveal the Mechanism of Related Protein Families

“…Conversely, the condition of muscle tissues of rPLO F240A- and rPLO N139K-infected mice did not show striking signs of damage compared to the PBS-treated group. Moreover, because PLO is a key promoter of host inflammation, tissue injury, morbidity, and mortality ( 34 ), we investigated the inflammatory response and structural destruction in muscle tissue of mice infected with rPLO, the two mutants, and PBS. Mice infected with rPLO F240A and rPLO N139K had significantly reduced muscle inflammation and less structural destruction, as evidenced by decreased expression of the inflammatory cytokines interleukin-1β (IL-1β) and interleukin-18 (IL-18) compared to mice infected with rPLO ( Fig.…”

“…Therefore, this study found for the first time the influence of the conserved amino acids Asn-139 and Phe-240 on the conformation and pore-forming activity of PLO and explain in detail the reasons for the changes in pore-forming activity and correlate pore formation with the conformation of the toxin. Additionally, some studies have reported that loss of indispensable cation-π interaction owing to replacement a residue in domain 1, associated with the conformational rigidity of domain 3, was found to be the major contributor to the loss of pore-forming ability of Ply-NH, which is another important member of the CDC family ( 34 ). Interestingly, these subtle substitutions accomplished by a single-residue mutation are adequate to efficaciously limit the mutant PLOs from performing their functions.…”