The molecular mechanism of manganese import in Streptococcus pneumoniae
- University of Adelaide, South Australia 5005, Australia.
- University of Queensland, Brisbane 4072, Australia.
- Australian National University, Canberra 2601, Australia.
- LaTrobe University, Melbourne 3080, Australia.
Streptococcus pneumoniae is a bacterial pathogen of global significance, responsible for more than 1 million deaths per year. Transition metal ion homeostasis is essential for S. pneumoniae pathogenicity. Consequently, the mechanisms associated with metal ion homeostasis are potential antimicrobial targets. The ATP binding cassette (ABC) transporter PsaBCA is the sole manganese acquisition pathway in vivo and is essential for pneumococcal virulence. The ABC permease complex comprises a homodimer of heterodimers (PsaB2C2), to which Mn2+ is delivered by the extra-cytoplasmic protein PsaA. Although PsaBCA only imports manganese ions, prior studies have shown that PsaA has highly promiscuous interactions with various divalent cations. Nonetheless, specificity of the transporter is achieved by slow release kinetics associated with non-cognate transition metal ions, such as Zn2+, from PsaA. However, these inferences are based on structural and in vitro experiments conducted in the absence of the ABC transporter. Building on this framework we sought to investigate how different PsaA-metal ion complexes influenced PsaBC function. Here, recombinant PsaBC was heterologously expressed in E. coli Lemo21(DE3). Membranes were isolated, the membrane protein complex solubilised by DDM and purified by affinity chromatography. Reconstitution of PsaBC was achieved using Triton X-100 mediated insertion into pre-formed liposomes, prior to detergent removal. Freeze-thaw cycling was employed to incorporate metal ions and PsaA into the proteoliposomes. PsaBCA complex was then assessed using an ATPase activity assay. This revealed that, in contrast to manganese, metal-PsaA complexes formed by non-cognate ions with slow release kinetics (i.e. zinc or copper), could not stimulate the activity of PsaBC. Hence, this work demonstrates how specificity of the S. pneumoniaemanganese transporter is achieved. This finding provides a mechanistic basis for how poorly abundant metal ions, such as manganese, can be selectively acquired from chemically complex extracellular environments.