Poking holes in pneumococci

Abstract

The idea of using bacteriophages as a tool for antibacterial therapy has been around ever since they were discovered >80 years ago. The specificity and efficiency with which they bind and lyse the cells of their bacterial hosts makes bacteriophages an attractive option for antibiotic development. Although bacteriophages have been indispensable in the establishment of molecular biology, significant progress towards their use as a functional antibiotic has been sporadic at best and was not rigorously pursued until recently.Loeffler et al. [1xRapid killing of Streptococcus pneumoniae with a bacteriophage cell wall hydrolase. Loeffler, J.M. et al. Science. 2001; 294: 2170–2172Crossref | PubMed | Scopus (245)See all References[1] have now presented a strategy to exploit the activity of a lytic enzyme, Pal, encoded by bacteriophage Dp-1, which specifically infects strains of Streptococcus pneumoniae. Pal is an amidase that cleaves N-acetylmuramoyl-l-alanine bonds in the pneumococcal cell wall and binds choline, which is uniquely present in the pneumococcal cell envelope, thus presumably accounting for its target specificity. The investigators expressed Pal in Escherichia coli and used affinity purification to obtain the enzyme in a phage-free form. This avoids the problems associated with using whole phage for therapeutic purposes. In an in vitro assay, purified Pal efficiently killed 15 clinical strains of pneumococci (including three penicillin-resistant strains), lowering the viable titer of log-phase cells in each case by 3–5 orders of magnitude (3–5 log10). The loss of viability is rapid and occurs within 30 seconds. The ability of Pal to target pneumococci specifically was demonstrated by subjecting eight other species of oral commensal streptococcal species to the same in vitro killing assay. Only Streptococcus oralis and Streptococcus mitis cultures showed lowered viability in this assay, but at a significantly lower rate (0.8 and 0.23 log10, respectively).Electron micrographs of Pal-treated pneumococcal cells allow stunning visualization of the Pal-mediated lytic events. After 1 min exposure to Pal, membrane protrusions, blebs and ruptures can be seen emanating from holes in the pneumococcal cell walls. After 5 min, only ghost cells are present, consisting of empty cell walls. These images are a dramatic demonstration that the purified amidase does not totally digest the entire cell wall in the killing assay, but rather creates holes in restricted locations where lethal membrane leakage takes place.Using a mouse model of nasopharyngeal pneumococcus colonization, the investigators then tested Pal antipneumococcal activity in vivo. Previously inoculated mice were treated with 700 and 1400 units of Pal in the nasopharyngeal cavity (100 units were used in the in vitro assays). Both dosages eliminated the pneumococci to non-detectable levels (although the higher dosage was more efficient). Even 8 days after the single-dosage Pal treatment, pneumococcal recolonization of the nasopharynx was dramatically reduced.This method of ‘lysis without’ (as opposed to ‘lysis within’ from whole lytic phage) using purified bacteriophage lytic enzymes is an exciting area in antibiotic development. Catalytic domains of lytic enzymes could be hooked to other binding domains to switch specificity, or other, more stable molecules could be designed with the same catalytic and binding sites. Such potential is promising in the face of growing resistance to current antibiotics.