Folklore across cultures is replete with images of skin- or cloak-based disguises. In Scottish mythology, the selkie can alternate between seal and human forms by means of a sealskin. In Norse mythology, the goddess Freyja has a cloak of feathers that can ferry her between human and falcon forms. The Aztec deity Tezcatlipoca sometimes wore a jaguar skin to disguise himself. Sneaking into the ranks of such mythological figures is the more tangible and much smaller Group A Streptococcus (GAS, aka Streptococcus pyogenes), cloaked via a similarly shifty stratagem.
As an efficient pathogen, GAS is a weighty burden on human health. This streptococcus is the pathogen behind diseases that range from minor offenders like strep throat to serious conditions like necrotizing fasciitis, altogether totaling 700 million infections and over 500,000 deaths annually. One reason for this infectious efficacy is steeped in deceit. It turns out that GAS co-opts pieces of red blood cell membranes to camouflage itself from the immune system, using a particularly tricky, sticky protein.
A collaboration between the LaRock lab at Emory and the Zhang and Gonzalez labs at UCSD identified this protein, via a trawl through GAS culture supernatant for proteins that bind to red blood cell membranes. By adding nanoparticles coated with red blood cell (RBC) membranes to GAS culture supernatants and then performing affinity-capture and mass-spectrometry, Wierzbicki et al. plucked out a protein highly conserved among streptococci. This 'S protein,' encoded by the genetic locus ess, is 158 amino acids long with a hydrophobic region at the N-terminus and a peptidoglycan-binding motif at the C-terminus. It was found both cell-associated and in culture supernatants.
Although overnight cultures of both wild-type and complemented Δess GAS (ess supplied on a plasmid) aggregated into clumps and settled at the bottom of the tubes, mutants lacking the S protein were instead dispersed through the medium. Between this observed dispersion and the finding that Δess strains bound less n-hexadecane, Wierzbicki et al. concluded that the protein was modulating cell surface hydrophobicity. Moreover, the lack of S protein increased phagocytosis by macrophages and extracellular killing by neutrophils. In one crafty way or another, the S protein clearly has a pivotal role in GAS cell surface properties and interaction with immune system cells.
We move onto unraveling the how. Several lines of evidence suggested that S protein is implicated in disguising GAS, that is, cloaking GAS in RBC membranes in a feat of molecular mimicry that conceals the bacteria from the host immune system. Firstly, Δess strains bound RBC-coated nanoparticles less frequently than both wild-type strains and complemented Δess strains. Secondly, incubation with lysed RBCs resulted in only a faint pink color as opposed to the bright red of wild-type and complemented strains. This indicated a link between lack of S protein and less binding to membrane fragments. The RBC-coated WT and complemented strains were also less likely to be internalized by macrophages than the mutant – the membrane-masquerading was indeed fooling the macrophages.
Next, profiling the protein composition of GAS lacking S protein revealed perturbations in both membrane and intracellular proteomes and virulence. Without S protein, key virulence factors such as M protein were downregulated, GAS virulence was attenuated, and inoculated mice exhibited a more robust immune response and immune memory. Thus, S protein is the moonlighting agent behind both membrane co-opting and broader proteome regulation, and GAS is the wolf in sheep's clothing of the microbial world.
Taking advantage of cell membrane-coated trickery is a specialty of the Liangfang Zhang lab at UCSD – a manipulation of manipulation that actually predates the 2019 Wierzbicki et al. paper. The Zhang lab first unveiled their red blood cell membrane coating technology in a 2011 paper titled "Erythrocyte membrane-camouflaged polymeric nanoparticles as a biomimetic delivery platform." They demonstrated that putting nanoparticles in membrane coatings reduced clearance and macrophage uptake, thereby opening up avenues for therapeutic agents with longer systemic circulation times. Since then, the lab and others have produced a panoply of variations on the membrane-coated theme.
Membrane-coated subterfuge has found its way into a series of collaborative efforts with the Nizet lab at UCSD. RBC membrane-coated nanoparticles have, in an ironic twist, been deployed against GAS itself, acting as "nanosponges" to sop up the pore-forming streptolysin O virulence factor. The nanoparticles have also been used against Group B Streptococcus (GBS), a similarly pathogenic relative of GAS. The primary pore-forming toxin produced by GBS is β-hemolysin/cytolysin (β-H/C), which, as the name suggests, pokes deadly holes into cell membranes. The nanosponges sopped up β-H/C and reduced hemolysis by GBS. The membrane-masquerading has not been limited to RBCs, however: there has also been a macrophage variation on the theme, with nanoparticles coated in macrophage membranes that bind and neutralize E. coli endotoxins to put a stopper on sepsis.
Typically, these artificial membrane constructs involve disguising bacteria-curbing compounds from the bacteria, but the tables have also been turned. One group camouflaged E. faecalis, S. aureus, and S. typhimurium themselves with cell membranes to create "stealth bacteria" for bacteria-based therapeutics (an area that seems a bold take on Paracelsus's adage, "all substances are poisons… and the right dose differentiates a poison from a remedy"). With an extruder device, they pushed a mixture of their bacteria and red blood cell membranes through a porous membrane to pop cell membrane jackets onto the bacterial cells. Taking a page from GAS's pathogenic book clearly has wide-ranging applications.
On Thursday, we continue the Strep pyogenes theme with another highlight of a repurposed tool for pathogenicity. Stay tuned…