Isolation and characterisation of cytokine-modulating proteins from bacteria implicated in infective endocarditis

The term infective endocarditis describes the inflammation of the heart caused by the colonisation of cardiac tissues, generally the valves, by microorganisms. Relatively little is known about the components of the causal bacteria that are responsible for perpetuating the inflammatory state. Soluble, exported material (EM) produced by Streptococcus sanguis, one of the most common causal agents of infective endocarditis, was investigated for a potential role in modulating inflammatory cytokine responses. Gentle saline extraction of blood agar-grown S. sanguis yielded proteinaceous molecules with the ability to induce inflammatory cytokine release (IL-1β, IL-6, IL-8 and TNFα) from human immune cells. A role for CD 14 in this activity was demonstrated. Use of a commercial cDNA array revealed that numerous pro- inflammatory cytokines were upregulated in PBMC in response to this material, including IL-1β, IL-8, IL-17 and G-CSF. Upregulation of cytokines purported to inhibit inflammation (e.g. IL-4, IL-10 and IL-1β) was not observed, suggesting that the protein(s) selectively modulate the inflammatory cytokine network in a damaging fashion. Two proteins with IL-6-inducing activity were isolated: one of approximately 190kDa, the other a 23kDa protein that was highly homologous to the manganese- dependent superoxide dismutases of S. mutans and S. pneumoniae. Exported material extracted from S. sanguis grown in the absence of blood was able to inhibit LPS-induced IL-6 production in PBMC, an activity that was not observed in EM from blood-grown bacteria. The active factor was partially purified from culture supernatant, and contained 34 and 39kDa proteins, and two proteins of greater than 66kDa, which may have been responsible for the activity. In conclusion, S. sanguis secretes material that is able to modulate inflammatory cytokine networks, shifting the balance towards a damaging inflammatory response in the presence of blood, but expressing cytokine-inhibitory activity in the absence of blood. Some progress has been made towards isolating the components responsible for these activities