Methicillin-resistantStaphylococcus aureus (MRSA) is a frequent cause of difficult-to-treat, often fatal infections in humans 1,2 . Most humans have antibodies against S. aureus, but these are highly variable and often not protective in immunocompromised patients 3 . Previous vaccine development programs have not been successful 4 . A large percentage of human antibodies against S. aureus target wall teichoic acid (WTA), a ribitol-phosphate (RboP) surface polymer modified with N-acetylglucosamine (GlcNAc) 5,6 . It is currently unknown whether the immune evasion capacities of MRSA are due to variation of dominant surface epitopes such as those associated with WTA. Here we show that a considerable proportion of the prominent healthcare-associated and livestock-associated MRSA clones CC5 and CC398, respectively, contain prophages that encode an alternative WTA glycosyltransferase. This enzyme, TarP, transfers GlcNAc to a different hydroxyl group of the WTA RboP than the standard enzyme TarS 7 , with important consequences for immune recognition. TarP-glycosylated WTA elicits 7.5-40-fold lower levels of immunoglobulin G in mice than TarS-modified WTA. Consistent with this, human sera contained only low levels of antibodies against TarP-modified WTA. Notably, mice immunized with TarS-modified WTA were not protected against infection with tarP-expressing MRSA, indicating that TarP is crucial for the capacity of S. aureus to evade host defences. High-resolution structural analyses of TarP bound to WTA components and uridine diphosphate GlcNAc (UDP-GlcNAc) explain the mechanism of altered RboP glycosylation and form a template for targeted inhibition of TarP. Our study reveals an immune evasion strategy of S. aureus based on averting the immunogenicity of its dominant glycoantigen WTA. These results will help with the identification of invariant S. aureus vaccine antigens and may enable the development of TarP inhibitors as a new strategy for rendering MRSA susceptible to human host defences.Novel prevention and treatment strategies against major antibioticresistant pathogens such as MRSA are urgently needed but are not within reach because some of the most critical virulence strategies of these pathogens are not understood 8 . The pathogenic potential of prominent healthcare-associated (HA)-MRSA and recently emerged livestock-associated (LA)-MRSA strains is thought to rely on particularly effective immune evasion strategies, whereas communityassociated (CA)-MRSA strains often produce more aggressive toxins 1,2 . Most humans have high overall levels of antibodies against S. aureus as a consequence of preceding infections, but antibody titres differ strongly for specific antigens and are often not protective in immunocompromised patients, for reasons that are not clear 3 . A large percentage of human antibodies against S. aureus is directed against WTA 5,9,10 , which is largely invariant. However, some S. aureus lineages produce altered WTA, which modulates, for instance, phage susceptibility 7,11 .To investigate whether ...

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Using 5% ethanol as a deciliating agent, 20 mm colchicine to prevent reciliation and 1 mm amiloride to affect ion fluxes in Paramecium we examined the compartmentation and function of Ca2+ fluxes employing the biosynthesis of cGMP and the stereotypic swimming behavior as indicators for Ca2+ entry. As a function of extracellular Ca2+ Paramecia responded to colchicine and amiloride with a short-lived ciliary augmentation (fast swimming) which indicated hyperpolarization, and formation of cGMP, i.e., the reported hyperpolarization-activated Ca2+ inward current in the somatic membrane is coupled to intracellular generation of cGMP. This is comparable to the coupling of the depolarization-activated, ciliary Ca2+ inward current and ciliary cGMP formation.Ethanol-deciliated cells and ethanol-treated, yet ciliated control cells did not respond to a depolarization with backward swimming or formation of cGMP. Both responses recovered with similar kinetics. A persistent effect of an ethanol exposure on the axonemal apparatus or on guanylyl cyclase activity of ciliated control cells was excluded using permeabilized cells and cell-free enzyme, respectively. Further, in the presence of 20 mm colchicine ethanol-treated cells only recovered the depolarization-dependent avoiding reaction whereas the formation of cGMP remained depressed, i.e., the drug dissected both responses. Similarly, ethanol exposure of Paramecia did not affect the fast swimming response towards the hyperpolarizing agent amiloride whereas the cGMP formation was abrogated and recovered over a period of 7 hr, i.e., amiloride dissected the hyperpolarization-elicited behavioral response from the intracellular cGMP formation. The data demonstrate that in Paramecium depolarization- and hyperpolarization-stimulated behavioral responses and cGMP formation are not coupled. The behavioral changes are triggered by smaller Ca2+ inward currents than the formation of intracellular cGMP.

The class IIIa adenylyl cyclase (AC) Rv1625c from Mycobacterium tuberculosis forms homodimers with two catalytic centres, whereas the Paramecium guanylyl and mammalian ACs operate as pseudoheterodimers with one catalytic centre. The functional and structural relationship of the catalytic domains of these related class III cyclases was investigated. Point mutations introduced into Rv1625c to engineer a forskolinbinding pocket created a single heterodimeric catalytic centre, yet did not result in forskolin activation. Chimerization of these Rv1625c point mutants with corresponding mammalian AC domains was impossible. However, it was successful using a complemental Paramecium guanylyl cyclase domain and resulted in an AC. The data signify a divergence of structural and functional evolution in class III Acs.

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