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Introduction: Kawasaki disease (KD) can be associated with concomitant viral or bacterial infections. Children with persistent or recurrent fever 36 hours after the end of intravenous immunoglobulin (IVIG) are considered to be resistant to treatment and are at increased risk for coronary complications. Although concomitant infection does not affect coronary outcome, it is unknown how it influences the response to IVIG treatment., Methodology: Retrospective cohort study between 2008 and 2016 in a tertiary pediatric university hospital, including 154 children, of which 59 (38%) had concomitant infection., Results: Children with concomitant infection were more likely to have fever 48 hours after initial IVIG treatment (36% vs 20%, p = 0.05) and to be treated with a second dose (33% vs 18%, p = 0.04). Children with infection had higher C-reactive protein at the time of diagnosis (148 vs 112 mg/L, p = 0.04), and 48 hours after IVIG administration (111 vs 59 mg/L, p = 0.003). Nevertheless, there was no statistically significant difference in the prevalence of coronary complications (Z-score > 2.5) between children with and without concomitant infection (36% vs 39%, p = 0.68)., Conclusion: Children with KD and concomitant infection are more likely to have persistent fever and elevated inflammatory markers after treatment. This association increases the likelihood of receiving a second dose of IVIG but not the risk of coronary complication. Accordingly, prospective studies to distinguish true IVIG resistance from infection induced persistent fever is warranted., Competing Interests: The authors have declared that no competing interests exist.

Differentiation of the protozoan parasite Toxoplasma gondii into its latent bradyzoite stage is a key event in the parasite's life cycle. Compound 2 is an imidazopyridine that was previously shown to inhibit the parasite lytic cycle, in part through inhibition of parasite cGMP-dependent protein kinase. We show here that Compound 2 can also enhance parasite differentiation, and we use yeast three-hybrid analysis to identify TgBRADIN/GRA24 as a parasite protein that interacts directly or indirectly with the compound. Disruption of the TgBRADIN/GRA24 gene leads to enhanced differentiation of the parasite, and the TgBRADIN/GRA24 knockout parasites show decreased susceptibility to the differentiation-enhancing effects of Compound 2. This study represents the first use of yeast three-hybrid analysis to study small-molecule mechanism of action in any pathogenic microorganism, and it identifies a previously unrecognized inhibitor of differentiation in T. gondii. A better understanding of the proteins and mechanisms regulating T. gondii differentiation will enable new approaches to preventing the establishment of chronic infection in this important human pathogen.

Motility of the protozoan parasite Toxoplasma gondii plays an important role in the parasite's life cycle and virulence within animal and human hosts. Motility is driven by a myosin motor complex that is highly conserved across the Phylum Apicomplexa. Two key components of this complex are the class XIV unconventional myosin, TgMyoA, and its associated light chain, TgMLC1. We previously showed that treatment of parasites with a small-molecule inhibitor of T. gondii invasion and motility, tachypleginA, induces an electrophoretic mobility shift of TgMLC1 that is associated with decreased myosin motor activity. However, the direct target(s) of tachypleginA and the molecular basis of the compound-induced TgMLC1 modification were unknown. We show here by "click" chemistry labelling that TgMLC1 is a direct and covalent target of an alkyne-derivatized analogue of tachypleginA. We also show that this analogue can covalently bind to model thiol substrates. The electrophoretic mobility shift induced by another structural analogue, tachypleginA-2, was associated with the formation of a 225.118 Da adduct on S57 and/or C58, and treatment with deuterated tachypleginA-2 confirmed that the adduct was derived from the compound itself. Recombinant TgMLC1 containing a C58S mutation (but not S57A) was refractory to click labelling and no longer exhibited a mobility shift in response to compound treatment, identifying C58 as the site of compound binding on TgMLC1. Finally, a knock-in parasite line expressing the C58S mutation showed decreased sensitivity to compound treatment in a quantitative 3D motility assay. These data strongly support a model in which tachypleginA and its analogues inhibit the motility of T. gondii by binding directly and covalently to C58 of TgMLC1, thereby causing a decrease in the activity of the parasite's myosin motor.