Lawton K. Chung

We have identified two novel proteins that colocalize with the subpellicular microtubules in the protozoan parasite Toxoplasma gondii and named these proteins SPM1 and SPM2. These proteins have basic isoelectric points and both have homologs in other apicomplexan parasites. SPM1 contains six tandem copies of a 32-amino-acid repeat, whereas SPM2 lacks defined protein signatures. Alignment of Toxoplasma SPM2 with apparent Plasmodium SPM2 homologs indicates that the greatest degree of conservation lies in the carboxy-terminal half of the protein. Analysis of Plasmodium homologs of SPM1 indicates that while the central 32-amino-acid repeats have expanded to different degrees (7, 8, 9, 12, or 13 repeats), the amino- and carboxy-terminal regions remain conserved. In contrast, although the Cryptosporidium SPM1 homolog has a conserved carboxy tail, the five repeats are considerably diverged, and it has a smaller amino-terminal domain. SPM1 is localized along the full length of the subpellicular microtubules but does not associate with the conoid or spindle microtubules. SPM2 has a restricted localization along the middle region of the subpellicular microtubules. Domain deletion analysis indicates that four or more copies of the SPM1 repeat are required for localization to microtubules, and the amino-terminal 63 residues of SPM2 are required for localization to the subpellicular microtubules. Gene deletion studies indicate that neither SPM1 nor SPM2 is essential for tachyzoite viability. However, loss of SPM1 decreases overall parasite fitness and eliminates the stability of subpellicular microtubules to detergent extraction.

YopM is a leucine-rich repeat (LRR)-containing effector in several Yersinia species, including Yersinia pestis and Y. pseudotuberculosis. Different Yersinia strains encode distinct YopM isoforms with variable numbers of LRRs but conserved C-terminal tails. A 15-LRR isoform in Y. pseudotuberculosis YPIII was recently shown to bind and inhibit caspase-1 via a YLTD motif in LRR 10, and attenuation of YopM(-) YPIII was reversed in mice lacking caspase-1, indicating that caspase-1 inhibition is a major virulence function of YopM(YPIII). To determine if other YopM proteins inhibit caspase-1, we utilized Y. pseudotuberculosis strains natively expressing a 21-LRR isoform lacking the YLTD motif (YopM(32777)) or ectopically expressing a Y. pestis 15-LRR version with a functional (YopM(KIM)) or inactivated (YopM(KIM) D271A) YLTD motif. Results of mouse and macrophage infections with these strains showed that YopM(32777), YopM(KIM), and YopM(KIM) D271A inhibit caspase-1 activation, indicating that the YLTD motif is dispensable for this activity. Analysis of YopM(KIM) deletion variants revealed that LRRs 6 to 15 and the C-terminal tail are required to inhibit caspase-1 activation. YopM(32777), YopM(KIM), and YopM(KIM) deletion variants were purified, and binding partners in macrophage lysates were identified. Caspase-1 bound to YopM(KIM) but not YopM(32777). Additionally, YopM(KIM) bound IQGAP1 and the use of Iqgap1(-/-) macrophages revealed that this scaffolding protein is important for caspase-1 activation upon infection with YopM(-) Y. pseudotuberculosis. Thus, while multiple YopM isoforms inhibit caspase-1 activation, their variable LRR domains bind different host proteins to perform this function and the LRRs of YopM(KIM) target IQGAP1, a novel regulator of caspase-1, in macrophages. Importance: Activation of caspase-1, mediated by macromolecular complexes termed inflammasomes, is important for innate immune defense against pathogens. Pathogens can, in turn, subvert caspase-1-dependent responses through the action of effector proteins. For example, the Yersinia effector YopM inhibits caspase-1 activation by arresting inflammasome formation. This caspase-1 inhibitory activity has been studied in a specific YopM isoform, and in this case, the protein was shown to act as a pseudosubstrate to bind and inhibit caspase-1. Different Yersinia strains encode distinct YopM isoforms, many of which lack the pseudosubstrate motif. We studied additional isoforms and found that these YopM proteins inhibit caspase-1 activation independently of a pseudosubstrate motif. We also identified IQGAP1 as a novel binding partner of the Yersinia pestis YopM(KIM) isoform and demonstrated that IQGAP1 is important for caspase-1 activation in macrophages infected with Yersinia. Thus, this study reveals new insights into inflammasome regulation during Yersinia infection.