Burette, M. | Search bioRxiv

bioRxiv2026

Two anchoring proteins control daughter apical complex assembly in Toxoplasma gondii

During Toxoplasma gondii division, the apical complex--comprising the conoid, apical polar ring (APR), and preconoidal rings--assembles with precise spatiotemporal coordination to form functional daughter buds. Despite their essential roles in invasion, motility, and division, the scaffolding proteins orchestrating this ordered assembly have remained largely unidentified. Here, we identify and characterize RCC1-2 and APR8 as essential factors directing distinct, sequential phases of daughter cell apical complex construction. Both proteins are recruited with precise spatial and temporal dynamics to the daughter buds, where they function as scaffolds rather than static structural components. APR8 transiently occupies the basal region of the APR specifically in early daughter cells. It is dispensable for conoid and PCR initiation, yet its loss causes APR collapse, abolishes SPMT anchoring, and eventually arrests conoid maturation. In contrast, RCC1-2 localizes beneath the APR basal layer and persists throughout daughter cell development, where it contributes to stabilizing the attachment of SPMTs to the APR. Notably, in situ cryo-electron tomography further reveals that the interspersed pillars bridging SPMTs ends to the APR fail to form properly in RCC1-2-depleted parasites. These findings map a hierarchical RCC1-2/APR8-dependent scaffolding process that advances our understanding of parasite replication.

microbiology↗

bioRxiv2024

The multifunction Coxiella effector Vice stimulates macropinocytosis and interferes with the ESCRT machinery

Intracellular bacterial pathogens divert multiple cellular pathways to establish their niche and persist inside their host. Coxiella burnetii, the causative agent of Q fever, secretes bacterial effector proteins via its Type 4 secretion system to generate a Coxiella-containing vacuole (CCV). Manipulation of lipid and protein trafficking by these effectors is essential for bacterial replication and virulence. Here, we have characterized the lipid composition of CCVs and discovered that the effector Vice interacts with phosphoinositides and membranes enriched in phosphatidylserine (PS) and lysobisphosphatidic acid (LBPA). Remarkably, eukaryotic cells ectopically expressing Vice present compartments that resemble early CCVs in both morphology and composition. We discovered that the biogenesis of these compartments relies on the double function of Vice. The effector protein initially localizes at the plasma membrane of eukaryotic cells where it triggers the internalization of large vacuoles by macropinocytosis. Then, Vice stabilizes these compartments by perturbing the ESCRT machinery and inhibiting the formation of intraluminal vesicles (ILVs). Collectively, our results reveal that Vice is an essential C. burnetii effector protein capable of hijacking two major cellular pathways to shape the bacterial replicative niche. Significance statementCoxiella burnetii is a unique bacterial pathogen that secretes more than a hundred effector proteins to manipulate cellular processes and establish a replicative niche, the Coxiella-containing vacuole (CCV). Our study identified host cell lipids that are actively recruited by the bacterium to the CCV. Using a library of effector mutants, we identified the protein Vice (for Vacuole-inducing Coxiella effector) as the first bacterial effector capable of interacting with lysobisphosphatydic acid-enriched membranes and accumulating this lipid to CCVs. We show that Vice is also capable of stimulating macropinocytosis and inhibiting the ESCRT machinery. Together, our data show how a single bacterial effector can manipulate different cellular processes to favor the biogenesis of a bacterial pathogens niche.

microbiology↗