Biology subjects

Balestra, A. C.

Publications and source records attributed to Balestra, A. C..

4 recordsLinked to original sources

bioRxiv2026

Cryptosporidium sex employs a complex male fertilization apparatus that is required for continued infection

Most apicomplexan parasites replicate asexually to colonize their mammalian hosts, and sexually in the context of transmission. In contrast, for Cryptosporidium sex is essential to sustain intestinal infection in vivo and therefore is a promising opportunity to prevent and treat the disease. However, the mechanism of sex, which occurs within host enterocytes, is poorly understood, limiting the discovery of targets for such intervention. Here we identify an elaborate fertilization apparatus and numerous of its molecular components in male gametes. We discern two elements, one displayed on the apex, that makes intimate contact with host cells containing female parasites, and a second internal structure, that harbors the membrane fusogen HAP2. Ablation of HAP2 by conditional mutagenesis blocked fertilization and oocyst production, rapidly cured infected mice and left them with enhanced resistance to subsequent challenge. Our studies provide insights into a remarkable fertilization mechanism and highlight specific molecules as targets for translation.

microbiology↗

bioRxiv2023

Structure and flexibility of the DNA polymerase holoenzyme of vaccinia virus

The year 2022 was marked by the mpox outbreak caused by human monkeypox virus (MPXV), which is about 98 % identical to vaccinia virus (VACV) at the sequence level regarding the proteins involved in DNA replication. We present the strategy for the production of the VACV DNA polymerase holoenzyme composed of the E9 polymerase associated with its co-factor, the A20-D4 heterodimer, which led to the 3.8 [A] cryo-electron microscopy (cryo-EM) structure of the DNA-free form of the holoenzyme. Model building used high-resolution structures of components of the complex and the A20 structure predicted by AlphaFold 2. The structure of E9 does not change in context of the holoenzyme compared to the crystal structure. As for the MPXV holoenzyme, a contact between E9 and D4 is mediated by a cluster of hydrophobic residues. The holoenzyme structure is quite compact and surprisingly similar to the MPXV holoenzyme in presence of a DNA template, with the exception of a movement of the finger domain and the thumb domain, which becomes ordered in presence of DNA. Even in absence of DNA, the VACV holoenzyme structure is too compact for an agreement with SAXS data. This suggests the presence of more open conformations in solution, which are also predicted by Alphafold 2 indicating hinge regions located within A20. Using biolayer interferometry we showed that indeed, the E9-D4 interaction is weak and transient although very important as it has not been possible to obtain viable viruses carrying mutations of key residues in the E9-D4 interface. Author SummaryThe 2022 outbreak of mpox is caused by monkeypox virus closely related to the best studied model, vaccinia virus. Genome replication, which takes place largely autonomously in the cytosol of the infected cell, is still not really understood. Viral DNA synthesis involves a DNA repair enzyme, the uracil-DNA glycosylase D4 linked to the structural protein A20 forming the processivity factor, which in turn binds to E9 forming the complex required for processive DNA synthesis. Here we present the first structure of the vaccinia virus polymerase holoenzyme E9-A20-D4 at 3.8 [A] obtained by cryo-electron microscopy. This structure, together with several recent structures from monkeypox virus, provide a static view of the complex with a previously undescribed contact between E9 and D4. Our small-angle scattering data show that other conformations, taking advantage of 2 hinge regions in A20, exist in solution. Using site-directed mutagenesis and binding studies we show that the contact between E9 and D4, which serves to encircle the template strand, is important, but transient. Thus the current model of the orientation of the holoenzyme on the replication fork may not be the only one possible.

molecular biology↗

bioRxiv2020

A multipass membrane protein interacts with the cGMP-dependent protein kinase to regulate critical calcium signals in malaria parasites

In malaria parasites, all cGMP-dependent signalling is mediated through a single cGMP-dependent protein kinase (PKG), a major function of which is to control essential calcium signals. However, how PKG transmits these signals in the absence of known second messenger-dependent calcium channels or scaffolding proteins is unknown. Here we identify a polytopic membrane protein, ICM1, with homology to transporters and calcium channels that is tightly-associated with PKG in both Plasmodium falciparum asexual blood stages and P. berghei gametocytes. Phosphoproteomic analyses in both Plasmodium species reveal multiple ICM1 phosphorylation events dependent upon PKG activity. Stage-specific depletion of P. berghei ICM1 blocks gametogenesis due to the inability of mutant parasites to mobilise intracellular calcium upon PKG activation, whilst conditional loss of P. falciparum ICM1 results in reduced calcium mobilisation, defective egress and lack of invasion. Our findings provide new insights into atypical calcium homeostasis in malaria parasites essential for pathology and disease transmission.

microbiology↗

bioRxiv2020

A divergent cyclin/cyclin-dependent kinase complex controls the atypical replication of Plasmodium berghei during gametogony and parasite transmission.

Cell cycle transitions are generally triggered by variation in the activity of cyclin-dependent kinases (CDKs) bound to cyclins. Malaria-causing parasites have a life cycle with unique cell-division cycles, and a repertoire of divergent CDKs and cyclins of poorly understood function and interdependency. We show that Plasmodium berghei CDK-related kinase 5 (CRK5), is a critical regulator of atypical mitosis in the gametogony and is required for mosquito transmission. It phosphorylates canonical CDK motifs of components in the pre-replicative complex and is essential for DNA replication. During a replicative cycle, CRK5 stably interacts with a single Plasmodium-specific cyclin (SOC2), although we obtained no evidence of SOC2 cycling by transcription, translation or degradation. Our results provide evidence that during Plasmodium male gametogony, this divergent cyclin/CDK pair fills the functional space of other eukaryotic cell-cycle kinases controlling DNA replication.

microbiology↗