Balancing Act: Tubulin Glutamylation and Microtubule Dynamics in Toxoplasma gondii.

The success of the intracellular parasite Toxoplasma gondii in invading host cells relies on the apical complex, a specialized microtubule cytoskeleton structure associated with secretory organelles. The T. gondii genome encodes three isoforms of both α- and ß-tubulin, which undergo specific post-translational modifications (PTMs), altering the biochemical and biophysical proprieties of microtubules and modulating their interaction with associated proteins. Tubulin PTMs represent a powerful and evolutionarily conserved mechanism for generating tubulin diversity, forming a biochemical 'tubulin code' interpretable by microtubule-interacting factors. T. gondii exhibits various tubulin PTMs, including α-tubulin acetylation, α-tubulin detyrosination, Δ5α-tubulin, Δ2α-tubulin, α- and ß-tubulin polyglutamylation, and α- and ß-tubulin methylation. Tubulin glutamylation emerges as a key player in microtubule remodeling in Toxoplasma, regulating stability, dynamics, interaction with motor proteins, and severing enzymes. The balance of tubulin glutamylation is maintained through the coordinated action of polyglutamylases and deglutamylating enzymes. This work reviews and discusses current knowledge on T. gondii tubulin glutamylation. Through in silico identification of protein orthologs, we update the recognition of putative proteins related to glutamylation, contributing to a deeper understanding of its role in T. gondii biology.

Impact of Endemic Besnoitiosis on the Performance of a Dairy Cattle Herd.

This study aimed to assess the effect of Besnoitia besnoiti infection on the reproductive and productive performance of a dairy cattle herd. A serological screening was performed by indirect fluorescent antibody test (IFAT) on every animal aged over one year (n = 262). Subsequently, 211 animals were clinically examined, with 96 of those being screened for detection of sclerocysts. The overall seroprevalence was 62.9% (CI95%: 56.1-69.5%). On clinical examination, 7.6% (16/211) of the animals presented chronic skin lesions, and 47.9% (46/96) had sclerocysts. Multivariate logistic regression showed that the time on herd represented a risk factor, and the odds of acquiring the infection increased 1.683× per additional year on herd, ranging from less than a year to 8 years. Seropositivity and the presence of sclerocysts revealed an association with a higher milk somatic cell count, which may have a considerable economic impact on dairy production. Regarding reproductive indicators, no negative impact could be associated with clinical besnoitiosis or positive serological results. In conclusion, our study highlights the need to thoroughly evaluate the economic impact of this emerging disease in dairy herd production to help with decision making at both herd and regional levels, particularly in endemic areas.

Characterization of a MOB1 Homolog in the Apicomplexan Parasite Toxoplasma gondii.

Monopolar spindle One Binder1 (MOB1) proteins are conserved components of the tumor-suppressing Hippo pathway, regulating cellular processes such as cytokinesis. Apicomplexan parasites present a life cycle that relies on the parasites' ability to differentiate between stages and regulate their proliferation; thus, Hippo signaling pathways could play an important role in the regulation of the apicomplexan life cycle. Here, we report the identification of one MOB1 protein in the apicomplexan Toxoplasma gondii. To characterize the function of MOB1, we generated gain-of-function transgenic lines with a ligand-controlled destabilization domain, and loss-of-function clonal lines obtained through CRISPR/Cas9 technology. Contrary to what has been characterized in other eukaryotes, MOB1 is not essential for cytokinesis in T. gondii. However, this picture is complex since we found MOB1 localized between the newly individualized daughter nuclei at the end of mitosis. Moreover, we detected a significant delay in the replication of overexpressing tachyzoites, contrasting with increased replication rates in knockout tachyzoites. Finally, using the proximity-biotinylation method, BioID, we identified novel members of the MOB1 interactome, a probable consequence of the observed lack of conservation of some key amino acid residues. Altogether, the results point to a complex evolutionary history of MOB1 roles in apicomplexans, sharing properties with other eukaryotes but also with divergent features, possibly associated with their complex life cycle.

MOB: Pivotal Conserved Proteins in Cytokinesis, Cell Architecture and Tissue Homeostasis.

The MOB family proteins are constituted by highly conserved eukaryote kinase signal adaptors that are often essential both for cell and organism survival. Historically, MOB family proteins have been described as kinase activators participating in Hippo and Mitotic Exit Network/ Septation Initiation Network (MEN/SIN) signaling pathways that have central roles in regulating cytokinesis, cell polarity, cell proliferation and cell fate to control organ growth and regeneration. In metazoans, MOB proteins act as central signal adaptors of the core kinase module MST1/2, LATS1/2, and NDR1/2 kinases that phosphorylate the YAP/TAZ transcriptional co-activators, effectors of the Hippo signaling pathway. More recently, MOBs have been shown to also have non-kinase partners and to be involved in cilia biology, indicating that its activity and regulation is more diverse than expected. In this review, we explore the possible ancestral role of MEN/SIN pathways on the built-in nature of a more complex and functionally expanded Hippo pathway, by focusing on the most conserved components of these pathways, the MOB proteins. We discuss the current knowledge of MOBs-regulated signaling, with emphasis on its evolutionary history and role in morphogenesis, cytokinesis, and cell polarity from unicellular to multicellular organisms.

The Origin and Diversity of Cpt1 Genes in Vertebrate Species.

The Carnitine palmitoyltransferase I (Cpt1) gene family plays a crucial role in energy homeostasis since it is required for the occurrence of fatty acid ß-oxidation in the mitochondria. The exact gene repertoire in different vertebrate lineages is variable. Presently, four genes are documented: Cpt1a, also known as Cpt1a1, Cpt1a2; Cpt1b and Cpt1c. The later is considered a mammalian innovation resulting from a gene duplication event in the ancestor of mammals, after the divergence of sauropsids. In contrast, Cpt1a2 has been found exclusively in teleosts. Here, we reassess the overall evolutionary relationships of Cpt1 genes using a combination of approaches, including the survey of the gene repertoire in basal gnathostome lineages. Through molecular phylogenetics and synteny studies, we find that Cpt1c is most likely a rapidly evolving orthologue of Cpt1a2. Thus, Cpt1c is present in other lineages such as cartilaginous fish, reptiles, amphibians and the coelacanth. We show that genome duplications (2R) and variable rates of sequence evolution contribute to the history of Cpt1 genes in vertebrates. Finally, we propose that loss of Cpt1b is the likely cause for the unusual energy metabolism of elasmobranch.

Mechanisms of transepithelial ammonia excretion and luminal alkalinization in the gut of an intestinal air-breathing fish, Misgurnus anguilliacaudatus.

The weatherloach, Misgurnus angulliacaudatus, is an intestinal air-breathing, freshwater fish that has the unique ability to excrete ammonia through gut volatilization when branchial and cutaneous routes are compromised during high environmental ammonia or air exposure. We hypothesized that transepithelial gut NH(4)(+) transport is facilitated by an apical Na(+)/H(+) (NH(4)(+)) exchanger (NHE) and a basolateral Na(+)/K(+)(NH(4)(+))-ATPase, and that gut boundary layer alkalinization (NH(4)(+) → NH(3) + H(+)) is facilitated by apical HCO(3)(-) secretion through a Cl(-)/HCO(3)(-) anion exchanger. This was tested using a pharmacological approach with anterior (digestive) and posterior (respiratory) intestine preparations mounted in pH-stat-equipped Ussing chambers. The anterior intestine had a markedly higher conductance, increased short-circuit current, and greater net base (J(base)) and ammonia excretion rates (J(amm)) than the posterior intestine. In the anterior intestine, HCO(3)(-) accounted for 70% of J(base). In the presence of an imposed serosal-mucosal ammonia gradient, inhibitors of both NHE (EIPA, 0.1 mmol l(-1)) and Na(+)/K(+)-ATPase (ouabain, 0.1 mmol l(-1)) significantly inhibited J(amm) in the anterior intestine, although only EIPA had an effect in the posterior intestine. In addition, the anion exchange inhibitor DIDS significantly reduced J(base) in the anterior intestine although only at a high dose (1 mmol l(-1)). Carbonic anhydrase does not appear to be associated with gut alkalinization under these conditions as ethoxzolamide was without effect on J(base). Membrane fluidity of the posterior intestine was low, suggesting low permeability, which was also reflected in a lower mucosal-serosal J(amm) in the presence of an imposed gradient, in contrast to that in the anterior intestine. To conclude, although the posterior intestine is highly modified for gas exchange, it is the anterior intestine that is the likely site of ammonia excretion and alkalinization leading to ammonia volatilization in the gut.

Roles of three branchial Na(+)-K(+)-ATPase α-subunit isoforms in freshwater adaptation, seawater acclimation, and active ammonia excretion in Anabas testudineus.

Three Na(+)-K(+)-ATPase (nka) α-subunit isoforms, nka α1a, nka α1b, and nka α1c, were identified from gills of the freshwater climbing perch Anabas testudineus. The cDNA sequences of nka α1a and nka α1b consisted of 3,069 bp, coding for 1,023 amino acids, whereas nka α1c was shorter by 22 nucleotides at the 5' end. In freshwater, the quantity of nka α1c mRNA transcripts present in the gills was the highest followed by nka α1a and nka α1b that was almost undetectable. The mRNA expression of nka α1a was downregulated in the gills of fish acclimated to seawater, indicating that it could be involved in branchial Na(+) absorption in a hypoosmotic environment. By contrast, seawater acclimation led to an upregulation of the mRNA expression of nka α1b and to a lesser extent nka α1c, indicating that they could be essential for ion secretion in a hyperosmotic environment. More importantly, ammonia exposure led to a significant upregulation of the mRNA expression of nka α1c, which might be involved in active ammonia excretion. Both seawater acclimation and ammonia exposure led to significant increases in the protein abundance and changes in the kinetic properties of branchial Na(+)-K(+)-ATPase (Nka), but they involved two different types of Nka-immunoreactive cells. Since there was a decrease in the effectiveness of NH(4)(+) to substitute for K(+) to activate branchial Nka from fish exposed to ammonia, Nka probably functioned to remove excess Na(+) and to transport K(+) instead of NH(4)(+) into the cell to maintain intracellular Na(+) and K(+) homeostasis during active ammonia excretion.

Cystic fibrosis transmembrane conductance regulator in the gills of the climbing perch, Anabas testudineus, is involved in both hypoosmotic regulation during seawater acclimation and active ammonia excretion during ammonia exposure.

This study aimed to clone and sequence the cystic fibrosis transmembrane conductance regulator (cftr) from, and to determine the effects of seawater acclimation or exposure to 100 mmol l⁻¹ NH4Cl in freshwater on its mRNA and protein expressions in, the gills of Anabas testudineus. There were 4,530 bp coding for 1,510 amino acids in the cftr cDNA sequence from A. testudineus. The branchial mRNA expression of cftr in fish kept in freshwater was low (<50 copies of transcript per ng cDNA), but significant increases were observed in fish acclimated to seawater for 1 day (92-fold) or 6 days (219-fold). Branchial Cftr expression was detected in fish acclimated to seawater but not in the freshwater control, indicating that Cl⁻ excretion through the apical Cftr of the branchial epithelium was essential to seawater acclimation. More importantly, fish exposed to ammonia also exhibited a significant increase (12-fold) in branchial mRNA expression of cftr, with Cftr being expressed in a type of Na⁺/K⁺-ATPase-immunoreactive cells that was apparently different from the type involved in seawater acclimation. It is probable that Cl⁻ excretion through Cftr generated a favorable electrical potential across the apical membrane to drive the excretion of NH4⁺ against a concentration gradient through a yet to be determined transporter, but it led to a slight loss of endogenous Cl⁻. Since ammonia exposure also resulted in significant decreases in blood pH, [HCO3⁻] and [total CO2] in A. testudineus, it can be deduced that active NH4⁺ excretion could also be driven by the exit of HCO3⁻ through the apical Cftr. Furthermore, A. testudineus uniquely responded to ammonia exposure by increasing the ambient pH and decreasing the branchial bafilomycin-sensitive V-type H⁺-ATPase activity, which suggests that its gills might have low NH3 permeability.