Apicomplexan parasite growth and replication relies on nutrient acquisition from host cells, in which intracellular multiplication occurs, yet the mechanisms that underlie the nutrient salvage remain elusive. Numerous ultrastructural studies have documented a plasma membrane invagination with a dense neck, termed the micropore, on the surface of intracellular parasites. However, the function of this structure remains unknown. Here we validate the micropore as an essential organelle for endocytosis of nutrients from the host cell cytosol and Golgi in the model apicomplexan Toxoplasma gondii. Detailed analyses demonstrated that Kelch13 is localized at the dense neck of the organelle and functions as a protein hub at the micropore for endocytic uptake. Intriguingly, maximal activity of the micropore requires the ceramide de novo synthesis pathway in the parasite. Thus, this study provides insights into the machinery underlying acquisition of host cell-derived nutrients by apicomplexan parasites that are otherwise sequestered from host cell compartments.

Background: Melanoma is a malignant tumor with a high mortality rate. Some microorganisms have been shown to activate the immune system and limit cancer progression. The objective of this study is to evaluate the anti-melanoma effect ofNeospora caninum, a livestock pathogen with no pathogenic activity in humans.Methods: Neospora caninumtachyzoites were inoculated into a C57BL/6 mouse melanoma model by intratumoral and distal subcutaneous injections. Tumor volumes were measured, and cell death areas were visualized by hematoxylin and eosin staining and quantified. Apoptosis in cell cultures and whole tumors was detected by propidium iodide (PI) and TUNEL staining, respectively. Cytokine and tumor-associated factor levels in tumors and spleens were detected by real-time quantitative polymerase chain reaction. Infiltration of macrophages and CD8⁺T cells in the tumor microenvironment (TME) were detected by immunohistochemistry with anti-CD68 and anti-CD8 antibodies, respectively. Finally, 16S rRNA sequencing of mice cecal contents was performed to evaluate the effect ofN. caninumon gut microbial diversity.Results: Intratumoral and distal subcutaneous injections ofN. caninumresulted in significant inhibition of tumor growth (P < 0.001), and more than 50% of tumor cells were dead without signs of apoptosis.Neospora caninumtreatment significantly increased the mRNA expression levels of IL-12, IFN-γ, IL-2, IL-10, TNF-α, and PD-L1 in the TME, and IL-12 and IFN-γ in the spleen of tumor-bearing mice (P < 0.05). An increase in the infiltration of CD8⁺T cells and macrophages in the TME was observed with these cytokine changes.Neospora caninumalso restored the abundance of gut microbiotaLactobacillus,Lachnospiraceae,Adlercreutzia, andPrevotellaceaeassociated with tumor growth, but the changes were not significant.Conclusion: Neospora caninuminhibits B16F10 melanoma by activating potent immune responses and directly destroying the cancer cells. The stable, non-toxic, and efficacious properties ofN. caninumdemonstrate the potential for its use as a cancer treatment.Graphical

Fusarium pseudograminearum has been identified as a significant pathogen. It causes Fusarium crown rot (FCR), which occurs in several major wheat-producing areas in China. Chemical control is the primary measure with which to control this disease. In this study, transcriptome sequencing (RNA-Seq) was used to determine the different mechanisms of action of four frequently used fungicides including carbendazim, pyraclostrobin, tebuconazole, and phenamacril on F. pseudograminearum. In brief, 381, 1896, 842, and 814 differentially expressed genes (DEGs) were identified under the carbendazim, pyraclostrobin, tebuconazole, and phenamacril treatments, respectively. After the joint analysis, 67 common DEGs were obtained, and further functional analysis showed that the ABC transported pathway was significantly enriched. Moreover, FPSE_04130 (FER6) and FPSE_11895 (MDR1), two important ABC multidrug transporter genes whose expression levels simultaneously increased, were mined under the different treatments, which unambiguously demonstrated the common effects. In addition, Mfuzz clustering analysis and WGCNA analysis revealed that the core DEGs are involved in several critical pathways in each of the four treatment groups. Taken together, these genes may play a crucial function in the mechanisms of F. pseudograminearum‘s response to the fungicides stress.

Balantioides coli plays an important role in the diarrhea of weaned piglets, but its pathogenic potential and interaction with gut microbes remain unclear. To investigate the impact of B. coli colonization on the gut bacterial structure and function of weaned piglets, a metagenomic analysis based on shotgun sequencing was performed on fresh fecal samples collected from ten B. coli-colonized piglets and eight B. coli-free ones in this study. The results showed that decreasing diversity and shifted composition and function of the bacterial community were detected in the weaned piglets infected by B. coli. In contrast to the B. coli-negative group, the relative abundances of some members of the Firmicutes phylum including Clostridium, Ruminococcus species, and Intestinimonas butyriciproducens, which produce short-chain fatty acids, were significantly reduced in the B. coli-positive group. Notably, some species of the Prevotella genus (such as Prevotella sp. CAG:604 and Prevotella stercorea) were significantly increased in abundance in the B. coli-positive piglets. A functional analysis of the gut microbiota demonstrated that the differential gene sets for the metabolism of carbohydrates and amino acids were abundant in both groups, and the more enriched pathways in B. coli-infected piglets were associated with the sugar-specific phosphotransferase system (PTS) and the two-component regulatory system, as well as lipopolysaccharide (LPS) biosynthesis. Furthermore, several species of Prevotella were significantly positively correlated to the synthesis of lipid A, leading to the exporting of endotoxins and, thereby, inducing inflammation in the intestines of weaned piglets. Taken together, these findings revealed that colonization by B. coli was distinctly associated with the dysbiosis of gut bacterial structure and function in weaned piglets. Lower relative abundances of Clostridiaceae and Ruminococcaceae and higher abundances of Prevotella species were biomarkers of B. coli infection in weaned piglets.

The apicoplast is a four-membrane plastid found in the apicomplexans, which harbors biosynthesis and organelle housekeeping activities in the matrix. However, the mechanism driving the flux of metabolites, in and out, remains unknown. Here, we used TurboID and genome engineering to identify apicoplast transporters in Toxoplasma gondii. Among the many novel transporters, we show that one pair of apicomplexan monocarboxylate transporters (AMTs) appears to have evolved from a putative host cell that engulfed a red alga. Protein depletion showed that AMT1 and AMT2 are critical for parasite growth. Metabolite analyses supported the notion that AMT1 and AMT2 are associated with biosynthesis of isoprenoids and fatty acids. However, stronger phenotypic defects were observed for AMT2, including in the inability to establish T. gondii parasite virulence in mice. This study clarifies, significantly, the mystery of apicoplast transporter composition and reveals the importance of the pair of AMTs in maintaining the apicoplast activity in apicomplexans.

Protein prenylation is an important protein modification that is responsible for diverse physiological activities in eukaryotic cells. This modification is generally catalyzed by three types of prenyl transferases, which include farnesyl transferase (FT), geranylgeranyl transferase (GGT-1) and Rab geranylgeranyl transferase (GGT-2). Studies in malaria parasites showed that these parasites contain prenylated proteins, which are proposed to play multiple functions in parasites. However, the prenyl transferases have not been functionally characterized in parasites of subphylum Apicomplexa. Here, we functionally dissected functions of three of the prenyl transferases in the Apicomplexa model organism Toxoplasma gondii (T. gondii) using a plant auxin-inducible degron system. The homologous genes of the beta subunit of FT, GGT-1 and GGT-2 were endogenously tagged with AID at the C-terminus in the TIR1 parental line using a CRISPR-Cas9 approach. Upon depletion of these prenyl transferases, GGT-1 and GGT-2 had a strong defect on parasite replication. Fluorescent assay using diverse protein markers showed that the protein markers ROP5 and GRA7 were diffused in the parasites depleted with GGT-1 and GGT-2, while the mitochondrion was strongly affected in parasites depleted with GGT-1. Importantly, depletion of GGT-2 caused the stronger defect to the sorting of rhoptry protein and the parasite morphology. Furthermore, parasite motility was observed to be affected in parasites depleted with GGT-2. Taken together, this study functionally characterized the prenyl transferases, which contributed to an overall understanding of protein prenylation in T. gondii and potentially in other related parasites.