A Coccidia-Specific Phosphate Transporter Is Essential for the Growth of Toxoplasma gondii Parasites.

Toxoplasma gondii is an obligate intracellular parasite that acquires all necessary nutrients from the hosts, but the exact nutrient acquisition mechanisms are poorly understood. Here, we identified three putative phosphate transporters in T. gondii. TgPiT and TgPT2 are mainly on the plasma membrane, whereas TgmPT is localized to the mitochondrion. TgPiT and TgmPT are widely present and conserved in apicomplexan parasites that include Plasmodium and Eimeria species. Nonetheless, they are dispensable for the growth and virulence of Toxoplasma. TgPT2, on the other hand, is restricted to coccidia parasites and is essential for Toxoplasma survival. TgPT2 depletion led to reduced motility and invasion, as well as growth arrest of the parasites both in vitro and in vivo. Both TgPiT and TgPT2 have phosphate transport activities and contribute to parasites' inorganic phosphate (Pi) absorption. Interestingly, the Pi importing activity of Toxoplasma parasites could be competitively inhibited by ATP and AMP. Furthermore, direct uptake of 32P-ATP was also observed, indicating the parasites' ability to scavenge host ATP. Nonetheless, ATP/AMP import is not mediated by TgPiT or TgPT2, suggesting additional mechanisms. Together, these results show the complex pathways of phosphate transport in Toxoplasma, and TgPT2 is a potential target for antitoxoplasmic intervention design due to its essential role in parasite growth. IMPORTANCE To grow and survive within host cells, Toxoplasma must scavenge necessary nutrients from hosts to support its parasitism. Transporters located in the plasma membrane of the parasites play critical roles in nutrient acquisition. Toxoplasma encodes a large number of transporters, but so far, only a few have been characterized. In this study, we identified two phosphate transporters, TgPiT and TgPT2, to localize to the plasma membrane of Toxoplasma. Although both TgPiT and TgPT2 possess phosphate transport activities, only the novel transporter TgPT2 was essential for parasite growth, both in vitro and in vivo. In addition, TgPT2 and its orthologs are only present in coccidia parasites. As such, TgPT2 represents a potential target for drug design against toxoplasmosis. In addition, our data indicated that Toxoplasma can take up ATP and AMP from the environment, providing new insights into the energy metabolism of Toxoplasma.

High infectivity and unique genomic sequence characteristics of Cryptosporidium parvum in China.

Zoonotic Cryptosporidium parvum infections are mainly caused by IIa and IId subtypes. As most biological characterizations have been performed on IIa subtypes, the biological and genetic characteristics of IId subtypes in China are not clear. We evaluated the infection and genetic characteristics of IId isolates in interferon-γ-knockout mice using qPCR to quantify oocyst shedding, histological examination to monitor pathological changes and comparative genomic analyses to identify infectivity and virulence-associated differences. Compared with the reference IIa isolate, mice infected with the IId isolates had significantly higher and longer oocyst shedding and lower body weight gain. In addition, the four IId isolates examined differed significantly in infectivity (as indicated by the median infective dose), oocyst shedding duration, and pathogenicity. Comparative genomic analysis indicated that the IId isolates had three more subtelomeric genes than the reference IIa isolate and 5385-5548 nucleotide substitutions, with the hypervariable genes mostly in two blocks on chromosome 1. In contrast, the four IId isolates differed from each other by 77-1,452 nucleotides, with virulence-associated sequence differences mainly in nine genes within a 28-kb block on chromosome 6. These data indicate the newly emerged C. parvum IId subtypes in China have high animal infectivity and unique genomic characteristics.

Decline in Cryptosporidium Infection in Free-Ranging Rhesus Monkeys in a Park After Public Health Interventions.

Nonhuman primates (NHPs) are considered an important source of parasitic zoonoses. A study in 2010 revealed high prevalence of Cryptosporidium spp. in free-ranging rhesus monkeys (Macaca mulatta) in a public park in Guiyang, southwestern China, which called for the control of disease in animals and long-term epidemiological tracking of Cryptosporidium spp. After the initiation of a series of public health interventions, we collected 2,402 fecal samples from monkeys and 123 water samples from lakes in the park on six occasions during 2013-2019. They were analyzed and genotyped for Cryptosporidium spp. using PCR and sequence analyses of the small subunit rRNA gene. The C. hominis and C. parvum identified were further subtyped by sequence analysis of the 60 kDa glycoprotein gene. Compared with the high prevalence of Cryptosporidium spp. in fecal samples (10.9% or 45/411) and water samples (47.8% or 11/23) in 2010, only 18 (0.7%) fecal samples and 3 (2.4%) water samples collected in the present study were positive for Cryptosporidium spp., including C. hominis (n = 9) and C. parvum (n = 12). The former belonged to the NHP-adapted IfA17G2R3 subtype, while the latter mostly belonged to rodent-adapted IIpA9. Therefore, the detection rate and genetic diversity of Cryptosporidium spp. during this study period were much lower than those before the public health interventions, and there was a switch from common occurrence of anthroponotic C. hominis subtypes to sporadic occurrence of NHP-adapted C. hominis and rodent-adapted C. parvum subtypes.

In situ aerobic composting eliminates the toxicity of Ageratina adenophora to maize and converts it into a plant- and soil-friendly organic fertilizer.

Ageratina adenophora has invaded many subtropical and tropical countries and caused tremendous ecological and economic losses. This necessitates a new way to use the debris left after clearing this plant. Therefore, the allelochemicals in fresh and aerobically composted A. adenophora plants (FA and CA, respectively) were compared, and their allelopathy against maize was evaluated. The results showed that CA decreased the allelochemicals (6-hydroxy-5-isopropyl-3,8-dimethyl-4a,5,6,7,8,8a-hexahydronaphthalen-2(1H)-one and 4,7-dimethyl-1-(propan-2-ylidene)-1,4,4a,8a-tetrahydronaphthalene-2,6(1H, 7H)-dione) by over 95% compared to FA. In a seed germination test, CA aqueous solutions improved the seed germination and seedling growth, whereas FA solutions led to opposite results. Chemical fertilizers (CF) plus FA resulted in much lower plant biomass and nutrient uptake than CF in a greenhouse experiment. Compared with CF, CF+CA showed positive effects on maize, soil microbial biomass and diversity and enzyme activities in the field. However, the compositions of the predominant microbes were almost unaffected by the application of CA and CF+CA. These significant findings extended our knowledge regarding the elimination of A. adenophora toxicity against other plants and soil microbes through allelochemical degradation in the composting process. In situ aerobic composting provides a new, simple and economical method to convert A. adenophora into a plant- and soil-friendly organic fertilizer.

Expression and Functional Studies of INS-5, an Insulinase-Like Protein in Cryptosporidium parvum.

The small Cryptosporidium genome (∼9 Mb) has over 20 copies of genes encoding insulinase-like proteases (INS), suggesting that these enzymes may have important biological functions in the pathogen and could be developmentally regulated. In this study, INS-5, a unique member of the INS family in Cryptosporidium parvum, was cloned and expressed in Escherichia coli BL21 (DE3). In addition to the predicted INS-5 of ∼78 kDa, smaller fragments of ∼70, ∼55, and ∼30 kDa were simultaneously generated. After purification through a nickel-nitrilotriacetic acid affinity column, the full recombinant protein obtained was used to prepare polyclonal antibodies. Antibodies raised against INS-5 recognized the recombinant protein and native protein in sporozoite extracts. Further characterization of INS-5 included qRT-PCR assessment of gene expression; immunofluorescence localization of the protein expression in sporozoites, merozoites, and other developmental stages; and neutralization of invasion of C. parvum in vitro. The results obtained indicated that although INS-5 was expressed in sporozoites and merozoites, the high gene expression was from 36 to 48 h of the in vitro culture after invasion. Anti-INS-5 antibodies partially neutralized the invasion (inhibition rate = 38.5%). Results of this study suggest that INS-5 plays some role in the invasion and growth of C. parvum.

Preliminary Characterization of MEDLE-2, a Protein Potentially Involved in the Invasion of Cryptosporidium parvum.

Cryptosporidium spp. are important causes of diarrhea in humans, ruminants, and other mammals. Comparative genomic analysis indicated that genetically related and host-adapted Cryptosporidium species have different numbers of subtelomeric genes encoding the Cryptosporidium-specific MEDLE family of secreted proteins, which could contribute to differences in host specificity. In this study, a Cryptosporidium parvum-specific member of the protein family MEDLE-2 encoded by cgd5_4590 was cloned and expressed in Escherichia coli. Immunofluorescent staining with antibodies generated from the recombinant protein showed the expression of the protein in sporozoites and development stages. In vitro neutralization assay with the antibodies partially blocked the invasion of sporozoites. These results support the potential involvement of MEDLE-2 in the invasion of host cells.

[Effect of biopesticide Pythium oligandrum broth on gummy stem blight in cucumber seedlings, animal health and plant growth].

Objective: Biopesticides are safe and environment friendly. We evaluated the biocontrol effect of Pythium oligandrum broth (POB) and its toxicity to animals and plant growth. Methods: Animal, antagonist, pot, and field experiments with mice, Mycosphaerella melonis, and cucumber seedlings were carried out to study animal toxicity, control of gummy stem blight, plant growth, fruit yield and quality with POB produced from self-isolated P. oligandrum CQ2010. Results: Mouse showed normal weight, appearances, performances and no pathogenic changes in organs and tissues with a large amount of POB supplied by lavage. The inhibition rate of POB against M. melonis was 51.95%, similar to thiophanate methy (800 times dilution) but much higher than chlorothalonil (200 times dilution). Malondialdehyde concentration was reduced whereas activities of peroxidase and superoxide dismutase were stimulated in seedling leaves irrespective of POB supplied before and after pathogenic inoculation. POB also decreased the pathogenic incidence and disease index with relative control efficacy from 54.8% to 64.1%. Thus, POB could alleviate cell membrane damage caused by pathogenic microbes, stimulate physiological reactions related to disease defense, and increase disease-resistant abilities of plants. Moreover, POB increased chlorophyll content, root activity, and uptake of nitrogen, phosphorus and potassium, resulting in growth acceleration, fruit yield increment, and quality improvement. Conclusion: POB is safe to animals and could control gummy stem blight of cucumber seedlings, promote plant growth, increase fruit yield, and improve the qualities.