A uracil auxotroph Toxoplasma gondii exerting immunomodulation to inhibit breast cancer growth and metastasis

Background Breast cancer is the most common cause of cancer-related death among women, and prognosis is especially poor for patients with triple-negative breast cancer (TNBC); therefore, there is an urgent need for new effective therapies. Recent studies have demonstrated that the uracil auxotroph Toxoplasma gondii vaccine displays anti-tumor effects. Here, we examined the immunotherapy effects of an attenuated uracil auxotroph strain of T. gondii against 4T1 murine breast cancer. Methods We constructed a uracil auxotroph T. gondii RH strain via orotidine 5′-monophosphate decarboxylase gene deletion (RH-Δompdc) with CRISPR/Cas9 technology. The strain’s virulence in the T. gondii-infected mice was determined in vitro and in vivo by parasite replication assay, plaque assay, parasite burden detection in mice peritoneal fluids and survival analysis. The immunomodulation ability of the strain was evaluated by cytokine detection. Its anti-tumor effect was evaluated after its in situ inoculation into 4T1 tumors in a mouse model; the tumor volume was measured, and the 4T1 lung metastasis was detected by hematoxylin and eosin and Ki67 antibody staining, and the cytokine levels were measured by an enzyme-linked immunosorbent assay. Results The RH-Δompdc strain proliferated normally when supplemented with uracil, but it was unable to propagate without the addition of uracil and in vivo, which suggested that it was avirulent to the hosts. This mutant showed vaccine characteristics that could induce intense immune responses both in vitro and in vivo by significantly boosting the expression of inflammatory cytokines. Inoculation of RH-Δompdc in situ into the 4T1 tumor inhibited tumor growth, reduced lung metastasis, promoted the survival of the tumor-bearing mice and increased the secretion of Th1 cytokines, including interleukin-12 (IL-12) and interferon-γ (INF-δ), in both the serum and tumor microenvironment (TME). Conclusion Inoculation of the uracil auxotroph RH-Δompdc directly into the 4T1 tumor stimulated anti-infection and anti-tumor immunity in mice, and resulted in inhibition of tumor growth and metastasis, promotion of the survival of the tumor-bearing mice and increased secretion of IL-12 and IFN-γ in both the serum and TME. Our findings suggest that the immunomodulation caused by RH-Δompdc could be a potential anti-tumor strategy. Graphical abstract

Fur4 mediated uracil-scavenging to screen for surface protein regulators

Cell surface membrane proteins perform diverse and critical functions and are spatially and temporally regulated by membrane trafficking pathways. Although perturbations in these pathways underlie many pathologies, our understanding of these pathways at a mechanistic level remains incomplete. Using yeast as a model, we have developed an assay that reports on the surface activity of the Fur4 uracil permease in uracil auxotroph strains grown in the presence of limited uracil. This assay was used to screen a haploid deletion library that identified mutants with both diminished and enhanced comparative growth in restricted uracil media. Factors identified, including various multi-subunit complexes, were enriched for membrane trafficking and transcriptional functions, in addition to various uncharacterised genes. Bioinformatic analysis of expression profiles from many strains lacking identified transcription factors required for efficient uracil-scavenging revealed they control expression of other uracil-scavenging factors, in addition to membrane trafficking genes essential for viability, and therefore not represented in the screen. Finally, we performed a secondary mating factor secretion screen to functionally categorise factors implicated in uracil-scavenging, most of which are conserved throughout evolution.

A Uracil Auxotroph Toxoplasma gondii Exerting Immunomodulation to Inhibit Breast Cancer Growth and Metastasis

Background: Breast cancer is the most common cause of cancer-related death among women, and patients with triple-negative breast cancer (TNBC) have poor prognosis, so it is necessary to develop new effective therapies urgently. Recent studies have demonstrated that uracil auxotroph Toxoplasma gondii vaccine displays antitumor effects. Here, we examined the immunotherapy effects of an attenuated uracil auxotroph strain of T. gondii against 4T1 murine breast cancer.Methods: We constructed a uracil auxotroph strain, the orotidine 5′-monophosphate decarboxylase gene deleted strain of T. gondii (RH-Δompdc) with the CRISPR/Cas9 technology. Its virulence in vitro and in vivo was determined by parasite replication assay, plaque assay, the parasite burden detection in mice peritoneal fluids and the survival analysis of T. gondii infection mice. Its immune modulation ability was evaluated by cytokines detection. Its antitumor effect was evaluated after its in situ inoculation to 4T1 tumors in mouse model, the tumor volume was measured, the 4T1 lung metastasis was detected by H&E and Ki67 antibody staining, and the cytokines levels were measured by ELISA.Results: RH-Δompdc strain could proliferate normally with uracil supplement, however, it was unable to propagate without uracil and in vivo, which implicated that it is avirulent to the hosts. This mutant showed vaccine characteristics that it could induce intense immune responses both in vitro and in vivo by boosting the expression of inflammatory cytokines significantly. RH-Δompdc in situ inoculation to the 4T1 tumors in mice could inhibit the tumor growth, reduce the lung metastasis, promote the survival of the tumor-bearing mice, and also increase the secretion of Th1 cytokines IL-12 and IFN-γ both in serum and in the tumor microenvironment (TME). Conclusion: The uracil auxotroph RH-Δompdc inoculation to the 4T1 tumors stimulated the anti-infection and antitumor immunity in mice, resulted in the inhibition of tumor growth and metastasis, the promotion in survival of the tumor-bearing mice, and the increasing secretion of IL-12 and IFN-γ both in serum and in the TME. Our findings implied that the immunomodulation resulted by RH-Δompdc could be a potential antitumor strategy.

Identification of a 2′-O-Methyluridine Nucleoside Hydrolase Using the Metagenomic Libraries

Ribose methylation is among the most ubiquitous modifications found in RNA. 2′-O-methyluridine is found in rRNA, snRNA, snoRNA and tRNA of Archaea, Bacteria, and Eukaryota. Moreover, 2′-O-methylribonucleosides are promising starting materials for the production of nucleic acid-based drugs. Despite the countless possibilities of practical use for the metabolic enzymes associated with methylated nucleosides, there are very few reports regarding the metabolic fate and enzymes involved in the metabolism of 2′-O-alkyl nucleosides. The presented work focuses on the cellular degradation of 2′-O-methyluridine. A novel enzyme was found using a screening strategy that employs Escherichia coli uracil auxotroph and the metagenomic libraries. A 2′-O-methyluridine hydrolase (RK9NH) has been identified together with an aldolase (RK9DPA)—forming a part of a probable gene cluster that is involved in the degradation of 2′-O-methylated nucleosides. The RK9NH is functional in E. coli uracil auxotroph and in vitro. The RK9NH nucleoside hydrolase could be engineered to enzymatically produce 2′-O-methylated nucleosides that are of great demand as raw materials for production of nucleic acid-based drugs. Moreover, RK9NH nucleoside hydrolase converts 5-fluorouridine, 5-fluoro-2′-deoxyuridine and 5-fluoro-2′-O-methyluridine into 5-fluorouracil, which suggests it could be employed in cancer therapy.

Nonreplicating, Cyst-Defective Type II Toxoplasma gondii Vaccine Strains Stimulate Protective Immunity against Acute and Chronic Infection

Live attenuated vaccine strains, such as type I nonreplicating uracil auxotroph mutants, are highly effective in eliciting lifelong immunity to virulent acute infection byToxoplasma gondii. However, it is currently unknown whether vaccine-elicited immunity can provide protection against acute infection and also prevent chronic infection. To address this problem, we developed nonreverting, nonreplicating, live attenuated uracil auxotroph vaccine strains in the type II Δku80genetic background by targeting the deletion of the orotidine 5′-monophosphate decarboxylase (OMPDC) and uridine phosphorylase (UP) genes. Deletion ofOMPDCinduced a severe uracil auxotrophy with loss of replication, loss of virulence in mice, and loss of the ability to develop cysts and chronic infection. Vaccination of mice using type II Δku80Δompdcmutants stimulated a fully protective CD8+T cell-dependent immunity that prevented acute infection by type I and type II strains ofT. gondii, and this vaccination also severely reduced or prevented cyst formation after type II challenge infection. Nonreverting, nonreplicating, and non-cyst-forming Δompdcmutants provide new tools to examine protective immune responses elicited by vaccination with a live attenuated type II vaccine.

Development of pyrF-Based Genetic System for Targeted Gene Deletion in Clostridium thermocellum and Creation of a pta Mutant

ABSTRACT We report development of a genetic system for making targeted gene knockouts in Clostridium thermocellum, a thermophilic anaerobic bacterium that rapidly solubilizes cellulose. A toxic uracil analog, 5-fluoroorotic acid (5-FOA), was used to select for deletion of the pyrF gene. The ΔpyrF strain is a uracil auxotroph that could be restored to a prototroph via ectopic expression of pyrF from a plasmid, providing a positive genetic selection. Furthermore, 5-FOA was used to select against plasmid-expressed pyrF, creating a negative selection for plasmid loss. This technology was used to delete a gene involved in organic acid production, namely pta, which encodes the enzyme phosphotransacetylase. The C. thermocellum Δpta strain did not produce acetate. These results are the first examples of targeted homologous recombination and metabolic engineering in C. thermocellum, a microbe that holds an exciting and promising future in the biofuel industry and development of sustainable energy resources.

Characterization of the Exoglucanase-Encoding Gene PaEXG2 and Study of Its Role in the Biocontrol Activity of Pichia anomala Strain K

The PaEXG2 gene, encoding an exo-β-1,3-glucanase, was isolated from the biocontrol agent Pichia anomala strain K. PaEXG2 has the capacity for coding an acidic protein of 427 amino acids with a predicted molecular weight of 45.7 kDa, a calculated pI of 4.7, and one potential N-glycosylation site. PaEXG2 was disrupted by the insertion of the URA3 marker gene, encoding orotidine monophosphate decarboxylase in strain KU1, a uracil auxotroph derived from strain K. Strain KU1 showed inferior biocontrol activity and colonization of wounds on apples, compared to the prototrophic strain. Antagonism and colonization were recovered after the restoration of prototrophy by transformation with the URA3 gene. Integrative transformation was shown to be mostly ectopic in strain K descendants (only 4% of integration by homologous recombination). PaEXG2 disruption abolished all detectable extracellular exo-β-1,3-glucanase activity in vitro and in situ but did not affect biocontrol of Botrytis cinerea on wounded apples.