Person: Rico San Román, Laura
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First Name
Laura
Last Name
Rico San Román
Affiliation
Universidad Complutense de Madrid
Faculty / Institute
Veterinaria
Department
Sanidad Animal
Area
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Item TaqMan-quantitative PCR assays applied in Neospora caninum knock-outs generated through CRISPR-Cas9 allow to determine the copy numbers of integrated dihydrofolate reductase-thymidylate synthase drug selectable markers(Frontiers in Cellular and Infection Microbiology, 2024) Rico San Román, Laura; Hänggeli, Kai Pascal Alexander; Hemphill, Andrew; Horcajo Iglesias, María Del Pilar; Collantes Fernández, Esther; Ortega Mora, Luis Miguel; Boubaker, GhaliaAs for many other organisms, CRISPR-Cas9 mediated genetic modification has gained increasing importance for the identification of vaccine candidates and drug targets in Neospora caninum, an apicomplexan parasite causing abortion in cattle and neuromuscular disease in dogs. A widely used approach for generating knock-out (KO) strains devoid of virulence factors is the integration of a drug selectable marker such as mutated dihydrofolate reductase-thymidylate synthase (mdhfr-ts) into the target gene, thus preventing the synthesis of respective protein and mediating resistance to pyrimethamine. However, CRISPR-Cas9 mutagenesis is not free of off-target effects, which can lead to integration of multiple mdhfr-ts copies into other sites of the genome. To determine the number of integrated mdhfr-ts in N. caninum, a duplex quantitative TaqMan PCR was developed. For this purpose, primers were designed that amplifies a 106 bp fragment from wild-type (WT) parasites corresponding to the single copy wtdhfrs-ts gene, as well as the mutated mdhfrs-ts present in KO parasites that confers resistance and were used simultaneously with primers amplifying the diagnostic NC5 gene. Thus, the dhfr-ts to NC5 ratio should be approximately 1 in WT parasites, while in KO parasites with a single integrated mdhrf-ts gene this ratio is doubled, and in case of multiple integration events even higher. This approach was applied to the Neospora KO strains NcΔGRA7 and NcΔROP40. For NcΔGRA7, the number of tachyzoites determined by dhfr-ts quantification was twice the number of tachyzoites determined by NC5 quantification, thus indicating that only one mdhfr-ts copy was integrated. The results obtained with the NcΔROP40 strain, however, showed that the number of dhfr-ts copies per genome was substantially higher, indicating that at least three copies of the selectable mdhfr-ts marker were integrated into the genomic DNA during gene editing by CRISPR-Cas9. This duplex TaqMan-qPCR provides a reliable and easy-to-use tool for assessing CRISPR-Cas9 mediated mutagenesis in WT N. caninum strains.Item Immunization with a Multivalent Listeria monocytogenes Vaccine Leads to a Strong Reduction in Vertical Transmission and Cerebral Parasite Burden in Pregnant and Non-Pregnant Mice Infected with Neospora caninum(Vaccines, 2023) Imhof, Dennis; Pownall, William; Hänggeli, Kai Pascal Alexander; Monney, Camille; Rico San Román, Laura; Ortega Mora, Luis Miguel; Forterre, Franck; Oevermann, Anna; Hemphill, AndrewNeospora caninum is an apicomplexan parasite that causes abortion and stillbirth in cattle. We employed the pregnant neosporosis mouse model to investigate the efficacy of a modified version of the attenuated Listeria monocytogenes vaccine vector Lm3Dx_NcSAG1, which expresses the major N. caninum surface antigen SAG1. Multivalent vaccines were generated by the insertion of gra7 and/or rop2 genes into Lm3Dx_NcSAG1, resulting in the double mutants, Lm3Dx_NcSAG1_NcGRA7 and Lm3Dx_NcSAG1_NcROP2, and the triple mutant, Lm3Dx_NcSAG1_NcGRA7_NcROP2. Six experimental groups of female BALB/c mice were inoculated intramuscularly three times at two-week intervals with 1 × 107 CFU of the respective vaccine strains. Seven days post-mating, mice were challenged by the subcutaneous injection of 1 × 105 N. caninum NcSpain-7 tachyzoites. Non-pregnant mice, dams and their offspring were observed daily until day 25 post-partum. Immunization with Lm3Dx_NcSAG1 and Lm3Dx_NcSAG1_NcGRA7_NcROP2 resulted in 70% postnatal pup survival, whereas only 50% and 58% of pups survived in the double mutant-vaccinated groups. Almost all pups had died at the end of the experiment in the infection control. The triple mutant was the most promising vaccine candidate, providing the highest rate of protection against vertical transmission (65%) and CNS infection. Overall, integrating multiple antigens into Lm3Dx_SAG1 resulted in lower vertical transmission and enhanced protection against cerebral infection in dams and in non-pregnant mice.Item Characterization of Neospora caninum virulence factors NcGRA7 and NcROP40 in bovine target cells(Veterinary Parasitology, 2023) Rico San Román, Laura; Amieva, Rafael; Horcajo Iglesias, María Del Pilar; García Sánchez, Marta Eulalia; Pastor Fernández, Iván; Ortega Mora, Luis Miguel; Collantes Fernández, EstherBovine neosporosis is one of the major causes of reproductive failure in cattle worldwide, and differences in virulence between isolates have been widely shown. However, the molecular basis and mechanisms underlying virulence in Neospora caninum are mostly unknown. Recently, we demonstrated the involvement of NcGRA7 and NcROP40 in the virulence of N. caninum in a pregnant murine model using single knockout mutants in these genes generated by CRISR/Cas9 technology. In this study, the role of these proteins was investigated in two in vitro models using bovine target cells: trophoblast (F3 cell line) and monocyte-derived macrophages (BoMØ). The proliferation capacity of the single knockout mutant parasites was compared to the wild-type strain, the Nc-Spain7 isolate, using both cell populations. For the bovine trophoblast, no differences were observed in the growth of the defective parasites compared to the wild-type strain, neither in the proliferation kinetics nor in the competition assay. However, in naïve BoMØ, a significant decrease in the proliferation capacity of the mutant parasites was observed from 48 h pi onwards. Stimulation of BoMØ with IFN-γ showed a similar inhibition of tachyzoite growth in defective and wild-type strains in a dose-dependent manner. Finally, BoMØ infected with knockout parasites showed higher expression levels of TLR3, which is involved in pathogen recognition. These results suggest that NcGRA7 and NcROP40 may be involved in the manipulation of innate immune defense mechanisms against neosporosis and confirm the usefulness of the BoMØ model for the evaluation of N. caninum virulence mechanisms. However, the specific functions of these proteins remain unknown, opening the way for future research.Item NcGRA7 and NcROP40 Play a Role in the Virulence of Neospora caninum in a Pregnant Mouse Model(Pathogens, 2022) Rico San Román, Laura; Amieva, Rafael; Regidor Cerrillo, Javier; García Sánchez, Marta Eulalia; Collantes Fernández, Esther; Pastor Fernández, Iván; Saeij, Jeroen P. J.; Ortega Mora, Luis Miguel; Horcajo Iglesias, María Del PilarThe intraspecific variability among Neospora caninum isolates in their in vitro behaviour and in vivo virulence has been widely studied. In particular, transcriptomic and proteomic analyses have shown a higher expression/abundance of specific genes/proteins in high-virulence isolates. Consequently, the dense granule protein NcGRA7 and the rhoptry protein NcROP40 were proposed as potential virulence factors. The objective of this study was to characterize the role of these proteins using CRISPR/Cas9 knockout (KO) parasites in a well-established pregnant BALB/c mouse model of N. caninum infection at midgestation. The deletion of NcGRA7 and NcROP40 was associated with a reduction of virulence, as infected dams displayed milder clinical signs, lower parasite burdens in the brain, and reduced mortality rates compared to those infected with the wild-type parasite (Nc-Spain7). Specifically, those infected with the NcGRA7 KO parasites displayed significantly milder clinical signs and a lower brain parasite burden. The median survival time of the pups from dams infected with the two KO parasites was significantly increased, but differences in neonatal mortality rates were not detected. Overall, the present study indicates that the disruption of NcGRA7 considerably impairs virulence in mice, while the impact of NcROP40 deletion was more modest. Further research is needed to understand the role of these virulence factors during N. caninum infection.Item Whole-transcriptome analysis reveals virulence-specific pathogen−host interactions at the placenta in bovine neosporosis(Frontiers in Immunology, 2023) Horcajo Iglesias, María Del Pilar; Coronado, Montserrat; Pastor Fernández, Iván; Collantes Fernández, Esther; Rico San Román, Laura; Reyes Palomares, Armando Adolfo; Ortega Mora, Luis MiguelResearch on bovine neosporosis has achieved relevant milestones, but the mechanisms underlying the occurrence of foetal death or protection against foetal death remain unclear. In a recent study, placentas from heifers challenged with the high-virulence isolate Nc-Spain7 exhibited focal necrosis and inflammatory infiltrates as soon as 10 days post-infection (dpi), although parasite detection was minimal. These lesions were more frequent at 20 dpi, coinciding with higher rates of parasite detection and the occurrence of foetal death in some animals. In contrast, such lesions were not observed in placentas from animals infected with the low-virulence isolate Nc-Spain1H, where the parasite was detected only in placenta from one animal at 20 dpi. This work aimed to study which mechanisms are triggered in the placentas (caruncles and cotyledons) of these pregnant heifers at early stages of infection (10 and 20 dpi) through whole-transcriptome analysis. In caruncles, infection with the high-virulence isolate provoked a strong proinflammatory response at 10 dpi. This effect was not observed in heifers infected with the low-virulence isolate, where IL-6/JAK/STAT3 signalling and TNF-alpha signalling via NF-κB pathways were down-regulated. Interestingly, the expression of E2F target genes, related to restraining the inflammatory response, was higher in these animals. At 20 dpi, more pronounced proinflammatory gene signatures were detectable in heifers infected with the high-virulence isolate, being more intense in heifers carrying dead fetuses. However, the low-virulence isolate continued without activating the proinflammatory response. In cotyledons, the response to infection with the high-virulence isolate was similar to that observed in caruncles; however, the low-virulence isolate induced mild proinflammatory signals at 20 dpi. Finally, a deconvolutional analysis of gene signatures from both placentome tissues revealed a markedly higher fraction of activated natural killers, M1 macrophages and CD8+ T cells for the high-virulence isolate. Therefore, our transcriptomic analysis supports the hypothesis that an intense immune response probably triggered by parasite multiplication could be a key contributor to abortion. Further studies are required to determine the parasite effectors that govern the distinct interactions of high- and low-virulence isolates with the host, which could help elucidate the molecular processes underlying the pathogenesis of neosporosis in cattle.