Biology subjects

Clemente, M.

Publications and source records attributed to Clemente, M..

6 recordsLinked to original sources

bioRxiv2025

Kazal-type serine protease inhibitors from Arabidopsis thaliana and Toxoplasma gondii exhibit antimicrobial activity against plant pathogens

Kazal-type serine protease inhibitors (KPIs) interfere with microbial proteases and have been associated with antimicrobial activity, yet their specific role in plant protection remains poorly understood. In this work, we evaluated the antimicrobial potential of recombinant Arabidopsis thaliana KPI (rAtKPI-1T), recombinant Toxoplasma gondii KPI (rTgPI-1), and two truncated versions of rTgPI-1 (rTgPI-1NT and rTgPI-1CT). Both rAtKPI-1T and rTgPI-1 exhibited inhibitory effects against Pseudomonas syringae DC3000, P. syringae (AvRpm1), and P. viridiflava in a concentration-dependent manner, with significant inhibition at 3.5 M. The lower MIC50 values obtained for rTgPI-1 and its truncated forms compared to rAtKPI-1T suggest a higher antibacterial potency. Binding and immunofluorescence assays further revealed that rAtKPI-1T, rTgPI-1, and rTgPI-1NT associated with bacterial surfaces, while rTgPI-1CT displayed weaker or transient interactions. In addition to their antibacterial activity, rTgPI-1NT and rTgPI-1CT also inhibited the germination of Botrytis cinerea conidia. Both truncated proteins significantly reduced germination after 6 and 9 hours of incubation, with rTgPI-1CT exerting a markedly stronger antifungal effect than rTgPI-1NT. These findings suggest that specific Kazal-type domains, particularly those in the C-terminal region, could play a critical role in suppressing early infection processes of necrotrophic fungi. Overall, this study demonstrates that rKPIs display distinct and complementary antimicrobial profiles mediated by microbial binding and protease inhibition. The contrasting activities of rTgPI-1NT and rTgPI-1CT highlight the value of domain-level analysis to discern functional contributions within multidomain KPIs. These results expand current knowledge on plant-derived protease inhibitors and underscore their potential as biotechnological tools for sustainable crop protection.

microbiology↗

bioRxiv2025

Different kinetics of humoral response against individual antigens in the ovine model of Toxoplasma gondii infection and its pathological association

BackgroundToxoplasma gondii is a globally distributed protozoan parasite with major implications for human and animal health. In sheep, infection can cause reproductive failure and represents an important source of zoonotic transmission through contaminated meat. While cell-mediated immunity is the main protective mechanism, the contribution of humoral responses, particularly against individual recombinant antigens (rAGs), remains poorly characterized in ovine toxoplasmosis. MethodsWe analyzed the humoral response of lambs experimentally infected with two reference strains of T. gondii (RH and Me49) using different inoculation doses. Recombinant antigens rGra4Gra7, rGra8, rRop2, rCST9, and rBCLA were tested by IgG-ELISA to evaluate their kinetics during infection. Histopathological analyses of brain tissues were performed to assess lesion severity and cyst presence. Additionally, sera from naturally infected sheep were evaluated to test diagnostic performance. ResultsAn early IgG response against rGra4Gra7 and rGra8 was detected, consistent with markers of acute infections. Anti-rBCLA, rRop2, and rCST9 IgG responses were associated with the chronic phase and correlated with cyst burden. Notably, stronger anti-rGra4Gra7 IgG responses tended to associate with lower lesion scores, suggesting a potential protective role. In naturally infected sheep, IgG-ELISA against rGra4Gra7 and rGra8 demonstrated the highest diagnostic performance, outperforming total lysate antigen (TLA)-based assays. ConclusionOur findings highlight distinct kinetics of specific antibody responses against selected T. gondii antigens in sheep, suggesting their value as markers of infection stage and pathology. rGra4Gra7 and rGra8 appear particularly promising both as diagnostic candidates and for exploring mechanisms of humoral modulation in ovine toxoplasmosis.

pathology↗

bioRxiv2025

Optimization of a Cannabis sativa micropropagation protocol (chemotype III) to preserve its cannabinoid profile

A micropropagation protocol was developed using axillary buds of the Charlies Dream variety of Cannabis sativa (chemotype III, THC<<CBD), under the hypothesis that clonal propagation preserves the CBD/{Delta}9-THC profile between donor and micropropagated plants. The effects of plant growth regulators BAP and TDZ (0.5, 1, 2.5 M) in MS medium with or without vitamin supplementation (MS vs. MSS) were determined. The progress in the growth of the axillary buds was evaluated by analyzing the shoot length, the number of shoots per explant, and the foliar area of the shoots. The role of naphthaleneacetic acid (NAA) and indolebutyric acid (IBA) in the rooting process was also evaluated in vitro and ex vitro. Resins were extracted from dried flowers of donor and micropropagated plants using cold ethanol treatment. The CBD and {Delta}9-THC contents were analyzed by GC/MS. The results show that vitamin supplementation in the MS medium did not improve any of the growth parameters evaluated. However, significant differences were observed when comparing TDZ and BAP. At 15 days post-initiation of micropropagation (dpim) from axillary buds, the explants treated with 0.5 and 1 M TDZ showed a greater number of shoots per explant and larger foliar area, compared to those treated with any concentration of BAP. However, by 30 dpim, explants treated with BAP showed an improved performance, reaching shoot numbers and foliar area values similar to those observed with TDZ. It is worth mentioning that no differences were observed in the shoot length at all concentrations analyzed for both hormones. Likewise, the explants grown in 2.5 M TDZ showed greater callus production and vitrification. The effects of supplementation with the hormone gibberellin (7 {micro}M) or exposure of the shoots to red light were also evaluated. The results indicated that combining these treatments with BAP at 0.5 and 1 {micro}M significantly enhanced shoot elongation compared to combinations with TDZ at the same concentrations. On the other hand, in vitro rooting was unsuccessful. However, direct rooting in soil was observed, and root length was further enhanced when shoots were pretreated with 2.5 M IBA. The CBD/{Delta}9-THC ratio remained stable between donor and micropropagated plants, supporting the development of standardized micropropagation protocols. Our results confirm that clonal micropropagation is a reliable method to preserve cannabinoid profiles in C. sativa.

plant biology↗

bioRxiv2024

Immunization with Plant-based Vaccine Expressing Toxoplasma gondii SAG1 Fused to Plant HSP90 Elicits Protective Immune Response in Lambs

Toxoplasma gondii is a protozoan parasite causing toxoplasmosis, a principal concern for public health and livestock industries. Effective vaccination strategies are crucial for controlling toxoplasmosis, particularly in the lamb, which are significant reservoirs of T. gondii. In addition, ovine toxoplasmosis also causes economic losses due to abortions and reproductive complications. In this study, we evaluated two immunization strategies to elucidate the immune protective potential of SAG1 fused to the plant Hsp90 adjuvant against experimental toxoplasmosis in lambs. We performed an oral administration of AtHsp81.2-SAG1HC-infiltrated fresh leaves homogenate (Plant Vaccine) and a subcutaneous administration of recombinant NbHsp90.3-SAG1HC produced in Escherichia coli (Recombinant Vaccine). Our results showed that only the Recombinant Vaccine significantly increased anti-rSAG1 total IgG values. In addition, only lambs immunized with the Plant Vaccine showed a significant increase in IFN-{gamma} serum levels after the experimental infection (evaluated 8 days post-challenge). On the other hand, we also observed a statistically significant decrease in histopathological lesions (injury score) in challenged vaccinated lambs compared to challenged but not vaccinated animals (vehicle and control groups). Finally, to evaluate T. gondii infection, we choose the chimera rGra4-Gra7 as an acute phase protein marker. All lambs from the control and vehicle groups showed higher rates of serological reactivity than lambs from the vaccinated groups, concurrently with increased severity of lesions. These results suggest that both the plant-based and recombinant vaccines are promising candidates for controlling T. gondii infection in lambs, with potential benefits for enhancing public health and animal welfare.

molecular biology↗

bioRxiv2023

Safe plant Hsp90 adjuvants elicit an effective immune response against SARS-CoV2 derived RBD antigen

To better understand the role of pHsp90 adjuvant in immune response modulation, we proposed the use of the Receptor Binding Domain (RBD) of the Spike protein of SARS-CoV2, the principal candidate in the design of subunit vaccines. We evaluated the humoral and cellular immune responses against RBD through the strategy "protein mixture" (Adjuvant + Antigen). The rRBD adjuvanted with rAtHsp81.2 group showed a higher increase of the anti-rRBD IgG1, while the rRBD adjuvanted with rNbHsp90.3 group showed a significant increase of anti-rRBD IgG2b/2a. These results were consistent with the cellular immune response analysis. Spleen cell cultures from rRBD+rNbHsp90.3-immunized mice showed significantly increased IFN-{gamma} production. In contrast, spleen cell cultures from rRBD+rAtHsp81.2-immunized mice showed significant increased IL-4 levels. Finally, vaccines adjuvanted with rNbHsp90.3 induced higher neutralizing antibody responses compared to those adjuvanted with rAtHsp81.2. To know whether both chaperones must form complexes to generate an effective immune response, we performed co-immunoprecipitation (co-IP) assays. The results indicated that the greater neutralizing capacity observed in the rRBD adjuvanted with rNbHsp90.3 group would be given by the rRBD-rNbHsp90.3 interaction rather than by the quality of the immune response triggered by the adjuvants. These results, together with our previous results, provide a comparative benchmark of these two novel and safe vaccine adjuvants for their capacity to stimulate immunity to a subunit vaccine, demonstrating the capacity of adjuvanted SARS-CoV2 subunit vaccines. Furthermore, these results revealed differences in the ability to modulate the immune response between these two pHsp90s, highlighting the importance of adjuvant selection for future rational vaccine and adjuvant design.

molecular biology↗

bioRxiv2018

Non-intravenous, carbapenem-sparing antibiotics for the treatment of bacteremia due to ESBL or AMP-C β-lactamase: A propensity score study

IntroductionCarbapenems are considered the treatment of choice for extended-spectrum {beta}-lactamase (ESBL) or Amp-C {beta}-lactamase-producing Enterobacteriaceae bacteremia. Data on the effectiveness of non-intravenous carbapenem-sparing antibiotic options are limited.\n\nObjectiveTo compare the 30 day-mortality and clinical failures associated with the use of carbapenems vs an alternative non-intravenous antibiotic for the definitive treatment of ESBL/Amp-C positive Enterobacteriaceae bacteremia.\n\nMethodsThis is a 12-year retrospective study (2004 - 2015) including all patients with bacteremia due to ESBL/Amp-C-producing Enterobacteriaceae. Given the lack of randomization of the initial therapies, a propensity score for receiving carbapenems was calculated.\n\nResultsThere were 1115 patients with a first episode of bacteremia due to E. coli or K. pneumoniae, of which 123 were ESBL/Amp C-positive (11%). There were 101 eligible patients: 59 in the carbapenem group and 42 in the alternative treatment group (cotrimoxazole 59.5%, quinolones 21.4%). The most frequent sources of infection were urinary (63%) and biliary (15%). Compared to the carbapenem group, patients treated with the alternative regimen had a shorter hospital stay (median [IQR]: 7 days [5-10] vs 12 days [9-18], p<0,001). The use of an alternative non-IV treatment did not increase mortality (OR 27; 95% CI 0.05-1.61; p=.15). After controlling for confounding factors with the propensity score, the adjusted OR of carbapenem treatment was 4.95; 95% CI (0.9426.01, p=.059).\n\nConclusionAlternative non-IV carbapenem-sparing antibiotics could have a role in the definitive treatment of ESBL/Amp-C-positive Enterobacteriaceae bloodstream infections, allowing a reduction in carbapenem use. The use of cotrimoxazole in this setting has shown favourable results.\n\nSome of the data contained in this article were presented at the 55th Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC) and at the 28th International Congress of Chemotherapy Meeting (ICC), San Diego, USA, 2015.

pathology↗