Leishmaniasis, caused by the intracellular protozoan parasite Leishmania, remains an important neglected tropical infectious disease. Infection may be lethal if untreated. Currently, the available drugs for the disease are limited by high toxicity and drug resistance. There is an urgent need to develop novel anti-leishmanial strategies. Antimicrobial peptides (AMPs) have been described as the first-line immune defense against pathogenic microbes and are being developed as emerging anti-parasitic therapies. In the present study, we showed the anti-leishmanial activity of the synthetic 4-amino acid peptide lysine, aspartic acid, glutamic acid, and leucine (KDEL), the endoplasmic reticulum retention sequence, against Leishmania tarentolae promastigote and amastigote. Different concentrations of KDEL peptides were incubated with promastigotes, MTT viability assay, and promastigote assay were carried out. Macrophages infected with GFP-transfected L. tarentolae promastigotes were incubated with KDEL peptides, and the anti-amastigote activity of the KDEL peptides was measured by fluorescence microscopy. The damage of L. tarentolae was observed by light microscopy and electron microscopy. The cell apoptosis was analyzed using the Annexin V-FITC/PI apoptosis detection kit and mitochondrial membrane potential assay kit and by flow cytometry. Results showed that L. tarentolae was susceptible to KDEL peptides in a dose-dependent manner, and KDEL peptides disrupted the surface membrane integrity and caused cell apoptosis. In our study, we found for the first time an AMP KDEL from Pseudomonas aeruginosa and proved its significant therapeutic potential as a novel anti-leishmanial drug.
Background Infection with Trichomonas vaginalis can lead to cervicitis, urethritis, pelvic inflammatory disease, prostatitis and perinatal complications and increased risk of HIV transmission. Here, we used an RPA-based CRISPR-Cas12a assay system in combination with a lateral flow strip (LFS) (referred to as RPA-CRISPR-Cas12a) to establish a highly sensitive and field-ready assay and evaluated its ability to detect clinical samples. Methods We developed a one-pot CRISPR-Cas12a combined with RPA-based field detection technology for T. vaginalis, chose actin as the target gene to design crRNA and designed RPA primers based on the crRNA binding site. The specificity of the method was demonstrated by detecting genomes from nine pathogens. To improve the usability and visualize the RPA-CRISPR-Cas12a assay results, both fluorescence detection and LFS readouts were devised. Results The RPA-CRISPR-Cas12a assay platform was completed within 60 min and had a maximum detection limit of 1 copy/µl and no cross-reactivity with Candida albicans, Mycoplasma hominis, Neisseria gonorrhoeae, Escherichia coli, Cryptosporidium parvum, G. duodenalis or Toxoplasma gondii after specificity validation. Thirty human vaginal secretions were tested by RPA-CRISPR-Cas12a assays, and the results were read by a fluorescent reporter and LFS biosensors and then compared to the results from nested PCR detection of these samples. Both RPA-CRISPR-Cas12a assays showed 26.7% (8/30) T. vaginalis-positive samples and a consistency of 100% (8/8). The RPA-CRISPR-Cas12a assays had a higher sensitivity than nested PCR (only seven T. vaginalis-positive samples were detected). Conclusions The T. vaginalis RPA-CRISPR-Cas12a assay platform in this study can be used for large-scale field testing and on-site tests without the need for trained technicians or costly ancillary equipment. Graphical abstract
Background: Toxoplasma gondii is capable of infecting a wide range of warm-blooded animals, causing a worldwide epidemic of zoonotic toxoplasmosis. SAG1 protein is expressed at the proliferative tachyzoite stage, whereas MAG1 is expressed at the bradyzoite and tachyzoite stages. These two proteins display protective roles in previous studies, however, there synergetic protective e cacy as a DNA vaccine against toxoplasmosis has not been clari ed.Methods: In this study, we ampli ed TgMAG1 and TgSAG1 genes and inserted into eukaryotic expression vector pcDNA3.1(+). The pcDNA3.1(+)-TgMAG1 (pMAG1), pcDNA3.1(+)-TgSAG1 (pSAG1), pcDNA3.1(+)-TgMAG1-TgSAG1 (pMAG1-SAG1) plasmids were transfected into HEK-293 cells and each protein was veri ed in vitro through western blot. Then, mice were intramuscularly immunized with pMAG1, pSGA1 or pMAG1-SGA1, and anti-T. gondii IgG levels were measured in the serum. Cytokines levels of IL-4, IL-10 and IFN-γ in mice splenocytes culturing supernatants were measured using commercial ELISA kits. Immunized mice were challenged with T. gondii tachyzoites with lethal doses followed by determination of mortality, whereas mice infected with low dose tachyzoites followed by monitoring the parameters of survival rate and parasites burden analysis of brains and livers.Results: The pMAG1, pSGA1 or pMAG1-SGA1 exhibited well reactogenicity with expected band sizes of 17.843 kDa, 15.572 kDa, 33.415 kDa, respectively. The immunized mice triggered signi cantly high levels of anti-T. gondii IgG antibodies in comparison with that in the negative control groups, moreover pMAG1-SGA1 immune achieved the highest levels. The DNA vaccines also led to obvious IFN-γ release from splenocytes culturing supernatants, whereas had no role in the IL-4 and IL-10. The protective e cacy results showed that DNA vaccines immunization prolonged the acute infection mice survival time to 14 d, 16 d, 32 d. Consistently, the liver and brain parasites in each immunization group were signi cantly reduced comparing with PBS control group.Conclusions: This study revealed that bivalent TgMAG1 and TgSAG1 DNA vaccine displayed excellent protective immunity against toxoplasmosis in mice. These data provide new sight into the development of Toxoplasma gondii vaccines.
Background: Toxoplasma gondii is capable of infecting a wide range of warm-blooded animals, causing a worldwide epidemic of zoonotic toxoplasmosis. SAG1 protein is expressed at the proliferative tachyzoite stage, whereas MAG1 is expressed at the bradyzoite and tachyzoite stages. These two proteins display protective roles in previous studies, however, there synergetic protective efficacy as a DNA vaccine against toxoplasmosis has not been clarified.Methods: In this study, we amplified TgMAG1 and TgSAG1 genes and inserted into eukaryotic expression vector pcDNA3.1(+). The pcDNA3.1(+)-TgMAG1 (pMAG1), pcDNA3.1(+)-TgSAG1 (pSAG1), pcDNA3.1(+)-TgMAG1-TgSAG1 (pMAG1-SAG1) plasmids were transfected into HEK-293 cells and each protein was verified in vitro through western blot. Then, mice were intramuscularly immunized with pMAG1, pSGA1 or pMAG1-SGA1, and anti-T. gondii IgG levels were measured in the serum. Cytokines levels of IL-4, IL-10 and IFN-γ in mice splenocytes culturing supernatants were measured using commercial ELISA kits. Immunized mice were challenged with T. gondii tachyzoites with lethal doses followed by determination of mortality, whereas mice infected with low dose tachyzoites followed by monitoring the parameters of survival rate and parasites burden analysis of brains and livers.Results: The pMAG1, pSGA1 or pMAG1-SGA1 exhibited well reactogenicity with expected band sizes of 17.843 kDa, 15.572 kDa, 33.415 kDa, respectively. The immunized mice triggered significantly high levels of anti-T. gondii IgG antibodies in comparison with that in the negative control groups, moreover pMAG1-SGA1 immune achieved the highest levels. The DNA vaccines also led to obvious IFN-γ release from splenocytes culturing supernatants, whereas had no role in the IL-4 and IL-10. The protective efficacy results showed that DNA vaccines immunization prolonged the acute infection mice survival time to 14 d, 16 d, 32 d. Consistently, the liver and brain parasites in each immunization group were significantly reduced comparing with PBS control group.Conclusions: This study revealed that bivalent TgMAG1 and TgSAG1 DNA vaccine displayed excellent protective immunity against toxoplasmosis in mice. These data provide new sight into the development of Toxoplasma gondii vaccines.
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