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1.
Int J Mol Sci ; 23(10)2022 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-35628522

RESUMO

The sole currently approved malaria vaccine targets the circumsporozoite protein-the protein that densely coats the surface of sporozoites, the parasite stage deposited in the skin of the mammalian host by infected mosquitoes. However, this vaccine only confers moderate protection against clinical diseases in children, impelling a continuous search for novel candidates. In this work, we studied the importance of the membrane-associated erythrocyte binding-like protein (MAEBL) for infection by Plasmodium sporozoites. Using transgenic parasites and live imaging in mice, we show that the absence of MAEBL reduces Plasmodium berghei hemolymph sporozoite infectivity to mice. Moreover, we found that maebl knockout (maebl-) sporozoites display reduced adhesion, including to cultured hepatocytes, which could contribute to the defects in multiple biological processes, such as in gliding motility, hepatocyte wounding, and invasion. The maebl- defective phenotypes in mosquito salivary gland and liver infection were reverted by genetic complementation. Using a parasite line expressing a C-terminal myc-tagged MAEBL, we found that MAEBL levels peak in midgut and hemolymph parasites but drop after sporozoite entry into the salivary glands, where the labeling was found to be heterogeneous among sporozoites. MAEBL was found associated, not only with micronemes, but also with the surface of mature sporozoites. Overall, our data provide further insight into the role of MAEBL in sporozoite infectivity and may contribute to the design of future immune interventions.


Assuntos
Plasmodium berghei , Proteínas de Protozoários , Receptores de Superfície Celular , Animais , Culicidae , Eritrócitos/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Plasmodium berghei/genética , Plasmodium berghei/patogenicidade , Proteínas de Protozoários/metabolismo , Receptores de Superfície Celular/metabolismo , Esporozoítos/metabolismo
2.
Methods ; 127: 37-44, 2017 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-28522323

RESUMO

Hematogenous dissemination followed by tissue tropism is a characteristic of the infectious process of many pathogens including those transmitted by blood-feeding vectors. After entering into the blood circulation, these pathogens must arrest in the target organ before they infect a specific tissue. Here, we describe a non-invasive method to visualize and quantify the homing of pathogens to the host tissues. By using in vivo bioluminescence imaging we quantify the accumulation of luciferase-expressing parasites in the host organs during the first minutes following their intravascular inoculation in mice. Using this technique we show that in the malarial infection, once in the blood circulation, most of bioluminescent Plasmodium berghei sporozoites, the parasite stage transmitted to the host skin by a mosquito bite, rapidly home to the liver where they invade and develop inside hepatocytes. This homing is specific to this developmental stage since blood stage parasites do not accumulate in the liver, as well as extracellular Trypanosoma brucei bloodstream forms and liver-infecting Leishmania infantum amastigotes. Finally, this method can be used to study the dynamics of tissue tropism of parasites, dissect the molecular and cellular basis of their increased arrest in organs and to evaluate immune interventions designed to block this targeted interaction.


Assuntos
Interações Hospedeiro-Patógeno , Leishmania/fisiologia , Medições Luminescentes/métodos , Plasmodium berghei/fisiologia , Trypanosoma/fisiologia , Animais , Sangue/diagnóstico por imagem , Sangue/parasitologia , Fígado/diagnóstico por imagem , Fígado/parasitologia , Luciferases , Camundongos , Esporozoítos/fisiologia , Tropismo
3.
Parasitology ; 144(10): 1384-1393, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28534448

RESUMO

Canine leishmaniosis (CanL) is a major veterinary concern and a public health issue. Serological data are essential for disease management. Several antigens used in serological assays have specificity related problems preventing relevant seropositivity values establishment. Herein we report significant seropositivity level disparity in a study cohort with 384 dogs from eight countries, for antigens traditionally used in CanL - soluble promastigote Leishmania antigens (SPLA) and K39 recombinant protein (rK39): 43·8 and 2·9% for SPLA and rK39, respectively. To better understand the reasons for this disparity, CanL-associated serological response was characterized using, for complement serological evaluation, a ubiquitous antigen - soluble Escherichia coli antigens (SECAs). Using cohorts of CanL dogs and dogs without clinical evidences of CanL from non-endemic regions of Portugal, the serological response of CanL animals followed specific trend of seropositivity rK39 > SPLA > SECA absent in non-diseased animals. Using receiver operating characteristic curve analysis, these characteristic trends were converted in ratios, SPLA/SECA, rK39/SECA and rK39/SPLA, that presented high predictive for discriminating the CanL cohort that was potentiated when applied in a scoring system involving positivity to four out of five predictors (rK39, SPLA, SPLA/SECA, rK39/SECA and rK39/SPLA). In fact, this approach discriminated CanL with similar sensitivity/specificity as reference antigens, diminishing seropositivity in European cohort to 1·8%. Ultimately, non-related antigens like SECA and seropositivity ratios between antigens enable different perspectives into serological data focusing on the search of characteristic serological signatures and not simple absolute serology values contributing to comprehensive serological status characterization.


Assuntos
Adenosina Trifosfatases/sangue , Antígenos de Bactérias/sangue , Antígenos de Protozoários/sangue , Proteínas de Bactérias/sangue , Doenças do Cão/diagnóstico , Escherichia coli/imunologia , Leishmania infantum/isolamento & purificação , Leishmaniose Visceral/veterinária , Canais de Translocação SEC/sangue , Animais , Doenças do Cão/imunologia , Cães , Ensaio de Imunoadsorção Enzimática/veterinária , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/imunologia , Portugal , Proteínas de Protozoários/sangue , Proteínas Recombinantes/sangue , Proteínas SecA , Sensibilidade e Especificidade
4.
J Exp Med ; 221(9)2024 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-39141127

RESUMO

HIV-1 antiretroviral therapy is highly effective but fails to eliminate a reservoir of latent proviruses, leading to a requirement for life-long treatment. How the site of integration of authentic intact latent proviruses might impact their own or neighboring gene expression or reservoir dynamics is poorly understood. Here, we report on proviral and neighboring gene transcription at sites of intact latent HIV-1 integration in cultured T cells obtained directly from people living with HIV, as well as engineered primary T cells and cell lines. Proviral gene expression was correlated to the level of endogenous gene expression under resting but not activated conditions. Notably, latent proviral promoters were 100-10,000× less active than in productively infected cells and had little or no measurable impact on neighboring gene expression under resting or activated conditions. Thus, the site of integration has a dominant effect on the transcriptional activity of intact HIV-1 proviruses in the latent reservoir, thereby influencing cytopathic effects and proviral immune evasion.


Assuntos
Infecções por HIV , HIV-1 , Provírus , Transcrição Gênica , Integração Viral , Latência Viral , HIV-1/genética , HIV-1/fisiologia , Humanos , Provírus/genética , Latência Viral/genética , Integração Viral/genética , Infecções por HIV/virologia , Infecções por HIV/genética , Regulação Viral da Expressão Gênica , Regiões Promotoras Genéticas/genética , Linfócitos T CD4-Positivos/virologia , Linfócitos T/virologia , Linfócitos T/imunologia , Linhagem Celular
5.
bioRxiv ; 2024 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-38746186

RESUMO

HIV-1 anti-retroviral therapy is highly effective but fails to eliminate a reservoir of latent proviruses leading to a requirement for life-long treatment. How the site of integration of authentic intact latent proviruses might impact their own or neighboring gene expression or reservoir dynamics is poorly understood. Here we report on proviral and neighboring gene transcription at sites of intact latent HIV-1 integration in cultured T cells obtained directly from people living with HIV, as well as engineered primary T cells and cell lines. Proviral gene expression was correlated to the level of endogenous gene expression under resting but not activated conditions. Notably, latent proviral promoters were 10010,000X less active than in productively infected cells and had little or no measurable impact on neighboring gene expression under resting or activated conditions. Thus, the site of integration has a dominant effect on the transcriptional activity of intact HIV-1 proviruses in the latent reservoir thereby influencing cytopathic effects and proviral immune evasion.

6.
Front Immunol ; 13: 868305, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35669785

RESUMO

Malaria is a major public health concern, as a highly effective human vaccine remains elusive. The efficacy of a subunit vaccine targeting the most abundant protein of the sporozoite surface, the circumsporozoite protein (CSP) has been hindered by difficulties in generating an effective humoral response in both quantity and quality. Using the rodent Plasmodium yoelii model we report here that immunization with CSP adjuvanted with 5'ppp-dsRNA, a RIG-I agonist, confers early and long-lasting sterile protection in mice against stringent sporozoite and mosquito bite challenges. The immunization induced high levels of antibodies, which were functional in targeting and killing the sporozoites and were sustained over time through the accumulation of long-lived plasma cells in the bone marrow. Moreover, 5'ppp-dsRNA-adjuvanted immunization with the CSP of P. falciparum was also significantly protective against challenges using a transgenic PfCSP-expressing P. yoelii parasite. Conversely, using the TLR3 agonist poly(A:U) as adjuvant resulted in a formulation that despite inducing high antibody levels was unable to generate equally functional antibodies and was, consequently, less protective. In conclusion, we demonstrate that using 5'ppp-dsRNA as an adjuvant to vaccines targeting CSP induces effective anti-Plasmodium humoral immunity.


Assuntos
Vacinas Antimaláricas , Adjuvantes Imunológicos/farmacologia , Animais , Anticorpos Antiprotozoários , Imunização , Camundongos , Plasmodium falciparum , Proteínas de Protozoários , Esporozoítos
7.
Sci Rep ; 8(1): 15101, 2018 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-30305687

RESUMO

Plasmodium sporozoites deposited in the skin following a mosquito bite must migrate and invade blood vessels to complete their development in the liver. Once in the bloodstream, sporozoites arrest in the liver sinusoids, but the molecular determinants that mediate this specific homing are not yet genetically defined. Here we investigate the involvement of the thrombospondin-related sporozoite protein (TRSP) in this process using knockout Plasmodium berghei parasites and in vivo bioluminescence imaging in mice. Resorting to a homing assay, trsp knockout sporozoites were found to arrest in the liver similar to control parasites. Moreover, we found no defects in the establishment of infection in mice following inoculation of trsp knockout sporozoites via intravenous and cutaneous injection or mosquito bite. Accordingly, mutant sporozoites were also able to successfully invade hepatocytes in vitro. Altogether, these results suggest TRSP may have a redundant role in the completion of the pre-erythrocytic phase of the malaria parasite. Nonetheless, identifying molecules with paramount roles in this phase could aid in the search for new antigens needed for the design of a protective vaccine against malaria.


Assuntos
Eritrócitos/parasitologia , Plasmodium berghei/metabolismo , Proteínas de Protozoários/metabolismo , Trombospondinas/metabolismo , Animais , Culicidae/parasitologia , Feminino , Técnicas de Inativação de Genes , Células Hep G2 , Hepatócitos/parasitologia , Humanos , Mordeduras e Picadas de Insetos/parasitologia , Fígado/parasitologia , Camundongos Endogâmicos C57BL , Plasmodium berghei/patogenicidade , Esporozoítos/metabolismo , Esporozoítos/patogenicidade
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