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1.
Med Microbiol Immunol ; 213(1): 23, 2024 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-39441372

RESUMO

Giardia duodenalis, an important zoonotic protozoan parasite, adheres to host intestinal epithelial cells (IECs) via the ventral disc and causes giardiasis characterized mainly by diarrhea. To date, it remains elusive how excretory-secretory products (ESPs) of Giardia enter IECs and how the cells respond to the entry. Herein, we initially demonstrated that ESPs evoked IEC endocytosis in vitro. We indicated that ESPs contributed vitally in triggering intrinsic apoptosis, pro-inflammatory responses, tight junction (TJ) protein expressional changes, and autophagy in IECs. Endocytosis was further proven to be implicated in those ESPs-triggered IEC responses. Ten predicted virulent excretory-secretory proteins of G. duodenalis were investigated for their capability to activate clathrin/caveolin-mediated endocytosis (CME/CavME) in IECs. Pyridoxamine 5'-phosphate oxidase (PNPO) was confirmed to be an important contributor. PNPO was subsequently verified as a vital promoter in the induction of giardiasis-related IEC apoptosis, inflammation, and TJ protein downregulation. Most importantly, this process seemed to be involved majorly in PNPO-evoked CME pathway, rather than CavME. Collectively, this study identified Giardia ESPs, notably PNPO, as potentially important pathogenic factors during noninvasive infection. It was also noteworthy that ESPs-evoked endocytosis might play a role in triggering giardiasis-inducing cellular regulation. These findings would deepen our understanding about the role of ESPs, notably PNPO, in the pathogenesis of giardiasis and the potential attributed endocytosis mechanism.


Assuntos
Apoptose , Endocitose , Células Epiteliais , Giardia lamblia , Giardia lamblia/patogenicidade , Giardia lamblia/metabolismo , Humanos , Células Epiteliais/parasitologia , Células Epiteliais/metabolismo , Linhagem Celular , Proteínas de Protozoários/metabolismo , Giardíase/parasitologia , Autofagia , Clatrina/metabolismo
2.
Vet Parasitol ; 331: 110296, 2024 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-39217762

RESUMO

Coccidiosis is an important parasitic disease that has serious adverse effects on the global poultry industry. The mechanism by which the pathogenic factors of Eimeria tenella damage host cells is unknown. Some kinases from the rhoptry compartment can regulate apoptosis of host cells. This study focused on revealing the role and critical nodes of E. tenella rhoptry protein (EtROP) 38 in controlling the apoptosis of host cells via the P38 mitogen-activated protein kinase (MAPK) signaling pathway. The cells were treated with EtROP38 protein, siRNA p38MAPK, or both. The rate of infection, apoptosis, and the dynamic changes in the expression and activation of key factor genes of the P38MAPK signaling pathway in host cells infected with E. tenella were measured. The results showed that the addition of EtROP38 and/or knockdown of the host cells p38 gene reduced the apoptosis rate of cecal epithelial cells (CECS), decreased the mRNA expressions of p38, p53, c-myc, c-fos, and c-jun and increased the expression of p65, decreased the protein expressions of c-myc, c-fos, and c-jun, decreased the p38 protein phosphorylation level, and increased the p65 protein phosphorylation level in CECS. When E. tenella was inoculated for 4-96 h, the addition of Et ROP38 and/or host cell p38 knockdown both increased the infection rate of host cells, and this effect was more pronounced with the addition of EtROP38 with the host cell p38 knockdown. These observations indicate that E. tenella can inhibits the activation of the p38MAPK signaling pathway in host cells via EtROP38, which suppresses apoptosis in host cells.


Assuntos
Apoptose , Galinhas , Eimeria tenella , Proteínas Quinases p38 Ativadas por Mitógeno , Eimeria tenella/fisiologia , Animais , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Doenças das Aves Domésticas/parasitologia , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/genética , Coccidiose/parasitologia , Coccidiose/veterinária , Sistema de Sinalização das MAP Quinases , Células Epiteliais/parasitologia , Ceco/parasitologia , Transdução de Sinais
3.
Exp Parasitol ; 266: 108839, 2024 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-39265884

RESUMO

Trichomonas vaginalis is a protist parasite of the urogenital tract, responsible for human trichomoniasis, an infection sexually transmitted that affects approximately 156 million people worldwide. This pathology is more evident in females and can cause miscarriages, premature births, and infertility. The disease can also lead to a greater predisposition to HIV infection and cervical and prostate cancer. Metronidazole (MTZ) is a drug that treats human trichomoniasis. The data from studies involving human subjects are limited regarding MTZ use during pregnancy. In addition to the toxicity of the treatment, some isolates have become resistant to MTZ. Therefore, searching for new compounds active for treating trichomoniasis becomes necessary. In the present study, we report results obtained using new phospholipid analogs. Two cardanol-based compounds designated LDT117 and LDT134 were active against T. vaginalis with an IC50 of 4.58 and 10.24 µM, respectively. These compounds were not toxic to epithelial cells in culture. Scanning electron microscopy observations revealed a rounding of the cells, a shortening of the flagella, and protrusions on the surface of drug-treated cells. Transmission electron microscopy of treated cells revealed alterations in the plasma membrane with formations of blebs, protrusions, depressions, and vacuoles with myelin figures and vacuolization in the cytoplasm after incubation. Furthermore, after treatments with the compounds LDT117 and LDT134, the parasites presented a positive reaction for TUNEL, indicating death by a mechanism like apoptosis. Given the results obtained, further in vivo studies using animal experimental models are necessary to validate that these compounds are effective for treating human trichomoniasis.


Assuntos
Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Fosfolipídeos , Trichomonas vaginalis , Trichomonas vaginalis/efeitos dos fármacos , Trichomonas vaginalis/ultraestrutura , Humanos , Fosfolipídeos/química , Feminino , Concentração Inibidora 50 , Apoptose/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Animais , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/parasitologia , Antiprotozoários/farmacologia , Antiprotozoários/toxicidade , Antiprotozoários/uso terapêutico , Antiprotozoários/química , Metronidazol/farmacologia
4.
Infect Immun ; 92(10): e0029924, 2024 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-39194219

RESUMO

The obligate intracellular parasite Toxoplasma gondii can infect and replicate in any warm-blooded cell tested to date, but much of our knowledge about T. gondii cell biology comes from just one host cell type: human foreskin fibroblasts (HFFs). To expand our knowledge of host-parasite lipid interactions, we studied T. gondii in intestinal epithelial cells, the first site of host-parasite contact following oral infection and the exclusive site of parasite sexual development in feline hosts. We found that highly metabolic Caco-2 cells are permissive to T. gondii growth even when treated with high levels of linoleic acid (LA), a polyunsaturated fatty acid (PUFA) that kills parasites in HFFs. Caco-2 cells appear to sequester LA away from the parasite, preventing membrane disruptions and lipotoxicity that characterize LA-induced parasite death in HFFs. Our work is an important step toward understanding host-parasite interactions in feline intestinal epithelial cells, an understudied but important cell type in the T. gondii life cycle.


Assuntos
Fibroblastos , Interações Hospedeiro-Parasita , Ácido Linoleico , Toxoplasma , Toxoplasma/crescimento & desenvolvimento , Toxoplasma/metabolismo , Ácido Linoleico/metabolismo , Ácido Linoleico/farmacologia , Humanos , Células CACO-2 , Animais , Gatos , Fibroblastos/parasitologia , Fibroblastos/metabolismo , Células Epiteliais/parasitologia , Células Epiteliais/metabolismo
5.
mBio ; 15(8): e0172024, 2024 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-38995074

RESUMO

Infection with the apicomplexan parasite Cryptosporidium is a leading cause of diarrheal disease. Cryptosporidiosis is of particular importance in infants and shows a strong association with malnutrition, both as a risk factor and as a consequence. Cryptosporidium invades and replicates within the small intestine epithelial cells. This is a highly dynamic tissue that is developmentally stratified along the villus axis. New cells emerge from a stem cell niche in the crypt and differentiate into mature epithelial cells while moving toward the villus tip, where they are ultimately shed. Here, we studied the impact of Cryptosporidium infection on this dynamic architecture. Tracing DNA synthesis in pulse-chase experiments in vivo, we quantified the genesis and migration of epithelial cells along the villus. We found proliferation and epithelial migration to be elevated in response to Cryptosporidium infection. Infection also resulted in significant cell loss documented by imaging and molecular assays. Consistent with these observations, single-cell RNA sequencing of infected intestines showed a gain of young and a loss of mature cells. Interestingly, enhanced epithelial cell loss was not a function of enhanced apoptosis of infected cells. To the contrary, Cryptosporidium-infected cells were less likely to be apoptotic than bystanders, and experiments in tissue culture demonstrated that infection provided enhanced resistance to chemically induced apoptosis to the host but not bystander cells. Overall, this study suggests that Cryptosporidium may modulate cell apoptosis and documents pronounced changes in tissue homeostasis due to parasite infection, which may contribute to its long-term impact on the developmental and nutritional state of children. IMPORTANCE: The intestine must balance its roles in digestion and nutrient absorption with the maintenance of an effective barrier to colonization and breach by numerous potential pathogens. An important component of this balance is its constant turnover, which is modulated by a gain of cells due to proliferation and loss due to death or extrusion. Here, we report that Cryptosporidium infection changes the dynamics of this process increasing both gain and loss of enterocytes speeding up the villus elevator. This leads to a much more immature epithelium and a reduction of the number of those cells typically found toward the villus apex best equipped to take up key nutrients including carbohydrates and lipids. These changes in the cellular architecture and physiology of the small intestine may be linked to the profound association between cryptosporidiosis and malnutrition.


Assuntos
Criptosporidiose , Cryptosporidium , Células Epiteliais , Criptosporidiose/parasitologia , Animais , Células Epiteliais/parasitologia , Cryptosporidium/genética , Cryptosporidium/fisiologia , Camundongos , Mucosa Intestinal/parasitologia , Apoptose , Humanos , Proliferação de Células , Movimento Celular , Intestino Delgado/parasitologia
6.
Front Immunol ; 15: 1397117, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39040107

RESUMO

Intestinal epithelial cells possess the requisite molecular machinery to initiate cell-intrinsic defensive responses against intracellular pathogens, including intracellular parasites. Interferons(IFNs) have been identified as cornerstones of epithelial cell-intrinsic defense against such pathogens in the gastrointestinal tract. Long non-coding RNAs (lncRNAs) are RNA transcripts (>200 nt) not translated into protein and represent a critical regulatory component of mucosal defense. We report here that lncRNA Nostrill facilitates IFN-γ-stimulated intestinal epithelial cell-intrinsic defense against infection by Cryptosporidium, an important opportunistic pathogen in AIDS patients and a common cause of diarrhea in young children. Nostrill promotes transcription of a panel of genes controlled by IFN-γ through facilitating Stat1 chromatin recruitment and thus, enhances expression of several genes associated with cell-intrinsic defense in intestinal epithelial cells in response to IFN-γ stimulation, including Igtp, iNos, and Gadd45g. Induction of Nostrill enhances IFN-γ-stimulated intestinal epithelial defense against Cryptosporidium infection, which is associated with an enhanced autophagy in intestinal epithelial cells. Our findings reveal that Nostrill enhances the transcription of a set of genes regulated by IFN-γ in intestinal epithelial cells. Moreover, induction of Nostrill facilitates the IFN-γ-mediated epithelial cell-intrinsic defense against cryptosporidial infections.


Assuntos
Criptosporidiose , Interferon gama , Mucosa Intestinal , RNA Longo não Codificante , Interferon gama/metabolismo , RNA Longo não Codificante/genética , Criptosporidiose/imunologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/parasitologia , Mucosa Intestinal/metabolismo , Animais , Humanos , Transcrição Gênica , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Células Epiteliais/parasitologia , Camundongos , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT1/genética , Cryptosporidium/genética , Cryptosporidium/imunologia , Regulação da Expressão Gênica , Autofagia/imunologia
7.
Immunity ; 57(6): 1243-1259.e8, 2024 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-38744291

RESUMO

Epithelial cells secrete chloride to regulate water release at mucosal barriers, supporting both homeostatic hydration and the "weep" response that is critical for type 2 immune defense against parasitic worms (helminths). Epithelial tuft cells in the small intestine sense helminths and release cytokines and lipids to activate type 2 immune cells, but whether they regulate epithelial secretion is unknown. Here, we found that tuft cell activation rapidly induced epithelial chloride secretion in the small intestine. This response required tuft cell sensory functions and tuft cell-derived acetylcholine (ACh), which acted directly on neighboring epithelial cells to stimulate chloride secretion, independent of neurons. Maximal tuft cell-induced chloride secretion coincided with immune restriction of helminths, and clearance was delayed in mice lacking tuft cell-derived ACh, despite normal type 2 inflammation. Thus, we have uncovered an epithelium-intrinsic response unit that uses ACh to couple tuft cell sensing to the secretory defenses of neighboring epithelial cells.


Assuntos
Acetilcolina , Cloretos , Células Epiteliais , Mucosa Intestinal , Animais , Acetilcolina/metabolismo , Camundongos , Cloretos/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/parasitologia , Células Epiteliais/imunologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/parasitologia , Intestino Delgado/imunologia , Intestino Delgado/parasitologia , Intestino Delgado/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células em Tufo
8.
PLoS Negl Trop Dis ; 18(5): e0012212, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38787872

RESUMO

BACKGROUND: Cryptosporidium spp. cause watery diarrhea in humans and animals, especially in infants and neonates. They parasitize the apical surface of the epithelial cells in the intestinal lumen. However, the pathogenesis of Cryptosporidium-induced diarrhea is not fully understood yet. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we infected C57BL/6j neonatal mice with C. parvum IIa and IId subtypes, and examined oocyst burden, pathological changes, and intestinal epithelial permeability during the infection. In addition, transcriptomic analyses were used to study the mechanism of diarrhea induced by the C. parvum IId subtype. The neonatal mice were sensitive to both C. parvum IIa and IId infection, but the IId subtype caused a wide oocyst shedding window and maintained the high oocyst burden in the mice compared with the IIa subtype. In addition, the mice infected with C. parvum IId resulted in severe intestinal damage at the peak of infection, leading to increased permeability of the epithelial barrier. The KEGG, GO and GSEA analyses revealed that the downregulation of adherens junction and cell junction molecules at 11 dpi. Meanwhile, E-cadherin, which is associated with adherens junction, was reduced at the protein level in mouse ileum at peak and late infection. CONCLUSIONS/SIGNIFICANCE: C. parvum IId infection causes more severe pathological damage than C. parvum IIa infection in neonatal mice. Furthermore, the impairment of the epithelial barrier during C. parvum IId infection results from the downregulation of intestinal junction proteins.


Assuntos
Animais Recém-Nascidos , Criptosporidiose , Cryptosporidium parvum , Regulação para Baixo , Mucosa Intestinal , Camundongos Endogâmicos C57BL , Animais , Cryptosporidium parvum/genética , Criptosporidiose/parasitologia , Criptosporidiose/patologia , Camundongos , Mucosa Intestinal/parasitologia , Mucosa Intestinal/patologia , Caderinas/metabolismo , Caderinas/genética , Diarreia/parasitologia , Células Epiteliais/parasitologia , Feminino , Oocistos , Íleo/parasitologia , Íleo/patologia , Modelos Animais de Doenças
9.
Acta Trop ; 256: 107243, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38719083

RESUMO

Opisthorchis viverrini is a pathogenic liver fluke that is known to cause cholangiocarcinoma in chronic infections. The underlying mechanism for this carcinogenesis is believed to be multifactorial, with parasite-derived excretory-secretory (ES) products potentially playing major roles. A recent study on these ES products has identified microRNAs (miRNA) that originate from O. viverrini but their influence on carcinogenesis remains understudied. Hence, we aimed to investigate the role of these miRNAs in the carcinogenesis of O. viverrini-associated cholangiocarcinoma. The mature miRNA sequences were retrieved from published data. Bioinformatics analysis was employed to identify miRNA targets and to identify potentially mitogenic miRNAs. An in vitro study was conducted to test the effects of miRNA on the bile duct epithelial cell lines. The miRNA target prediction analysis revealed that Ov_miRNA_EV_36/ovi-miR-3479a targets cancer-associated pathways. Hence, it was selected and used to assess its effect on the cell proliferation rate of H69 and MMNK-1 cholangiocyte cell lines. The results showed that Ov_miRNA_EV_36/ovi-miR-3479a induced significant cell proliferation in both cell lines when compared to negative controls. These results indicate that Ov_miRNA_EV_36/ovi-miR-3479a may play an essential role in the carcinogenesis of O. viverrini and therefore warrant further investigations.


Assuntos
Proliferação de Células , Colangiocarcinoma , MicroRNAs , Opisthorchis , MicroRNAs/genética , MicroRNAs/metabolismo , Animais , Opisthorchis/genética , Humanos , Colangiocarcinoma/parasitologia , Colangiocarcinoma/genética , Células Epiteliais/parasitologia , Biologia Computacional , Linhagem Celular , Opistorquíase/parasitologia , Opistorquíase/complicações , Carcinogênese/genética , Neoplasias dos Ductos Biliares/parasitologia , Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/patologia
10.
Exp Parasitol ; 262: 108788, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38759775

RESUMO

Giardiasis is a common waterborne zoonotic disease caused by Giardia intestinalis. Upon infection, Giardia releases excretory and secretory products (ESPs) including secreted proteins (SPs) and extracellular vesicles (EVs). Although the interplay between ESPs and intestinal epithelial cells (IECs) has been previously described, the functions of EVs in these interactions and their differences from those of SPs require further exploration. In the present study, EVs and EV-depleted SPs were isolated from Giardia ESPs. Proteomic analyses of isolated SPs and EVs showed 146 and 91 proteins, respectively. Certain unique and enriched proteins have been identified in SPs and EVs. Transcriptome analysis of Caco-2 cells exposed to EVs showed 96 differentially expressed genes (DEGs), with 56 upregulated and 40 downregulated genes. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSEA) indicated that Caco-2 genes related to metabolic processes, the HIF-1 signaling pathway, and the cAMP signaling pathway were affected. This study provides new insights into host-parasite interactions, highlighting the potential significance of EVs on IECs during infections.


Assuntos
Vesículas Extracelulares , Giardia lamblia , Mucosa Intestinal , Humanos , Células CACO-2 , Giardia lamblia/genética , Giardia lamblia/metabolismo , Vesículas Extracelulares/metabolismo , Mucosa Intestinal/parasitologia , Mucosa Intestinal/metabolismo , Perfilação da Expressão Gênica , Células Epiteliais/parasitologia , Células Epiteliais/metabolismo , Proteômica , Interações Hospedeiro-Parasita , Expressão Gênica , Transcriptoma , Giardíase/parasitologia
11.
PLoS Pathog ; 20(5): e1011820, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38718306

RESUMO

The production of IFN-γ is crucial for control of multiple enteric infections, but its impact on intestinal epithelial cells (IEC) is not well understood. Cryptosporidium parasites exclusively infect epithelial cells and the ability of interferons to activate the transcription factor STAT1 in IEC is required for parasite clearance. Here, the use of single cell RNA sequencing to profile IEC during infection revealed an increased proportion of mid-villus enterocytes during infection and induction of IFN-γ-dependent gene signatures that was comparable between uninfected and infected cells. These analyses were complemented by in vivo studies, which demonstrated that IEC expression of the IFN-γ receptor was required for parasite control. Unexpectedly, treatment of Ifng-/- mice with IFN-γ showed the IEC response to this cytokine correlates with a delayed reduction in parasite burden but did not affect parasite development. These data sets provide insight into the impact of IFN-γ on IEC and suggest a model in which IFN-γ signalling to uninfected enterocytes is important for control of Cryptosporidium.


Assuntos
Criptosporidiose , Interferon gama , Mucosa Intestinal , Camundongos Knockout , Animais , Interferon gama/metabolismo , Interferon gama/imunologia , Criptosporidiose/imunologia , Criptosporidiose/parasitologia , Camundongos , Mucosa Intestinal/parasitologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/imunologia , Cryptosporidium , Células Epiteliais/parasitologia , Células Epiteliais/metabolismo , Células Epiteliais/imunologia , Enterócitos/parasitologia , Enterócitos/metabolismo , Enterócitos/imunologia , Camundongos Endogâmicos C57BL , Receptor de Interferon gama , Fator de Transcrição STAT1/metabolismo , Receptores de Interferon/metabolismo , Receptores de Interferon/genética , Transdução de Sinais
12.
J Microbiol Immunol Infect ; 57(4): 638-646, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38744621

RESUMO

INTRODUCTION: Lung cancer is one of the most prevalent malignancies worldwide. Substantial research has illuminated the intricate interplay between microorganisms and human health, revealing their role in disease regulation. Trichomonads is a flagellated protozoan in the human cavity and have been previously identified as a pathogen associated with pneumonia, contributing to tissue chronic inflammation and carcinogenesis. METHODS: Nested polymerase chain reaction methods were employed to scrutinize the prevalence of trichomonads in the bronchovesicular fluid of patients diagnosed with lung cancer. Subsequently, the influence of Trichomonas tenax invasion on lung cancer cells was elucidated through proliferation assays, migration assays, and transcription analysis. RESULTS: Bronchoalveolar fluid samples from lung cancer patients yielded positive nested PCR results for eight out of twenty-seven samples. Seven of these samples were identified as Trichomonas tenax, while one was identified as Tetratrichomonas spp. Our findings revealed a significant upregulation of pathways associated with carcinogenesis, including cellular proliferation, migration, and drug resistance, in response to T. tenax invasion. CONCLUSIONS: This study underscores the importance of recognizing the presence of trichomonads and the influence of T. tenax invasion on host responses to respiratory diseases. The identified pathways implicated in cancer development may pave the way for developing targeted treatment strategies for pulmonary diseases. These findings hold promise for informing and improving the precision of therapeutic interventions in the context of pulmonary ailments.


Assuntos
Neoplasias Pulmonares , Tricomoníase , Trichomonas , Humanos , Neoplasias Pulmonares/parasitologia , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/genética , Trichomonas/genética , Tricomoníase/parasitologia , Células Epiteliais/parasitologia , Líquido da Lavagem Broncoalveolar/parasitologia , Masculino , Movimento Celular , Proliferação de Células , Feminino , Pessoa de Meia-Idade , Idoso , Pulmão/parasitologia , Pulmão/patologia , Reação em Cadeia da Polimerase
13.
Parasit Vectors ; 17(1): 242, 2024 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-38812022

RESUMO

BACKGROUND: Proteases produced by Acanthamoeba spp. play an important role in their virulence and may be the key to understanding Acanthamoeba pathogenesis; thus, increasing attention has been directed towards these proteins. The present study aimed to investigate the lytic factors produced by Acanthamoeba castellanii during the first hours of in vitro co-culture with human corneal epithelial cells (HCECs). METHODS: We used one old and one recent Acanthamoeba isolate, both from patients with severe keratitis, and subsets of these strains with enhanced pathogenic potential induced by sequential passaging over HCEC monolayers. The proteolytic profiles of all strains and substrains were examined using 1D in-gel zymography. RESULTS: We observed the activity of additional proteases (ranging from 33 to 50 kDa) during the early interaction phase between amoebae and HCECs, which were only expressed for a short time. Based on their susceptibilities to protease inhibitors, these proteases were characterized as serine proteases. Protease activities showed a sharp decline after 4 h of co-incubation. Interestingly, the expression of Acanthamoeba mannose-binding protein did not differ between amoebae in monoculture and those in co-culture. Moreover, we observed the activation of matrix metalloproteinases in HCECs after contact with Acanthamoeba. CONCLUSIONS: This study revealed the involvement of two novel serine proteases in Acanthamoeba pathogenesis and suggests a pivotal role of serine proteases during Acanthamoeba-host cell interaction, contributing to cell adhesion and lysis.


Assuntos
Acanthamoeba castellanii , Técnicas de Cocultura , Células Epiteliais , Epitélio Corneano , Peptídeo Hidrolases , Humanos , Acanthamoeba castellanii/enzimologia , Acanthamoeba castellanii/genética , Células Epiteliais/parasitologia , Epitélio Corneano/parasitologia , Epitélio Corneano/enzimologia , Peptídeo Hidrolases/metabolismo , Peptídeo Hidrolases/genética , Ceratite por Acanthamoeba/parasitologia , Serina Proteases/metabolismo , Serina Proteases/genética , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/genética , Virulência
14.
Eur J Protistol ; 94: 126086, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38688045

RESUMO

Acanthamoeba castellanii, a free-living amoeba, can be pathogenic to humans causing a corneal infection named Acanthamoeba keratitis (AK). The mannose-binding protein (MBP) is well established as the major factor related to Acanthamoeba pathogenesis. However, additional factors that participate in the adhesion process and protect trophozoites from cytolytic effects caused by host immune responses remain unknown. Ectonucleotidases, including 3'-nucleotidase/nuclease (3'-NT/NU), a bifunctional enzyme that was recently reported in A. castellanii, are frequently related to the establishment of parasitic infections. We verified that trophozoites can hydrolyze 3'-AMP, and this activity is similar to that observed in other protists. The addition of 3'-AMP increases the adhesion of trophozoites to LLC-MK2 epithelial cells, and this stimulation is completely reversed by DTT, an inhibitor of ecto-3'-nucleotidase activity. Lesions in corneal cells caused by AK infection may elevate the extracellular level of 3'-AMP. We believe that ecto-3'-nucleotidase activity can modulate the host immune response, thus facilitating the establishment of parasitic infection. This activity results from the generation of extracellular adenosine, which can bind to purinergic receptors present in host immune cells. Positive feedback may occur in this cascade of events once the ecto-3'-nucleotidase activity of trophozoites is increased by the adhesion of trophozoites to LLC-MK2 cells.


Assuntos
Acanthamoeba castellanii , Adenosina , Adesão Celular , Trofozoítos , Acanthamoeba castellanii/enzimologia , Adenosina/metabolismo , Linhagem Celular , Animais , Nucleotidases/metabolismo , Células Epiteliais/parasitologia
15.
Parasitol Int ; 101: 102898, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38648880

RESUMO

Abortion caused by the parasite Neospora caninum is an important threat to the livestock industry worldwide. Trophoblasts and caruncular cells play major roles in initiating innate immune responses and controlling parasite infection at the fetal-maternal interface. In the present study, bovine uterine epithelial cells (BUECs) and bovine trophoblastic (BT) cells treated with bovine interferon-gamma (IFN-γ), IFN-alpha (IFN-α) and IFN-tau (IFN-τ) followed by infection with N. caninum were examined by measuring the mRNA expression levels of numerous pregnancy-associated proteins and observing parasite growth to elucidate the host-parasite interaction at the uteroplacental region. N. caninum infection increased the expression of prolactin-related protein 1 (PRP1), pregnancy-associated glycoprotein 1 (PAG1), and cytokines (TNF-α, IL-8 and IL-10) in BUECs and of IL-8 in BT cells. Bovine IFN-γ inhibited IL-8 and TNF-α expression in BUECs and IL-8 in BT cells. In contrast, the expression of the interferon-stimulated gene OAS1 was significantly increased by treatment of the infected BT cells with IFN-γ. However, treatment with bovine IFNs did not inhibit N. caninum growth in either cell line. In conclusion, our results suggest that bovine IFN-γ plays a crucial role in control of pathogenesis in uterus and induction of inflammatory response in the placental region following N. caninum infection, rather than growth inhibition of the parasites.


Assuntos
Coccidiose , Citocinas , Endométrio , Células Epiteliais , Neospora , Proteínas da Gravidez , Trofoblastos , Animais , Bovinos , Neospora/fisiologia , Trofoblastos/parasitologia , Trofoblastos/metabolismo , Feminino , Citocinas/metabolismo , Citocinas/genética , Células Epiteliais/parasitologia , Endométrio/parasitologia , Endométrio/metabolismo , Endométrio/citologia , Coccidiose/parasitologia , Coccidiose/veterinária , Proteínas da Gravidez/genética , Proteínas da Gravidez/farmacologia , Gravidez , Doenças dos Bovinos/parasitologia , Regulação da Expressão Gênica , Interações Hospedeiro-Parasita
16.
Acta Trop ; 249: 107076, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37977254

RESUMO

The research aimed to describe a new Trichinella spiralis dipeptidyl peptidase 1 (TsDPP1) and investigate its functions in the larval invasion of intestinal epithelial cells (IECs). The gene TsDPP1 was successfully replicated and produced in Escherichia coli BL21 (DE3), showing a strong immune response. TsDPP1 was detected in diverse stages of T. spiralis and showed significant expression in the intestine infective larvae (IIL) and adult worms at 6 days post infection, as confirmed by qPCR and Western blot analysis. The primary localization of TsDPP1 in this parasite was observed in cuticles, stichosomes, and embryos by using the indirect immunofluorescence assay (IIFA). rTsDPP1 exhibited the enzymatic function of natural dipeptidyl peptidase and showed specific binding to IECs, and the binding site was found to be localized on cell membrane. Following transfection with dsRNA-TsDPP1, the expression of TsDPP1 mRNA and protein in muscle larvae (ML) were decreased by approximately 63.52 % and 58.68 %, correspondingly. The activity of TsDPP1 in the ML and IIL treated with dsRNA-TsDPP1 was reduced by 42.98 % and 45.07 %, respectively. The acceleration of larval invasion of IECs was observed with rTsDPP1, while the invasion was suppressed by anti-rTsDPP1 serum. The ability of the larvae treated with dsRNA-TsDPP1 to invade IECs was hindered by 31.23 %. In mice infected with dsRNA-treated ML, the intestinal IIL, and adults experienced a significant decrease in worm burdens and a noticeable reduction in adult female length and fecundity compared to the PBS group. These findings indicated that TsDPP1 significantly impedes the invasion, growth, and reproductive capacity of T. spiralis in intestines, suggesting its potential as a target for anti-Trichinella vaccines.


Assuntos
Catepsina C , Proteínas de Helminto , Mucosa Intestinal , Trichinella spiralis , Triquinelose , Animais , Feminino , Camundongos , Células Epiteliais/parasitologia , Proteínas de Helminto/genética , Proteínas de Helminto/metabolismo , Larva/patogenicidade , Camundongos Endogâmicos BALB C , Trichinella spiralis/genética , Trichinella spiralis/patogenicidade , Triquinelose/parasitologia , Catepsina C/genética , Catepsina C/metabolismo , Mucosa Intestinal/parasitologia
17.
PLoS Negl Trop Dis ; 17(12): e0011816, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-38048314

RESUMO

BACKGROUND: Cathepsin L, a lysosomal enzyme, participates in diverse physiological processes. Recombinant Trichinella spiralis cathepsin L domains (rTsCatL2) exhibited natural cysteine protease activity and hydrolyzed host immunoglobulin and extracellular matrix proteins in vitro, but its functions in larval invasion are unknown. The aim of this study was to explore its functions in T. spiralis invasion of the host's intestinal epithelial cells. METHODOLOGY/PRINCIPAL FINDINGS: RNAi significantly suppressed the expression of TsCatL mRNA and protein with TsCatL specific siRNA-302. T. spiralis larval invasion of Caco-2 cells was reduced by 39.87% and 38.36%, respectively, when anti-TsCatL2 serum and siRNA-302 were used. Mice challenged with siRNA-302-treated muscle larvae (ML) exhibited a substantial reduction in intestinal infective larvae, adult worm, and ML burden compared to the PBS group, with reductions of 44.37%, 47.57%, and 57.06%, respectively. The development and fecundity of the females from the mice infected with siRNA-302-treated ML was significantly inhibited. After incubation of rTsCatL2 with Caco-2 cells, immunofluorescence test showed that the rTsCatL2 gradually entered into the cells, altered the localization of cellular tight junction proteins (claudin 1, occludin and zo-1), adhesion junction protein (e-cadherin) and extracellular matrix protein (laminin), and intercellular junctions were lost. Western blot showed a 58.65% reduction in claudin 1 expression in Caco-2 cells treated with rTsCatL2. Co-IP showed that rTsCatL2 interacted with laminin and collagen I but not with claudin 1, e-cadherin, occludin and fibronectin in Caco-2 cells. Moreover, rTsCatL2 disrupted the intestinal epithelial barrier by inducing cellular autophagy. CONCLUSIONS: rTsCatL2 disrupts the intestinal epithelial barrier and facilitates T. spiralis larval invasion.


Assuntos
Catepsina L , Junções Íntimas , Trichinella spiralis , Triquinelose , Animais , Feminino , Humanos , Camundongos , Células CACO-2 , Caderinas/metabolismo , Catepsina L/genética , Catepsina L/metabolismo , Claudina-1/genética , Claudina-1/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/parasitologia , Laminina/genética , Laminina/metabolismo , Larva/parasitologia , Camundongos Endogâmicos BALB C , Ocludina/genética , Ocludina/metabolismo , RNA de Cadeia Dupla , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Junções Íntimas/parasitologia , Junções Íntimas/patologia , Trichinella spiralis/genética
18.
PLoS Negl Trop Dis ; 17(1): e0011016, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36595499

RESUMO

Cytoadherence of Trichomonas vaginalis to human vaginal epithelial cells (hVECs) was previously shown to involve surface lipoglycans and several reputed adhesins on the parasite. Herein, we report some new observations on the host-parasite interactions of adherent versus nonadherent T. vaginalis isolates to hVECs. The binding of the TH17 adherent isolate to hVECs exhibited an initial discrete phase followed by an aggregation phase inhibited by lactose. T. vaginalis infection immediately induced surface expression of galectin-1 and -3, with extracellular amounts in the spent medium initially decreasing and then increasing thereafter over the next 60 min. Extracellular galectin-1 and -3 were detected on the parasite surface but only the TH17 adherent isolate could uptake galectin-3 via the lysosomes. Only the adherent isolate could morphologically transform from the round-up flagellate with numerous transient protrusions into a flat amoeboid form on contact with the solid surface. Cytochalasin D challenge revealed that actin organization was essential to parasite morphogenesis and cytoadherence. Real-time microscopy showed that parasite exploring and anchoring on hVECs via the axostyle may be required for initial cytoadherence. Together, the parasite cytoskeleton behaviors may collaborate with cell surface adhesion molecules for cytoadherence. The nonadherent isolate migrated faster than the adherent isolate, with motility transiently increasing in the presence of hVECs. Meanwhile, differential histone acetylation was detected between the two isolates. Also, TH17 without Mycoplasma symbiosis suggests that symbiont might not determine TH17 innate cytoadherence. Our findings regarding distinctive host-parasite interactions of the isolates may provide novel insights into T. vaginalis infection.


Assuntos
Trichomonas vaginalis , Feminino , Humanos , Galectina 1 , Interações Hospedeiro-Parasita , Adesão Celular , Células Epiteliais/parasitologia , Moléculas de Adesão Celular
19.
Exp Parasitol ; 242: 108376, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36089006

RESUMO

Aminopeptidases P are metalloproteases belonging to the M24 peptidase family. It specifically hydrolyzes the N-terminus of polypeptides free of acidic amino acids, and plays an important role in the nutrition, metabolism and growth of parasites. The aim of this study was to characterize a novel Trichinella spiralis aminopeptidase P (TsAPP) and to investigate its functions in the invasion of T. spiralis. TsAPP contained two domains of creatinase (a creatinase N and creatinase N2) and a domain of peptidase M24C and APP. The complete TsAPP sequence was cloned and expressed in Escherichia coli BL21 cells. The recombinantly produced TsAPP was used to raise polyclonal antibodies that were subsequently used to detect the expression of the protein in the different life stages of T. spiralis. TsAPP was expressed in various T. spiralis stages. TsAPP was primarily localized in the cuticle, stichosome and intrauterine embryos of this nematode. rTsAPP has an enzymatic activity of a natural aminopeptidase P to hydrolyze the substrate H-Ala-Pro-OH. rTsAPP promoted the larval intrusion of intestinal epithelium cells (IECs). The results showed that TsAPP is involved in the T. spiralis intrusion of IECs and it might be a potential candidate vaccine target against Trichinella infection.


Assuntos
Trichinella spiralis , Triquinelose , Vacinas , Camundongos , Animais , Proteínas de Helminto , Camundongos Endogâmicos BALB C , Triquinelose/parasitologia , Aminopeptidases/genética , Aminopeptidases/metabolismo , Células Epiteliais/parasitologia , Larva
20.
Exp Parasitol ; 240: 108329, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35868574

RESUMO

Intestinal epithelial cells (IECs) reside in a highly anaerobic environment that is subject to daily fluctuations in partial oxygen pressure (pO2), depending on intestinal tissue perfusion. This condition, known as physiological hypoxia, has a major impact on the maintenance of gut homeostasis, such as effects on the integrity and function of the intestinal epithelial barrier. Giardia lamblia is a microaerophilic protozoan parasite that infects and colonizes the small intestine of its host, causing watery diarrhea. The disease, known as giardiasis, is associated with enhanced intestinal permeability and disruption or reorganization of tight junction (TJ) proteins between IECs. Given the central role of oxygen in gut homeostasis, in this study, we aimed to evaluate whether pO2 affects intestinal permeability (flux of ions and macromolecules) and TJ protein expression in human IECs during G. lamblia infection. Using human cell lines HuTu-80 and Caco-2 as models of "loose" (low resistance) and "tight" (high resistance) intestines, respectively, we elucidated that low pO2 drives intestinal barrier dysfunction in IECs infected with trophozoites through dephosphorylation of protein kinase C (PKC α/ß II). Additionally, we demonstrated that IECs infected with trophozoites in the presence of a pharmacological PKC activator (phorbol 12-myristate 13-acetate) partially restored the barrier function, which was correlated with increased protein expression levels of zonula occludens (ZO)-2 and occludin. Collectively, these results support the emerging theory that molecular oxygen impacts gut homeostasis during Giardia infection via direct host signaling pathways. These findings further our knowledge regarding Giardia-host interactions and the pathophysiological mechanisms of human giardiasis.


Assuntos
Giardia lamblia , Giardíase , Células CACO-2 , Células Epiteliais/parasitologia , Giardia lamblia/metabolismo , Giardíase/parasitologia , Humanos , Mucosa Intestinal/parasitologia , Oxigênio/metabolismo , Permeabilidade , Junções Íntimas/metabolismo , Proteína da Zônula de Oclusão-1/metabolismo
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