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1.
Microbes Infect ; 26(4): 105299, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38224944

RESUMO

This study aimed to develop aptamers targeting LipL32, a most abundant lipoprotein in pathogenic Leptospira, to hinder bacterial invasion. The objectives were to identify high-affinity aptamers through SELEX and evaluate their specificity and inhibitory effects. SELEX was employed to generate LipL32 aptamers (L32APs) over 15 rounds of selection. L32APs' binding affinity and specificity for pathogenic Leptospira were assessed. Their ability to inhibit LipL32-ECM interaction and Leptospira invasion was investigated. Animal studies were conducted to evaluate the impact of L32AP treatment on survival rates, Leptospira colonization, and kidney damage. Three L32APs with strong binding affinity were identified. They selectively detected pathogenic Leptospira, sparing non-pathogenic strains. L32APs inhibited LipL32-ECM interaction and Leptospira invasion. In animal studies, L32AP administration significantly improved survival rates, reduced Leptospira colonies, and mitigated kidney damage compared to infection alone. This pioneering research developed functional aptamers targeting pathogenic Leptospira. The identified L32APs exhibited high affinity, pathogen selectivity, and inhibition of invasion and ECM interaction. L32AP treatment showed promising results, enhancing survival rates and reducing Leptospira colonization and kidney damage. These findings demonstrate the potential of aptamers to impede pathogenic Leptospira invasion and aid in recovery from Leptospira-induced kidney injury (190 words).


Assuntos
Aptâmeros de Nucleotídeos , Proteínas da Membrana Bacteriana Externa , Leptospira , Leptospirose , Lipoproteínas , Técnica de Seleção de Aptâmeros , Animais , Camundongos , Aptâmeros de Nucleotídeos/farmacologia , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Modelos Animais de Doenças , Rim/microbiologia , Rim/patologia , Leptospira/efeitos dos fármacos , Leptospira/patogenicidade , Leptospira/metabolismo , Leptospirose/microbiologia , Leptospirose/tratamento farmacológico , Lipoproteínas/antagonistas & inibidores , Lipoproteínas/metabolismo
2.
FEBS J ; 290(18): 4513-4532, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37243454

RESUMO

Leptospirosis is a commonly overlooked zoonotic disease that occurs in tropical and subtropical regions. Recent studies have divided the Leptospira spp. into three groups based on virulence, including pathogenic, intermediate, and saprophytic species. Pathogenic species express a protein family with leucine-rich repeat (LRR) domains, which are less expressed or absent in nonpathogenic species, highlighting the importance of this protein family in leptospirosis. However, the role of LRR domain proteins in the pathogenesis of leptospirosis is still unknown and requires further investigation. In this study, the 3D structure of LSS_01692 (rLRR38) was obtained using X-ray crystallography at a resolution of 3.2 Å. The results showed that rLRR38 forms a typical horseshoe structure with 11 α-helices and 11 ß-sheets and an antiparallel dimeric structure. The interactions of rLRR38 with extracellular matrix and cell surface receptors were evaluated using ELISA and single-molecule atomic force microscopy. The results showed that rLRR38 interacted with fibronectin, collagen IV, and Toll-like receptor 2 (TLR2). Incubating HK2 cells with rLRR38 induced two downstream inflammation responses (IL-6 and MCP-1) in the TLR2 signal transduction pathway. The TLR2-TLR1 complex showed the most significant upregulation effects under rLRR38 treatment. Inhibitors also significantly inhibited nuclear factor κB and mitogen-activated protein kinases signals transduction under rLRR38 stimulation. In conclusion, rLRR38 was determined to be a novel LRR domain protein in 3D structure and demonstrated as a TLR2-binding protein that induces inflammatory responses. These structural and functional studies provide a deeper understanding of the pathogenesis of leptospirosis.


Assuntos
Leptospira , Leptospirose , Humanos , Leptospira/genética , Leptospira/química , Leptospira/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismo , Transdução de Sinais , Leptospirose/genética , Leptospirose/metabolismo
3.
Biomed J ; 46(4): 100595, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37142093

RESUMO

Leptospirosis is a neglected bacterial disease caused by leptospiral infection that carries a substantial mortality risk in severe cases. Research has shown that acute, chronic, and asymptomatic leptospiral infections are closely linked to acute and chronic kidney disease (CKD) and renal fibrosis. Leptospires affect renal function by infiltrating kidney cells via the renal tubules and interstitium and surviving in the kidney by circumventing the immune system. The most well-known pathogenic molecular mechanism of renal tubular damage caused by leptospiral infection is the direct binding of the bacterial outer membrane protein LipL32 to toll-like receptor-2 expressed in renal tubular epithelial cells (TECs) to induce intracellular inflammatory signaling pathways. These pathways include the production of tumor necrosis factor (TNF)-α and nuclear factor kappa activation, resulting in acute and chronic leptospirosis-related kidney injury. Few studies have investigated the relationship between acute and chronic renal diseases and leptospirosis and further evidence is necessary. In this review, we intend to discuss the roles of acute kidney injury (AKI) to/on CKD in leptospirosis. This study reviews the molecular pathways underlying the pathogenesis of leptospirosis kidney disease, which will assist in concentrating on potential future research directions.


Assuntos
Injúria Renal Aguda , Leptospira , Leptospirose , Insuficiência Renal Crônica , Humanos , Insuficiência Renal Crônica/complicações , Insuficiência Renal Crônica/patologia , Rim/microbiologia , Rim/patologia , Leptospira/metabolismo
4.
Genomics ; 115(3): 110624, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-37062365

RESUMO

Leptospirosis can cause chronic kidney damage, putting patients at risk of additional kidney injury due to other factors that can lead to renal failure. To understand the combined effect, the transcriptome profiles of kidneys of mice with adenine-induced and chronically Leptospira-infected kidneys were analysed. Chronic inflammation and T-helper 17 immune responses were activated and a high-level expression of Indoleamine 2,3-dioxygenase 1 protein was found. The results indicate that the combination may predispose patients to chronic inflammation, kidney function disruption, and symptoms seen in progressive chronic kidney disease (CKD). Furthermore, immunometabolic regulation may contribute to renal injury caused by chronic leptospirosis with secondary nephrotoxic injury. This study identified several significantly disrupted genes that could serve as potential targets for the diagnosis or treatment of CKD. Our work provides insight into the combined effect of leptospirosis and secondary kidney damage and the molecular basis for rapid progression of CKD.


Assuntos
Anti-Infecciosos , Leptospirose , Insuficiência Renal Crônica , Animais , Camundongos , Transcriptoma , Leptospirose/complicações , Rim , Insuficiência Renal Crônica/complicações , Inflamação
5.
Int J Mol Sci ; 23(15)2022 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-35955634

RESUMO

The aberrant activation of the purinergic signaling pathway has been shown to promote cyst growth and fluid secretion in autosomal dominant polycystic kidney disease (ADPKD). Suramin is an anti-parasitic drug that has strong anti-purinergic properties. Whether suramin could have a therapeutic effect on ADPKD has not been fully investigated. We examined the effect of suramin on cyst progression in a Pkd1 microRNAs transgenic mouse model that presented stable Pkd1 knockdown and moderate disease progression. The Pkd1-deficient mice were treated with suramin (60 mg/kg) by intraperitoneal injection twice a week from postnatal days 35 to 90. Kidney-to-body weight ratios, cyst indices, and blood urea nitrogen (BUN) levels were measured. Cell proliferation and macrophage infiltration were determined by immunohistochemistry. The suramin-treated group had significantly lower renal cyst densities, cell proliferation, and macrophage infiltration compared with saline-treated controls. Suramin significantly inhibited ERK phosphorylation and the expression of Il1b, Il6, Nlrp3, Tgfb, Fn1, P2rx7, and P2ry2 mRNAs in the kidneys. However, BUN levels remained high despite the reduction in cyst growth. Furthermore, plasma cystatin C and neutrophil gelatinase-associated lipocalin (NGAL) levels were significantly higher in the suramin-treated group compared with the control group. Periodic acid-Schiff staining revealed degenerative changes and epithelial cell vacuolation in the non-cystic renal tubules, which indicated phospholipidosis following suramin treatment. These results suggest that suramin may reduce renal cyst growth and inflammation, but the associated tubular cell injuries could limit its therapeutic potential. Other purinergic receptor antagonists with less nephrotoxicity may deserve further investigation for the treatment of ADPKD.


Assuntos
Cistos , Doenças Renais Policísticas , Rim Policístico Autossômico Dominante , Canais de Cátion TRPP/metabolismo , Animais , Proliferação de Células , Cistos/tratamento farmacológico , Modelos Animais de Doenças , Rim/metabolismo , Camundongos , Camundongos Transgênicos , Doenças Renais Policísticas/metabolismo , Rim Policístico Autossômico Dominante/tratamento farmacológico , Rim Policístico Autossômico Dominante/genética , Rim Policístico Autossômico Dominante/metabolismo , Suramina/farmacologia , Suramina/uso terapêutico , Canais de Cátion TRPP/genética
6.
Microbiol Spectr ; 10(3): e0259521, 2022 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-35638785

RESUMO

Leptospirosis, an emerging infectious disease caused by pathogenic Leptospira spp., occurs in ecoregions with heavy rainfall and has public health implications. Macrophages are the major anti-Leptospira phagocytes that infiltrate the kidneys during renal leptospirosis, which is caused by leptospires residing in the renal tubules. The pathogenicity of Leptospira spp. in immune effector cells such as macrophages is not well understood. To evaluate this pathogenesis, we characterized and compared the transcriptome-wide alterations in macrophages infected with pathogenic and nonpathogenic Leptospira spp. Using transcriptome data and quantitative reverse transcription PCR analysis, at 2 h postinfection, the hypoxia-inducible factor-1α-dependent glycolysis pathway was implicated in pathogenic Leptospira-infected macrophages but not in nonpathogenic leptospiral infections. Immune-related biological processes were mostly activated in pathogenic Leptospira-infected macrophages, and flow cytometry investigations revealed that classically activated macrophages represent the predominant polarization status. At 24 h after infection, biological pathways associated with interleukin-10, IL-10, signaling the induction of macrophage tolerance, as well as higher levels of IL-10 mRNA and protein expression, were observed in nonpathogenic Leptospira-infected macrophages compared to in pathogenic leptospiral infection. Following leptospiral infection of macrophages, strong IL-10-expressing transcriptome signatures were observed following nonpathogenic leptospiral infection. The transcriptional programs generated in Leptospira-infected macrophages revealed an inflammatory milieu following the production of a critical anti-inflammatory cytokine, IL-10, which is implicated in controlling the pathogenicity of activated macrophages. These findings imply that IL-10-mediated anti-inflammatory responses and tolerance in activated macrophages induced by nonpathogenic Leptospira spp. infection reduce inflammation and tissue damage, thus providing a potential therapeutic target for leptospirosis. IMPORTANCE Activation of macrophages by Leptospira spp. infection is thought to be involved in the pathogenesis of leptospirosis. To evaluate the innate macrophage responses to Leptospira spp., specifically pathogenic versus nonpathogenic Leptospira spp., we characterized the entire transcriptome-wide alterations in infected macrophages. We showed that hypoxia-inducible factor-1α and immune-related pathways are activated in pathogenic leptospiral-infected macrophages. We confirmed the significantly high levels of IL-10-expressing signatures and tolerance in activated macrophages caused by nonpathogenic Leptospira infection. Furthermore, nonpathogenic leptospiral infections attenuated macrophage activation responses. These findings suggest a potential therapeutic strategy for the immune microenvironment caused by macrophage activation driven by IL-10 overexpression, which may contribute to regulating inflammation in leptospirosis.


Assuntos
Leptospira , Leptospirose , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Inflamação/metabolismo , Interleucina-10/genética , Interleucina-10/metabolismo , Leptospira/genética , Leptospirose/genética , Macrófagos , Virulência
7.
Membranes (Basel) ; 12(3)2022 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-35323775

RESUMO

Leptospirosis is a widespread zoonosis that frequently occurs in tropical and subtropical countries. Leptospira enters the host through wounds or mucous membranes and spreads to the whole body through the blood, causing systemic infection. Kidneys are the preferential site where Leptospira accumulates, especially in the renal interstitium and renal tubule epithelial cells. Clinical symptoms in humans include high fever, jaundice, renal failure, and severe multiple-organ failure (Weil's syndrome). Surface-exposed antigens are located at the outermost layer of Leptospira and these potential virulence factors are likely involved in primary host-pathogen interactions, adhesion, and/or invasion. Using the knockout/knockdown techniques to the evaluation of pathogenicity in the virulence factor are the most direct and effective methods and many virulence factors are evaluated including lipopolysaccharides (LPS), Leptospira lipoprotein 32 (LipL32), Leptospira ompA domain protein 22 (Loa22), LipL41, LipL71, Leptospira immunoglobulin-like repeat A (LigA), LigB, and LipL21. In this review, we will discuss the structure, functions, and dynamics of these virulence factors and the roles of these virulence factors in Leptospira pathogenicity. In addition, a protein family with special Leucine-rich repeat (LRR) will also be discussed for their vital role in Leptospira pathogenicity. Finally, these surface-exposed antigens are discussed in the application of the diagnosis target for leptospirosis and compared with the serum microscope agglutination test (MAT), the gold standard for leptospirosis.

8.
Hum Mol Genet ; 31(10): 1560-1573, 2022 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-34957500

RESUMO

Metabolic reprogramming is a potential treatment strategy for autosomal dominant polycystic kidney disease (ADPKD). Metformin has been shown to inhibit the early stages of cyst formation in animal models. However, metformin can lead to lactic acidosis in diabetic patients with advanced chronic kidney disease, and its efficacy in ADPKD is still not fully understood. Here, we investigated the effect of metformin in an established hypomorphic mouse model of PKD that presents stable and heritable knockdown of Pkd1. The Pkd1 miRNA transgenic mice of both genders were randomized to receive metformin or saline injections. Metformin was administrated through daily intraperitoneal injection from postnatal day 35 for 4 weeks. Unexpectedly, metformin treatment at a concentration of 150 mg/kg increased disease severity, including kidney-to-body weight ratio, cystic index and plasma BUN levels, and was associated with increased renal tubular cell proliferation and plasma lactate levels. Functional enrichment analysis for cDNA microarrays from kidney samples revealed significant enrichment of several pro-proliferative pathways including ß-catenin, hypoxia-inducible factor-1α, protein kinase Cα and Notch signaling pathways in the metformin-treated mutant mice. The plasma metformin concentrations were still within the recommended therapeutic range for type 2 diabetic patients. Short-term metformin treatment in a second Pkd1 hypomorphic model (Pkd1RC/RC) was however neutral. These results demonstrate that metformin may exacerbate late-stage cyst growth associated with the activation of lactate-related signaling pathways in Pkd1 deficiency. Our findings indicate that using metformin in the later stage of ADPKD might accelerate disease progression and call for the cautious use of metformin in these patients.


Assuntos
Cistos , Metformina , Rim Policístico Autossômico Dominante , Animais , Cistos/metabolismo , Modelos Animais de Doenças , Feminino , Rim/metabolismo , Ácido Láctico/metabolismo , Masculino , Metformina/metabolismo , Metformina/farmacologia , Camundongos , Camundongos Transgênicos , Doenças Renais Policísticas , Rim Policístico Autossômico Dominante/tratamento farmacológico , Rim Policístico Autossômico Dominante/genética , Rim Policístico Autossômico Dominante/metabolismo , Canais de Cátion TRPP/genética , Canais de Cátion TRPP/metabolismo
9.
Int J Mol Sci ; 22(23)2021 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-34884937

RESUMO

Approximately 1 million cases of leptospirosis, an emerging infectious zoonotic disease, are reported each year. Pathogenic Leptospira species express leucine-rich repeat (LRR) proteins that are rarely expressed in non-pathogenic Leptospira species. The LRR domain-containing protein family is vital for the virulence of pathogenic Leptospira species. In this study, the biological mechanisms of an essential LRR domain protein from pathogenic Leptospira were examined. The effects of Leptospira and recombinant LRR20 (rLRR20) on the expression levels of factors involved in signal transduction were examined using microarray, quantitative real-time polymerase chain reaction, and western blotting. The secreted biomarkers were measured using an enzyme-linked immunosorbent assay. rLRR20 colocalized with E-cadherin on the cell surface and activated the downstream transcription factor ß-catenin, which subsequently promoted the expression of MMP7, a kidney injury biomarker. Additionally, MMP7 inhibitors were used to demonstrate that the secreted MMP7 degrades surface E-cadherin. This feedback inhibition mechanism downregulated surface E-cadherin expression and inhibited the colonization of Leptospira. The degradation of surface E-cadherin activated the NF-κB signal transduction pathway. Leptospirosis-associated acute kidney injury is associated with the secretion of NGAL, a downstream upregulated biomarker of the NF-κB signal transduction pathway. A working model was proposed to illustrate the crosstalk between E-cadherin/ß-catenin and NF-κB signal transduction pathways during Leptospira infection. Thus, rLRR20 of Leptospira induces kidney injury in host cells and inhibits the adhesion and invasion of Leptospira through the upregulation of MMP7 and NGAL.


Assuntos
Antígenos CD/metabolismo , Caderinas/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , Leptospirose/metabolismo , NF-kappa B/metabolismo , beta Catenina/metabolismo , Antígenos CD/genética , Caderinas/genética , Regulação da Expressão Gênica , Humanos , Leptospira/metabolismo , Leptospira/patogenicidade , Leptospirose/microbiologia , Proteínas de Repetições Ricas em Leucina/genética , Proteínas de Repetições Ricas em Leucina/metabolismo , Lipocalina-2/metabolismo , Metaloproteinase 7 da Matriz/metabolismo , Transporte Proteico , Transdução de Sinais , beta Catenina/genética
10.
Am J Physiol Renal Physiol ; 320(5): F1001-F1018, 2021 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-33779314

RESUMO

High-incidence regions of leptospirosis caused by Leptospira spp. coincide with chronic kidney disease. This study investigated whether asymptomatic leptospirosis is an emerging culprit that predisposes to progressive chronic kidney disease when superimposed on secondary nephrotoxic injury. Kidney histology/function and whole transcriptomic profiles were evaluated for Leptospira-infected C57/BL6 mice with adenine-induced kidney injury. The extent of tubulointerstitial kidney lesions and expression of inflammation/fibrosis genes in infected mice with low-dose (0.1%) adenine, particularly in high-dose (0.2%) adenine-fed superimposed on Leptospira-infected mice, were significantly increased compared with mice following infection or adenine diet alone, and the findings are consistent with renal transcriptome analysis. Pathway enrichment findings showed that integrin-ß- and fibronectin-encoding genes had distinct expression within the integrin-linked kinase-signaling pathway, which were upregulated in 0.2% adenine-fed Leptospira-infected mice but not in 0.2% adenine-fed mice, indicating that background subclinical Leptospiral infection indeed enhanced subsequent secondary nephrotoxic kidney injury and potential pathogenic molecules associated with secondary nephrotoxic leptospirosis. Comparative analysis of gene expression patterns with unilateral ureteric obstruction-induced mouse renal fibrosis and patients with chronic kidney disease showed that differentially expressed orthologous genes such as hemoglobin-α2, PDZ-binding kinase, and DNA topoisomerase II-α were identified in infected mice fed with low-dose and high-dose adenine, respectively, revealing differentially expressed signatures identical to those found in the datasets and may serve as markers of aggravated kidney progression. This study indicates that background subclinical leptospirosis, when subjected to various degrees of subsequent secondary nephrotoxic injury, may predispose to exacerbated fibrosis, mimicking the pathophysiological process of progressive chronic kidney disease.NEW & NOTEWORTHYLeptospira-infected mice followed by secondary nephrotoxic injury exacerbated immune/inflammatory responses and renal fibrosis. Comparison with the murine model revealed candidates involved in the progression of renal fibrosis in chronic kidney disease (CKD). Comparative transcriptome study suggests that secondary nephrotoxic injury in Leptospira-infected mice recapitulates the gene expression signatures found in CKD patients. This study indicates that secondary nephrotoxic injury may exacerbate CKD in chronic Leptospira infection implicating in the progression of CKD of unknown etiology.


Assuntos
Leptospirose/complicações , Insuficiência Renal Crônica/complicações , Transcriptoma , Animais , Doença Crônica , Fibrose/etiologia , Humanos , Inflamação , Leptospirose/metabolismo , Leptospirose/patologia , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Insuficiência Renal Crônica/metabolismo , Insuficiência Renal Crônica/patologia
11.
Sci Rep ; 11(1): 1064, 2021 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-33441663

RESUMO

Leptospirosis is an overlooked zoonotic disease caused by pathogenic Leptospira depended on virulence of Leptospira and the host-pathogen interaction. Kidney is the major organ infected by Leptospira which causes tubulointerstitial nephritis. Leptospira outer membrane contains several virulence factors and an outer membrane protein A (OmpA) like protein (Loa22) is essential for virulence. Pull-down assays suggested that Loa22 was a potential Toll-Like Receptor 2 (TLR2) binding candidates from pathogenic Leptospira. Confocal microscopy was employed to observe the co-localization of TLR2 and Loa22-LPGN (Leptospira peptidoglycan) complexes. Atomic force microscopy (AFM), side-directed mutagenesis, and enzyme-linked immunosorbent assay (ELISA) were performed to investigate the affinity between rLoa22, LPGN, and TLR2. Real time PCR was applied to measure the cytokines expression. Downstream signal transduction components were verified by western blot to evaluate the gene regulations. Mutation of two Loa22 key residues (Asp122 and Arg143) attenuated the affinities for LPGN. rLoa22-LPGN complexes were observed to co-localize with TLR2 and provoked inflammatory responses including CXCL8/IL8, hCCL2/MCP-1, and hTNF-α. Affinity studies suggested that Loa22-LPGN complexes elevated the affinity to TLR2 as compared to Loa22 protein. Downstream signals from TLR2 including p38, ERK, and JNK were regulated under rLoa22-LPGN complexes treatments. This study identified LPGN mediates interactions between Loa22 and TLR2 and induces downstream signals to trigger inflammatory responses. rLoa22-LPGN-TLR2 complexes reveal a novel binding mechanism for the innate immune system.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Imunidade Inata , Leptospira/metabolismo , Leptospirose/imunologia , Peptidoglicano/metabolismo , Receptor 2 Toll-Like/metabolismo , Proteínas da Membrana Bacteriana Externa/imunologia , Células HEK293 , Humanos , Inflamação/imunologia , Inflamação/parasitologia , Leptospira/imunologia , Leptospirose/metabolismo , Microscopia Confocal , Reação em Cadeia da Polimerase em Tempo Real
12.
Biomedicines ; 10(1)2021 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-35052695

RESUMO

In contrast to Western counties, the incidence of urothelial carcinoma (UC) remains mar-edly elevated in Taiwan. Regulatory T cells (Tregs) play a crucial role in limiting immune responses within the tumor microenvironment. To elucidate the relationship between immune checkpoints in the tumor immune microenvironment and UC progression, we utilize the Gene Expression Omnibus (GEO) to analyze a microarray obtained from 308 patients with UC. We observed that the expression level of CD276 or TIM-3 was positively correlated with late-stage UC and poor prognosis. Patients with simultaneously high CD276 and TIM-3 expression in tumors have significantly reduced both univariate and multivariate survival, indicating that mRNA levels of these immune checkpoints could be independent prognostic biomarkers for UC overall survival and recurrence. Our cohort study showed rare CD8+ cytotoxic T-cells and Tregs infiltration during early-stage UC-known as cold tumors. Approximately 30% of late-stage tumors exhibited highly infiltrated cytotoxic T cells with high PD-1 and FOXP3 expression, which implied that cytotoxic T cells were inhibited in the advanced UC microenvironment. Collectively, our findings provide a better prognosis prediction by combined immune checkpoint biomarkers and a basis for early-stage UC standard treatment to convert cold tumors into hot tumors, followed by immune checkpoint therapy.

13.
Biochem J ; 477(21): 4313-4326, 2020 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-33094809

RESUMO

Leptospirosis is the most common zoonotic disease caused by pathogenic Leptospira, which is classified into three groups according to virulence. Its pathogenic and intermediate species contain leucine-rich repeat (LRR) proteins that are rarely expressed in non-pathogenic strains. In this study, we presented the crystal structure of LSS_11580 (rLRR20) from pathogenic L. santarosai serovar Shermani. X-ray diffraction at a resolution of 1.99 Šrevealed a horseshoe-shaped structure containing seven α-helices and five ß-sheets. Affinity assays indicated that rLRR20 interacts with E-cadherin on the cell surface. Interestingly, its binds to the extracellular (EC) 1 domain in human epithelial (E)-cadherin, which is responsible for binding to another E-cadherin molecule in neighboring cells. Several charged residues on the concave face of LRR20 were predicted to interact with EC1 domain. In the affinity assays, these charged residues were replaced by alanine, and their affinities to E-cadherin were measured. Three vital residues and mutation variants of LRR20, namely D56A, E59A, and E123A, demonstrated significantly reduced affinity to E-cadherin compared with the control. Besides, we also demonstrated that rLRR20 induced the expression of neutrophil gelatinase-associated lipocalin (NGAL) in HK2 cells. The low ability of the three mutation variants to induce NGAL expression further demonstrates this induction. The present findings indicate that LRR20 from pathogenic Leptospira binds to E-cadherin and interacts with its EC1 domain. In addition, its induction of NGAL expression in HK2 cells is associated with acute kidney injury in human.


Assuntos
Caderinas/metabolismo , Cristalografia por Raios X/métodos , Leptospira/metabolismo , Proteínas/química , Proteínas/metabolismo , Proteínas de Transporte/metabolismo , Linhagem Celular , Humanos , Leptospirose/metabolismo , Proteínas de Repetições Ricas em Leucina
14.
J Photochem Photobiol B ; 198: 111560, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31336216

RESUMO

Previous studies revealed significant impact on cancer cell by mid-infrared (MIR) radiation. However, the effects of narrow band MIR on immune reaction and infectious disease are still unknown. In this study, an enhanced innate immune response was observed through the interaction between Leptospiral outer membrane protein (LipL32) and toll-like receptor 2 (TLR2). Thereafter, human kidney proximal tubular cells (HK-2 cells) initiated a serial reaction of enhanced MCP-1 production. The 6 µm narrow bandwidth light source emitted by waveguide thermal emitter (WTE) was applied to induce carbonyl group (CO bond) stretching vibration during the stage of antigen-receptor complex formation. The amount of MCP-1 gene expression had 2.5 folds increase after narrow band MIR illumination comparing to non-MIR illumination at low dose LipL32 condition. Besides, both ELISA and confocal microscopy results also revealed that the chemokine concentration increased significantly after narrow band MIR illumination either at low or high concentration of LipL32. Furthermore, a specific phenomenon that narrow band MIR can amplify the signal of weak immune response by enhancing sensitivity of the interaction between antigen and receptor was observed. This study exhibits clear evidence that the narrow band MIR exposure can modulate the early immune response of infectious disease and play a potential role to develop host-directed therapy in the future.


Assuntos
Proteínas da Membrana Bacteriana Externa/farmacologia , Raios Infravermelhos , Lipoproteínas/farmacologia , Proteínas da Membrana Bacteriana Externa/imunologia , Linhagem Celular , Sobrevivência Celular/efeitos da radiação , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos da radiação , Humanos , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , Leptospira/metabolismo , Lipoproteínas/imunologia
15.
J Infect Dis ; 218(9): 1411-1423, 2018 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-29868892

RESUMO

Background: Leptospirosis caused by pathogenic Leptospira spp leads to kidney damage that may progress to chronic kidney disease. However, how leptospiral infections induced renal damage is unclear. Methods: We apply microarray and next-generation sequencing technologies to investigate the first murine transcriptome-wide, leptospires-mediated changes in renal gene expression to identify biological pathways associated with kidney damage. Results: Leptospiral genes were detected in renal transcriptomes of mice infected with Leptospira interrogans at day 28 postinfection, suggesting colonization of leptospires within the kidney with propensity of chronicity. Comparative differential gene expression and pathway analysis were investigated in renal transcriptomes of mice infected with pathogens and nonpathogens. Pathways analysis showed that Toll-like receptor signaling, complements activation, T-helper 1 type immune response, and T cell-mediated immunity/chemotaxis/proliferation were strongly associated with progressive tubulointerstitial damage caused by pathogenic leptospiral infection. In addition, 26 genes related with complement system, immune function, and cell-cell interactions were found to be significantly up-regulated in the L interrogans-infected renal transcriptome. Conclusions: Our results provided comprehensive knowledge regarding the host transcriptional response to leptospiral infection in murine kidneys, particularly the involvement of cell-to-cell interaction in the immune response. It would provide valuable resources to explore functional studies of chronic renal damage caused by leptospiral infection.


Assuntos
Rim/fisiologia , Leptospira interrogans/imunologia , Leptospirose/genética , Insuficiência Renal Crônica/genética , Transcriptoma/genética , Animais , Feminino , Expressão Gênica/genética , Expressão Gênica/imunologia , Rim/imunologia , Leptospirose/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Insuficiência Renal Crônica/imunologia , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Receptores Toll-Like/genética
16.
Sci Rep ; 7(1): 8363, 2017 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-28827637

RESUMO

Proteins belonging to the toll-like receptor (TLR) family, particularly TLR2, are the major components of innate immunity against Leptospira infection. The ligands for TLR2 harbor several conserved patterns such as lipidation molecules, leucine-rich repeat (LRR) domains, TLR2 binding motifs, and TLR2 binding structure. In Leptospira, LipL32 interacts with TLR2 on human kidney cells concomitantly stimulating inflammatory responses. However, the binding mechanism of LipL32 to TLR2 is unknown. The computational prediction suggests that ß1ß2, loop-α3-loop, and α4 domains of LipL32 play vital roles in LipL32-TLR2 complex formation. To test these predictions, protein truncation experiments revealed that LipL32ΔNß1ß2 significantly decreased the affinity to TLR2 while LipL32ΔCα4 slightly reduced it. Interestingly, LipL32ΔCenα3 retained affinity to TLR2 in the absence of Ca2+ ions, indicating that Cenα3 play a role preventing the interaction between LipL32 and TLR2. Furthermore, the critical residues of LipL32 involved in interacting with TLR2 suggested that V35S, L36S and L263S variants significantly decreased the affinity to TLR2. The results further confirm that LipL32 interacts with TLR2 through Nß1ß2 and Cα4 domains of LipL32 as well as LipL32-TLR2 complex formation results from hydrophobic interactions. This study provides a detailed mechanism of the interaction between LipL32 and TLR2 and the residues involved in complex formation.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Leptospira/imunologia , Lipoproteínas/metabolismo , Receptor 2 Toll-Like/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Humanos , Interações Hidrofóbicas e Hidrofílicas , Lipoproteínas/química , Lipoproteínas/genética , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Ligação Proteica , Deleção de Sequência , Receptor 2 Toll-Like/química
17.
Sci Rep ; 6: 27838, 2016 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-27278903

RESUMO

Leptospirosis is an often overlooked cause of acute kidney injury that can lead to multiple organ failure and even death. The principle protein that conserved in many pathogenic leptospires is the outer membrane protein LipL32. However, the role of LipL32 in the pathogenesis of renal injury in leptospirosis is not entirely clear. Here we studied the effects of LipL32 on the developing kidney in zebrafish larvae. Incubation of zebrafish larvae with Leptospira santarosai serovar Shermani induced acute tubular injury predominantly in the proximal pronephric ducts. Furthermore, microinjection of lipl32 mRNA or recombinant LipL32 protein into zebrafish larvae increased macrophage accumulation and disrupted the basolateral location of NA-K-ATPase in pronephric ducts. These changes led to substantial impairment of the pronephric kidney structure. We further demonstrated that morpholino knockdown of tlr2, but not tlr4, reduced the LipL32-induced leukocyte infiltration and kidney injury. These data demonstrate that LipL32 contributes to the renal pathology in leptospirosis and gives some clues to the potential virulence of LipL32. Our results support the use of zebrafish as a model organism for studying the disease mechanism of leptospiral infection. This model might permit the future exploration of the virulence and molecular pathways of different leptospiral outer membrane proteins.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Nefropatias , Rim , Leptospira/metabolismo , Lipoproteínas/metabolismo , Pronefro , Peixe-Zebra , Animais , Proteínas da Membrana Bacteriana Externa/genética , Inflamação/embriologia , Inflamação/genética , Inflamação/microbiologia , Rim/embriologia , Rim/microbiologia , Nefropatias/embriologia , Nefropatias/genética , Nefropatias/microbiologia , Leptospira/genética , Lipoproteínas/genética , Pronefro/embriologia , Pronefro/microbiologia , Peixe-Zebra/embriologia , Peixe-Zebra/microbiologia
18.
J Biol Chem ; 290(2): 1197-209, 2015 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-25451931

RESUMO

Single molecule atomic force microscopy (smAFM) was employed to unfold transmembrane domain interactions of a unique vacuolar H(+)-pyrophosphatase (EC 3.6.1.1) from Vigna radiata. H(+)-Pyrophosphatase is a membrane-embedded homodimeric protein containing a single type of polypeptide and links PPi hydrolysis to proton translocation. Each subunit consists of 16 transmembrane domains with both ends facing the lumen side. In this investigation, H(+)-pyrophosphatase was reconstituted into the lipid bilayer in the same orientation for efficient fishing out of the membrane by smAFM. The reconstituted H(+)-pyrophosphatase in the lipid bilayer showed an authentically dimeric structure, and the size of each monomer was ∼4 nm in length, ∼2 nm in width, and ∼1 nm in protrusion height. Upon extracting the H(+)-pyrophosphatase out of the membrane, force-distance curves containing 10 peaks were obtained and assigned to distinct domains. In the presence of pyrophosphate, phosphate, and imidodiphosphate, the numbers of interaction curves were altered to 7, 8, and 10, respectively, concomitantly with significant modification in force strength. The substrate-binding residues were further replaced to verify these domain changes upon substrate binding. A working model is accordingly proposed to show the interactions between transmembrane domains of H(+)-pyrophosphatase in the presence and absence of substrate and its analog.


Assuntos
Pirofosfatase Inorgânica/química , Pirofosfatase Inorgânica/ultraestrutura , Transporte de Íons , Vacúolos/enzimologia , Fabaceae/química , Fabaceae/enzimologia , Hidrólise , Pirofosfatase Inorgânica/metabolismo , Cinética , Bicamadas Lipídicas/química , Microscopia de Força Atômica , Estrutura Terciária de Proteína , Prótons , Especificidade por Substrato
19.
J Biol Chem ; 288(17): 12335-44, 2013 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-23486465

RESUMO

Leptospirosis is the most widespread zoonosis caused by the pathogenic Leptospira worldwide. LipL32, a 32-kDa lipoprotein, is the most abundant protein on the outer membrane of Leptospira and has an atypical poly(Asp) motif ((161)DDDDDGDD(168)). The x-ray crystallographic structure of LipL32 revealed that the calcium-binding cluster of LipL32 includes several essential residues Asp(132), Thr(133), Asp(164), Asp(165), and Tyr(178). The goals of this study were to determine possible roles of the Ca(2+)-binding cluster for the interaction of LipL32 and Toll-like receptor 2 (TLR2) in induced inflammatory responses of human kidney cells. Site-directed mutagenesis was employed to individually mutate Ca(2+)-binding residues of LipL32 to Ala, and their effects subsequently were observed. These mutations abolished primarily the structural integrity of the calcium-binding cluster in LipL32. The binding assay and atomic force microscopy analysis further demonstrated the decreased binding capability of LipL32 mutants to TLR2. Inflammatory responses induced by LipL32 variants, as determined by TLR2 pathway intermediates hCXCL8/IL-8, hCCL2/MCP-1, hMMP7, and hTNF-α, were also lessened. In conclusion, the calcium-binding cluster of LipL32 plays essential roles in presumably sustaining LipL32 conformation for its proper association with TLR2 to elicit inflammatory responses in human renal cells.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Rim/metabolismo , Leptospira/metabolismo , Leptospirose/metabolismo , Lipoproteínas/metabolismo , Transdução de Sinais , Receptor 2 Toll-Like/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Linhagem Celular , Quimiocina CCL2/biossíntese , Quimiocina CCL2/genética , Humanos , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Interleucina-8/biossíntese , Interleucina-8/genética , Rim/patologia , Leptospira/genética , Leptospirose/genética , Leptospirose/patologia , Lipoproteínas/genética , Metaloproteinase 7 da Matriz/biossíntese , Metaloproteinase 7 da Matriz/genética , Mutagênese Sítio-Dirigida , Receptor 2 Toll-Like/genética , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/genética
20.
J Biol Chem ; 286(14): 11970-6, 2011 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-21292767

RESUMO

H+-translocating pyrophosphatase (H+-PPase; EC 3.6.1.1) drives proton transport against an electrochemical potential gradient by hydrolyzing pyrophosphate (PPi) and is found in various endomembranes of higher plants, bacteria, and some protists. H+-PPase contains seven highly conserved lysines. We examined the functional roles of these lysines, which are, for the most part, found in the cytosolic regions of mung bean H+-PPase by site-directed mutagenesis. Construction of mutants that each had a cytosolic and highly conserved lysine substituted with an alanine resulted in dramatic drops in the PPi hydrolytic activity. The effects caused by ions on the activities of WT and mutant H+-PPases suggest that Lys-730 may be in close proximity to the Mg2+-binding site, and the great resistance of the K694A and K695A mutants to fluoride inhibition suggests that these lysines are present in the active site. The modifier fluorescein 5'-isothiocyanate (FITC) labeled a lysine at the H+-PPase active site but did not inhibit the hydrolytic activities of K250A, K250N, K250T, and K250S, which suggested that Lys-250 is essential for substrate binding and may be involved in proton translocation. Analysis of tryptic digests indicated that Lys-711 and Lys-717 help maintain the conformation of the active site. Proteolytic evidence also demonstrated that Lys-250 is the primary target of trypsin and confirmed its crucial role in H+-PPase hydrolysis.


Assuntos
Pirofosfatase Inorgânica/química , Pirofosfatase Inorgânica/metabolismo , Vacúolos/enzimologia , Western Blotting , Eletroforese em Gel de Poliacrilamida , Pirofosfatase Inorgânica/genética , Lisina/genética , Lisina/metabolismo , Mutagênese Sítio-Dirigida , Ligação Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
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