M16-Type Metallopeptidases Are Involved in Virulence for Invasiveness and Diffusion of Leptospira interrogans and Transmission of Leptospirosis

2020 ◽  
Vol 222 (6) ◽  
pp. 1008-1020
Author(s):  
Yu-Mei Ge ◽  
Ai-Hua Sun ◽  
David M Ojcius ◽  
Shi-Jun Li ◽  
Wei-Lin Hu ◽  
...  
Keyword(s):  
Lethal Dose ◽  
Matrix Proteins ◽  
Leptospira Interrogans ◽  
Internal Organs ◽  
Western Blot Assay ◽  
Cell Monolayers ◽  
Genes Encoding ◽  
Encoding Genes ◽  
And Diffusion

Abstract Background Leptospirosis is a global zoonotic infectious disease caused by Leptospira interrogans. The pathogen rapidly invades into hosts and diffuses from bloodstream into internal organs and excretes from urine to cause transmission of leptospirosis. However, the mechanism of leptospiral invasiveness remains poorly understood. Methods Proteolytic activity of M16-type metallopeptidases (Lep-MP1/2/3) of L. interrogans was determined by spectrophotometry. Expression and secretion of Lep-MP1/2/3 during infection of cells were detected by quantitative reverse-transcription polymerase chain reaction, Western blot assay, and confocal microscopy. Deletion and complementation mutants of the genes encoding Lep-MP1/2/3 were generated to determine the roles of Lep-MP1/2/3 in invasiveness using transwell assay and virulence in hamsters. Results Leptospira interrogans but not saprophytic Leptospira biflexa strains were detectable for Lep-MP-1/2/3-encoding genes. rLep-MP1/2/3 hydrolyzed extracellular matrix proteins, but rLep-MP1/3 displayed stronger proteolysis than rLep-MP2, with 123.179/340.136 μmol/L Km and 0.154/0.159 s-1 Kcat values. Expression, secretion and translocation of Lep-MP1/2/3 during infection of cells were increased. ΔMP1/3 but not ΔMP2 mutant presented attenuated transmigration through cell monolayers, decreased leptospiral loading in the blood, lungs, liver, kidneys, and urine, and 10/13-fold decreased 50% lethal dose and milder histopathologic injury in hamsters. Conclusions Lep-MP1 and 3 are involved in virulence of L. interrogans in invasion into hosts and diffusion in vivo, and transmission of leptospirosis.

scholarly journals GspD, The Type II Secretion System Secretin of Leptospira, Protects Hamsters against Lethal Infection with a Virulent L. interrogans Isolate

Vaccines ◽  
2020 ◽  
Vol 8 (4) ◽  
pp. 759
Author(s):  
Samantha Paulina Llanos Salinas ◽  
Luz Olivia Castillo Sánchez ◽  
Giselle Castañeda Miranda ◽  
Ernesto Armando Rodríguez Reyes ◽  
Liliana Ordoñez López ◽  
...  
Keyword(s):  
Virulent Strain ◽  
Lethal Dose ◽  
Secretion System ◽  
Type Ii Secretion ◽  
Leptospira Interrogans ◽  
Type Ii ◽  
Pathogenic Leptospira ◽  
Exact Test ◽  
Type Ii Secretion System

The wide variety of pathogenic Leptospira serovars and the weak protection offered by the available vaccines encourage the search for protective immunogens against leptospirosis. We found that the secretin GspD of the type II secretion system (T2S) of Leptospira interrogans serovar Canicola was highly conserved amongst pathogenic serovars and was expressed in vivo during infection, as shown by immunohistochemistry. Convalescent sera of hamsters, dogs, and cows showed the presence of IgG antibodies, recognizing a recombinant version of this protein expressed in Escherichia coli (rGspDLC) in Western blot assays. In a pilot vaccination study, a group of eight hamsters was immunized on days zero and 14 with 50 µg of rGspDLC mixed with Freund’s incomplete adjuvant (FIA). On day 28 of the study, 1,000 LD50 (Lethal Dose 50%) of a virulent strain of Leptospira interrogans serovar Canicola (LOCaS46) were inoculated by an intraoral submucosal route (IOSM). Seventy-five percent protection against disease (p = 0.017573, Fisher’s exact test) and 50% protection against infection were observed in this group of vaccinated hamsters. In contrast, 85% of non-vaccinated hamsters died six to nine days after the challenge. These results suggest the potential usefulness of the T2S secretin GspD of Leptospira as a protective recombinant vaccine against leptospirosis.

scholarly journals Characterization of leptospiral proteins that afford partial protection in hamsters against lethal challenge with Leptospira interrogans

2010 ◽  
Vol 59 (9) ◽  
pp. 1005-1015 ◽  
Author(s):  
Marina V. Atzingen ◽  
Amane P. Gonçales ◽  
Zenaide M. de Morais ◽  
Eduardo R. Araújo ◽  
Thales De Brito ◽  
...  
Keyword(s):  
Virulent Strain ◽  
Lethal Dose ◽  
Whole Genome Sequence ◽  
Leptospira Interrogans ◽  
Lethal Challenge ◽  
T Helper 2 ◽  
Bioinformatic Tools ◽  
Genes Encoding ◽  
Antibody Titres

Leptospirosis is a worldwide zoonosis caused by pathogenic Leptospira. The whole-genome sequence of Leptospira interrogans serovar Copenhageni together with bioinformatic tools allow us to search for novel antigen candidates suitable for improved vaccines against leptospirosis. This study focused on three genes encoding conserved hypothetical proteins predicted to be exported to the outer membrane. The genes were amplified by PCR from six predominant pathogenic serovars in Brazil. The genes were cloned and expressed in Escherichia coli strain BL21-SI using the expression vector pDEST17. The recombinant proteins tagged with N-terminal 6×His were purified by metal-charged chromatography. The proteins were recognized by antibodies present in sera from hamsters that were experimentally infected. Immunization of hamsters followed by challenge with a lethal dose of a virulent strain of Leptospira showed that the recombinant protein rLIC12730 afforded statistically significant protection to animals (44 %), followed by rLIC10494 (40 %) and rLIC12922 (30 %). Immunization with these proteins produced an increase in antibody titres during subsequent boosters, suggesting the involvement of a T-helper 2 response. Although more studies are needed, these data suggest that rLIC12730 and rLIC10494 are promising candidates for a multivalent vaccine for the prevention of leptospirosis.

scholarly journals Lack of salidroside impact on selected cytochromes encoding genes transcription in the liver of ethanol induced rats

2021 ◽  
Vol 67 (3) ◽  
pp. 53-65
Author(s):  
Radosław Kujawski ◽  
Michał Szulc ◽  
Maria Toboła ◽  
Marcin Graczyk ◽  
Kamila Czora-Poczwardowska ◽  
...  
Keyword(s):  
Real Time Pcr ◽  
Liver Tissue ◽  
Molecular Basis ◽  
Statistical Significance ◽  
Mrna Level ◽  
Alcohol Tolerance ◽  
Qrt Pcr ◽  
Genes Encoding ◽  
Encoding Genes

Summary Introduction: The molecular basis of in vivo metabolism of selected representatives of phenylethanoids in the presence of ethanol has not been fully elucidated. Objective: The aim was to estimate a salidroside (Sal) metabolism in the liver tissue in rats with induced alcohol tolerance by assessing changes in the transcription of genes encoding cytochromes: CYP1A2, 2D2, 3A1, 2C23. Methods: cDNA was synthesized from total RNA isolated from rat liver samples. mRNA level changes were evaluated using real-time PCR (qRT-PCR) technique. Results: Ethanol caused a significant induction of the CYP1A2 and CYP2C23 genes transcription, and a decrease in the CYP3A1 mRNA level, predominantly without statistical significance. A statistically significant increase of the CYP1A2 mRNA level was observed in the group receiving only Sal (4.5 mg/kg b.w.; p.o.) (p<0.01). Conclusions: There was no unequivocal effect of salidroside on the transcription of investigated cytochrome genes in the liver of rats with induced alcohol tolerance.

scholarly journals Identification of Specific In Vivo-Induced (ivi) Genes in Yersinia ruckeri and Analysis of Ruckerbactin, a Catecholate Siderophore Iron Acquisition System

2004 ◽  
Vol 70 (9) ◽  
pp. 5199-5207 ◽  
Author(s):  
L. Fernández ◽  
I. Márquez ◽  
J. A. Guijarro
Keyword(s):  
Iron Acquisition ◽  
Lethal Dose ◽  
Yersinia Ruckeri ◽  
Secretion Systems ◽  
Regulation Of Expression ◽  
Significant Similarity ◽  
Technology System ◽  
Type Iv ◽  
Genes Encoding

ABSTRACT This work reports the utilization of an in vivo expression technology system to identify in vivo-induced (ivi) genes in Yersinia ruckeri after determination of the conditions needed for its selection in fish. Fourteen clones were selected, and the cloned DNA fragments were analyzed after partial sequencing. In addition to sequences with no significant similarity, homology with genes encoding proteins putatively involved in two-component and type IV secretion systems, adherence, specific metabolic functions, and others were found. Among these sequences, four were involved in iron acquisition through a catechol siderophore (ruckerbactin). Thus, unlike other pathogenic yersiniae producing yersiniabactin, Y. ruckeri might be able to produce and utilize only this phenolate. The genetic organization of the ruckerbactin biosynthetic and uptake loci was similar to that of the Escherichia coli enterobactin gene cluster. Genes rucC and rupG, putative counterparts of E. coli entC and fepG, respectively, involved in the biosynthesis and transport of the iron siderophore complex, respectively, were analyzed further. Thus, regulation of expression by iron and temperature and their presence in other Y. ruckeri siderophore-producing strains were confirmed for these two loci. Moreover, 50% lethal dose values 100-fold higher than those of the wild-type strain were obtained with the rucC isogenic mutant, showing the importance of ruckerbactin in the pathogenesis caused by this microorganism.

scholarly journals Characterization of a Six-Subunit Holo-Elongator Complex Required for the Regulated Expression of a Group of Genes in Saccharomyces cerevisiae

2001 ◽  
Vol 21 (23) ◽  
pp. 8203-8212 ◽  
Author(s):  
Nevan J. Krogan ◽  
Jack F. Greenblatt
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase Ii ◽  
Expression Profiles ◽  
Affinity Purification ◽  
Gene Expression Profiles ◽  
Elongator Complex ◽  
Genes Encoding ◽  
The Individual ◽  
Encoding Genes

ABSTRACT The Elongator complex associated with elongating RNA polymerase II in Saccharomyces cerevisiae was originally reported to have three subunits, Elp1, Elp2, and Elp3. Using the tandem affinity purification (TAP) procedure, we have purified a six-subunit yeast Holo-Elongator complex containing three additional polypeptides, which we have named Elp4, Elp5, and Elp6. TAP tapping and subsequent purification of any one of the six subunits result in the isolation of all six components. Purification of Elongator in higher salt concentrations served to demonstrate that the complex could be separated into two subcomplexes: one consisted of Elp1, -2, and -3, and the other consisted of Elp4, -5, and -6. Deletions of the individual genes encoding the new Elongator subunits showed that only theELP5 gene is essential for growth. Disruption of the two nonessential new Elongator-encoding genes, ELP4 andELP6, caused the same phenotypes observed with knockouts of the original Elongator-encoding genes. Results of microarray analyses demonstrated that the gene expression profiles of strains containing deletions of genes encoding subunits of either Elongator subcomplex, in which we detected significantly altered mRNA expression levels for 96 genes, are very similar, implying that all the Elongator subunits likely function together to regulate a group of S. cerevisiae genes in vivo.

scholarly journals Endocytic recycling and vesicular transport systems mediate transcytosis of Leptospira interrogans across cell monolayer

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Yang Li ◽  
Kai-Xuan Li ◽  
Wei-Lin Hu ◽  
David M Ojcius ◽  
Jia-Qi Fang ◽  
...  
Keyword(s):  
Intracellular Transport ◽  
Vesicular Transport ◽  
Cell Monolayer ◽  
Snare Complex ◽  
Transport Systems ◽  
Leptospira Interrogans ◽  
Renal Tubules ◽  
Internal Organs ◽  
Endocytic Recycling

Many bacterial pathogens can cause septicemia and spread from the bloodstream into internal organs. During leptospirosis, individuals are infected by contact with Leptospira-containing animal urine-contaminated water. The spirochetes invade internal organs after septicemia to cause disease aggravation, but the mechanism of leptospiral excretion and spreading remains unknown. Here, we demonstrated that Leptospira interrogans entered human/mouse endothelial and epithelial cells and fibroblasts by caveolae/integrin-β1-PI3K/FAK-mediated microfilament-dependent endocytosis to form Leptospira (Lep)-vesicles that did not fuse with lysosomes. Lep-vesicles recruited Rab5/Rab11 and Sec/Exo-SNARE proteins in endocytic recycling and vesicular transport systems for intracellular transport and release by SNARE-complex/FAK-mediated microfilament/microtubule-dependent exocytosis. Both intracellular leptospires and infected cells maintained their viability. Leptospiral propagation was only observed in mouse fibroblasts. Our study revealed that L. interrogans utilizes endocytic recycling and vesicular transport systems for transcytosis across endothelial or epithelial barrier in blood vessels or renal tubules, which contributes to spreading in vivo and transmission of leptospirosis.

Consumption of Hageman Factor Activity in the Generalized Shwartzman Reaction Induced by Liquoid

1970 ◽  
Vol 23 (02) ◽  
pp. 386-404 ◽  
Author(s):  
G Müller-Berghaus ◽  
H. G Lasch
Keyword(s):  
Partial Thromboplastin Time ◽  
Lethal Dose ◽  
Control Group ◽  
Factor V ◽  
Consumption Coagulopathy ◽  
Factor Activity ◽  
Hageman Factor ◽  
Intravascular Coagulation ◽  
Shwartzman Reaction

SummaryThe role of Hageman factor in triggering intravascular coagulation has been studied in rabbits injected intravenously with Liquoid. Besides changes of coagulation parameters characteristic of consumption coagulopathy (e.g. decrease in platelet counts, fibrinogen levels, factor V activity), a pronounced drop in Hageman factor activity was observed after injection of Liquoid. Likewise, the partial thromboplastin time became prolonged.The activation of Hageman factor in vivo could be prevented by intravenous infusion of lysozyme. Twenty min after starting the lysozyme infusion, the partial thromboplastin time became prolonged from a mean of 29 sec to 108 sec. Animals infused with lysozyme and injected with a lethal dose of Liquoid did not develop a consumption coagulopathy. In the same manner, none of 10 animals treated with lysozyme developed the generalized Shwartzman reaction, whereas in the control group 19 out of 20 animals showed fibrin thrombi in the glomerular capillaries.From the present study it may be concluded that the intravascular coagulation process after intravenous injection of Liquoid is triggered by Hageman factor activation.

Hepato- and Nephroprotective Effects of the Polyphenol Concentrate From Cabernet Sauvignon Grape Varieties Cultivated in Kazakhstan in the Experimental Model Of CCL4-Induced Toxicity

2017 ◽  
Vol 9 (03) ◽  
Author(s):  
Nurgozhin T. ◽  
Sergazy S. H. ◽  
Adilgozhina G. ◽  
Gulyayev A. ◽  
Shulgau Z. ◽  
...  
Keyword(s):  
Carbon Tetrachloride ◽  
Experimental Model ◽  
Lethal Dose ◽  
Radical Scavenging ◽  
Cabernet Sauvignon ◽  
Intragastric Administration ◽  
Liver And Kidney ◽  
Antioxidant Stress

Objective:This study investigates the hepatoprotective effect and the antioxidant role of polyphenol concentrate in the experimental model of carbon tetrachloride (CCl4) induced toxicity. Methods: Antioxidant activity of Cabernet Sauvignon grape polyphenol were evaluated by radical scavenging of 1,1-diphenyl-2-picryl hydrazyl radical (DPPH), 2,2’-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS.+). In addition, the effects of polyphenol concentrate on the survival of Wistar rats in the toxicity model, was also investigated. The polyphenol concentrate was administered for 5 five days prior to injection of carbon tetrachloride in a sub-lethal dose of 300 mg/kg of animal body weight in order to perform histological examinations of the liver and kidney, and detect the levels of AST, ALT and bilirubin. Results: Administration of polyphenol concentrate increased animal survival in the experimental model. Moreover, the intragastric administration of polyphenol concentrate prior to the initiation of the experimental model of toxicity, which was caused by a sub-lethal CCl4 dose, reduced morphological injuries in the liver and kidney, decreased the AST and ALT levels of the blood serum. Discussion and conclusion: Our data demonstrate that polyphenol concentrate possesses an antioxidant potential both in vitro and in vivo by reducing antioxidant stress that was caused by CCl4 administration into rats.

The Anti-tumor Activity and Mechanisms of rLj-RGD3 on Human Laryngeal Squamous Carcinoma Hep2 Cells

2020 ◽  
Vol 19 (17) ◽  
pp. 2108-2119
Author(s):  
Yang Jin ◽  
Li Lv ◽  
Shu-Xiang Ning ◽  
Ji-Hong Wang ◽  
Rong Xiao
Keyword(s):  
Wound Healing ◽  
Western Blot ◽  
Morphological Changes ◽  
In Vivo Studies ◽  
Migration And Invasion ◽  
Tunel Staining ◽  
Dna Ladder ◽  
Western Blot Assay

Background: Laryngeal Squamous Cell Carcinoma (LSCC) is a malignant epithelial tumor with poor prognosis and its incidence rate increased recently. rLj-RGD3, a recombinant protein cloned from the buccal gland of Lampetra japonica, contains three RGD motifs that could bind to integrins on the tumor cells. Methods: MTT assay was used to detect the inhibitory rate of viability. Giemsa’s staining assay was used to observe the morphological changes of cells. Hoechst 33258 and TUNEL staining assay, DNA ladder assay were used to examine the apoptotic. Western blot assay was applied to detect the change of the integrin signal pathway. Wound-healing assay, migration, and invasion assay were used to detect the mobility of Hep2 cells. H&E staining assay was used to show the arrangement of the Hep2 cells in the solid tumor tissues. Results: In the present study, rLj-RGD3 was shown to inhibit the viability of LSCC Hep2 cells in vitro by inducing apoptosis with an IC50 of 1.23µM. Western blot showed that the apoptosis of Hep2 cells induced by rLj- RGD3 was dependent on the integrin-FAK-Akt pathway. Wound healing, transwells, and western blot assays in vitro showed that rLj-RGD3 suppressed the migration and invasion of Hep2 cells by integrin-FAKpaxillin/ PLC pathway which could also affect the cytoskeleton arrangement in Hep2 cells. In in vivo studies, rLj-RGD3 inhibited the growth, tumor volume, and weight, as well as disturbed the tissue structure of the solid tumors in xenograft models of BALB/c nude mice without reducing their body weights. Conclusion: hese results suggested that rLj-RGD3 is an effective and safe suppressor on the growth and metastasis of LSCC Hep2 cells from both in vitro and in vivo experiments. rLj-RGD3 might be expected to become a novel anti-tumor drug to treat LSCC patients in the near future.

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