scholarly journals Clonamiento y expresión de la proteína LipL21 desde Leptospira interrogans serovar canicola aislado en Chile

2020 ◽  
Vol 4 (1) ◽  
Author(s):  
Daniel A. Soto ◽  
Paula Palma ◽  
Patricia González ◽  
Jesús Badilla ◽  
Guillemo Muñoz ◽  
...  
Keyword(s):  
Leptospira Interrogans ◽  
E Coli ◽  
Santiago De Chile ◽  
Hanta Virus

Leptospira es el agente etiológico de la leptospirosis, que es una zoonosis bacteriana distribuida en todo el mundo. Las infecciones zoonóticas en humanos con Leptospiras son un importante problema de salud pública en los países en desarrollo. Se están desarrollando vacunas y metodologías de detección de Leptospira sp, donde proteínas con un alto nivel de conservación en su secuencia de aminoácidos se están utilizando y representan una buena alternativa. La proteína LipL21 se ha descrito como un buen blanco para estos objetivos. Aquí se describe la purificación de la proteína LipL21 recombinante. Purificamos el ADN cromosómico de Leptospira interrogans serovar Canicola aislado en Santiago de Chile. Utilizamos una reacción en cadena de la polimerasa para la amplificación del gen lipl21 y se clonó en el plásmido pET21a y la proteína recombinante se expresó en cultivo bacteriano E. coli BL21 (DE3). La proteína LipL21 recombinante se purificó en un alto grado pureza. Nuestra proteína LipL21 recombinante podría usarse en el desarrollo de pruebas inmunológicas para la detección de IgM e IgG en sueros humanos, considerando que es una enfermedad zoonótica, que además tiene diagnóstico diferencial con Hanta virus.

scholarly journals Oral Immunization with Escherichia coli Expressing a Lipidated Form of LigA Protects Hamsters against Challenge with Leptospira interrogans Serovar Copenhageni

2013 ◽  
Vol 82 (2) ◽  
pp. 893-902 ◽  
Author(s):  
Kristel Lourdault ◽  
Long-Chieh Wang ◽  
Ana Vieira ◽  
James Matsunaga ◽  
Rita Melo ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Oral Vaccine ◽  
Oral Immunization ◽  
Reservoir Host ◽  
Leptospira Interrogans ◽  
Renal Tubules ◽  
Hamster Model ◽  
Content Type ◽  
E Coli ◽  
Humoral Immune

ABSTRACTLeptospirosis is a potentially fatal zoonosis transmitted by reservoir host animals that harbor leptospires in their renal tubules and shed the bacteria in their urine.Leptospira interrogansserovar Copenhageni transmitted fromRattus norvegicusto humans is the most prevalent cause of urban leptospirosis. We examinedL. interrogansLigA, domains 7 to 13 (LigA7-13), as an oral vaccine delivered byEscherichia colias a lipidated, membrane-associated protein. The efficacy of the vaccine was evaluated in a susceptible hamster model in terms of the humoral immune response and survival from leptospiral challenge. Four weeks of oral administration of liveE. coliexpressing LigA7-13 improved survival from intraperitoneal (i.p.) and intradermal (i.d.) challenge byL. interrogansserovar Copenhageni strain Fiocruz L1-130 in Golden Syrian hamsters. Immunization withE. coliexpressing LigA7-13 resulted in a systemic antibody response, and a significant LigA7-13 IgG level after the first 2 weeks of immunization was completely predictive of survival 28 days after challenge. As in previous LigA vaccine studies, all immunized hamsters that survived infection had renal leptospiral colonization and histopathological changes. In summary, an oral LigA-based vaccine improved survival from leptospiral challenge by either the i.p. or i.d. route.

scholarly journals Identification of σE-Dependent Promoter Upstream of clpB from the Pathogenic Spirochaete Leptospira interrogans by Applying an E. coli Two-Plasmid System

2019 ◽  
Vol 20 (24) ◽  
pp. 6325
Author(s):  
Sabina Kędzierska-Mieszkowska ◽  
Katarzyna Potrykus ◽  
Zbigniew Arent ◽  
Joanna Krajewska
Keyword(s):  
Gene Expression ◽  
Gene Expression Regulation ◽  
Transcriptional Start Site ◽  
Leptospira Interrogans ◽  
Start Site ◽  
Limited Information ◽  
E Coli ◽  
Oxidative Stresses ◽  
Genetic Mechanisms ◽  
Site Identification

There is limited information on gene expression in the pathogenic spirochaete Leptospira interrogans and genetic mechanisms controlling its virulence. Transcription is the first step in gene expression that is often determined by environmental effects, including infection-induced stresses. Alterations in the environment result in significant changes in the transcription of many genes, allowing effective adaptation of Leptospira to mammalian hosts. Thus, promoter and transcriptional start site identification are crucial for determining gene expression regulation and for the understanding of genetic regulatory mechanisms existing in Leptospira. Here, we characterized the promoter region of the L. interrogans clpB gene (clpBLi) encoding an AAA+ molecular chaperone ClpB essential for the survival of this spirochaete under thermal and oxidative stresses, and also during infection of the host. Primer extension analysis demonstrated that transcription of clpB in L. interrogans initiates at a cytidine located 41 bp upstream of the ATG initiation codon, and, to a lesser extent, at an adenine located 2 bp downstream of the identified site. Transcription of both transcripts was heat-inducible. Determination of clpBLi transcription start site, combined with promoter transcriptional activity assays using a modified two-plasmid system in E. coli, revealed that clpBLi transcription is controlled by the ECF σE factor. Of the ten L. interrogans ECF σ factors, the factor encoded by LIC_12757 (LA0876) is most likely to be the key regulator of clpB gene expression in Leptospira cells, especially under thermal stress. Furthermore, clpB expression may be mediated by ppGpp in Leptospira.

scholarly journals Isoleucine Biosynthesis in Leptospira interrogans Serotype lai Strain 56601 Proceeds via a Threonine-Independent Pathway

2004 ◽  
Vol 186 (16) ◽  
pp. 5400-5409 ◽  
Author(s):  
Hai Xu ◽  
Yuzhen Zhang ◽  
Xiaokui Guo ◽  
Shuangxi Ren ◽  
Andreas A. Staempfli ◽  
...  
Keyword(s):  
Alternative Pathway ◽  
Temperature Sensitive ◽  
Leptospira Interrogans ◽  
Mrna Levels ◽  
Protein Coding ◽  
Isopropylmalate Synthase ◽  
E Coli ◽  
Isoleucine Biosynthesis

ABSTRACT Three leuA-like protein-coding sequences were identified in Leptospira interrogans. One of these, the cimA gene, was shown to encode citramalate synthase (EC 4.1.3.-). The other two encoded α-isopropylmalate synthase (EC 4.1.3.12). Expressed in Escherichia coli, the citramalate synthase was purified and characterized. Although its activity was relatively low, it was strictly specific for pyruvate as the keto acid substrate. Unlike the citramalate synthase of the thermophile Methanococcus jannaschii, the L. interrogans enzyme is temperature sensitive but exhibits a much lower Km (0.04 mM) for pyruvate. The reaction product was characterized as (R)-citramalate, and the proposed β-methyl-d-malate pathway was further confirmed by demonstrating that citraconate was the substrate for the following reaction. This alternative pathway for isoleucine biosynthesis from pyruvate was analyzed both in vitro by assays of leptospiral isopropylmalate isomerase (EC 4.2.1.33) and β-isopropylmalate dehydrogenase (EC 1.1.1.85) in E. coli extracts bearing the corresponding clones and in vivo by complementation of E. coli ilvA, leuC/D, and leuB mutants. Thus, the existence of a leucine-like pathway for isoleucine biosynthesis in L. interrogans under physiological conditions was unequivocally proven. Significant variations in either the enzymatic activities or mRNA levels of the cimA and leuA genes were detected in L. interrogans grown on minimal medium supplemented with different levels of the corresponding amino acids or in cells grown on serum-containing rich medium. The similarity of this metabolic pathway in leptospires and archaea is consistent with the evolutionarily primitive status of the eubacterial spirochetes.

scholarly journals Leptospira interrogans triosephosphate isomerase: exploring the structural determinants of stability, high reaction rate and specificity.

2020 ◽  
Author(s):  
VIDHI PAREEK ◽  
Vaigundan Dhayabarn ◽  
Hemalatha Balaram ◽  
Patnam R Krishnaswamy
Keyword(s):  
Metabolic Flux ◽  
Triosephosphate Isomerase ◽  
Guanidine Hydrochloride ◽  
Decisive Role ◽  
Zero Point ◽  
Effect Of Temperature ◽  
Leptospira Interrogans ◽  
Structural Determinants ◽  
E Coli ◽  
Transition State Stabilization

Leptospires are zoonotic pathogens that cause significant socio-economic burden in developing countries, world-wide. The pathogenic species Leptospira interrogans (Li) is an important and interesting target for investigating the enzymes essential to its metabolic needs and adaptations. We cloned and expressed triosephosphate isomerase (LiTIM), a central metabolic flux regulator of Li, in AA200, E. coli TIM null strain. LiTIM was obtained as an active dimer (D-GAP to DHAP, kcat = 1740 /s and Km (D-GAP) = 0.21 mM, at 25 C) with mid-transition concentrations, Cm, 0.8 mM and 2.6 mM, respectively, for guanidine hydrochloride and urea induced equilibrium unfolding. We report the high resolution X-ray structures of LiTIM in apo and substrate (DHAP) bound forms. Our analysis highlights key features of TIM that regulate the mode of substrate binding and transition state stabilization and thus play a decisive role in attainment of high proficiency for the isomerisation reaction while avoiding the elimination reaction. Unexpected differences in the effect of temperature on stability and activity were observed for the three mesophilic pathogenic TIMs viz. from Li, Plasmodium falciparum (Pf) and Trypanosoma brucei (Tb). LiTIM and TbTIM (Tm = 46.5 C) were more susceptible to unfolding and precipitation compared to PfTIM (Tm = 67 C). In contrast, the initial (or zero point) activity of PfTIM rises till 50 C and saturates unlike LiTIM and TbTIM which show a rise till 55 C and 60 C, respectively. These observations could be rationalized by sequence comparison and examination of the structures of the three TIMs.

scholarly journals Identification of a Novel Prophage-Like Gene Cluster Actively Expressed in Both Virulent and Avirulent Strains of Leptospira interrogans Serovar Lai

2008 ◽  
Vol 76 (6) ◽  
pp. 2411-2419 ◽  
Author(s):  
Jin-Hong Qin ◽  
Qing Zhang ◽  
Zhi-Ming Zhang ◽  
Yi Zhong ◽  
Yang Yang ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Dna Sequences ◽  
Transcription Initiation ◽  
Gene Annotation ◽  
Specific Binding ◽  
Expression Profiles ◽  
Regulatory Sequences ◽  
Leptospira Interrogans ◽  
E Coli

ABSTRACT DNA microarray analysis was used to compare the differential gene expression profiles between Leptospira interrogans serovar Lai type strain 56601 and its corresponding attenuated strain IPAV. A 22-kb genomic island covering a cluster of 34 genes (i.e., genes LA0186 to LA0219) was actively expressed in both strains but concomitantly upregulated in strain 56601 in contrast to that of IPAV. Reverse transcription-PCR assays proved that the gene cluster comprised five transcripts. Gene annotation of this cluster revealed characteristics of a putative prophage-like remnant with at least 8 of 34 sequences encoding prophage-like proteins, of which the LA0195 protein is probably a putative prophage CI-like regulator. The transcription initiation activities of putative promoter-regulatory sequences of transcripts I, II, and III, all proximal to the LA0195 gene, were further analyzed in the Escherichia coli promoter probe vector pKK232-8 by assaying the reporter chloramphenicol acetyltransferase (CAT) activities. The strong promoter activities of both transcripts I and II indicated by the E. coli CAT assay were well correlated with the in vitro sequence-specific binding of the recombinant LA0195 protein to the corresponding promoter probes detected by the electrophoresis mobility shift assay. On the other hand, the promoter activity of transcript III was very low in E. coli and failed to show active binding to the LA0195 protein in vitro. These results suggested that the LA0195 protein is likely involved in the transcription of transcripts I and II. However, the identical complete DNA sequences of this prophage remnant from these two strains strongly suggests that possible regulatory factors or signal transduction systems residing outside of this region within the genome may be responsible for the differential expression profiling in these two strains.

scholarly journals Penicillin-Binding Proteins inLeptospira interrogans

2001 ◽  
Vol 45 (3) ◽  
pp. 870-877 ◽  
Author(s):  
Audrey Brenot ◽  
Daren Trott ◽  
Isabelle Saint Girons ◽  
Richard Zuerner
Keyword(s):  
Escherichia Coli ◽  
Binding Proteins ◽  
Sequence Variation ◽  
Protein Sequences ◽  
Leptospira Interrogans ◽  
Homologous Proteins ◽  
Penicillin Binding Proteins ◽  
E Coli

ABSTRACT The Leptospira interrogans ponA andpbpB genes were isolated and characterized.ponA and pbpB encode the penicillin-binding proteins (PBPs) 1 and 3, respectively. There is little sequence variation between the PBP genes from two L. interrogans strains (serovar icterohaemorrhagiae strain Verdun and serovar pomona strain RZ11). The deduced L. interrogans PBP 1 and PBP 3 protein sequences from the two strains shared over 50% similarity to homologous proteins fromEscherichia coli. It was demonstrated for strain Verdun that ponA and pbpB are transcribed individually from their own promoter. The ponA andpbpB genes from both strains are separated by 8 to 10 kb and oriented such that their transcription is convergent. The L. interrogans PBP 1 and PBP 3 proteins were synthesized inE. coli and were modified with ampicillin using a digoxigenin-ampicillin conjugate. These data show that both genes encode functional PBPs.

Endotoxin Alteration of the Mucopolysaccharide Blood Vessel Coating in Rats

1974 ◽  
Vol 32 ◽  
pp. 148-149
Author(s):  
D. E. Philpott ◽  
A. Takahashi
Keyword(s):  
Skeletal Muscle ◽  
Endothelial Cells ◽  
Salivary Gland ◽  
Carotid Artery ◽  
Basement Membrane ◽  
Coronary Vessels ◽  
Ruthenium Red ◽  
E Coli ◽  
Old Rats ◽  
The Brain

Two month, eight month and two year old rats were treated with 10 or 20 mg/kg of E. Coli endotoxin I. P. The eight month old rats proved most resistant to the endotoxin. During fixation the aorta, carotid artery, basil arartery of the brain, coronary vessels of the heart, inner surfaces of the heart chambers, heart and skeletal muscle, lung, liver, kidney, spleen, brain, retina, trachae, intestine, salivary gland, adrenal gland and gingiva were treated with ruthenium red or alcian blue to preserve the mucopolysaccharide (MPS) coating. Five, 8 and 24 hrs of endotoxin treatment produced increasingly marked capillary damage, disappearance of the MPS coating, edema, destruction of endothelial cells and damage to the basement membrane in the liver, kidney and lung.

Ribosome Structural Organization

1974 ◽  
Vol 32 ◽  
pp. 332-333
Author(s):  
James A. Lake
Keyword(s):  
Ribosomal Proteins ◽  
Structural Organization ◽  
Three Dimensional ◽  
Small Subunit ◽  
Structural Studies ◽  
X Ray Diffraction ◽  
Specific Antibodies ◽  
X Ray ◽  
E Coli ◽  
Complete Sequences

The understanding of ribosome structure has advanced considerably in the last several years. Biochemists have characterized the constituent proteins and rRNA's of ribosomes. Complete sequences have been determined for some ribosomal proteins and specific antibodies have been prepared against all E. coli small subunit proteins. In addition, a number of naturally occuring systems of three dimensional ribosome crystals which are suitable for structural studies have been observed in eukaryotes. Although the crystals are, in general, too small for X-ray diffraction, their size is ideal for electron microscopy.

Sites of Adsorption of the Bacteriophages T3 and T5 on the Wall of Escherichia Coli B.

1967 ◽  
Vol 25 ◽  
pp. 96-97
Author(s):  
Manfred E. Bayer
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Electron Microscope ◽  
Uranyl Acetate ◽  
E Coli ◽  
Receptor Sites ◽  
Rigid Cell Wall ◽  
Host Cell Surface ◽  
Bacterial Viruses ◽  
Escherichia Coli B

Bacterial viruses adsorb specifically to receptors on the host cell surface. Although the chemical composition of some of the cell wall receptors for bacteriophages of the T-series has been described and the number of receptor sites has been estimated to be 150 to 300 per E. coli cell, the localization of the sites on the bacterial wall has been unknown.When logarithmically growing cells of E. coli are transferred into a medium containing 20% sucrose, the cells plasmolize: the protoplast shrinks and becomes separated from the somewhat rigid cell wall. When these cells are fixed in 8% Formaldehyde, post-fixed in OsO4/uranyl acetate, embedded in Vestopal W, then cut in an ultramicrotome and observed with the electron microscope, the separation of protoplast and wall becomes clearly visible, (Fig. 1, 2). At a number of locations however, the protoplasmic membrane adheres to the wall even under the considerable pull of the shrinking protoplast. Thus numerous connecting bridges are maintained between protoplast and cell wall. Estimations of the total number of such wall/membrane associations yield a number of about 300 per cell.

Emigration of neutrophils in the central nervous system

1983 ◽  
Vol 41 ◽  
pp. 788-789
Author(s):  
John L.Beggs ◽  
John D. Waggener ◽  
Wanda Miller ◽  
Jane Watkins
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Osmium Tetroxide ◽  
White Blood Cells ◽  
Arterial Perfusion ◽  
E Coli ◽  
Intracisternal Injection ◽  
The Central Nervous System ◽  
Brain And Spinal Cord ◽  
Interendothelial Junctions

Studies using mesenteric and ear chamber preparations have shown that interendothelial junctions provide the route for neutrophil emigration during inflammation. The term emigration refers to the passage of white blood cells across the endothelium from the vascular lumen. Although the precise pathway of transendo- thelial emigration in the central nervous system (CNS) has not been resolved, the presence of different physiological and morphological (tight junctions) properties of CNS endothelium may dictate alternate emigration pathways.To study neutrophil emigration in the CNS, we induced meningitis in guinea pigs by intracisternal injection of E. coli bacteria.In this model, leptomeningeal inflammation is well developed by 3 hr. After 3 1/2 hr, animals were sacrificed by arterial perfusion with 3% phosphate buffered glutaraldehyde. Tissues from brain and spinal cord were post-fixed in 1% osmium tetroxide, dehydrated in alcohols and propylene oxide, and embedded in Epon. Thin serial sections were cut with diamond knives and examined in a Philips 300 electron microscope.

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