scholarly journals Molecular and Functional Characterization of a ToxR-Regulated Lipoprotein from a Clinical Isolate of Aeromonas hydrophila

2006 ◽  
Vol 74 (7) ◽  
pp. 3742-3755 ◽  
Author(s):  
Lakshmi Pillai ◽  
Jian Sha ◽  
Tatiana E. Erova ◽  
Amin A. Fadl ◽  
Bijay K. Khajanchi ◽  
...  
Keyword(s):  
Virulence Factor ◽  
Aeromonas Hydrophila ◽  
Functional Characterization ◽  
Affinity Column ◽  
Serum Resistance ◽  
Classical Pathway ◽  
Dependent Manner ◽  
T7 Promoter

ABSTRACT Human diseases caused by species of Aeromonas have been classified into two major groups: septicemia and gastroenteritis. In this study, we reported the molecular and functional characterization of a new virulence factor, ToxR-regulated lipoprotein, or TagA, from a diarrheal isolate, SSU, of Aeromonas hydrophila. The tagA gene of A. hydrophila exhibited 60% identity with that of a recently identified stcE gene from Escherichia coli O157:H7, which encoded a protein (StcE) that provided serum resistance to the bacterium and prevented erythrocyte lysis by controlling classical pathway of complement activation by cleaving the complement C1-esterase inhibitor (C1-INH). We purified A. hydrophila TagA as a histidine-tagged fusion protein (rTagA) from E. coli DE3 strain using a T7 promoter-based pET30 expression vector and nickel affinity column chromatography. rTagA cleaved C1-INH in a time-dependent manner. The tagA isogenic mutant of A. hydrophila, unlike its corresponding wild-type (WT) or the complemented strain, was unable to cleave C1-INH, which is required to potentiate the C1-INH-mediated lysis of host and bacterial cells. We indeed demonstrated colocalization of C1-INH and TagA on the bacterial surface by confocal fluorescence microscopy, which ultimately resulted in increased serum resistance of the WT bacterium. Likewise, we delineated the role of TagA in contributing to the enhanced ability of C1-INH to inhibit the classical complement-mediated lysis of erythrocytes. Importantly, we provided evidence that the tagA mutant was significantly less virulent in a mouse model of infection (60%) than the WT bacterium at two 50% lethal doses, which resulted in 100% mortality within 48 h. Taken together, our data provided new information on the role of TagA as a virulence factor in bacterial pathogenesis. This is the first report of TagA characterization from any species of Aeromonas.

scholarly journals Role of GntR Family Regulatory Gene SCO1678 in Gluconate Metabolism in Streptomyces coelicolor M145

2017 ◽  
Vol 2017 ◽  
pp. 1-9 ◽  
Author(s):  
Olga Tsypik ◽  
Roman Makitrynskyy ◽  
Agnieszka Bera ◽  
Lijiang Song ◽  
Wolfgang Wohlleben ◽  
...  
Keyword(s):  
Antibiotic Production ◽  
Streptomyces Coelicolor ◽  
Regulatory Gene ◽  
Functional Characterization ◽  
Bioinformatic Analysis ◽  
Transcriptional Analysis ◽  
Dependent Manner ◽  
Putative Operon

Here we report functional characterization of the Streptomyces coelicolor M145 gene SCO1678, which encodes a GntR-like regulator of the FadR subfamily. Bioinformatic analysis suggested that SCO1678 is part of putative operon (gnt) involved in gluconate metabolism. Combining the results of SCO1678 knockout, transcriptional analysis of gnt operon, and Sco1678 protein-DNA electromobility shift assays, we established that Sco1678 protein controls the gluconate operon. It does so via repression of its transcription from a single promoter located between genes SCO1678 and SCO1679. The knockout also influenced, in a medium-dependent manner, the production of secondary metabolites by S. coelicolor. In comparison to the wild type, on gluconate-containing minimal medium, the SCO1678 mutant produced much less actinorhodin and accumulated a yellow-colored pigment, likely to be the cryptic polyketide coelimycin. Possible links between gluconate metabolism and antibiotic production are discussed.

scholarly journals Functional Characterization of LcpA, a Surface-Exposed Protein of Leptospira spp. That Binds the Human Complement Regulator C4BP

2010 ◽  
Vol 78 (7) ◽  
pp. 3207-3216 ◽  
Author(s):  
Angela S. Barbosa ◽  
Denize Monaris ◽  
Ludmila B. Silva ◽  
Zenaide M. Morais ◽  
Sílvio A. Vasconcellos ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Functional Characterization ◽  
Lectin Pathway ◽  
Classical Pathway ◽  
Dependent Manner ◽  
Human Complement ◽  
Cofactor Activity ◽  
Complement Regulator

ABSTRACT We have previously shown that pathogenic leptospiral strains are able to bind C4b binding protein (C4BP). Surface-bound C4BP retains its cofactor activity, indicating that acquisition of this complement regulator may contribute to leptospiral serum resistance. In the present study, the abilities of seven recombinant putative leptospiral outer membrane proteins to interact with C4BP were evaluated. The protein encoded by LIC11947 interacted with this human complement regulator in a dose-dependent manner. The cofactor activity of C4BP bound to immobilized recombinant LIC11947 (rLIC11947) was confirmed by detecting factor I-mediated cleavage of C4b. rLIC11947 was therefore named LcpA (for leptospiral complement regulator-acquiring protein A). LcpA was shown to be an outer membrane protein by using immunoelectron microscopy, cell surface proteolysis, and Triton X-114 fractionation. The gene coding for LcpA is conserved among pathogenic leptospiral strains. This is the first characterization of a Leptospira surface protein that binds to the human complement regulator C4BP in a manner that allows this important regulator to control complement system activation mediated either by the classical pathway or by the lectin pathway. This newly identified protein may play a role in immune evasion by Leptospira spp. and may therefore represent a target for the development of a human vaccine against leptospirosis.

scholarly journals Functional Characterization of Spliceosomal Introns and Identification of U2, U4, and U5 snRNAs in the Deep-Branching Eukaryote Entamoeba histolytica

2007 ◽  
Vol 6 (6) ◽  
pp. 940-948 ◽  
Author(s):  
Carrie A. Davis ◽  
Michael P. S. Brown ◽  
Upinder Singh
Keyword(s):  
Entamoeba Histolytica ◽  
Splice Site ◽  
Expressed Sequence Tag ◽  
Functional Characterization ◽  
Molecular Evidence ◽  
Spliceosomal Introns ◽  
Accurate Expression ◽  
Eukaryotic Organisms

ABSTRACT Pre-mRNA splicing is essential to ensure accurate expression of many genes in eukaryotic organisms. In Entamoeba histolytica, a deep-branching eukaryote, approximately 30% of the annotated genes are predicted to contain introns; however, the accuracy of these predictions has not been tested. In this study, we mined an expressed sequence tag (EST) library representing 7% of amoebic genes and found evidence supporting splicing of 60% of the testable intron predictions, the majority of which contain a GUUUGU 5′ splice site and a UAG 3′ splice site. Additionally, we identified several splice site misannotations, evidence for the existence of 30 novel introns in previously annotated genes, and identified novel genes through uncovering their spliced ESTs. Finally, we provided molecular evidence for the E. histolytica U2, U4, and U5 snRNAs. These data lay the foundation for further dissection of the role of RNA processing in E. histolytica gene expression.

Functional analysis of a stable transcription arrest site in the first intron of the murine adenosine deaminase gene

1993 ◽  
Vol 13 (5) ◽  
pp. 2718-2729
Author(s):  
S F Kash ◽  
J W Innis ◽  
A U Jackson ◽  
R E Kellems
Keyword(s):  
Rna Polymerase Ii ◽  
Adenosine Deaminase ◽  
Functional Characterization ◽  
Gel Filtration ◽  
Point Mutations ◽  
Elongation Factor ◽  
Dependent Manner ◽  
First Intron ◽  
Intron 1

Transcription arrest plays a role in regulating the expression of a number of genes, including the murine adenosine deaminase (ADA) gene. We have previously identified two prominent arrest sites at the 5' end of the ADA gene: one in the first exon and one in the first intron (J. W. Innis and R. E. Kellems, Mol. Cell. Biol. 11:5398-5409, 1991). Here we report the functional characterization of the intron 1 arrest site, located 137 to 145 nucleotides downstream of the cap site. We have determined, using gel filtration, that the intron 1 arrest site is a stable RNA polymerase II pause site and that the transcription elongation factor SII promotes read-through at this site. Additionally, the sequence determinants for the pause are located within a 37-bp fragment encompassing this site (+123 to +158) and can direct transcription arrest in an orientation-dependent manner in the context of the ADA and adenovirus major late promoters. Specific point mutations in this region increase or decrease the relative pausing efficiency. We also show that the sequence determinants for transcription arrest can function when placed an additional 104 bp downstream of their natural position.

scholarly journals Schistosome tegumental ecto-apyrase (SmATPDase1) degrades exogenous pro-inflammatory and pro-thrombotic nucleotides.

2014 ◽  
Author(s):  
Akram A Da'dara ◽  
Rita Bhardwaj ◽  
Yasser MB Ali ◽  
Patrick Skelly
Keyword(s):  
Thrombus Formation ◽  
Functional Characterization ◽  
Host Cells ◽  
Dependent Manner ◽  
Host Immune Responses ◽  
Genes Encoding ◽  
Molecular Patterns ◽  
Parasitic Worms ◽  
Damage Associated Molecular Patterns

Schistosomes are parasitic worms that can survive in the hostile environment of the human bloodstream where they appear refractory to both immune elimination and thrombus formation. We hypothesize that parasite migration in the bloodstream can stress the vascular endothelium causing this tissue to release chemicals alerting responsive host cells to the stress. Such chemicals are called damage associated molecular patterns (DAMPs) and among the most potent is the proinflammatory mediator, adenosine triphosphate (ATP). Furthermore, the ATP derivative ADP is a pro-thrombotic molecule that acts as a strong activator of platelets. Schistosomes are reported to possess at their host interactive tegumental surface a series of enzymes that could, like their homologs in mammals, degrade extracellular ATP and ADP. These are alkaline phosphatase (SmAP), phosphodiesterase (SmNPP-5) and ATP diphosphohydrolase (SmATPDase1). In this work we employ RNAi to knock down expression of the genes encoding these enzymes in the intravascular life stages of the parasite. We then compare the abilities of these parasites to degrade exogenously added ATP and ADP. . We find that only SmATPDase1-suppressed parasites are significantly impaired in their ability to degrade these nucleotides. Suppression of SmAP or SmNPP-5 does not appreciably affect the worms’ ability to catabolize ATP or ADP. These findings are confirmed by the functional characterization of the enzymatically active, full-length recombinant SmATPDase1 expressed in CHO-S cells. The enzyme is a true apyrase; SmATPDase1 degrades ATP and ADP in a cation dependent manner. Optimal activity is seen at alkaline pH. The Km of SmATPDase1 for ATP is 0.4 ±0.02 mM and for ADP, 0.252 ± 0.02 mM. The results confirm the role of tegumental SmATPDase1 in the degradation of the exogenous pro-inflammatory and pro-thrombotic nucleotides ATP and ADP by live intravascular stages of the parasite. By degrading host inflammatory signals like ATP, and pro-thrombotic signals like ADP, these parasite enzymes may minimize host immune responses, inhibit blood coagulation and promote schistosome survival.)

scholarly journals Identification of an isoflavonoid transporter required for the nodule establishment of the Rhizobium-Fabaceae symbiotic interaction

2021 ◽  
Author(s):  
Wanda Biala-Leonhard ◽  
Laura Zanin ◽  
Stefano Gottardi ◽  
Rita de Brito Francisco ◽  
Silvia Venuti ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Crop Productivity ◽  
Signaling Cascades ◽  
Nutritional Deficiencies ◽  
White Lupin ◽  
Symbiotic Interaction ◽  
Phosphate Availability ◽  
Rhizobial Symbiosis

Nitrogen (N) as well as Phosphorus (P) are key nutrients determining crop productivity. Legumes have developed strategies to overcome nutrient limitation by e.g., forming a symbiotic relationship with N-fixing rhizobia and the release of P-mobilizing exudates and are thus able to grow without supply of N or P fertilizers. The legume-rhizobial symbiosis starts with root release of isoflavonoids, that act as signaling molecules perceived by compatible bacteria. Subsequently, bacteria release nod factors, which induce signaling cascades allowing the formation of functional N-fixing nodules. We report here the identification and functional characterization of a plasma membrane-localized MATE-type transporter (LaMATE2) involved in the release of genistein from white lupin roots. The LaMATE2 expression in the root is upregulated under N deficiency as well as low phosphate availability, two nutritional deficiencies that induce the release of this isoflavonoid. LaMATE2 silencing reduced genistein efflux and even more the formation of symbiotic nodules, supporting the crucial role of LaMATE2 in isoflavonoid release and nodulation. Furthermore, silencing of LaMATE2 limited the P-solubilization activity of lupin root exudates. Transport assays in yeast vesicles demonstrated that LaMATE2 acts as a proton-driven isoflavonoid transporter.

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