scholarly journals Identification and Functional Characterization of the Novel Edwardsiella tarda Effector EseJ

2015 ◽  
Vol 83 (4) ◽  
pp. 1650-1660 ◽  
Author(s):  
Hai-Xia Xie ◽  
Jin-Fang Lu ◽  
Ying Zhou ◽  
Jia Yi ◽  
Xiu-Jun Yu ◽  
...  
Keyword(s):  
Mutant Strain ◽  
Bacterial Adhesion ◽  
Sequence Similarity ◽  
Functional Characterization ◽  
Lethal Dose ◽  
Edwardsiella Tarda ◽  
Bacterial Cells ◽  
Wild Type ◽  
High Sequence Similarity ◽  
Content Type

Edwardsiella tardais a Gram-negative enteric pathogen that causes hemorrhagic septicemia in fish and gastro- and extraintestinal infections in humans. The type III secretion system (T3SS) ofE. tardahas been identified as a key virulence factor that contributes to pathogenesis in fish. However, little is known about the associated effectors translocated by this T3SS. In this study, by comparing the profile of secreted proteins of the wild-type PPD130/91 and its T3SS ATPase ΔesaNmutant, we identified a new effector by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry. This effector consists of 1,359 amino acids, sharing high sequence similarity with Orf29/30 ofE. tardastrain EIB202, and is renamed EseJ. The secretion and translocation of EseJ depend on the T3SS. A ΔeseJmutant strain adheres to epithelioma papillosum of carp (EPC) cells 3 to 5 times more extensively than the wild-type strain does. EseJ inhibits bacterial adhesion to EPC cells from within bacterial cells. Importantly, the ΔeseJmutant strain does not replicate efficiently in EPC cells and fails to replicate in J774A.1 macrophages. In infected J774A.1 macrophages, the ΔeseJmutant elicits higher production of reactive oxygen species than wild-typeE. tarda. The replication defect is consistent with the attenuation of the ΔeseJmutant in the blue gourami fish model: the 50% lethal dose (LD50) of the ΔeseJmutant is 2.34 times greater than that of the wild type, and the ΔeseJmutant is less competitive than the wild type in mixed infection. Thus, EseJ represents a novel effector that contributes to virulence by reducing bacterial adhesion to EPC cells and facilitating intracellular bacterial replication.

scholarly journals Edwardsiella tarda EscE (Orf13 Protein) Is a Type III Secretion System-Secreted Protein That Is Required for the Injection of Effectors, Secretion of Translocators, and Pathogenesis in Fish

2015 ◽  
Vol 84 (1) ◽  
pp. 2-10 ◽  
Author(s):  
Jin Fang Lu ◽  
Wei Na Wang ◽  
Gai Ling Wang ◽  
He Zhang ◽  
Ying Zhou ◽  
...  
Keyword(s):  
Type Iii Secretion ◽  
Bacterial Species ◽  
Lethal Dose ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Edwardsiella Tarda ◽  
Dependent Manner ◽  
Wild Type ◽  
Content Type ◽  
Type Iii

The type III secretion system (T3SS) ofEdwardsiella tardais crucial for its intracellular survival and pathogenesis in fish. Theorf13gene (escE) ofE. tardais located 84 nucleotides (nt) upstream ofesrCin the T3SS gene cluster. We found that EscE is secreted and translocated in a T3SS-dependent manner and that amino acids 2 to 15 in the N terminus were required for a completely functional T3SS inE. tarda. Deletion ofescEabolished the secretion of T3SS translocators, as well as the secretion and translocation of T3SS effectors, but did not influence their intracellular protein levels inE. tarda. Complementation of theescEmutant with a secretion-incompetent EscE derivative restored the secretion of translocators and effectors. Interestingly, the effectors that were secreted and translocated were positively correlated with the EscE protein level inE. tarda. TheescEmutant was attenuated in the blue gourami fish infection model, as its 50% lethal dose (LD50) increased to 4 times that of the wild type. The survival rate of theescEmutant-strain-infected fish was 69%, which was much higher than that of the fish infected with the wild-type bacteria (6%). Overall, EscE represents a secreted T3SS regulator that controls effector injection and translocator secretion, thus contributing toE. tardapathogenesis in fish. The homology of EscE within the T3SSs of other bacterial species suggests that the mechanism of secretion and translocation control used byE. tardamay be commonly used by other bacterial pathogens.

scholarly journals Two Unrelated 8-Vinyl Reductases Ensure Production of Mature Chlorophylls in Acaryochloris marina

2016 ◽  
Vol 198 (9) ◽  
pp. 1393-1400 ◽  
Author(s):  
Guangyu E. Chen ◽  
Andrew Hitchcock ◽  
Philip J. Jackson ◽  
Roy R. Chaudhuri ◽  
Mark J. Dickman ◽  
...  
Keyword(s):  
Transcriptional Control ◽  
Sequence Similarity ◽  
Open Reading Frames ◽  
High Sequence Similarity ◽  
Content Type ◽  
Ethyl Group ◽  
Acaryochloris Marina ◽  
Vinyl Group ◽  
Vinyl Reductase ◽  
Reading Frames

ABSTRACTThe major photopigment of the cyanobacteriumAcaryochloris marinais chlorophylld, while its direct biosynthetic precursor, chlorophylla, is also present in the cell. These pigments, along with the majority of chlorophylls utilized by oxygenic phototrophs, carry an ethyl group at the C-8 position of the molecule, having undergone reduction of a vinyl group during biosynthesis. Two unrelated classes of 8-vinyl reductase involved in the biosynthesis of chlorophylls are known to exist, BciA and BciB. The genome ofAcaryochloris marinacontains open reading frames (ORFs) encoding proteins displaying high sequence similarity to BciA or BciB, although they are annotated as genes involved in transcriptional control (nmrA) and methanogenesis (frhB), respectively. These genes were introduced into an 8-vinyl chlorophylla-producing ΔbciBstrain ofSynechocystissp. strain PCC 6803, and both were shown to restore synthesis of the pigment with an ethyl group at C-8, demonstrating their activities as 8-vinyl reductases. We propose thatnmrAandfrhBbe reassigned asbciAandbciB, respectively; transcript and proteomic analysis ofAcaryochloris marinareveal that bothbciAandbciBare expressed and their encoded proteins are present in the cell, possibly in order to ensure that all synthesized chlorophyll pigment carries an ethyl group at C-8. Potential reasons for the presence of two 8-vinyl reductases in this strain, which is unique for cyanobacteria, are discussed.IMPORTANCEThe cyanobacteriumAcaryochloris marinais the best-studied phototrophic organism that uses chlorophylldfor photosynthesis. Unique among cyanobacteria sequenced to date, its genome contains ORFs encoding two unrelated enzymes that catalyze the reduction of the C-8 vinyl group of a precursor molecule to an ethyl group. Carrying a reduced C-8 group may be of particular importance to organisms containing chlorophylld. Plant genomes also contain orthologs of both of these genes; thus, the bacterial progenitor of the chloroplast may also have contained bothbciAandbciB.

scholarly journals Activation of 2,4-Diaminoquinazoline in Mycobacterium tuberculosis by Rv3161c, a Putative Dioxygenase

2018 ◽  
Vol 63 (1) ◽  
Author(s):  
Eduard Melief ◽  
Shilah A. Bonnett ◽  
Edison S. Zuniga ◽  
Tanya Parish
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mutant Strain ◽  
Type Strain ◽  
Wild Type Strain ◽  
Type A ◽  
Wild Type ◽  
Metabolite Analysis ◽  
Content Type ◽  
Data Support ◽  
In The Wild

ABSTRACT The diaminoquinazoline series has good potency against Mycobacterium tuberculosis. Resistant isolates have mutations in Rv3161c, a putative dioxygenase. We carried out metabolite analysis on a wild-type strain and an Rv3161c mutant strain after exposure to a diaminoquinazoline. The parental compound was found in intracellular extracts from the mutant but not the wild type. A metabolite consistent with a monohydroxylated form was identified in the wild type. These data support the hypothesis that Rv3161c metabolizes diaminoquinazolines in M. tuberculosis.

scholarly journals An NADH-Dependent Reductase from Eubacterium ramulus Catalyzes the Stereospecific Heteroring Cleavage of Flavanones and Flavanonols

2019 ◽  
Vol 85 (19) ◽  
Author(s):  
Annett Braune ◽  
Michael Gütschow ◽  
Michael Blaut
Keyword(s):  
Sequence Similarity ◽  
Bacterial Species ◽  
Functional Characterization ◽  
Intestinal Bacteria ◽  
Cyclic Ether ◽  
Content Type ◽  
Oxygen Sensitive ◽  
Key Enzyme ◽  
Catalyzed Reactions ◽  
Dietary Flavonoids

ABSTRACT The human intestinal anaerobe Eubacterium ramulus is known for its ability to degrade various dietary flavonoids. In the present study, we demonstrate the cleavage of the heterocyclic C-ring of flavanones and flavanonols by an oxygen-sensitive NADH-dependent reductase, previously described as enoate reductase, from E. ramulus. This flavanone- and flavanonol-cleaving reductase (Fcr) was purified following its heterologous expression in Escherichia coli and further characterized. Fcr cleaved the flavanones naringenin, eriodictyol, liquiritigenin, and homoeriodictyol. Moreover, the flavanonols taxifolin and dihydrokaempferol served as substrates. The catalyzed reactions were stereospecific for the (2R)-enantiomers of the flavanone substrates and for the (2S,3S)-configured flavanonols. The enantioenrichment of the nonconverted stereoisomers allowed for the determination of hitherto unknown flavanone racemization rates. Fcr formed the corresponding dihydrochalcones and hydroxydihydrochalcones in the course of an unusual reductive cleavage of cyclic ether bonds. Fcr did not convert members of other flavonoid subclasses, including flavones, flavonols, and chalcones, the latter indicating that the reaction does not involve a chalcone intermediate. This view is strongly supported by the observed enantiospecificity of Fcr. Cinnamic acids, which are typical substrates of bacterial enoate reductases, were also not reduced by Fcr. Based on the presence of binding motifs for dinucleotide cofactors and a 4Fe-4S cluster in the amino acid sequence of Fcr, a cofactor-mediated hydride transfer from NADH onto C-2 of the respective substrate is proposed. IMPORTANCE Gut bacteria play a crucial role in the metabolism of dietary flavonoids, thereby contributing to their activation or inactivation after ingestion by the human host. Thus, bacterial activities in the intestine may influence the beneficial health effects of these polyphenolic plant compounds. While an increasing number of flavonoid-converting gut bacterial species have been identified, knowledge of the responsible enzymes is still limited. Here, we characterized Fcr as a key enzyme involved in the conversion of flavonoids of several subclasses by Eubacterium ramulus, a prevalent human gut bacterium. Sequence similarity of this enzyme to hypothetical proteins from other flavonoid-degrading intestinal bacteria in databases suggests a more widespread occurrence of this enzyme. Functional characterization of gene products of human intestinal microbiota enables the assignment of metagenomic sequences to specific bacteria and, more importantly, to certain activities, which is a prerequisite for targeted modulation of gut microbial functionality.

scholarly journals Cryptococcus neoformans Evades Pulmonary Immunity by Modulating Xylose Precursor Transport

2020 ◽  
Vol 88 (8) ◽  
Author(s):  
Lucy X. Li ◽  
Camaron R. Hole ◽  
Javier Rangel-Moreno ◽  
Shabaana A. Khader ◽  
Tamara L. Doering
Keyword(s):  
Cryptococcus Neoformans ◽  
Mutant Strain ◽  
Fungal Pathogen ◽  
Wild Type ◽  
Content Type ◽  
Helper Cell Type ◽  
Lymphoid Structures ◽  
Type Infection

ABSTRACT Cryptococcus neoformans is a fungal pathogen that kills almost 200,000 people each year and is distinguished by abundant and unique surface glycan structures that are rich in xylose. A mutant strain of C. neoformans that cannot transport xylose precursors into the secretory compartment is severely attenuated in virulence in mice yet surprisingly is not cleared. We found that this strain failed to induce the nonprotective T helper cell type 2 (Th2) responses characteristic of wild-type infection, instead promoting sustained interleukin 12p40 (IL-12p40) induction and increased IL-17A (IL-17) production. It also stimulated dendritic cells to release high levels of proinflammatory cytokines, a behavior we linked to xylose expression. We further discovered that inducible bronchus-associated lymphoid tissue (iBALT) forms in response to infection with either wild-type cryptococci or the mutant strain with reduced surface xylose; although iBALT formation is slowed in the latter case, the tissue is better organized. Finally, our temporal studies suggest that lymphoid structures in the lung restrict the spread of mutant fungi for at least 18 weeks after infection, which is in contrast to ineffective control of the pathogen after infection with wild-type cells. These studies demonstrate the role of xylose in modulation of host response to a fungal pathogen and show that cryptococcal infection triggers iBALT formation.

scholarly journals A Family of Small Intrinsically Disordered Proteins Involved in Flagellum-Dependent Motility inSalmonella enterica

2018 ◽  
Vol 201 (2) ◽  
Author(s):  
Tamiko Oguri ◽  
Youjeong Kwon ◽  
Jerry K. K. Woo ◽  
Gerd Prehna ◽  
Hyun Lee ◽  
...  
Keyword(s):  
Intrinsically Disordered Proteins ◽  
Expression Profiles ◽  
Functional Characterization ◽  
Disordered Proteins ◽  
Wild Type ◽  
Content Type ◽  
Cellular Pathway ◽  
Intrinsically Disordered ◽  
Small Proteins ◽  
Wide Range

ABSTRACTBy screening a collection ofSalmonellamutants deleted for genes encoding small proteins of ≤60 amino acids, we identified three paralogous small genes (ymdF,STM14_1829, andyciG) required for wild-type flagellum-dependent swimming and swarming motility. TheymdF,STM14_1829, andyciGgenes encode small proteins of 55, 60, and 60 amino acid residues, respectively. A bioinformatics analysis predicted that these small proteins are intrinsically disordered proteins, and circular dichroism analysis of purified recombinant proteins confirmed that all three proteins are unstructured in solution. A mutant deleted for STM14_1829 showed the most severe motility defect, indicating that among the three paralogs, STM14_1829 is a key protein required for wild-type motility. We determined that relative to the wild type, the expression of the flagellin protein FliC is lower in the ΔSTM14_1829mutant due to the downregulation of theflhDCoperon encoding the FlhDC master regulator. By comparing the gene expression profiles between the wild-type and ΔSTM14_1829strains via RNA sequencing, we found that the gene encoding the response regulator PhoP is upregulated in the ΔSTM14_1829mutant, suggesting the indirect repression of theflhDCoperon by the activated PhoP. Homologs of STM14_1829 are conserved in a wide range of bacteria, includingEscherichia coliandPseudomonas aeruginosa. We showed that the inactivation of STM14_1829 homologs inE. coliandP. aeruginosaalso alters motility, suggesting that this family of small intrinsically disordered proteins may play a role in the cellular pathway(s) that affects motility.IMPORTANCEThis study reports the identification of a novel family of small intrinsically disordered proteins that are conserved in a wide range of flagellated and nonflagellated bacteria. Although this study identifies the role of these small proteins in the scope of flagellum-dependent motility inSalmonella, they likely play larger roles in a more conserved cellular pathway(s) that indirectly affects flagellum expression in the case of motile bacteria. Small intrinsically disordered proteins have not been well characterized in prokaryotes, and the results of our study provide a basis for their detailed functional characterization.

scholarly journals Hypothetical Protein Avin_16040 as the S-Layer Protein of Azotobacter vinelandii and Its Involvement in Plant Root Surface Attachment

2015 ◽  
Vol 81 (21) ◽  
pp. 7484-7495 ◽  
Author(s):  
Pauline Woan Ying Liew ◽  
Bor Chyan Jong ◽  
Nazalan Najimudin
Keyword(s):  
Cell Surface ◽  
Deletion Mutant ◽  
Azotobacter Vinelandii ◽  
Plant Root ◽  
Hypothetical Protein ◽  
Surface Hydrophobicity ◽  
Root Surface ◽  
Bacterial Cells ◽  
Wild Type ◽  
Content Type

ABSTRACTA proteomic analysis of a soil-dwelling, plant growth-promotingAzotobacter vinelandiistrain showed the presence of a protein encoded by the hypotheticalAvin_16040gene when the bacterial cells were attached to theOryza sativaroot surface. AnAvin_16040deletion mutant demonstrated reduced cellular adherence to the root surface, surface hydrophobicity, and biofilm formation compared to those of the wild type. By atomic force microscopy (AFM) analysis of the cell surface topography, the deletion mutant displayed a cell surface architectural pattern that was different from that of the wild type.Escherichia colitransformed with the wild-typeAvin_16040gene displayed on its cell surface organized motifs which looked like the S-layer monomers ofA. vinelandii. The recombinantE. colialso demonstrated enhanced adhesion to the root surface.

scholarly journals Engineering and Preclinical Evaluation of Attenuated Nontyphoidal Salmonella Strains Serving as Live Oral Vaccines and as Reagent Strains

2011 ◽  
Vol 79 (10) ◽  
pp. 4175-4185 ◽  
Author(s):  
Sharon M. Tennant ◽  
Jin-Yuan Wang ◽  
James E. Galen ◽  
Raphael Simon ◽  
Marcela F. Pasetti ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Lethal Dose ◽  
Invasive Disease ◽  
Oral Immunization ◽  
Oral Vaccines ◽  
Wild Type ◽  
Lethal Challenge ◽  
Antibiotic Resistant ◽  
Content Type ◽  
Serovar Typhimurium

ABSTRACTWhile nontyphoidalSalmonella(NTS) has long been recognized as a cause of self-limited gastroenteritis, it is becoming increasingly evident that multiple-antibiotic-resistant strains are also emerging as important causes of invasive bacteremia and focal infections, resulting in hospitalizations and deaths. We have constructed attenuatedSalmonella entericaserovar Typhimurium andSalmonella entericaserovar Enteritidis strains that can serve as live oral vaccines and as “reagent strains” for subunit vaccine production in a safe and economical manner. Prototype attenuated vaccine strains CVD 1921 and CVD 1941, derived from the invasive wild-type strainsS. TyphimuriumI77 andS. EnteritidisR11, respectively, were constructed by deletingguaBA, encoding guanine biosynthesis, andclpP, encoding a master protease regulator. TheclpPmutation resulted in a hyperflagellation phenotype. An additional deletion infliDyielded reagent strains CVD 1923 and CVD 1943, respectively, which export flagellin monomers. Oral 50% lethal dose (LD50) analyses showed that the NTS vaccine strains were all highly attenuated in mice. Oral immunization with CVD 1921 or CVD 1923 protected mice against lethal challenge with wild-typeS. TyphimuriumI77. Immunization with CVD 1941 but not CVD 1943 protected mice against lethal infection withS. EnteritidisR11. Immune responses induced by these strains included high levels of serum IgG anti-lipopolysaccharide (LPS) and anti-flagellum antibodies, with titers increasing progressively during the immunization schedule. SinceS. TyphimuriumandS. Enteritidisare the most common NTS serovars associated with invasive disease, these findings can pave the way for development of a highly effective, broad-spectrum vaccine against invasive NTS.

Sinomicrobium oceani gen. nov., sp. nov., a member of the family Flavobacteriaceae isolated from marine sediment

2013 ◽  
Vol 63 (Pt_3) ◽  
pp. 1045-1050 ◽  
Author(s):  
Ying Xu ◽  
Xin-Peng Tian ◽  
Yu-Juan Liu ◽  
Jie Li ◽  
Chang-Jin Kim ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Marine Sediment ◽  
Sequence Similarity ◽  
Rrna Gene ◽  
High Sequence Similarity ◽  
Respiratory Quinone ◽  
Content Type ◽  
Link Type ◽  
The Family

A marine bacterium, designated SCSIO 03483T, was isolated from a marine sediment sample collected from the Nansha Islands in the South China Sea. The strain produced roundish colonies with diffusible yellow-coloured pigment on nutrient agar medium or marine agar 2216. Optimal growth occurred in the presence of 0–4 % (w/v) NaCl, at pH 7.0 and a temperature range of 28–37 °C. 16S rRNA gene sequence analysis indicated that the isolate belonged to the family Flavobacteriaceae and showed relatively high sequence similarity with Imtechella halotolerans K1T (92.7 %). Phylogenetic analysis based on nearly complete 16S rRNA gene sequences revealed that the isolate shared a lineage with members of the genera Imtechella , Joostella and Zhouia . Phospholipids were phosphatidylethanolamine, two unidentified aminolipids and three unknown polar lipids. The major respiratory quinone was MK-6 and the major fatty acids were iso-C15 : 0, iso-C17 : 0 3-OH and summed feature 3 (C16 : 1ω6c/C16 : 1ω7c). The DNA G+C content of strain SCSIO 03483T was 38.4 mol%. On the basis of phenotypic, chemotaxonomic and molecular data, strain SCSIO 03483T represents a novel species in a new genus in the family Flavobacteriaceae , for which the name Sinomicrobium oceani gen. nov., sp. nov. is proposed. The type strain of Sinobacterium oceani is SCSIO 03483T ( = KCTC 23994T = CGMCC 1.12145T).

scholarly journals Proline Metabolism IncreaseskatGExpression and Oxidative Stress Resistance in Escherichia coli

2014 ◽  
Vol 197 (3) ◽  
pp. 431-440 ◽  
Author(s):  
Lu Zhang ◽  
James R. Alfano ◽  
Donald F. Becker
Keyword(s):  
Oxidative Stress ◽  
Escherichia Coli ◽  
Hydrogen Peroxide ◽  
Mutant Strain ◽  
Stress Resistance ◽  
Proline Metabolism ◽  
Wild Type ◽  
Content Type ◽  
Oxidative Stress Resistance ◽  
Proline Utilization

The oxidation ofl-proline to glutamate in Gram-negative bacteria is catalyzed by the proline utilization A (PutA) flavoenzyme, which contains proline dehydrogenase (PRODH) and Δ1-pyrroline-5-carboxylate (P5C) dehydrogenase domains in a single polypeptide. Previous studies have suggested that aside from providing energy, proline metabolism influences oxidative stress resistance in different organisms. To explore this potential role and the mechanism, we characterized the oxidative stress resistance of wild-type andputAmutant strains ofEscherichia coli. Initial stress assays revealed that theputAmutant strain was significantly more sensitive to oxidative stress than the parental wild-type strain. Expression of PutA in theputAmutant strain restored oxidative stress resistance, confirming that depletion of PutA was responsible for the oxidative stress phenotype. Treatment of wild-type cells with proline significantly increased hydroperoxidase I (encoded bykatG) expression and activity. Furthermore, the ΔkatGstrain failed to respond to proline, indicating a critical role for hydroperoxidase I in the mechanism of proline protection. The global regulator OxyR activates the expression ofkatGalong with several other genes involved in oxidative stress defense. In addition tokatG, proline increased the expression ofgrxA(glutaredoxin 1) andtrxC(thioredoxin 2) of the OxyR regulon, implicating OxyR in proline protection. Proline oxidative metabolism was shown to generate hydrogen peroxide, indicating that proline increases oxidative stress tolerance inE. colivia a preadaptive effect involving endogenous hydrogen peroxide production and enhanced catalase-peroxidase activity.

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