scholarly journals Arsenite Oxidase Also Functions as an Antimonite Oxidase

2015 ◽  
Vol 81 (6) ◽  
pp. 1959-1965 ◽  
Author(s):  
Qian Wang ◽  
Thomas P. Warelow ◽  
Yoon-Suk Kang ◽  
Christine Romano ◽  
Thomas H. Osborne ◽  
...  
Keyword(s):  
Environmental Protection Agency ◽  
Large Subunit ◽  
Environmental Pollutants ◽  
Transcriptional Responses ◽  
Oxidation Activity ◽  
Content Type ◽  
Genes Encoding ◽  
Basic Features

ABSTRACTArsenic and antimony are toxic metalloids and are considered priority environmental pollutants by the U.S. Environmental Protection Agency. Significant advances have been made in understanding microbe-arsenic interactions and how they influence arsenic redox speciation in the environment. However, even the most basic features of how and why a microorganism detects and reacts to antimony remain poorly understood. Previous work withAgrobacterium tumefaciensstrain 5A concluded that oxidation of antimonite [Sb(III)] and arsenite [As(III)] required different biochemical pathways. Here, we show within vivoexperiments that a mutation inaioA[encoding the large subunit of As(III) oxidase] reduces the ability to oxidize Sb(III) by approximately one-third relative to the ability of the wild type. Further,in vitrostudies with the purified As(III) oxidase fromRhizobiumsp. strain NT-26 (AioA shares 94% amino acid sequence identity with AioA ofA. tumefaciens) provide direct evidence of Sb(III) oxidation but also show a significantly decreasedVmaxcompared to that of As(III) oxidation. TheaioBAgenes encoding As(III) oxidase are induced by As(III) but not by Sb(III), whereasarsRgene expression is induced by both As(III) and Sb(III), suggesting that detection and transcriptional responses for As(III) and Sb(III) differ. While Sb(III) and As(III) are similar with respect to cellular extrusion (ArsB or Acr3) and interaction with ArsR, they differ in the regulatory mechanisms that control the expression of genes encoding the different Ars or Aio activities. In summary, this study documents an enzymatic basis for microbial Sb(III) oxidation, although additional Sb(III) oxidation activity also is apparent in this bacterium.

scholarly journals Mutations of the Listeria monocytogenes PeptidoglycanN-Deacetylase andO-Acetylase Result in Enhanced Lysozyme Sensitivity, Bacteriolysis, and Hyperinduction of Innate Immune Pathways

2011 ◽  
Vol 79 (9) ◽  
pp. 3596-3606 ◽  
Author(s):  
Chris S. Rae ◽  
Aimee Geissler ◽  
Paul C. Adamson ◽  
Daniel A. Portnoy
Keyword(s):  
Listeria Monocytogenes ◽  
Transcriptional Responses ◽  
Gram Positive ◽  
Content Type ◽  
Growth Defects ◽  
Gram Positive Bacteria ◽  
Host Cell Death

ABSTRACTListeria monocytogenesis a Gram-positive intracellular pathogen that is naturally resistant to lysozyme. Recently, it was shown that peptidoglycan modification by N-deacetylation or O-acetylation confers resistance to lysozyme in various Gram-positive bacteria, includingL. monocytogenes.L. monocytogenespeptidoglycan is deacetylated by the action ofN-acetylglucosamine deacetylase (Pgd) and acetylated byO-acetylmuramic acid transferase (Oat). We characterized Pgd−, Oat−, and double mutants to determine the specific role ofL. monocytogenespeptidoglycan acetylation in conferring lysozyme sensitivity during infection of macrophages and mice. Pgd−and Pgd−Oat−double mutants were attenuated approximately 2 and 3.5 logs, respectively,in vivo. In bone-marrow derived macrophages, the mutants demonstrated intracellular growth defects and increased induction of cytokine transcriptional responses that emanated from a phagosome and the cytosol. Lysozyme-sensitive mutants underwent bacteriolysis in the macrophage cytosol, resulting in AIM2-dependent pyroptosis. Each of thein vitrophenotypes was rescued upon infection of LysM−macrophages. The addition of extracellular lysozyme to LysM−macrophages restored cytokine induction, host cell death, andL. monocytogenesgrowth inhibition. This surprising observation suggests that extracellular lysozyme can access the macrophage cytosol and act on intracellular lysozyme-sensitive bacteria.

scholarly journals Adaptive Strategies and Pathogenesis of Clostridium difficile fromIn VivoTranscriptomics

2013 ◽  
Vol 81 (10) ◽  
pp. 3757-3769 ◽  
Author(s):  
Claire Janoir ◽  
Cécile Denève ◽  
Sylvie Bouttier ◽  
Frédéric Barbut ◽  
Sandra Hoys ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Stress Responses ◽  
Differentially Expressed ◽  
Content Type ◽  
Host Interactions ◽  
Colonization Factor ◽  
Genes Encoding ◽  
Colonization Process

ABSTRACTClostridium difficileis currently the major cause of nosocomial intestinal diseases associated with antibiotic therapy in adults. In order to improve our knowledge ofC. difficile-host interactions, we analyzed the genome-wide temporal expression ofC. difficile630 genes during the first 38 h of mouse colonization to identify genes whose expression is modulatedin vivo, suggesting that they may play a role in facilitating the colonization process. In the ceca of theC. difficile-monoassociated mice, 549 genes of theC. difficilegenome were differentially expressed compared to their expression duringin vitrogrowth, and they were distributed in several functional categories. Overall, our results emphasize the roles of genes involved in host adaptation. Colonization results in a metabolic shift, with genes responsible for the fermentation as well as several other metabolic pathways being regulated inversely to those involved in carbon metabolism. In addition, several genes involved in stress responses, such as ferrous iron uptake or the response to oxidative stress, were regulatedin vivo. Interestingly, many genes encoding conserved hypothetical proteins (CHP) were highly and specifically upregulatedin vivo. Moreover, genes for all stages of sporulation were quickly inducedin vivo, highlighting the observation that sporulation is central to the persistence ofC. difficilein the gut and to its ability to spread in the environment. Finally, we inactivated two genes that were differentially expressedin vivoand evaluated the relative colonization fitness of the wild-type and mutant strains in coinfection experiments. We identified a CHP as a putative colonization factor, supporting the suggestion that thein vivotranscriptomic approach can unravel newC. difficilevirulence genes.

scholarly journals Evolutionary Adaptation of an AraC-Like Regulatory Protein in Citrobacter rodentium and Escherichia Species

2015 ◽  
Vol 83 (4) ◽  
pp. 1384-1395 ◽  
Author(s):  
Aimee Tan ◽  
Nicola K. Petty ◽  
Dianna Hocking ◽  
Vicki Bennett-Wood ◽  
Matthew Wakefield ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Regulatory Protein ◽  
Gene Repertoire ◽  
Citrobacter Rodentium ◽  
Environmental Strain ◽  
Content Type ◽  
E Coli ◽  
Genes Encoding

The evolution of pathogenic bacteria is a multifaceted and complex process, which is strongly influenced by the horizontal acquisition of genetic elements and their subsequent expression in their new hosts. A well-studied example is the RegA regulon of the enteric pathogenCitrobacter rodentium. The RegA regulatory protein is a member of the AraC/XylS superfamily, which coordinates the expression of a gene repertoire that is necessary for full pathogenicity of this murine pathogen. Upon stimulation by an exogenous, gut-associated signal, namely, bicarbonate ions, RegA activates the expression of a series of genes, including virulence factors, such as autotransporters, fimbriae, a dispersin-like protein, and thegrlRAoperon on the locus of enterocyte effacement pathogenicity island. Interestingly, the genes encoding RegA homologues are distributed across the genusEscherichia, encompassing pathogenic and nonpathogenic subtypes. In this study, we carried out a series of bioinformatic, transcriptional, and functional analyses of the RegA regulons of these bacteria. Our results demonstrated thatregAhas been horizontally transferred toEscherichiaspp. andC. rodentium. Comparative studies of two RegA homologues, namely, those fromC. rodentiumandE. coliSMS-3-5, a multiresistant environmental strain ofE. coli, showed that the two regulators acted similarlyin vitrobut differed in terms of their abilities to activate the virulence ofC. rodentiumin vivo, which evidently was due to their differential activation ofgrlRA. Our data indicate that RegA fromC. rodentiumhas strain-specific adaptations that facilitate infection of its murine host. These findings shed new light on the development of virulence byC. rodentiumand on the evolution of virulence-regulatory genes of bacterial pathogens in general.

scholarly journals Evaluation of CpxRA as a Therapeutic Target for UropathogenicEscherichia coliInfections

2018 ◽  
Vol 86 (3) ◽  
pp. e00798-17 ◽  
Author(s):  
Lana Dbeibo ◽  
Julia J. van Rensburg ◽  
Sara N. Smith ◽  
Kate R. Fortney ◽  
Dharanesh Gangaiah ◽  
...  
Keyword(s):  
Phosphatase Activity ◽  
Virulence Determinants ◽  
Membrane Repair ◽  
Single Strain ◽  
Content Type ◽  
Phosphatase Inhibitors ◽  
Genes Encoding ◽  
Tract Infections

ABSTRACTCpxRA is an envelope stress response system found in all members of the familyEnterobacteriaceae; CpxA has kinase activity for CpxR and phosphatase activity for phospho-CpxR, a transcription factor. CpxR also accepts phosphate groups from acetyl phosphate, a glucose metabolite. Activation of CpxR increases the transcription of genes encoding membrane repair and downregulates virulence determinants. We hypothesized that activation of CpxR could serve as an antimicrobial/antivirulence strategy and discovered compounds that activate CpxR inEscherichia coliby inhibiting CpxA phosphatase activity. As a prelude to testing such compoundsin vivo, here we constructedcpxA(in the presence of glucose, CpxR is activated because of a lack of CpxA phosphatase) andcpxR(system absent) deletion mutants of uropathogenicE. coli(UPEC) CFT073. By RNA sequencing, few transcriptional differences were noted between thecpxRmutant and its parent, but in thecpxAmutant, several UPEC virulence determinants were downregulated, including thefimandpapoperons, and it exhibited reduced mannose-sensitive hemagglutination of guinea pig red blood cellsin vitro. In competition experiments with mice, both mutants were less fit than the parent in the urine, bladder, and kidney; these fitness defects were complemented intrans. Unexpectedly, in single-strain challenges, only thecpxAmutant was attenuated for virulence in the kidney but not in the bladder or urine. For thecpxAmutant, this may be due to the preferential use of amino acids over glucose as a carbon source in the bladder and urine by UPEC. These studies suggest that CpxA phosphatase inhibitors may have some utility for treating complex urinary tract infections.

scholarly journals The INO80 Complex Requires the Arp5-Ies6 Subcomplex for Chromatin Remodeling and Metabolic Regulation

2016 ◽  
Vol 36 (6) ◽  
pp. 979-991 ◽  
Author(s):  
Wei Yao ◽  
Devin A. King ◽  
Sean L. Beckwith ◽  
Graeme J. Gowans ◽  
Kuangyu Yen ◽  
...  
Keyword(s):  
Chromatin Remodeling ◽  
Metabolic Regulation ◽  
Chromatin Remodeler ◽  
Transcriptional Responses ◽  
Mitochondrial Potential ◽  
Content Type ◽  
Expression Of Genes ◽  
Genomic Studies

ATP-dependent chromatin remodeling complexes are essential for transcription regulation, and yet it is unclear how these multisubunit complexes coordinate their activities to facilitate diverse transcriptional responses. In this study, we found that the conserved Arp5 and Ies6 subunits of theSaccharomyces cerevisiaeINO80 chromatin-remodeler form an abundant and distinct subcomplexin vivoand stimulate INO80-mediated activityin vitro. Moreover, our genomic studies reveal that the relative occupancy of Arp5-Ies6 correlates with nucleosome positioning at transcriptional start sites and expression levels of >1,000 INO80-regulated genes. Notably, these genes are significantly enriched in energy metabolism pathways. Specifically,arp5Δ,ies6Δ, andino80Δ mutants demonstrate decreased expression of genes involved in glycolysis and increased expression of genes in the oxidative phosphorylation pathway. Deregulation of these metabolic pathways results in constitutively elevated mitochondrial potential and oxygen consumption. Our results illustrate the dynamic nature of the INO80 complex assembly and demonstrate for the first time that a chromatin remodeler regulates glycolytic and respiratory capacity, thereby maintaining metabolic stability.

scholarly journals TheIn VitroRedundant Enzymes PurN and PurT Are Both Essential for Systemic Infection of Mice in Salmonella enterica Serovar Typhimurium

2016 ◽  
Vol 84 (7) ◽  
pp. 2076-2085 ◽  
Author(s):  
Lotte Jelsbak ◽  
Mie I. B. Mortensen ◽  
Mogens Kilstrup ◽  
John E. Olsen
Keyword(s):  
Salmonella Enterica ◽  
Single Gene ◽  
Systemic Infection ◽  
Gene Deletions ◽  
Content Type ◽  
Serovar Typhimurium ◽  
Genes Encoding ◽  
Glycine Cleavage

Metabolic enzymes show a high degree of redundancy, and for that reason they are generally ignored in searches for novel targets for anti-infective substances. The enzymes PurN and PurT are redundantin vitroinSalmonella entericaserovar Typhimurium, in which they perform the third step of purine synthesis. Surprisingly, the results of the current study demonstrated that single-gene deletions of each of the genes encoding these enzymes caused attenuation (competitive infection indexes [CI] of <0.03) in mouse infections. While the ΔpurTmutant multiplied as fast as the wild-type strain in cultured J774A.1 macrophages, net multiplication of the ΔpurNmutant was reduced approximately 50% in 20 h. The attenuation of the ΔpurTmutant was abolished by simultaneous removal of the enzyme PurU, responsible for the formation of formate, indicating that the attenuation was related to formate accumulation or wasteful consumption of formyl tetrahydrofolate by PurU. In the process of further characterization, we disclosed that the glycine cleavage system (GCV) was the most important for formation of C1unitsin vivo(CI = 0.03 ± 0.03). In contrast, GlyA was the only important enzyme for the formation of C1unitsin vitro. The results with the ΔgcvTmutant further revealed that formation of serine by SerA and further conversion of serine into C1units and glycine by GlyA were not sufficient to ensure C1formation inS. Typhimuriumin vivo. The results of the present study call for reinvestigations of the concept of metabolic redundancy inS. Typhimuriumin vivo.

scholarly journals An Improved Recombination-BasedIn VivoExpression Technology-Like Reporter System Reveals DifferentialcyaAGene Activation in Bordetella Species

2013 ◽  
Vol 81 (4) ◽  
pp. 1295-1305 ◽  
Author(s):  
Matthew S. Byrd ◽  
Eliza Mason ◽  
Michael W. Henderson ◽  
Erich V. Scheller ◽  
Peggy A. Cotter
Keyword(s):  
Virulence Factors ◽  
Gene Activation ◽  
Regulatory System ◽  
Reporter System ◽  
Fluorescent Reporter ◽  
Content Type ◽  
Genes Encoding ◽  
Species Specific

ABSTRACTBordetella pertussisandBordetella bronchisepticarely on the global two-component regulatory system BvgAS to control expression of distinct phenotypic phases. In the Bvg−phase, expression ofvrggenes, including those required for motility inB. bronchiseptica, is activated and genes encoding virulence factors are not expressed. Conversely, in the Bvg+phase, genes encoding virulence factors are highly expressed while genes necessary for motility are repressed. Although several genetic analyses have demonstrated the importance of the Bvg+phase during respiratory infection, Bvg-regulated gene activation inB. bronchisepticahas not been investigatedin vivo. To address this, we developed a plasmid, pGFLIP, that encodes a sensitive Flp recombinase-based fluorescent reporter system able to document gene activation bothin vitroandin vivo. Using pGFLIP, we demonstrated thatcyaA, considered to be a “late” Bvg+phase gene, is activated substantially earlier inB. bronchisepticathanB. pertussisfollowing a switch from Bvg−to Bvg+phase conditions. We show that the altered activation ofcyaAis not due to differences in thecyaApromoter or in thebvgASalleles ofB. bronchisepticacompared toB. pertussis, but appears to be species specific. Finally, we used pGFLIP to show thatflaAremains repressed during infection, confirming thatB. bronchisepticadoes not modulate to the Bvg−phasein vivo.

scholarly journals Novel Phage Lysin Capable of Killing the Multidrug-Resistant Gram-Negative Bacterium Acinetobacter baumannii in a Mouse Bacteremia Model

2015 ◽  
Vol 59 (4) ◽  
pp. 1983-1991 ◽  
Author(s):  
Rolf Lood ◽  
Benjamin Y. Winer ◽  
Adam J. Pelzek ◽  
Roberto Diez-Martinez ◽  
Mya Thandar ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Genomic Library ◽  
Multidrug Resistant ◽  
Clinical Isolates ◽  
Gram Negative ◽  
Content Type ◽  
Highly Active ◽  
Genes Encoding

ABSTRACTAcinetobacter baumannii, a Gram-negative multidrug-resistant (MDR) bacterium, is now recognized as one of the more common nosocomial pathogens. Because most clinical isolates are found to be multidrug resistant, alternative therapies need to be developed to control this pathogen. We constructed a bacteriophage genomic library based on prophages induced from 13A. baumanniistrains and screened it for genes encoding bacteriolytic activity. Using this approach, we identified 21 distinct lysins with different activities and sequence diversity that were capable of killingA. baumannii. The lysin (PlyF307) displaying the greatest activity was further characterized and was shown to efficiently kill (>5-log-unit decrease) all testedA. baumanniiclinical isolates. Treatment with PlyF307 was able to significantly reduce planktonic and biofilmA. baumanniibothin vitroandin vivo. Finally, PlyF307 rescued mice from lethalA. baumanniibacteremia and as such represents the first highly active therapeutic lysin specific for Gram-negative organisms in an array of native lysins found inAcinetobacterphage.

scholarly journals In vivo transcriptome analysis provides insights into host-dependent expression of virulence factors by Yersinia entomophaga MH96, during infection of Galleria mellonella

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Amber R Paulson ◽  
Maureen O’Callaghan ◽  
Xue-Xian Zhang ◽  
Paul B Rainey ◽  
Mark R H Hurst
Keyword(s):  
Galleria Mellonella ◽  
Functional Enrichment ◽  
Natural Environments ◽  
Insecticidal Toxin ◽  
Heat Stable ◽  
Expression Of Genes ◽  
Genes Encoding ◽  
Cell Densities

Abstract The function of microbes can be inferred from knowledge of genes specifically expressed in natural environments. Here, we report the in vivo transcriptome of the entomopathogenic bacterium Yersinia entomophaga MH96, captured during initial, septicemic, and pre-cadaveric stages of intrahemocoelic infection in Galleria mellonella. A total of 1285 genes were significantly upregulated by MH96 during infection; 829 genes responded to in vivo conditions during at least one stage of infection, 289 responded during two stages of infection, and 167 transcripts responded throughout all three stages of infection compared to in vitro conditions at equivalent cell densities. Genes upregulated during the earliest infection stage included components of the insecticidal toxin complex Yen-TC (chi1, chi2, and yenC1), genes for rearrangement hotspot element containing protein yenC3, cytolethal distending toxin cdtAB, and vegetative insecticidal toxin vip2. Genes more highly expressed throughout the infection cycle included the putative heat-stable enterotoxin yenT and three adhesins (usher-chaperone fimbria, filamentous hemagglutinin, and an AidA-like secreted adhesin). Clustering and functional enrichment of gene expression data also revealed expression of genes encoding type III and VI secretion system-associated effectors. Together these data provide insight into the pathobiology of MH96 and serve as an important resource supporting efforts to identify novel insecticidal agents.

scholarly journals Designing P. aeruginosa synthetic phages with reduced genomes

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Diana P. Pires ◽  
Rodrigo Monteiro ◽  
Dalila Mil-Homens ◽  
Arsénio Fialho ◽  
Timothy K. Lu ◽  
...  
Keyword(s):  
Galleria Mellonella ◽  
Antibacterial Properties ◽  
Genetically Engineered ◽  
In Vivo Studies ◽  
Infection Model ◽  
Promising Therapeutic Approach ◽  
Genes Encoding ◽  
First Time

AbstractIn the era where antibiotic resistance is considered one of the major worldwide concerns, bacteriophages have emerged as a promising therapeutic approach to deal with this problem. Genetically engineered bacteriophages can enable enhanced anti-bacterial functionalities, but require cloning additional genes into the phage genomes, which might be challenging due to the DNA encapsulation capacity of a phage. To tackle this issue, we designed and assembled for the first time synthetic phages with smaller genomes by knocking out up to 48% of the genes encoding hypothetical proteins from the genome of the newly isolated Pseudomonas aeruginosa phage vB_PaeP_PE3. The antibacterial efficacy of the wild-type and the synthetic phages was assessed in vitro as well as in vivo using a Galleria mellonella infection model. Overall, both in vitro and in vivo studies revealed that the knock-outs made in phage genome do not impair the antibacterial properties of the synthetic phages, indicating that this could be a good strategy to clear space from phage genomes in order to enable the introduction of other genes of interest that can potentiate the future treatment of P. aeruginosa infections.

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