Characterization of Haemophilus ducreyi cdtA, cdtB, and cdtC Mutants in In Vitro and In Vivo Systems

ABSTRACT Haemophilus ducreyi expresses a soluble cytolethal distending toxin (CDT) that is encoded by the cdtABC gene cluster and can be detected in culture supernatant fluid by its ability to kill HeLa cells. The cdtA, cdtB, and cdtCgenes of H. ducreyi were cloned independently into plasmid vectors, and their encoded proteins expressed singly or in various combinations in an Escherichia coli background. All three gene products had to be expressed in order for E. coli-derived culture supernatant fluids to demonstrate cytotoxicity for HeLa cells. Isogenic H. ducreyi cdtA and cdtB mutants were constructed and used in combination with the wild-type parent strain and a previously describedH. ducreyi cdtC mutant (M. K. Stevens, J. L. Latimer, S. R. Lumbley, C. K. Ward, L. D. Cope, T. Lagergard, and E. J. Hansen, Infect. Immun. 67:3900–3908, 1999) to determine the relative contributions of the CdtA, CdtB, and CdtC proteins to CDT activity. Expression of CdtA, CdtB, and CdtC appeared necessary for H. ducreyi-derived culture supernatant fluid to exhibit cytotoxicity for HeLa cells. Whole-cell sonicates and periplasmic extracts from the cdtB and cdtC mutants had no effect on HeLa cells, whereas these same fractions from a cdtA mutant had a very modest cytotoxic effect on these same human cells. CdtA appeared to be primarily associated with the H. ducreyi cell envelope, whereas both CdtB and CdtC were present primarily in the soluble fraction from sonicated cells. Both the cdtAmutant and the cdtB mutant were found to be fully virulent in the temperature-dependent rabbit model for experimental chancroid.

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Characterization of a Haemophilus ducreyi Mutant Deficient in Expression of Cytolethal Distending Toxin

Infection and Immunity ◽

10.1128/iai.67.8.3900-3908.1999 ◽

1999 ◽

Vol 67 (8) ◽

pp. 3900-3908 ◽

Cited By ~ 49

Author(s):

Marla K. Stevens ◽

Jo L. Latimer ◽

Sheryl R. Lumbley ◽

Christine K. Ward ◽

Leslie D. Cope ◽

...

Keyword(s):

Gene Cluster ◽

Hela Cells ◽

Culture Supernatant ◽

Haemophilus Ducreyi ◽

Cytolethal Distending Toxin ◽

Supernatant Fluid ◽

Hacat Keratinocytes ◽

Wild Type ◽

Insertional Inactivation

ABSTRACT Haemophilus ducreyi expresses a soluble cytolethal distending toxin (CDT) that kills HeLa, HEp-2, and other human epithelial cells in vitro. H. ducreyi CDT activity is encoded by a three-gene cluster (cdtABC), and antibody to the cdtC gene product can neutralize CDT activity in vitro (L. D. Cope, S. R. Lumbley, J. L. Latimer, J. Klesney-Tait, M. K. Stevens, L. S. Johnson, M. Purven, R. S. Munson, Jr., T. Lagergard, J. D. Radolf, and E. J. Hansen, Proc. Natl. Acad. Sci. USA 94:4056–4061, 1997). Culture supernatant fluid from a recombinant Escherichia colistrain containing the H. ducreyi cdtABC gene cluster readily killed both HeLa cells and HaCaT keratinocytes and had a modest inhibitory effect on the growth of human foreskin fibroblasts. Insertional inactivation of the cdtC gene in this recombinant E. coli strain eliminated the ability of this strain to kill HeLa cells and HaCaT keratinocytes. This mutatedH. ducreyi cdtABC gene cluster was used to construct an isogenic H. ducreyi cdtC mutant. Monoclonal antibodies against the H. ducreyi CdtA, CdtB, and CdtC proteins were used to characterize protein expression by this cdtCmutant. Culture supernatant fluid from this H. ducreyi cdtCmutant did not detectably affect any of the human cells used in this study. The presence of the wild-type H. ducreyi cdtC gene in trans in this H. ducreyi mutant restored its ability to express a CDT that killed both HeLa cells and HaCaT keratinocytes. The isogenic H. ducreyi cdtC mutant was shown to be as virulent as its wild-type parent strain in the temperature-dependent rabbit model for experimental chancroid. Lack of expression of the H. ducreyi CdtC protein also did not affect the ability of this H. ducreyi mutant to survive in the skin of rabbits.

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The LspB Protein Is Involved in the Secretion of the LspA1 and LspA2 Proteins by Haemophilus ducreyi

Infection and Immunity ◽

10.1128/iai.72.4.1874-1884.2004 ◽

2004 ◽

Vol 72 (4) ◽

pp. 1874-1884 ◽

Cited By ~ 25

Author(s):

Christine K. Ward ◽

Jason R. Mock ◽

Eric J. Hansen

Keyword(s):

Membrane Protein ◽

Outer Membrane ◽

Outer Membrane Protein ◽

Culture Supernatant ◽

Cell Envelope ◽

Rabbit Model ◽

Haemophilus Ducreyi ◽

Pcr Analysis ◽

Supernatant Fluid ◽

Reading Frame

ABSTRACT The LspA1 and LspA2 proteins of Haemophilus ducreyi 35000 are two very large macromolecules that can be detected in concentrated culture supernatant fluid. Both of these proteins exhibit homology with the N-terminal region of the Bordetella pertussis filamentous hemagglutinin (FHA), which is involved in secretion of the latter macromolecule. The lspA2 open reading frame is flanked upstream by a gene, lspB, that encodes a predicted protein with homology to the B. pertussis FhaC outer membrane protein that is involved in secretion of FHA across the outer membrane. The H. ducreyi lspB gene encodes a protein with a predicted molecular mass of 66,573 Da. Reverse transcription-PCR analysis suggested that the lspB gene was transcribed together with the lspA2 gene on a single mRNA transcript. Polyclonal H. ducreyi LspB antiserum reacted with a 64-kDa antigen present in the Sarkosyl-insoluble cell envelope fraction of H. ducreyi 35000, which indicated that the LspB protein is likely an outer membrane protein. Concentrated culture supernatant fluids from H. ducreyi lspB and lspA1 lspB mutants did not contain detectable LspA1 and detectable LspA2, respectively. However, complementation of the lspB mutant with the wild-type lspB gene on a plasmid restored LspB protein expression and resulted in release of detectable amounts of the LspA1 protein into culture supernatant fluid. When evaluated in the temperature-dependent rabbit model of infection, the lspB mutant was attenuated in the ability to cause lesions and was never recovered in a viable form from lesions. These results indicated that the H. ducreyi LspB protein is involved in secretion of the LspA1 and LspA2 proteins across the outer membrane.

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Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

10.26434/chemrxiv.7725002.v1 ◽

2019 ◽

Author(s):

Priya Prakash ◽

Travis Lantz ◽

Krupal P. Jethava ◽

Gaurav Chopra

Keyword(s):

Amyloid Beta ◽

Western Blots ◽

Force Microscopy ◽

E Coli ◽

Atomic Force ◽

Set Up ◽

Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

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Functional identification of ygiP as a positive regulator of the ttdA-ttdB-ygjE operon

Microbiology ◽

10.1099/mic.0.28753-0 ◽

2006 ◽

Vol 152 (7) ◽

pp. 2129-2135 ◽

Cited By ~ 17

Author(s):

Taku Oshima ◽

Francis Biville

Keyword(s):

Functional Characterization ◽

Anaerobic Growth ◽

Functional Identification ◽

New Members ◽

E Coli ◽

Inner Membrane Protein ◽

Tartrate Dehydratase

Functional characterization of unknown genes is currently a major task in biology. The search for gene function involves a combination of various in silico, in vitro and in vivo approaches. Available knowledge from the study of more than 21 LysR-type regulators in Escherichia coli has facilitated the classification of new members of the family. From sequence similarities and its location on the E. coli chromosome, it is suggested that ygiP encodes a lysR regulator controlling the expression of a neighbouring operon; this operon encodes the two subunits of tartrate dehydratase (TtdA, TtdB) and YgiE, an integral inner-membrane protein possibly involved in tartrate uptake. Expression of tartrate dehydratase, which converts tartrate to oxaloacetate, is required for anaerobic growth on glycerol as carbon source in the presence of tartrate. Here, it has been demonstrated that disruption of ygiP, ttdA or ygjE abolishes tartrate-dependent anaerobic growth on glycerol. It has also been shown that tartrate-dependent induction of the ttdA-ttdB-ygjE operon requires a functional YgiP.

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Characterization of in vitro and in vivo murine models of human enteropathogenic E. coli (EPEC) infection

Gastroenterology ◽

10.1016/s0016-5085(03)82826-3 ◽

2003 ◽

Vol 124 (4) ◽

pp. A558

Author(s):

Suzana D. Savkovic ◽

Farol L. Tomson ◽

Michelle Muza ◽

Gail Hecht

Keyword(s):

Murine Models ◽

E Coli

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Characterization of MazFSa, an Endoribonuclease from Staphylococcus aureus

Journal of Bacteriology ◽

10.1128/jb.01272-07 ◽

2007 ◽

Vol 189 (24) ◽

pp. 8871-8879 ◽

Cited By ~ 57

Author(s):

Zhibiao Fu ◽

Niles P. Donegan ◽

Guido Memmi ◽

Ambrose L. Cheung

Keyword(s):

Staphylococcus Aureus ◽

Environmental Stress Response ◽

Binding Studies ◽

Cell Growth Arrest ◽

Consensus Sequences ◽

E Coli ◽

Endoribonuclease Activity

ABSTRACT The mazEF homologs of Staphylococcus aureus, designated mazEFsa , have been shown to cotranscribe with the sigB operon under stress conditions. In this study, we showed that MazEF Sa , as with their Escherichia coli counterparts, compose a toxin-antitoxin module wherein MazF Sa leads to rapid cell growth arrest and loss in viable CFU upon overexpression. MazF Sa is a novel sequence-specific endoribonuclease which cleaves mRNA to inhibit protein synthesis. Using ctpA mRNA as the model substrate both in vitro and in vivo, we demonstrated that MazF Sa cleaves single-strand RNA preferentially at the 5′ side of the first U or 3′ side of the second U residue within the consensus sequences VUUV′ (where V and V′ are A, C, or G and may or may not be identical). Binding studies confirmed that the antitoxin MazE Sa binds MazF Sa to form a complex to inhibit the endoribonuclease activity of MazF Sa . Contrary to the system in E. coli, exposure to selected antibiotics augmented mazEFsa transcription, akin to what one would anticipate from the environmental stress response of the sigB system. These data indicate that the mazEF system of S. aureus differs from the gram-negative counterparts with respect to mRNA cleavage specificity and antibiotic stresses.

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Peptidoglycan Association of Murein Lipoprotein Is Required for KpsD-Dependent Group 2 Capsular Polysaccharide Expression and Serum Resistance in a UropathogenicEscherichia coliIsolate

mBio ◽

10.1128/mbio.00603-17 ◽

2017 ◽

Vol 8 (3) ◽

Cited By ~ 11

Author(s):

Jingyu Diao ◽

Catrien Bouwman ◽

Donghong Yan ◽

Jing Kang ◽

Anand K. Katakam ◽

...

Keyword(s):

Outer Membrane ◽

Capsular Polysaccharide ◽

Cell Envelope ◽

Bloodstream Infections ◽

Serum Resistance ◽

E Coli ◽

Serum Killing ◽

Group 2

ABSTRACTMurein lipoprotein (Lpp) and peptidoglycan-associated lipoprotein (Pal) are major outer membrane lipoproteins inEscherichia coli. Their roles in cell-envelope integrity have been documented inE. colilaboratory strains, and while Lpp has been linked to serum resistancein vitro, the underlying mechanism has not been established. Here,lppandpalmutants of uropathogenicE. colistrain CFT073 showed reduced survival in a mouse bacteremia model, but only thelppmutant was sensitive to serum killingin vitro. The peptidoglycan-bound Lpp form was specifically required for preventing complement-mediated bacterial lysisin vitroand complement-mediated clearancein vivo. Compared to the wild-type strain, thelppmutant had impaired K2 capsular polysaccharide production and was unable to respond to exposure to serum by elevating capsular polysaccharide amounts. These properties correlated with altered cellular distribution of KpsD, the predicted outer membrane translocon for “group 2” capsular polysaccharides. We identified a novel Lpp-dependent association between functional KpsD and peptidoglycan, highlighting important interplay between cell envelope components required for resistance to complement-mediated lysis in uropathogenicE. coliisolates.IMPORTANCEUropathogenicE. coli(UPEC) isolates represent a significant cause of nosocomial urinary tract and bloodstream infections. Many UPEC isolates are resistant to serum killing. Here, we show that a major cell-envelope lipoprotein (murein lipoprotein) is required for serum resistancein vitroand for complement-mediated bacterial clearancein vivo. This is mediated, in part, through a novel mechanism by which murein lipoprotein affects the proper assembly of a key component of the machinery involved in production of “group 2” capsules. The absence of murein lipoprotein results in impaired production of the capsule layer, a known participant in complement resistance. These results demonstrate an important role for murein lipoprotein in complex interactions between different outer membrane biogenesis pathways and further highlight the importance of lipoprotein assembly and transport in bacterial pathogenesis.

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Characterization of P. sativum chloroplast psbA transcripts produced in vivo, in vitro and in E. coli

Plant Molecular Biology ◽

10.1007/bf00015229 ◽

1986 ◽

Vol 6 (4) ◽

pp. 229-243 ◽

Cited By ~ 38

Author(s):

Scott K. Boyer ◽

John E. Mullet

Keyword(s):

E Coli

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Identification and Partial Characterization of an L-Tyrosine Aminotransferase (TAT) fromArabidopsis thaliana

Biochemistry Research International ◽

10.1155/2010/549572 ◽

2010 ◽

Vol 2010 ◽

pp. 1-11 ◽

Cited By ~ 25

Author(s):

Pranav R. Prabhu ◽

André O. Hudson

Keyword(s):

Arabidopsis Thaliana ◽

Gene Family ◽

Tyrosine Aminotransferase ◽

Functional Complementation ◽

E Coli ◽

Full Length Cdna ◽

Putative Aminotransferase

The aminotransferase gene family in the model plantArabidopsis thalianaconsists of 44 genes. Twenty six of these enzymes are classified as characterized meaning that the reaction(s) that the enzyme catalyzes are documented using experimental means. The remaining 18 enzymes are uncharacterized and are therefore deemed putative. Our laboratory is interested in elucidating the function(s) of the remaining putative aminotransferase enzymes. To this end, we have identified and partially characterized an aminotransferase (TAT) enzyme from Arabidopsis annotated by the locus tag At5g36160. The full-length cDNA was cloned and the purified recombinant enzyme was characterized usingin vitroandin vivoexperiments.In vitroanalysis showed that the enzyme is capable of interconverting L-Tyrosine and 4-hydroxyphenylpyruvate, and L-Phenylalanine and phenylpyruvate.In vivoanalysis by functional complementation showed that the gene was able to complement anE. coliwith a background of aminotransferase mutations that confers auxotrophy for L-Tyrosine and L-Phenylalanine.

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