scholarly journals Mucosal Adjuvanticity and Immunogenicity of LTR72, a Novel Mutant of Escherichia coli Heat-labile Enterotoxin with Partial Knockout of ADP-ribosyltransferase Activity

1998 ◽  
Vol 187 (7) ◽  
pp. 1123-1132 ◽  
Author(s):  
Marzia Monica Giuliani ◽  
Giuseppe Del Giudice ◽  
Valentina Giannelli ◽  
Gordon Dougan ◽  
Gill Douce ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Enzymatic Activity ◽  
Wild Type ◽  
Ileal Loop ◽  
Heat Labile ◽  
A Subunit ◽  
Reduced Toxicity ◽  
Adp Ribosyltransferase

Heat-labile Escherichia coli enterotoxin (LT) has the innate property of being a strong mucosal immunogen and adjuvant. In the attempt to reduce toxicity and maintain the useful immunological properties, several LT mutants have been produced. Some of these are promising mucosal adjuvants. However, so far, only those that were still toxic maintained full adjuvanticity. In this paper we describe a novel LT mutant with greatly reduced toxicity that maintains most of the adjuvanticity. The new mutant (LTR72), that contains a substitution Ala → Arg in position 72 of the A subunit, showed only 0.6% of the LT enzymatic activity, was 100,000-fold less toxic than wild-type LT in Y1 cells in vitro, and was at least 20 times less effective than wild-type LT in the rabbit ileal loop assay in vivo. At a dose of 1 μg, LTR72 exhibited a mucosal adjuvanticity, similar to that observed with wild-type LT, better than that induced by the nontoxic, enzymatically inactive LTK63 mutant, and much greater than that of the recombinant B subunit. This trend was consistent for both the amounts and kinetics of the antibody induced, and priming of antigen-specific T lymphocytes. The data suggest that the innate high adjuvanticity of LT derives from the independent contribution of the nontoxic AB complex and the enzymatic activity. LTR72 optimizes the use of both properties: the enzymatic activity for which traces are enough, and the nontoxic AB complex, the effect of which is dose dependent. In fact, in dose–response experiments in mice, 20 μg of LTR72 were a stronger mucosal adjuvant than wild-type LT. This suggests that LTR72 may be an excellent candidate to be tested in clinical trials.

scholarly journals A genetically detoxified derivative of heat-labile Escherichia coli enterotoxin induces neutralizing antibodies against the A subunit.

1994 ◽  
Vol 180 (6) ◽  
pp. 2147-2153 ◽  
Author(s):  
M Pizza ◽  
M R Fontana ◽  
M M Giuliani ◽  
M Domenighini ◽  
C Magagnoli ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cholera Toxin ◽  
Neutralizing Antibodies ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
E Coli ◽  
Heat Labile ◽  
A Subunit ◽  
Adp Ribosyltransferase ◽  
Derivatives Of

Escherichia coli enterotoxin (LT) and the homologous cholera toxin (CT) are A-B toxins that cause travelers' diarrhea and cholera, respectively. So far, experimental live and killed vaccines against these diseases have been developed using only the nontoxic B portion of these toxins. The enzymatically active A subunit has not been used because it is responsible for the toxicity and it is reported to induce a negligible titer of toxin neutralizing antibodies. We used site-directed mutagenesis to inactivate the ADP-ribosyltransferase activity of the A subunit and obtained nontoxic derivatives of LT that elicited a good titer of neutralizing antibodies recognizing the A subunit. These LT mutants and equivalent mutants of CT may be used to improve live and killed vaccines against cholera and enterotoxinogenic E. coli.

scholarly journals Evading the Proteasome: Absence of Lysine Residues Contributes to Pertussis Toxin Activity by Evasion of Proteasome Degradation

2007 ◽  
Vol 75 (6) ◽  
pp. 2946-2953 ◽  
Author(s):  
Zoë E. V. Worthington ◽  
Nicholas H. Carbonetti
Keyword(s):  
Pertussis Toxin ◽  
Intracellular Transport ◽  
Cellular Activity ◽  
Wild Type ◽  
Proteasome Degradation ◽  
Lysine Residues ◽  
Arginine Residues ◽  
A Subunit ◽  
Adp Ribosyltransferase

ABSTRACT Pertussis toxin (PT) is an important virulence factor produced by Bordetella pertussis. PT holotoxin comprises one enzymatically active A subunit (S1), associated with a pentamer of B subunits. PT is an ADP-ribosyltransferase that modifies several mammalian heterotrimeric G proteins. Some bacterial toxins are believed to undergo retrograde intracellular transport through the Golgi apparatus to the endoplasmic reticulum (ER). The ER-associated degradation (ERAD) pathway involves the removal of misfolded proteins from the ER and degradation upon their return to the cytosol; this pathway may be exploited by PT and other toxins. In the cytosol, ERAD substrates are ubiquitinated at lysine residues, targeting them to the proteasome for degradation. We hypothesize that S1 avoids ubiquitination and proteasome degradation due to its lack of lysine residues. We predicted that the addition of lysine residues would reduce PT toxicity by allowing ubiquitination and degradation to occur. Variant forms of PT were engineered, replacing one, two, or three arginines with lysines in a variety of locations on S1. Several variants were identified with wild-type in vitro enzymatic activity but reduced cellular activity, consistent with our hypothesis. Significant recovery of the cellular activity of these variants was observed when CHO cells were pretreated with a proteasome inhibitor. We concluded that the replacement of arginine residues with lysine in the S1 subunit of PT renders the toxin subject to proteasomal degradation, suggesting that wild-type PT avoids proteasome degradation due to an absence of lysine residues.

scholarly journals Fimbria-Encoding GeneyadCHas a Pleiotropic Effect on Several Biological Characteristics and Plays a Role in Avian Pathogenic Escherichia coli Pathogenicity

2015 ◽  
Vol 84 (1) ◽  
pp. 187-193 ◽  
Author(s):  
Renu Verma ◽  
Thaís Cabrera Galvão Rojas ◽  
Renato Pariz Maluta ◽  
Janaína Luisa Leite ◽  
Livia Pilatti Mendes da Silva ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Soft Agar ◽  
Pleiotropic Effect ◽  
Biological Characteristics ◽  
Wild Type ◽  
Content Type ◽  
Pathogenic Escherichia Coli

The extraintestinal pathogen termed avian pathogenicEscherichia coli(APEC) is known to cause colibacillosis in chickens. The molecular basis of APEC pathogenesis is not fully elucidated yet. In this work, we deleted a component of the Yad gene cluster (yadC) in order to understand the role of Yad in the pathogenicity of the APEC strain SCI-07.In vitro, the transcription level ofyadCwas upregulated at 41°C and downregulated at 22°C. TheyadCexpressionin vivowas more pronounced in lungs than in spleen, suggesting a role in the early steps of the infection. Chicks infected with the wild-type and mutant strains presented, respectively, 80% and 50% mortality rates. The ΔyadCstrain presented a slightly decreased ability to adhere to HeLa cells with or without thed-mannose analog compared with the wild type. Real-time PCR (RT-PCR) assays showed thatfimHwas downregulated (P< 0.05) andcsgAandecpAwere slightly upregulated in the mutant strain, showing thatyadCmodulates expression of other fimbriae. Bacterial internalization studies showed that the ΔyadCstrain had a lower number of intracellular bacteria recovered from Hep-2 cells and HD11 cells than the wild-type strain (P< 0.05). Motility assays in soft agar demonstrated that the ΔyadCstrain was less motile than the wild type (P< 0.01). Curiously, flagellum-associated genes were not dramatically downregulated in the ΔyadCstrain. Taken together, the results show that the fimbrial adhesin Yad contributes to the pathogenicity and modulates different biological characteristics of the APEC strain SCI-07.

scholarly journals In vivo and in vitro adjuvant activities of the B subunit of Type IIb heat-labile enterotoxin (LT-IIb-B5) from Escherichia coli

Vaccine ◽  
2009 ◽  
Vol 27 (32) ◽  
pp. 4302-4308 ◽  
Author(s):  
Shuang Liang ◽  
Kavita B. Hosur ◽  
Hesham F. Nawar ◽  
Michael W. Russell ◽  
Terry D. Connell ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
B Subunit ◽  
Heat Labile

scholarly journals Cooperative Role of Antibodies against Heat-Labile Toxin and the EtpA Adhesin in Preventing Toxin Delivery and Intestinal Colonization by Enterotoxigenic Escherichia coli

2012 ◽  
Vol 19 (10) ◽  
pp. 1603-1608 ◽  
Author(s):  
Koushik Roy ◽  
David J. Hamilton ◽  
James M. Fleckenstein
Keyword(s):  
Escherichia Coli ◽  
Diarrheal Disease ◽  
Vaccine Development ◽  
Enterotoxigenic Escherichia Coli ◽  
Intestinal Colonization ◽  
Content Type ◽  
Heat Labile

ABSTRACTEnterotoxigenicEscherichia coli(ETEC) is an important cause of diarrheal disease in developing countries, where it is responsible for hundreds of thousands of deaths each year. Vaccine development for ETEC has been hindered by the heterogeneity of known molecular targets and the lack of broad-based sustained protection afforded by existing vaccine strategies. In an effort to explore the potential role of novel antigens in ETEC vaccines, we examined the ability of antibodies directed against the ETEC heat-labile toxin (LT) and the recently described EtpA adhesin to prevent intestinal colonizationin vivoand toxin delivery to epithelial cellsin vitro. We demonstrate that EtpA is required for the optimal delivery of LT and that antibodies against this adhesin play at least an additive role in preventing delivery of LT to target intestinal cells when combined with antibodies against either the A or B subunits of the toxin. Moreover, vaccination with a combination of LT and EtpA significantly impaired intestinal colonization. Together, these results suggest that the incorporation of recently identified molecules such as EtpA could be used to enhance current approaches to ETEC vaccine development.

scholarly journals Mutational Analysis of Residues in PriA and PriC Affecting Their Ability To Interact with SSB in Escherichia coli K-12

2020 ◽  
Vol 202 (23) ◽  
Author(s):  
Anastasiia N. Klimova ◽  
Steven J. Sandler
Keyword(s):  
Escherichia Coli ◽  
Mutational Analysis ◽  
Wild Type ◽  
Content Type ◽  
Growth Defects ◽  
C Terminus ◽  
K 12 ◽  
And Function

ABSTRACT Escherichia coli PriA and PriC recognize abandoned replication forks and direct reloading of the DnaB replicative helicase onto the lagging-strand template coated with single-stranded DNA-binding protein (SSB). Both PriA and PriC have been shown by biochemical and structural studies to physically interact with the C terminus of SSB. In vitro, these interactions trigger remodeling of the SSB on ssDNA. priA341(R697A) and priC351(R155A) negated the SSB remodeling reaction in vitro. Plasmid-carried priC351(R155A) did not complement priC303::kan, and priA341(R697A) has not yet been tested for complementation. Here, we further studied the SSB-binding pockets of PriA and PriC by placing priA341(R697A), priA344(R697E), priA345(Q701E), and priC351(R155A) on the chromosome and characterizing the mutant strains. All three priA mutants behaved like the wild type. In a ΔpriB strain, the mutations caused modest increases in SOS expression, cell size, and defects in nucleoid partitioning (Par−). Overproduction of SSB partially suppressed these phenotypes for priA341(R697A) and priA344(R697E). The priC351(R155A) mutant behaved as expected: there was no phenotype in a single mutant, and there were severe growth defects when this mutation was combined with ΔpriB. Analysis of the priBC mutant revealed two populations of cells: those with wild-type phenotypes and those that were extremely filamentous and Par− and had high SOS expression. We conclude that in vivo, priC351(R155A) identified an essential residue and function for PriC, that PriA R697 and Q701 are important only in the absence of PriB, and that this region of the protein may have a complicated relationship with SSB. IMPORTANCE Escherichia coli PriA and PriC recruit the replication machinery to a collapsed replication fork after it is repaired and needs to be restarted. In vitro studies suggest that the C terminus of SSB interacts with certain residues in PriA and PriC to recruit those proteins to the repaired fork, where they help remodel it for restart. Here, we placed those mutations on the chromosome and tested the effect of mutating these residues in vivo. The priC mutation completely abolished function. The priA mutations had no effect by themselves. They did, however, display modest phenotypes in a priB-null strain. These phenotypes were partially suppressed by SSB overproduction. These studies give us further insight into the reactions needed for replication restart.

scholarly journals In Vivo Titration of Mitomycin C Action by Four Escherichia coli Genomic Regions on Multicopy Plasmids

2001 ◽  
Vol 183 (7) ◽  
pp. 2259-2264 ◽  
Author(s):  
Yan Wei ◽  
Amy C. Vollmer ◽  
Robert A. LaRossa
Keyword(s):  
Escherichia Coli ◽  
Mitomycin C ◽  
Transcriptional Activation ◽  
Efflux Pump ◽  
Wild Type ◽  
Potent Inducer ◽  
E Coli ◽  
Genomic Regions

ABSTRACT Mitomycin C (MMC), a DNA-damaging agent, is a potent inducer of the bacterial SOS response; surprisingly, it has not been used to select resistant mutants from wild-type Escherichia coli. MMC resistance is caused by the presence of any of four distinctE. coli genes (mdfA, gyrl, rob, andsdiA) on high-copy-number vectors. mdfAencodes a membrane efflux pump whose overexpression results in broad-spectrum chemical resistance. The gyrI (also called sbmC) gene product inhibits DNA gyrase activity in vitro, while the rob protein appears to function in transcriptional activation of efflux pumps. SdiA is a transcriptional activator of ftsQAZ genes involved in cell division.

scholarly journals Differential Biological and Adjuvant Activities of Cholera Toxin and Escherichia coli Heat-Labile Enterotoxin Hybrids

2001 ◽  
Vol 69 (3) ◽  
pp. 1528-1535 ◽  
Author(s):  
Christal C. Bowman ◽  
John D. Clements
Keyword(s):  
Escherichia Coli ◽  
Cyclic Amp ◽  
Cholera Toxin ◽  
The Other ◽  
Toxin Gene ◽  
Wild Type ◽  
B Subunit ◽  
Heat Labile ◽  
A Subunit ◽  
Ifn Γ

ABSTRACT Two bacterial products that have been demonstrated to function as mucosal adjuvants are cholera toxin (CT), produced by various strains of Vibrio cholerae, and the heat-labile enterotoxin (LT) produced by some enterotoxigenic strains of Escherichia coli. Although LT and CT have many features in common, they are clearly distinct molecules with biochemical and immunologic differences which make them unique. The goal of this study was to determine the basis for these biological differences by constructing and characterizing chimeric CT-LT molecules. Toxin gene fragments were subcloned to create two constructs, each expressing the enzymatically active A subunit of one toxin and the receptor binding B subunit of the other toxin. These hybrid toxins were purified, and the composition and assembly of CT A subunit (CT-A)-LT B subunit (LT-B) and LT A subunit (LT-A)-CT B subunit (CT-B) were confirmed. Hybrids were evaluated for enzymatic activity, as measured by the accumulation of cyclic AMP in Caco-2 cells, and the enterotoxicity of each toxin was assessed in a patent-mouse assay. The results demonstrated that LT-A–CT-B induces the accumulation of lower levels of cyclic AMP and has less enterotoxicity than either wild-type toxin or the other hybrid. Nonetheless, this hybrid retains adjuvant activity equivalent to or greater than that of either wild-type toxin or the other hybrid when used in conjunction with tetanus toxoid for intranasal immunization of BALB/c mice. Importantly, the ability of LT to induce a type 1 cytokine response was found to be a function of LT-A. Specifically, LT-A–CT-B was able to augment the levels of antigen-specific gamma interferon (IFN-γ) and interleukin 5 to levels comparable to those achieved with native LT, while CT-A–LT-B and native CT both produced lower levels of antigen-specific IFN-γ. Thus, these toxin hybrids possess unique biological characteristics and provide information about the basis for differences in the biological activities observed for CT and LT.

scholarly journals Cnu, a Novel oriC-Binding Protein of Escherichia coli

2005 ◽  
Vol 187 (20) ◽  
pp. 6998-7008 ◽  
Author(s):  
Myung Suk Kim ◽  
Sung-Hun Bae ◽  
Sang Hoon Yun ◽  
Hee Jung Lee ◽  
Sang Chun Ji ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid Identity ◽  
Replication Initiation ◽  
In Vitro Assays ◽  
Wild Type ◽  
Genetic Method ◽  
Dna Replication Initiation ◽  
Pair Sequence

ABSTRACT We have found, using a newly developed genetic method, a protein (named Cnu, for oriC-binding nucleoid-associated) that binds to a specific 26-base-pair sequence (named cnb) in the origin of replication of Escherichia coli, oriC. Cnu is composed of 71 amino acids (8.4 kDa) and shows extensive amino acid identity to a group of proteins belonging to the Hha/YmoA family. Cnu was previously discovered as a protein that, like Hha, complexes with H-NS in vitro. Our in vivo and in vitro assays confirm the results and further suggest that the complex formation with H-NS is involved in Cnu/Hha binding to cnb. Unlike the hns mutants, elimination of either the cnu or hha gene did not disturb the growth rate, origin content, and synchrony of DNA replication initiation of the mutants compared to the wild-type cells. However, the cnu hha double mutant was moderately reduced in origin content. The Cnu/Hha complex with H-NS thus could play a role in optimal activity of oriC.

scholarly journals Contributions of O Island 48 to Adherence of Enterohemorrhagic Escherichia coli O157:H7 to Epithelial Cells In Vitro and in Ligated Pig Ileal Loops

2009 ◽  
Vol 75 (18) ◽  
pp. 5779-5786 ◽  
Author(s):  
Xianhua Yin ◽  
Roger Wheatcroft ◽  
James R. Chambers ◽  
Bianfang Liu ◽  
Jing Zhu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Epithelial Cells ◽  
Cluster Formation ◽  
Gene Clusters ◽  
Enterohemorrhagic Escherichia Coli ◽  
Wild Type ◽  
The Difference ◽  
Large Clusters

ABSTRACT O island 48 (OI-48) of Escherichia coli consists of three functional gene clusters that encode urease, tellurite resistance (Ter), and putative adhesins Iha and AIDA-1. The functions of these clusters in enterohemorrhagic E. coli (EHEC) O157:H7 infection are unknown. Deletion mutants for these three regions were constructed and evaluated for their ability to adhere to epithelial cells in vitro and in ligated pig ileal loops. Deletion of the Ter gene cluster reduced the ability of the organism to adhere to and form large clusters on IPEC-J2 and HEp-2 cells. Complementation of the mutation by introducing the wild-type ter genes restored adherence and large-cluster formation. Tests in ligated pig ileal loops showed a decrease in colonization by the Ter-negative mutant, but the difference was not significant compared to colonization by the wild type (26.4% ± 21.2% versus 40.1% ± 19.1%; P = 0.168). The OI-48 aidA gene deletion had no effect on adherence in vitro or in vivo. Deletion of the iha and ureC genes had no effect on adherence in vitro but significantly reduced the colonization of EHEC O157:H7 in the ligated pig intestine. These data suggest that Ter, Iha, and urease may contribute to EHEC O157:H7 pathogenesis by promoting adherence of the pathogen to the host intestinal epithelium.

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