scholarly journals Fine-Tuning Synthesis of Yersinia pestis LcrV from Runaway-Like Replication Balanced-Lethal Plasmid in a Salmonella enterica Serovar Typhimurium Vaccine Induces Protection against a Lethal Y. pestis Challenge in Mice

2010 ◽  
Vol 78 (6) ◽  
pp. 2529-2543 ◽  
Author(s):  
Ascención Torres-Escobar ◽  
María Dolores Juárez-Rodríguez ◽  
Bronwyn M. Gunn ◽  
Christine G. Branger ◽  
Steven A. Tinge ◽  
...  
Keyword(s):  
Yersinia Pestis ◽  
Salmonella Enterica ◽  
Copy Number ◽  
Expression System ◽  
Fine Tuning ◽  
High Copy Number ◽  
Genetic Switch ◽  
Th2 Immune Response ◽  
Serovar Typhimurium

ABSTRACT A balanced-lethal plasmid expression system that switches from low-copy-number to runaway-like high-copy-number replication (pYA4534) was constructed for the regulated delayed in vivo synthesis of heterologous antigens by vaccine strains. This is an antibiotic resistance-free maintenance system containing the asdA gene (essential for peptidoglycan synthesis) as a selectable marker to complement the lethal chromosomal ΔasdA allele in live recombinant attenuated Salmonella vaccines (RASVs) such as Salmonella enterica serovar Typhimurium strain χ9447. pYA4534 harbors two origins of replication, pSC101 and pUC (low and high copy numbers, respectively). The pUC replication origin is controlled by a genetic switch formed by the operator/promoter of the P22 cro gene (O/Pcro) (PR), which is negatively regulated by an arabinose-inducible P22 c2 gene located on both the plasmid and the chromosome (araC PBAD c2). The absence of arabinose, which is unavailable in vivo, triggers replication to a high-copy-number plasmid state. To validate these vector attributes, the Yersinia pestis virulence antigen LcrV was used to develop a vaccine against plague. An lcrV sequence encoding amino acids 131 to 326 (LcrV196) was optimized for expression in Salmonella, flanked with nucleotide sequences encoding the signal peptide (SS) and the carboxy-terminal domain (CT) of β-lactamase, and cloned into pYA4534 under the control of the Ptrc promoter to generate plasmid pYA4535. Our results indicate that the live Salmonella vaccine strain χ9447 harboring pYA4535 efficiently stimulated a mixed Th1/Th2 immune response that protected mice against lethal challenge with Y. pestis strain CO92 introduced through either the intranasal or subcutaneous route.

Isolation of STD1, a high-copy-number suppressor of a dominant negative mutation in the yeast TATA-binding protein

1993 ◽  
Vol 13 (6) ◽  
pp. 3650-3659
Author(s):  
R W Ganster ◽  
W Shen ◽  
M C Schmidt
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Copy Number ◽  
Slow Growth ◽  
Tata Binding Protein ◽  
Dominant Negative ◽  
High Copy Number ◽  
N Terminus ◽  
Growth Phenotype

The TATA-binding protein (TBP) is an essential component of the transcriptional machinery of all three nuclear RNA polymerase enzymes. Comparison of the amino acid sequence of TBPs from a number of species reveals a highly conserved 180-residue C-terminal domain. In contrast, the N terminus is variable in both size and amino acid sequence. Overexpression of a TBP protein with a deletion of the nonconserved N terminus (TBP delta 57) in Saccharomyces cerevisiae results in a dominant negative phenotype of extremely slow growth. Associated with the slow-growth phenotype are defects in RNA polymerase II transcription in vivo. We have screened a high-copy-number yeast genomic library for suppression of the slow-growth phenotype and have isolated plasmids which encode suppressors of TBP delta 57 overexpression. Here we report the sequence and initial characterization of one suppressor, designated STD1 for suppressor of TBP deletion. The STD1 gene contains a single continuous open reading frame with the potential to encode a 50.2-kDa protein. Disruption of the STD1 gene indicates that it is not essential for vegetative growth, mating, or sporulation. High-copy-number suppression by the STD1 gene is not the result of a decrease in TBP delta 57 protein accumulation or DNA-binding activity; instead, STD1 suppression is coincident with the elimination of TBP delta 57-induced RNA polymerase II defects in both uninduced and induced transcription in vivo.

scholarly journals Role of sapA and yfgA in Susceptibility to Antibody-Mediated Complement-Dependent Killing and Virulence of Salmonella enterica Serovar Typhimurium

2017 ◽  
Vol 85 (9) ◽  
Author(s):  
Edna M. Ondari ◽  
Jennifer N. Heath ◽  
Elizabeth J. Klemm ◽  
Gemma Langridge ◽  
Lars Barquist ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Protein Translocation ◽  
Invasive Disease ◽  
Immune Serum ◽  
Insertion Mutant ◽  
Content Type ◽  
Serovar Typhimurium ◽  
Serum Killing ◽  
Increased Sensitivity

ABSTRACT The ST313 pathovar of Salmonella enterica serovar Typhimurium contributes to a high burden of invasive disease among African infants and HIV-infected adults. It is characterized by genome degradation (loss of coding capacity) and has increased resistance to antibody-dependent complement-mediated killing compared with enterocolitis-causing strains of S. Typhimurium. Vaccination is an attractive disease-prevention strategy, and leading candidates focus on the induction of bactericidal antibodies. Antibody-resistant strains arising through further gene deletion could compromise such a strategy. Exposing a saturating transposon insertion mutant library of S. Typhimurium to immune serum identified a repertoire of S. Typhimurium genes that, when interrupted, result in increased resistance to serum killing. These genes included several involved in bacterial envelope biogenesis, protein translocation, and metabolism. We generated defined mutant derivatives using S. Typhimurium SL1344 as the host. Based on their initial levels of enhanced resistance to killing, yfgA and sapA mutants were selected for further characterization. The S. Typhimurium yfgA mutant lost the characteristic Salmonella rod-shaped appearance, exhibited increased sensitivity to osmotic and detergent stress, lacked very long lipopolysaccharide, was unable to invade enterocytes, and demonstrated decreased ability to infect mice. In contrast, the S. Typhimurium sapA mutants had similar sensitivity to osmotic and detergent stress and lipopolysaccharide profile and an increased ability to infect enterocytes compared with the wild type, but it had no increased ability to cause in vivo infection. These findings indicate that increased resistance to antibody-dependent complement-mediated killing secondary to genetic deletion is not necessarily accompanied by increased virulence and suggest the presence of different mechanisms of antibody resistance.

scholarly journals Terminal Deoxynucleotidyl Transferase Is Not Required for Antibody Response to Polysaccharide Vaccines againstStreptococcus pneumoniaeandSalmonella entericaSerovar Typhi

2018 ◽  
Vol 86 (9) ◽  
Author(s):  
Vivek Belde ◽  
Matthew P. Cravens ◽  
Dania Gulandijany ◽  
Justin A. Walker ◽  
Isabel Palomo-Caturla ◽  
...  
Keyword(s):  
Antibody Response ◽  
B Cell ◽  
Salmonella Enterica ◽  
Passive Immunization ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Human Infants ◽  
Content Type ◽  
Serovar Typhimurium

ABSTRACTB cell antigen receptor (BCR) diversity increases by several orders of magnitude due to the action of terminal deoxynucleotidyl transferase (TdT) during V(D)J recombination. Unlike adults, infants have limited BCR diversity, in part due to reduced expression of TdT. Since human infants and young mice respond poorly to polysaccharide vaccines, such as the pneumococcal polysaccharide vaccine Pneumovax23 and Vi polysaccharide (ViPS) ofSalmonella entericaserovar Typhi, we tested the contribution of TdT-mediated BCR diversity in response to these vaccines. We found that TdT+/−and TdT−/−mice generated comparable antibody responses to Pneumovax23 and survivedStreptococcus pneumoniaechallenge. Moreover, passive immunization of B cell-deficient mice with serum from Pneumovax23-immunized TdT+/−or TdT−/−mice conferred protection. TdT+/−and TdT−/−mice generated comparable levels of anti-ViPS antibodies and antibody-dependent, complement-mediated bactericidal activity againstS. Typhiin vitro. To test the protective immunity conferred by ViPS immunizationin vivo, TdT+/−and TdT−/−mice were challenged with a chimericSalmonella entericaserovar Typhimurium strain expressing ViPS, since mice are nonpermissive hosts forS. Typhi infection. Compared to their unimmunized counterparts, immunized TdT+/−and TdT−/−mice challenged with ViPS-expressingS. Typhimurium exhibited a significant reduction in the bacterial burden and liver pathology. These data suggest that the impaired antibody response to the Pneumovax23 and ViPS vaccines in the young is not due to limited TdT-mediated BCR diversification.

scholarly journals Attenuation of Salmonella enterica Serovar Typhimurium by Altering Biological Functions of Murein Lipoprotein and Lipopolysaccharide

2005 ◽  
Vol 73 (12) ◽  
pp. 8433-8436 ◽  
Author(s):  
A. A. Fadl ◽  
J. Sha ◽  
G. R. Klimpel ◽  
J. P. Olano ◽  
C. L. Galindo ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Salmonella Enterica Serovar Typhimurium ◽  
Biological Functions ◽  
Double Knockout ◽  
In Vivo Models ◽  
Background Strain ◽  
Knockout Mutants ◽  
Serovar Typhimurium

ABSTRACT We constructed Salmonella enterica serovar Typhimurium double-knockout mutants in which either the lipoprotein A (lppA) or the lipoprotein B (lppB) gene was deleted from an msbB-negative background strain by marker exchange mutagenesis. These mutants were highly attenuated when tested with in vitro and in vivo models of Salmonella pathogenesis.

scholarly journals Differences in Enzymatic Properties Allow SodCI but Not SodCII To Contribute to Virulence in Salmonella enterica Serovar Typhimurium Strain 14028

2004 ◽  
Vol 186 (16) ◽  
pp. 5230-5238 ◽  
Author(s):  
Radha Krishnakumar ◽  
Maureen Craig ◽  
James A. Imlay ◽  
James M. Slauch
Keyword(s):  
Salmonella Enterica ◽  
Osmotic Shock ◽  
Acid Resistance ◽  
Noncovalent Interaction ◽  
Open Reading Frames ◽  
Striking Difference ◽  
Significant Difference ◽  
Serovar Typhimurium

ABSTRACT Salmonella enterica serovar Typhimurium produces two Cu/Zn cofactored periplasmic superoxide dismutases, SodCI and SodCII. While mutations in sodCI attenuate virulence eightfold, loss of SodCII does not confer a virulence phenotype, nor does it enhance the defect observed in a sodCI background. Despite this in vivo phenotype, SodCI and SodCII are expressed at similar levels in vitro during the stationary phase of growth. By exchanging the open reading frames of sodCI and sodCII, we found that SodCI contributes to virulence when placed under the control of the sodCII promoter. In contrast, SodCII does not contribute to virulence even when expressed from the sodCI promoter. Thus, the disparity in virulence phenotypes is due primarily to some physical difference between the two enzymes. In an attempt to identify the unique property of SodCI, we have tested factors that might affect enzyme activity inside a phagosome. We found no significant difference between SodCI and SodCII in their resistance to acid, resistance to hydrogen peroxide, or ability to obtain copper in a copper-limiting environment. Both enzymes are synthesized as apoenzymes in the absence of copper and can be fully remetallated when copper is added. The one striking difference that we noted is that, whereas SodCII is released normally by an osmotic shock, SodCI is “tethered” within the periplasm by an apparently noncovalent interaction. We propose that this novel property of SodCI is crucial to its ability to contribute to virulence in serovar Typhimurium.

scholarly journals Novel Salmonella enterica Serovar Typhimurium Protein That Is Indispensable for Virulence and Intracellular Replication

2001 ◽  
Vol 69 (12) ◽  
pp. 7413-7418 ◽  
Author(s):  
Tahar van der Straaten ◽  
Angela van Diepen ◽  
Kitty Kwappenberg ◽  
Sjaak van Voorden ◽  
Kees Franken ◽  
...  
Keyword(s):  
Amino Acid ◽  
Protein Function ◽  
Salmonella Enterica ◽  
Host Cells ◽  
Wild Type ◽  
Intracellular Replication ◽  
Oxygen Intermediates ◽  
Serovar Typhimurium

ABSTRACT Upon contact with host cells, the intracellular pathogenSalmonella enterica serovar Typhimurium promotes its uptake, targeting, and survival in intracellular niches. In this process, the bacterium evades the microbicidal effector mechanisms of the macrophage, including oxygen intermediates. This study reports the phenotypic and genotypic characterization of an S. enterica serovar Typhimurium mutant that is hypersusceptible to superoxide. The susceptible phenotype is due to a MudJ insertion-inactivation of a previously undescribedSalmonella gene designated sspJ that is located between 54.4 and 64 min of the Salmonellachromosome and encodes a 392-amino-acid protein. In vivo, upon intraperitoneal injection of 104 to 107bacteria in C3H/HeN and 101 to 104 bacteria in BALB/c mice, the mutant strain was less virulent than the wild type. Consistent with this finding, during the first hour after ingestion by macrophage-like J774 and RAW264.7 cells in vitro, the intracellular killing of the strain carrying sspJ::MudJ is enhanced fivefold over that of wild-type microorganisms. Wild-type salmonellae displayed significant intracellular replication during the first 24 h after uptake, but sspJ::MudJ mutants failed to do so. This phenotype could be restored to that of the wild type by sspJ complementation. The SspJ protein is found in the cytoplasmic membrane and periplasmic space. Amino acid sequence homology analysis did reveal a leader sequence and putative pyrroloquinoline quinone-binding domains, but no putative protein function. We excluded the possibility that SspJ is a scavenger of superoxide or has superoxide dismutase activity.

scholarly journals Yersinia pestis Is Viable with Endotoxin Composed of Only Lipid A

2005 ◽  
Vol 187 (18) ◽  
pp. 6599-6600 ◽  
Author(s):  
Li Tan ◽  
Creg Darby
Keyword(s):  
Escherichia Coli ◽  
Yersinia Pestis ◽  
Outer Membrane ◽  
Salmonella Enterica ◽  
Lipid A ◽  
Gram Negative Bacteria ◽  
Membrane Component ◽  
Gram Negative ◽  
Serovar Typhimurium

ABSTRACT Lipopolysaccharide (LPS) is the major outer membrane component of gram-negative bacteria. The minimal LPS structure for viability of Escherichia coli and Salmonella enterica serovar Typhimurium is lipid A glycosylated with 3-deoxy-D-manno-octulosonic acid (Kdo) residues. Here we show that another member of the Enterobacteriaceae, Yersinia pestis, can survive without Kdo in its LPS.

scholarly journals In Vitro and In Vivo Assessment of Salmonella enterica Serovar Typhimurium DT104 Virulence

2001 ◽  
Vol 69 (7) ◽  
pp. 4673-4677 ◽  
Author(s):  
Chris A. Allen ◽  
Paula J. Fedorka-Cray ◽  
Andrés Vazquez-Torres ◽  
Mitsu Suyemoto ◽  
Craig Altier ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Phage Type ◽  
Multidrug Resistant ◽  
Peritoneal Macrophages ◽  
Lethal Infection ◽  
Animal Infection ◽  
Serovar Typhimurium ◽  
Clinical Illness

ABSTRACT Multidrug-resistant Salmonella enterica serovar Typhimurium phage type DT104 has become a widespread cause of human and other animal infection worldwide. The severity of clinical illness inS. enterica serovar Typhimurium DT104 outbreaks has led to the suggestion that this strain possesses enhanced virulence. In the present study, in vitro and in vivo virulence-associated phenotypes of several clinical isolates of S. enterica serovar Typhimurium DT104 were examined and compared to S. entericaserovar Typhimurium ATCC 14028s. The ability of these DT104 isolates to survive within murine peritoneal macrophages, invade cultured epithelial cells, resist antimicrobial actions of reactive oxygen and nitrogen compounds, and cause lethal infection in mice were assessed. Our results failed to demonstrate that S. enterica serovar Typhimurium DT104 isolates are more virulent than S. enterica serovar Typhimurium ATCC 14028s.

scholarly journals Increased gene copy number of DEFA1/DEFA3 worsens sepsis by inducing endothelial pyroptosis

2019 ◽  
Vol 116 (8) ◽  
pp. 3161-3170 ◽  
Author(s):  
QiXing Chen ◽  
Yang Yang ◽  
JinChao Hou ◽  
Qiang Shu ◽  
YiXuan Yin ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Copy Number ◽  
Treatment Options ◽  
Gene Copy Number ◽  
High Copy Number ◽  
Gene Copy ◽  
Dependent Manner ◽  
Barrier Dysfunction ◽  
Specific Expression

Sepsis claims an estimated 30 million episodes and 6 million deaths per year, and treatment options are rather limited. Human neutrophil peptides 1–3 (HNP1–3) are the most abundant neutrophil granule proteins but their neutrophil content varies because of unusually extensive gene copy number polymorphism. A genetic association study found that increased copy number of the HNP-encoding gene DEFA1/DEFA3 is a risk factor for organ dysfunction during sepsis development. However, direct experimental evidence demonstrating that these risk alleles are pathogenic for sepsis is lacking because the genes are present only in some primates and humans. Here, we generate DEFA1/DEFA3 transgenic mice with neutrophil-specific expression of the peptides. We show that mice with high copy number of DEFA1/DEFA3 genes have more severe sepsis-related vital organ damage and mortality than mice with low copy number of DEFA1/DEFA3 or wild-type mice, resulting from more severe endothelial barrier dysfunction and endothelial cell pyroptosis after sepsis challenge. Mechanistically, HNP-1 induces endothelial cell pyroptosis via P2X7 receptor-mediating canonical caspase-1 activation in a NLRP3 inflammasome-dependent manner. Based on these findings, we engineered a monoclonal antibody against HNP-1 to block the interaction with P2X7 and found that the blocking antibody protected mice carrying high copy number of DEFA1/DEFA3 from lethal sepsis. We thus demonstrate that DEFA1/DEFA3 copy number variation strongly modulates sepsis development in vivo and explore a paradigm for the precision treatment of sepsis tailored by individual genetic information.

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