High efficiency transformation of Salmonella typhimurium and Salmonella typhi by electroporation

O’Callaghan, David; Charbit, Alain

doi:10.1007/bf00315809

Cited by 111 publications

(74 citation statements)

References 7 publications

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“…The Pseudomonas wild type toxin was cloned by PCR using primers 1 and 2. When the complete construct was confirmed by DNA sequencing using primers 3–10, the plasmids were transformed into electroporation competent JR501 (O’Callaghan and Charbit, 1990; Tsai et al, 1989) to provide the appropriate methylation. Plasmid DNA was then prepared from the JR501 clones and then transformed by electroporation into VNP20009.…”

Section: Methodsmentioning

confidence: 99%

EGFR‐targeted Chimeras of Pseudomonas ToxA released into the extracellular milieu by attenuated Salmonella selectively kill tumor cells

Quintero

Carrafa²,

Vincent³

et al. 2016

Biotech & Bioengineering

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Tumor-targeted Salmonella VNP20009 preferentially replicate within tumor tissue and partially suppress tumor growth in murine tumor models. These Salmonella have the ability to locally induce apoptosis when they are in direct contact with cancer cells but they lack significant bystander killing, which may correlate with their overall lack of antitumor activity in human clinical studies. In order to compensate for this deficiency without enhancing overall toxicity, we engineered the bacteria to express epidermal growth factor receptor (EGFR)-targeted cytotoxic proteins that are released into the extracellular milieu. In this study, we demonstrate the ability of the Salmonella strain VNP20009 to produce three different forms of the Pseudomonas exotoxin A (ToxA) chimeric with a tumor growth factor alpha (TGFα) which results in its producing culture supernatants that are cytotoxic and induce apoptosis in EGFR positive cancer cells as measured by the tetrazolium dye reduction, and Rhodamine 123 and JC-10 mitochondrial depolarization assays. In addition, exchange of the ToxA REDLK endoplasmic reticulum retention signal for KDEL and co-expression of the ColE3 lysis protein resulted in an overall increased cytotoxicity compared to the wild type toxin. This approach has the potential to significantly enhance the antitumor activity of VNP20009 while maintaining its previously established safety profile.

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Section: Methodsmentioning

confidence: 99%

EGFR‐targeted Chimeras of Pseudomonas ToxA released into the extracellular milieu by attenuated Salmonella selectively kill tumor cells

Quintero

Carrafa²,

Vincent³

et al. 2016

Biotech & Bioengineering

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“…When required, antibiotics, amino acids or supplements were added at the following concentrations: 50 mg ampicilin ml 21 , 50 mg diaminopimelic acid (DAP) ml 21 , 34 mg chloramphenicol ml 21 , 22 mg tryptophan ml 21 , 22 mg cysteine ml 21 and 22 mg arginine ml 21 . Transformation of bacterial strains was routinely done by using the calcium/manganese-based (CCMB) or electroporation methods, as described by O'Callaghan & Charbit (1990).…”

Section: Methodsmentioning

confidence: 99%

A novel PhoP-regulated locus encoding the cytolysin ClyA and the secreted invasin TaiA of Salmonella enterica serovar Typhi is involved in virulence

2009

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Salmonella enterica serovar Typhi causes a human-restricted systemic infection called typhoid fever. We have identified a Typhi genomic region encoding two ORFs, STY1498 and STY1499, that are expressed during infection of human macrophages and organized in an operon. STY1498 corresponds to clyA, which encodes a pore-forming cytolysin, and STY1499 encodes a 27 kDa protein, without any attributed function, which we have named TaiA (Typhi-associated invasin A). In order to evaluate the roles of these genes in Typhi pathogenesis, isogenic Typhi strains harbouring a non-polar mutation of either clyA or taiA were constructed. In macrophages, taiA was involved in increasing phagocytosis, as taiA deletion reduced bacterial uptake, whereas clyA reduced or controlled bacterial growth, as clyA deletion enhanced Typhi survival within macrophages without affecting cytotoxicity. In epithelial cells, deletion of taiA had no effect on invasion, whereas deletion of clyA enhanced the Typhi invasion rate, and reduced cytotoxicity. Overexpression of taiA in Typhi or in Escherichia coli resulted in a higher invasion rate of epithelial cells. We have demonstrated that TaiA is secreted independently of both the Salmonella pathogenicity island (SPI)-1 and the SPI-2 type three secretion systems. We have shown that this operon is regulated by the virulence-associated regulator PhoP. Moreover, our results revealed that products of this operon might be involved in promoting the use of macrophages as a sheltered reservoir for Typhi and allowing long-term persistence inside the host. INTRODUCTIONThe genus Salmonella is composed of two distinct species, Salmonella bongori and Salmonella enterica. While S. bongori is rarely involved in human infections, S. enterica is a major human pathogen. Out of the 2000 serovars of S. enterica, only a small fraction is associated with human infections (Porwollik et al., 2004). Serovars Typhimurium and Enteritidis cause a localized infection, gastroenteritis, whereas serovar Typhi causes a severe systemic infection called typhoid fever, which kills an estimated 600 000 people annually (Parry et al., 2002). Because Typhi is restricted to humans, Typhimurium has been used for many years to study typhoid fever pathogenesis using a murine infection model in which Typhimurium causes a systemic infection. This model has been crucial in understanding systemic infections by Salmonella.S. enterica and S. bongori possess a type three secretion system (T3SS), encoded by Salmonella pathogenicity island 1 (SPI-1), which mediates invasion of host cells (Galan, 1999;Marcus et al., 2000). Only S. enterica possesses a second T3SS located in SPI-2, which is required for survival inside macrophages and the infection of mammalian hosts . The T3SS injects bacterial proteins directly into the host cell and disturbs normal cell function. Induction of the SPI-1-encoded genes requires high osmolarity and low aeration, conditions present in the small intestine where the SPI-1 T3SS initiates cell invasion (Altier, 2005;Lostroh & Lee,...

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“…When required, antibiotics, amino acids, or supplements were added at the following concentrations: kanamycin, ampicillin, and diaminopimelic acid (DAP), 50 g/ml; chloramphenicol, 34 g/ml; and tryptophan, cysteine, and arginine, 22 g/ml. Transformation of bacterial strains was routinely done by using the calcium/manganesebased or electroporation method as described previously (27).…”

Section: Methodsmentioning

confidence: 99%

Contribution of thestgFimbrial Operon ofSalmonella entericaSerovar Typhi during Interaction with Human Cells

et al. 2007

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Salmonella serovars contain a wide variety of putative fimbrial systems that may contribute to colonization of specific niches. Salmonella enterica serovar Typhi is the etiologic agent of typhoid fever and is a pathogen specific to humans. In a previous study, we identified a gene, STY3920 (stgC), encoding the predicted usher of the stg fimbrial operon, that was expressed by serovar Typhi during infection of human macrophages. The stg genes are located in the glmS-pstS intergenic region in serovar Typhi and certain Escherichia coli strains, but they are absent in other S. enterica serovars. We cloned the stg fimbrial operon into a nonfimbriate E. coli K-12 strain and into S. enterica serovar Typhimurium. We demonstrated that the stg fimbrial operon contributed to increased adherence to human epithelial cells. Transcriptional fusion assays with serovar Typhi suggested that stg is preferentially expressed in minimal medium. Deletion of stg reduced adherence of serovar Typhi to epithelial cells. However, deletion of stg increased uptake of serovar Typhi by human macrophages, and overexpression of stg in serovar Typhi and serovar Typhimurium strains reduced phagocytosis by human macrophages. These strains survived inside macrophages as well as the wild-type parent. Although the stgC gene contains a premature stop codon that disrupts the expected open reading frame encoding the usher and is therefore considered a pseudogene, our results show that the stg operon may encode a functional fimbria. Thus, this serovar Typhi-specific fimbrial operon contributes to interactions with host cells, and further characterization is important for understanding the role of the stg fimbrial cluster in typhoid fever pathogenesis.

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High efficiency transformation of Salmonella typhimurium and Salmonella typhi by electroporation

Cited by 111 publications

References 7 publications

EGFR‐targeted Chimeras of Pseudomonas ToxA released into the extracellular milieu by attenuated Salmonella selectively kill tumor cells

EGFR‐targeted Chimeras of Pseudomonas ToxA released into the extracellular milieu by attenuated Salmonella selectively kill tumor cells

A novel PhoP-regulated locus encoding the cytolysin ClyA and the secreted invasin TaiA of Salmonella enterica serovar Typhi is involved in virulence

Contribution of thestgFimbrial Operon ofSalmonella entericaSerovar Typhi during Interaction with Human Cells

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