Site-Directed Mutagenesis and Its Application in Studying the Interactions of T3S Components

Francis, Matthew S.; Amer, Ayad A. A.; Milton, Debra L.; Costa, Tiago

doi:10.1007/978-1-4939-6649-3_2

Cited by 13 publications

(11 citation statements)

References 88 publications

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“…The bacterial strains and plasmids used in this study are listed in Table 1. The gene deletion mutant strains were constructed by double exchange of homologous recombination as described previously [77]. The upstream and downstream fragments of target genes were amplified using PCR by employing the primers listed in Table 2.…”

Section: Bacterial Strainsmentioning

confidence: 99%

Essential role of Salmonella Enteritidis DNA adenine methylase in modulating inflammasome activation

Guo

Huang

et al. 2020

BMC Microbiol

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Background: Salmonella Enteritidis (SE) is one of the major foodborne zoonotic pathogens of worldwide importance which can induce activation of NLRC4 and NLRP3 inflammasomes during infection. Given that the inflammasomes play an essential role in resisting bacterial infection, Salmonella has evolved various strategies to regulate activation of the inflammasome, most of which largely remain unclear. Results: A transposon mutant library in SE strain C50336 was screened for the identification of the potential factors that regulate inflammasome activation. We found that T3SS-associated genes invC, prgH, and spaN were required for inflammasome activation in vitro. Interestingly, C50336 strains with deletion or overexpression of Dam were both defective in activation of caspase-1, secretion of IL-1β and phosphorylation of c-Jun N-terminal kinase (Jnk). Transcriptome sequencing (RNA-seq) results showed that most of the differentially expressed genes and enriched KEGG pathways between the C50336-VS-C50336Δdam and C50336-VS-C50336::dam groups overlapped, which includes multiple signaling pathways related to the inflammasome. C50336Δdam and C50336::dam were both found to be defective in suppressing the expression of several anti-inflammasome factors. Moreover, overexpression of Dam in macrophages by lentiviral infection could specifically enhance the activation of NLRP3 inflammasome independently via promoting the Jnk pathway. Conclusions: These data indicated that Dam was essential for modulating inflammasome activation during SE infection, there were complex and dynamic interplays between Dam and the inflammasome under different conditions. New insights were provided about the battle between SE and host innate immunological mechanisms.

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Section: Bacterial Strainsmentioning

confidence: 99%

Essential role of Salmonella Enteritidis DNA adenine methylase in modulating inflammasome activation

Guo

Huang

et al. 2020

BMC Microbiol

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“…All the plasmid used in the study were cloned based on conventional PCR, restriction digestion, and ligation. Mutated alleles were generated by overlap PCR adapted from the previous protocol (reviewed in Francis et al, 2017). In essence of overlap PCR is based on four strategically designed primers.…”

Section: Construction Of Plasmidsmentioning

confidence: 99%

Inhibition of PLK1-dependent EBNA2 phosphorylation promotes lymphomagenesis in EBV-infected mice

Zhang

Schuhmachers

Mourão

et al. 2021

Preprint

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While Epstein-Barr virus (EBV) establishes a life-long latent infection in apparently healthy human immunocompetent hosts, immunodeficient individuals are at particular risk to develop lymphoproliferative B cell malignancies caused by EBV. A key EBV protein is the transcription factor EBV nuclear antigen 2 (EBNA2), which initiates B cell proliferation. Here, we combine biochemical, cellular and in vivo experiments demonstrating that the mitotic polo-like kinase 1 (PLK1) binds to EBNA2, phosphorylates its transactivation domain and thereby inhibits its biological activity. EBNA2 mutants that impair PLK1 binding or prevent EBNA2 phosphorylation are gain-of-function mutants. They have enhanced transactivation capacities, accelerate the proliferation of infected B cells and promote the development of monoclonal B cell lymphomas in infected mice. Thus, PLK1 coordinates the activity of EBNA2 to attenuate the risk of tumor incidences in favor of the establishment of latency in the infected but healthy host.

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“…The bacterial strains and plasmids used in this study are listed in Table 1. The gene deletion mutant strains were constructed by double exchange of homologous recombination as described previously [54]. The upstream and downstream fragments of target genes were amplified using PCR by employing the primers listed in Table 2.…”

Section: Bacterial Strainsmentioning

confidence: 99%

Essential role of Salmonella Enteritidis DNA adenine methylase in modulating inflammasome activation

Guo

Huang

et al. 2020

Preprint

View full text Add to dashboard Cite

Background: Salmonella Enteritidis (SE) is one of the major foodborne zoonotic pathogens of worldwide importance which can induce activation of NLRC4 and NLRP3 inflammasomes during infection. Given that the inflammasomes play an essential role in resisting bacterial infection, Salmonella has evolved various strategies to regulate activation of the inflammasome, most of which largely remain unclear.Results: A transposon mutant library in SE strain C50336 was screened for the identification of the potential factors that regulate inﬂammasome activation. We found that T3SS-associated genes invC, prgH, and spaN were required for inﬂammasome activation in vitro. Interestingly, C50336 strains with deletion or overexpression of Dam were both defective in activation of caspase-1 and secretion of IL-1β. Transcriptome sequencing (RNA-seq) results suggest that the absence and overexpression of Dam had similar effects on infected cells, as high overlapping rates of differentially expressed genes and enriched pathways were found between the C50336-VS-C50336Δdam and C50336-VS-C50336::dam groups, including multiple signaling pathways related to the inﬂammasome. Moreover, overexpression of Dam in macrophages by lentiviral infection could specifically promote the activation of the NLRP3 inflammasome independently.Conclusions: These data indicate that Dam was essential for modulating inflammasome activation during SE infection, there are complex and dynamic interplays between Dam and the inflammasome under different conditions. New insights were provided about the battle between SE and host innate immunological mechanisms.

show abstract

Site-Directed Mutagenesis and Its Application in Studying the Interactions of T3S Components

Cited by 13 publications

References 88 publications

Essential role of Salmonella Enteritidis DNA adenine methylase in modulating inflammasome activation

Essential role of Salmonella Enteritidis DNA adenine methylase in modulating inflammasome activation

Inhibition of PLK1-dependent EBNA2 phosphorylation promotes lymphomagenesis in EBV-infected mice

Essential role of Salmonella Enteritidis DNA adenine methylase in modulating inflammasome activation

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