Host cell type-dependent translocation and PhoP-mediated positive regulation of the effector SseK1 of Salmonella enterica

Baisón-Olmo, Fernando; Galindo-Moreno, María; Ramos‐Morales, Francisco

doi:10.3389/fmicb.2015.00396

Cited by 14 publications

(13 citation statements)

References 71 publications

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“…To determine the binding of PhoP to DNA probe of hlyF promoter, EMSAs were conducted using the commercialized EMSA kit (Invitrogen, California) according to the manufacturer’s protocol ( 15 ). To obtain phosphorylated PhoP, purified PhoP was phosphorylated with acetylphosphate (Sigma) according to the previous study ( 37 ). PhoP (10 µM) was incubated in 100 µl of phosphorylation buffer (20 mM HEPES, pH 7.5, 100 mM NaCl, and 5 mM MgCl 2 ) containing 20 mM acetylphosphate for 2 h at 37°C.…”

Section: Methodsmentioning

confidence: 99%

A Novel PhoP/PhoQ Regulation Pathway Modulates the Survival of Extraintestinal Pathogenic Escherichia coli in Macrophages

et al. 2018

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The extraintestinal pathogenic Escherichia coli (ExPEC) is a typical facultative intracellular bacterial pathogen. Sensing the environmental stimuli and undertaking adaptive change are crucial for ExPEC to successfully colonize in specific extraintestinal niches. The previous studies show that pathogens exploit two-component systems (TCSs) in response to the host environments during its infection. The PhoP/PhoQ is a typical TCS which is ubiquitous in Gram-negative bacteria. However, there is an incompletely understanding about critical regulatory roles of PhoP/PhoQ in ExPEC pathogenesis. Conjugative ColV-related plasmids are responsible for ExPEC virulence, which is associated with ExPEC zoonotic risk. In this study, the molecular characteristics of HlyF, Mig-14 ortholog (Mig-14p), and OmpT variant (OmpTp) encoded by ColV plasmids were identified. Mig-14p and OmpTp played important roles in conferring ExPEC resistance to cationic antimicrobial peptides (CAMPs) during the infection. Moreover, HlyF and Mig-14p acted as intracellular survival factors to promote ExPEC resistance to macrophages killing. The hlyF and Mig-14p formed an operon in ExPEC ColV plasmid, and PhoP acted as a transcriptional activator of hlyF operon by directly binding to the PhlyF promoter. The acidic pH and CAMPs could additively stimulate ExPEC PhoQ/PhoP activities to upregulate the expression of HlyF and Mig-14p. Our studies revealed that the novel PhoP/PhoQ-HlyF signaling pathway directly upregulates the production of ExPEC outer membrane vesicles. Furthermore, our study first clarified that this PhoP/PhoQ-HlyF pathway was essential for ExPEC intracellular survival in macrophages. It was required to prevent the fusion of ExPEC-containing phagosomes with lysosomes. Moreover, PhoP/PhoQ-HlyF pathway facilitated the inhibition of the phagolysosomal acidification and disruption of the phagolysosomal membranes. In addition, this pathway might promote the formation of ExPEC-containing autophagosome during ExPEC replication in macrophages. Collectively, our studies suggested that PhoP/PhoQ system and CloV plasmids could facilitate ExPEC survival and replication within macrophages.

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Section: Methodsmentioning

confidence: 99%

A Novel PhoP/PhoQ Regulation Pathway Modulates the Survival of Extraintestinal Pathogenic Escherichia coli in Macrophages

et al. 2018

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“…An example is SseK1: when expressed from a constitutive promoter it can be secreted through T3SS1 at earlier time points p.i. than when expressed from its own promoter ( Baisón-Olmo et al, 2015 ). Coexpression of T3SS1 and srfJ from P iolE may take place in plants due to the presence of MI, making it possible the delivery of the effector through this way.…”

Section: Discussionmentioning

confidence: 99%

Dual Expression of the Salmonella Effector SrfJ in Mammalian Cells and Plants

et al. 2017

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SrfJ is an effector of the Salmonella pathogenicity island 2-encoded type III secretion system. Salmonella enterica serovar Typhimurium expresses srfJ under two disparate sets of conditions: media with low Mg2+ and low pH, imitating intravacuolar conditions, and media with myo-inositol (MI), a carbohydrate that can be used by Salmonella as sole carbon source. We investigated the molecular basis for this dual regulation. Here, we provide evidence for the existence of two distinct promoters that control the expression of srfJ. A proximal promoter, PsrfJ, responds to intravacuolar signals and is positively regulated by SsrB and PhoP and negatively regulated by RcsB. A second distant promoter, PiolE, is negatively regulated by the MI island repressor IolR. We also explored the in vivo activity of these promoters in different hosts. Interestingly, our results indicate that the proximal promoter is specifically active inside mammalian cells whereas the distant one is expressed upon Salmonella colonization of plants. Importantly, we also found that inappropriate expression of srfJ leads to reduced proliferation inside macrophages whereas lack of srfJ expression increases survival and decreases activation of defense responses in plants. These observations suggest that SrfJ is a relevant factor in the interplay between Salmonella and hosts of different kingdoms.

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“…Since most of the isolated clones corresponded to TBCB, we focused our study on the interaction of SseK1 with this host protein. To confirm the results obtained with the yeast two-hybrid approach, the bacterial glutathione S-transferase (GST) expression system was used to produce GST or GST-TBCB in a strain of Salmonella expressing a chromosomal SseK1-3xFLAG fusion (strain SV7071) [15]. Then, we performed affinity purification using glutathione-agarose beads to isolate GST proteins, and we further analyzed the presence of SseK1 by western blot.…”

Section: Ssek1 Copurifies and Coimmunoprecipitates With Tbcbmentioning

confidence: 96%

Tubulin Folding Cofactor TBCB is a Target of the Salmonella Effector Protein SseK1

Araujo-Garrido

Baisón-Olmo

Bernal-Bayard

et al. 2020

IJMS

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Salmonella enterica serovar Typhimurium is a human and animal pathogen that uses type III secretion system effectors to manipulate the host cell and fulfill infection. SseK1 is a Salmonella effector with glycosyltransferase activity. We carried out a yeast two-hybrid screen and have identified tubulin-binding cofactor B (TBCB) as a new binding partner for this effector. SseK1 catalyzed the addition of N-acetylglucosamine to arginine on TBCB, and its expression promoted the stabilization of the microtubule cytoskeleton of HEK293T cells. The conserved Asp-x-Asp (DxD) motif that is essential for the activity of SseK1 was required for the binding and modification of TBCB and for the effect on the cytoskeleton. Our study has identified a novel target for SseK1 and suggests that this effector may have a role in the manipulation of the host cell microtubule network to provide a safe niche for this pathogen.

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Host cell type-dependent translocation and PhoP-mediated positive regulation of the effector SseK1 of Salmonella enterica

Cited by 14 publications

References 71 publications

A Novel PhoP/PhoQ Regulation Pathway Modulates the Survival of Extraintestinal Pathogenic Escherichia coli in Macrophages

A Novel PhoP/PhoQ Regulation Pathway Modulates the Survival of Extraintestinal Pathogenic Escherichia coli in Macrophages

Dual Expression of the Salmonella Effector SrfJ in Mammalian Cells and Plants

Tubulin Folding Cofactor TBCB is a Target of the Salmonella Effector Protein SseK1

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