Guanosine tetraphosphate relieves the negative regulation of Salmonella pathogenicity island-2 gene transcription exerted by the AT-rich ssrA discriminator region

Timothy Tapscott,Jessica Jones-Carson,Matthew A Crawford,Andrés Vázquez-Torres,Ju-Sim Kim,Lin Liu,Liam Fitzsimmons

doi:10.1038/s41598-018-27780-9

Timothy Tapscott, Jessica Jones-Carson + Show 5 more

Open Access

https://doi.org/10.1038/s41598-018-27780-9

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Abstract

The repressive activity of ancestral histone-like proteins helps integrate transcription of foreign genes with discrepant AT content into existing regulatory networks. Our investigations indicate that the AT-rich discriminator region located between the −10 promoter element and the transcription start site of the regulatory gene ssrA plays a distinct role in the balanced expression of the Salmonella pathogenicity island-2 (SPI2) type III secretion system. The RNA polymerase-binding protein DksA activates the ssrAB regulon post-transcriptionally, whereas the alarmone guanosine tetraphosphate (ppGpp) relieves the negative regulation imposed by the AT-rich ssrA discriminator region. An increase in the GC-content of the ssrA discriminator region enhances ssrAB transcription and SsrB translation, thus activating the expression of downstream SPI2 genes. A Salmonella strain expressing a GC-rich ssrA discriminator region is attenuated in mice and grows poorly intracellularly. The combined actions of ppGpp and DksA on SPI2 expression enable Salmonella to grow intracellularly, and cause disease in a murine model of infection. Collectively, these findings indicate that (p)ppGpp relieves the negative regulation associated with the AT-rich discriminator region in the promoter of the horizontally-acquired ssrA gene, whereas DksA activates ssrB gene expression post-transcriptionally. The combined effects of (p)ppGpp and DksA on the ssrAB locus facilitate a balanced SPI2 virulence gene transcription that is essential for Salmonella pathogenesis.

Highlights

Nontyphoidal Salmonella enterica serovar Typhimurium is a common cause of gastroenteritis in immunocompetent individuals and a life-threatening disseminated complication in immunocompromised hosts unable to mount CD4+ T cell immunity or IFNγ host responses[1,2]
The stringent response is controlled by the RNA polymerase-binding protein DksA and the nucleotide alarmones guanosine tetra/pentaphosphate [(p) ppGpp] that are synthesized in Salmonella by the RelA and SpoT proteins
Microarrays and differential RNA sequencing indicate that the stringent response regulators DksA and (p) ppGpp are required for the activation of Salmonella pathogenicity island-2 (SPI2) gene transcription[33,34,35,36,37,38]

Summary

Introduction

Nontyphoidal Salmonella enterica serovar Typhimurium is a common cause of gastroenteritis in immunocompetent individuals and a life-threatening disseminated complication in immunocompromised hosts unable to mount CD4+ T cell immunity or IFNγ host responses[1,2]. Compared to wild-type controls, both ΔdksA and ΔrelA ΔspoT Salmonella expressed low levels of the SPI2 effector sifA in J774A.1 macrophage-like cells (Fig. 1E). As shown previously[33,34,35,37,38], wild-type Salmonella grown for 3 h in 8 μM MgCl2 N9 medium expressed all SPI2 promoters tested; ΔdksA or ΔrelA ΔspoT Salmonella did not stimulate expression of any SPI2 genes examined (Fig. S1B).

Results

Conclusion