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RNA-seq analysis of Ralstonia solanacearum treated with nitrosative and oxidative stress

ABSTRACT: Ralstonia solancearum causes bacterial wilt disease on diverse plant hosts. R. solanacearum cells enter a host from soil or infested water through the roots, then multiply and spread in the water-transporting xylem vessels. Despite the low nutrient content of xylem sap, R. solanacearum grows extremely well inside the host, using denitrification to respire in this hypoxic environment. R. solanacearum growth in planta also depends on the successful deployment of protein effectors into host cells using a Type III Secretion System (T3SS). The T3SS is absolutely required for R. solanacearum virulence, but it is metabolically costly and can trigger host defenses. Thus, the pathogen’s success depends on optimized regulation of the T3SS. We found that a byproduct of denitrification, the toxic free-radical nitric oxide (NO), positively regulates the R. solanacearum T3SS both in vitro and in planta. Using chemical treatments and R. solanacearum mutants with altered NO levels, we show that the expression of a key T3SS regulator is induced by NO in culture. Analyzing the transcriptome of R. solanacearum responding to varying levels of NO both in culture and in planta revealed that the T3SS and effectors were broadly upregulated with increasing levels of NO. This regulation was specific to the T3SS and was not shared by other stressors. Our results suggest that R. solanacearum experiences an NO-rich environment in the plant host and may use this NO as a signal to activate T3SS during infection.

ORGANISM(S): Ralstonia solanacearum

PROVIDER: GSE160024 | GEO | 2020/10/26

REPOSITORIES: GEO

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Project description:Many Gram-negative bacterial pathogens interact with mammalian cells by using type III secretion systems (T3SS) to inject virulence proteins directly into infected host cells. A subset of these injected protein ‘effectors’ are enzymes that modify the structure and inhibit the function of human proteins by catalyzing the addition of unusual post-translational modifications. T3SS effectors play essential roles in bacterial virulence and their modes of action have provided great insight into the functions and the components of the innate immune system. The E. coli and Citrobacter rodentium NleB effectors, as well as the Salmonella enterica SseK effectors are glycosyltransferases that modify host protein substrates with N-acetyl glucosamine (GlcNAc) on arginine residues. Arginine glycosylation is unusual because it occurs on the guanidinium groups of arginines, which are poor nucleophiles. This post-translational modification disrupts the normal functioning of host immune response proteins. T3SS effectors are chaperoned in the bacterium to keep the effectors partially unfolded and competent for secretion, as well as for targeting the effectors to the T3SS sorting platform. The chaperones are then stripped from their effector substrates at the sorting platform and the effectors are secreted in an unfolded conformation. T3SS effectors are thought to be inactive within the bacterium and fold into their active conformations after they are injected into host cells. While performing mass spectrometry experiments to identify glycosylation substrates of NleB orthologs, we unexpectedly observed that the E. coli glutathione synthetase (GshB) is glycosylated on an arginine residue (R256) by NleB. NleB glycosyltransferase activity is essential to C. rodentium survival in oxidative stress conditions because glycosylation of GshB results in enhanced glutathione production. These data represent, to our knowledge, the first intra-bacterial activity for a T3SS effector and show that effector activities thought to be restricted to host cell compartments additionally play important roles in regulating bacterial physiology.

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RNA-seq analysis of Ralstonia solanacearum treated with nitrosative and oxidative stress

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