Date Published: August 31, 2018
Publisher: Public Library of Science
Author(s): Kaiyu Yin, Yunpeng Guan, Ruiqing Ma, Lifan Wei, Bing Liu, Xiaohong Liu, Xiangshan Zhou, Yue Ma, Yuanxing Zhang, Matthew K. Waldor, Qiyao Wang, Brian K. Coombes.
Edwardsiella piscicida is a leading fish pathogen that causes significant economic loses in the aquaculture industry. The pathogen depends on type III and type VI secretion systems (T3/T6SS) for growth and virulence in fish and the expression of both systems is controlled by the EsrB transcription activator. Here, we performed a Tn-seq-based screen to uncover factors that govern esrB expression. Unexpectedly, we discovered that RpoS antagonizes esrB expression and thereby inhibits production of E. piscicida’s T3/T6SS. Using in vitro transcription assays, we showed that RpoS can block RpoD-mediated transcription of esrB. ChIP-seq- and RNA-seq-based profiling, as well as mutational and biochemical analyses revealed that RpoS-repressed promoters contain a -6G in their respective discriminator sequences; moreover, this -6G proved critical for RpoS to inhibit esrB expression. Mutation of the RpoS R99 residue, an amino acid that molecular modeling predicts interacts with -6G in the esrB discriminator, abolished RpoS’ capacity for repression. In a turbot model, an rpoS deletion mutant was attenuated early but not late in infection, whereas a mutant expressing RpoSR99A exhibited elevated fitness throughout the infection period. Collectively, these findings deepen our understanding of how RpoS can inhibit gene expression and demonstrate the temporal variation in the requirement for this sigma factor during infection.
Edwardsiella piscicida (formerly included in Edwardsiella tarda) belongs to the enterobacteriaceae family  and is phylogenetically related to Salmonella enterica . Like some species of Salmonella, E. piscicida can also infect a broad range of animal hosts including, fish, amphibians, mammals and humans . The organism is a bane of the aquaculture industry because it infects over 20 species of fish, including important farmed species such as turbot, flounder, eel and catfish, resulting in significant economic losses globally [4–6]. Several E. piscicida virulence determinants, such as adhesins, siderophores, and hemolysin EthA have been uncovered using single mutants (reviewed in ) and in genome-wide transposon insertion sequencing (Tn-seq)-based studies .
Here, we used a genome-wide loss-of-function Tn-seq screen to identify regulators controlling the expression of EsrB, a key activator of E. piscicida virulence. Unexpectedly, we discovered that RpoS inhibits esrB expression, and thus limits production of the pathogen’s T3SS/T6SS. Comparisons of the global transcription profiles of wt and ΔrpoS strains showed that RpoS controls expression, directly or indirectly, of more than 700 genes. Several stress stimuli modulate RpoS abundance and thus likely control esrB expression and E. piscicida virulence. Notably, in vitro transcription of esrB by the RpoD-core RNAP complex (Eσ70) was blocked by RpoS. Furthermore, this inhibitory effect, likely mediated by Eσ38, was abrogated by mutations in the esrB promoter discriminator or by a single amino acid substitution in RpoS R99, a residue in the sigma 1.2 region (the first part of RpoS conserved region 2) that molecular modeling predicted to be in close proximity to the -6G nucleotide of the esrB promoter discriminator. Collectively, these observations strongly suggest that direct interactions of Eσ38 with the esrB promoter impede transcription of this virulence regulator. In a turbot model, RpoS was required for robust E. piscicida growth during the first few days of infection, whereas EsrB was not; conversely, by 5 dpi RpoS becomes dispensable and EsrB becomes critical. By mediating expression of genes promoting stress responses  and inhibiting expression of esrB-controlled virulence genes, RpoS activity allows E. piscicida to co-ordinate expression of diverse cellular pathways (Fig 8). Thus, our findings suggest that the pathogen interprets variations in host-derived signals during the course of infection to modulate RpoS abundance/activity and thereby fine tunes its physiology for growth in different host environments.