Critical role for a promoter discriminator in RpoS control of virulence in Edwardsiella piscicida

Abstract

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.

Genome-Wide Identification of Fitness Factors in Seawater for Edwardsiella piscicida

“…Limited availabilities of iron, nutrients, and cofactors in seawater (34) likely represent significant stress cues for bacterial growth. Consistent with this, RpoS, an alternative sigma factor enabling transcription of genes associated with the general stress response and stationary-phase metabolism in Gram-negative bacteria, including E. piscicida (31), and other waterborne pathogens, including Legionella pneumophila (35), was highly essential (log 2 FC ϭ Ϫ7.86, P ϭ 0.0006119) during growth in 28°C seawater (Table S4). In L. pneumophila, the disruption of rpoS resulted in a strong survival defect in defined water medium compared to the wild type due to an aberrant regulation of the stringent response to exclude successful adaptation into starvation (35).…”

“…Previous time-resolved TIS analysis indicates that ϳ540 and ϳ417 genes, respectively, are essential for E. piscicida grown in LB medium and in turbot (19). T3SS and T6SS genes are essential for in vivo colonization in the bacterium (11,(31)(32)(33). Here, T3SS genes linked in a genomic island and the nuoA-nuoN genes in the same cistron (6) were shown to be involved in the growth fitness in lower and higher temperatures of seawater ( Fig.…”

“…In L. pneumophila, the disruption of rpoS resulted in a strong survival defect in defined water medium compared to the wild type due to an aberrant regulation of the stringent response to exclude successful adaptation into starvation (35). In addition, coenzyme metabolism (thiL, ETAE_0728), inorganic ion transport and metabolism (corA), and nucleotide metabolism and transport (purR), as well as stress adaptationrelated genes (uspA and hflCK), are also significantly underrepresented in 28°C seawater, and most of these genes are regulated by RpoS (31). However, none of these genes were included in the candidate essential gene list for E. piscicida treated with 16°C seawater (Table S3).…”

The role of rpoS in the regulation of Vibrio alginolyticus virulence and the response to diverse stresses

“…Among these, RpoS was proved to enable transcription of genes associated with the general stress response and stationary phase metabolism (Campos-Gomez & Benitez, 2018;Park, Nam, Kweon, & Shin, 2018). Meanwhile, rpoS was closely related to the virulence regulation of pathogenic bacteria (Xiao et al, 2009;Yin et al, 2018). Therefore, as a stresses responsor and a virulence regulator, the rpoS gene probably plays a crucial role required for adhesion regulation under adverse conditions.…”

“…For example, rpoS mutants of Salmonella enterica serovar Typhimurium showed significant attenuation in virulence (Fang et al., ), whereas an rpoS mutant of Yersinia enterocolitica appeared the same virulence as the wild type (Badger & Miller, ). Nevertheless, more and more evidences show that rpoS plays an important role in the virulence regulation of pathogenic bacteria (Yin et al., ), such as chondroitinase activity, serum resistance, biofilm production and autoinducer synthetases expression (Xiao et al., ). However, the relationships between rpoS and V. alginolyticus virulence factors have not been fully illustrated yet.…”