Alternative Sigma Factor RpoE Is Important for Vibrio parahaemolyticus Cell Envelope Stress Response and Intestinal Colonization

Abstract

Vibrio parahaemolyticus is a halophile that inhabits brackish waters and a wide range of hosts, including crustaceans, fish, mollusks, and humans. In humans, it is the leading cause of bacterial seafood-borne gastroenteritis. The focus of this work was to determine the role of alternative sigma factors in the stress response of V. parahaemolyticus RIMD2210633, an O3:K6 pandemic isolate. Bioinformatics identified five putative extracytoplasmic function (ECF) family of alternative sigma factors: VP0055, VP2210, VP2358, VP2578, and VPA1690. ECF factors typically respond to cell wall/cell envelope stress, iron levels, and the oxidation state of the cell. We have demonstrated here that one such sigma factor, VP2578, a homologue of RpoE from Escherichia coli, is important for survival under a number of cell envelope stress conditions and in gastrointestinal colonization of a streptomycin-treated adult mouse. In this study, we determined that an rpoE deletion mutant strain BHM2578 compared to the wild type (WT) was significantly more sensitive to polymyxin B, ethanol, and high-temperature stresses. We demonstrated that in in vivo competition assays between the rpoE mutant and the WT marked with the ␤-galactosidase gene lacZ (WBWlacZ), the mutant strain was defective in colonization compared to the WT. In contrast, deletion of the rpoS stress response regulator did not affect in vivo survival. In addition, we examined the role of the outer membrane protein, OmpU, which in V. cholerae is proposed to be the sole activator of RpoE. We found that an ompU deletion mutant was sensitive to bile salt stress but resistant to polymyxin B stress, indicating OmpU is not essential for the cell envelope stress responses or RpoE function. Overall, these data demonstrate that RpoE is a key cell envelope stress response regulator and, similar to E. coli, RpoE may have several factors that stimulate its function. Vibrio parahaemolyticus is a Gram-negative organism, commonly found within brackish waters, such as coastal marine and estuarine waters, worldwide (1-3). V. parahaemolyticus is frequently associated with bacterial seafood-borne gastroenteritis following the consumption of raw or undercooked fish and shellfish, resulting from this organism's ability to colonize shellfish in high numbers (3-5). Disease caused by V. parahaemolyticus is marked by diarrhea and abdominal pain. In addition, septicemia and mortality have been documented in immunocompromised individuals and in cases following exposure of open wounds to the organism (6). In the United States the number of incidences of illness associated with Vibrio, including V. parahaemolyticus, has increased. According to the Centers for Disease Control and Prevention, the rate of infections caused by Vibrio species in 2012 had increased 43% since 2006-2008, whereas the rates of infections caused by other enteric species, such as Escherichia coli O157 and Salmonella enterica, showed no change during that same time period (7).V. parahaemolyticus must be able to alternate between natural...

Quorum Sensing Regulators Are Required for Metabolic Fitness in Vibrio parahaemolyticus

“…Water was restored immediately upon inoculation, and food was restored at 2 h postinfection. The V. parahaemolyticus strain used for in vivo experiments is the ␤-galactosidase knock-in designated WBWlacZ, which allows for blue-white colony screening (13)(14)(15). Overnight cultures were diluted 1:50 with LBS-streptomycin medium and grown for 4 h at 37°C with aeration.…”

“…For growth curves in individual mucus sugars and amino acids, 4-h cultures were pelleted, washed, and diluted 1:40 in M9G (10 mM) medium or M9 medium with D-gluconate (10 mM), D-mannose (10 mM), D-ribose (10 mM), L-arabinose (10 mM), D-galactose (10 mM), D-glucosamine (10 mM), pyruvic acid (10 mM), D-trehalose (10 mM), fructose (10 mM), L-glutamic acid (5 mM), or L-aspartic acid (30 mM). Mouse intestinal mucus was extracted as described previously (14,15). Mouse gastrointestinal tracts were harvested and then flushed with PBS to remove intestinal contents.…”

“…The importance of ToxR for in vivo survival also was demonstrated in an infant rabbit model of infection (16). The alternative sigma factor RpoE, required for the cell envelope stress response, was shown to be essential for in vivo survival as deletion of the rpoE gene resulted in attenuation of mouse colonization (15). In contrast, analysis of a deletion mutant of sigma-54, rpoN, showed that in in vivo competition assays, the mutant colonized significantly more proficiently than the wild-type strain.…”

“…This lack of knowledge is in part due to a lack of animal models to study colonization and infection in vivo. The development of a streptomycin-pretreated adult mouse model that removes microbiota colonization resistance and allows V. parahaemolyticus to colonize has uncovered a number of bacterial colonization factors required for colonization (13)(14)(15). Examination of a mutant deficient in the global regulator ToxR demonstrated a significant defect in intestinal colonization compared to that of the wild type (13).…”

Microbial Surface Colonization and Biofilm Development in Marine Environments

“…Association with insoluble materials may be the preferred lifestyle of vibrios, including deep-sea hydrothermal vent species (15,403,524), and regulatory systems involving TCSs, chemotaxis, QS, sRNAs, cAMP, c-di-GMP, alternative sigma factors, and the stringent response enable vibrios to optimize resource utilization and survival (Fig. 2) (49, 339, 472, 475,511,518,[525][526][527][528][529].…”