Loss of Sigma Factor RpoN Increases Intestinal Colonization of Vibrio parahaemolyticus in an Adult Mouse Model

Abstract

bVibrio parahaemolyticus is the leading cause of bacterial seafood-borne gastroenteritis worldwide, yet little is known about how this pathogen colonizes the human intestine. The alternative sigma factor RpoN/sigma-54 is a global regulator that controls flagellar synthesis, as well as a wide range of nonflagellar genes. We constructed an in-frame deletion mutation in rpoN (VP2670) in V. parahaemolyticus RIMD2210633, a clinical serogroup O3:K6 isolate, and examined the effects in vivo using a streptomycintreated mouse model of colonization. We confirmed that deletion of rpoN rendered V. parahaemolyticus nonmotile, and it caused reduced biofilm formation and an apparent defect in glutamine synthetase production. In in vivo competition assays between the rpoN mutant and a wild-type RIMD2210633 strain marked with the ␤-galactosidase gene lacZ (WBWlacZ), the mutant colonized significantly more proficiently. Intestinal persistence competition assays also demonstrated that the rpoN mutant had enhanced fitness and outcompeted WBWlacZ. Mutants defective in the polar flagellum biosynthesis FliAP sigma factor also outcompeted WBWlacZ but not to the same level as the rpoN mutant, which suggested that lack of motility is not the sole cause of the fitness effect. In an in vitro growth competition assay in mouse intestinal mucus, the rpoN mutant also outcompeted the wild type and exhibited faster doubling times when grown in mucus and on individual components of mucus. Genes in the pathways for the catabolism of mucus sugars also had significantly higher expression levels in a ⌬rpoN mutant than in the wild type. These data suggest that in V. parahaemolyticus, RpoN plays an important role in carbon utilization regulation, which may significantly affect host colonization. Vibrio parahaemolyticus is a Gram-negative bacterium ubiquitous in the marine and estuarine environments worldwide (1-4). Vibrio parahaemolyticus is also the leading cause of seafoodassociated bacterial gastroenteritis in the United States and Asia (5, 6), which usually stems from the consumption of raw or undercooked shellfish (7,8). Typically, infection by this organism leads to nausea, vomiting, fever, and a diarrhea distinct from that of the related Vibrio cholerae. Less commonly, infection by V. parahaemolyticus can cause wound infection and septicemia, leading to mortality in immunocompromised individuals (9-12).Much effort has gone into understanding the mechanisms that contribute to V. parahaemolyticus pathogenesis, with a particular focus on virulence factors produced by this bacterium. Strains that caused disease often possessed either the thermostable direct hemolysin (TDH) or the TDH-related hemolysin (TRH), while nonpathogenic strains typically lacked these two markers (13-15). Additionally, sequence analysis of RIMD2210633, an O3:K6 isolate (TDH ϩ TRH Ϫ ), revealed the presence of two type 3 secretion systems (T3SS), one on each chromosome (T3SS-1 and T3SS-2) (16, 17). T3SS-1 is common to both clinical and nonclinical strains of V. parahaemolyticu...

Quorum Sensing Regulators Are Required for Metabolic Fitness in Vibrio parahaemolyticus

“…The in vivo defect observed for the luxO mutant is in contrast to the superior colonization phenotype that we had previously showed for an rpoN deletion mutant (14). According to the quorum sensing pathway in V. parahaemolyticus, deletion of both luxO and rpoN should have the same effect on the expression of the two master regulators aphA and opaR.…”

“…To determine the role of the QS regulators in V. parahaemolyticus pathogenesis, we examined each of the QS mutants for their ability to colonize the adult mouse intestine. We examined luxO, aphA, and opaR deletion mutant strains as well as luxO opaR and opaR aphA double deletion mutant strains in in vivo competition assays with the wild type using the streptomycin-pretreated adult mouse model of intestinal colonization (13,14). Mice pretreated with streptomycin were orogastrically coinoculated with an equal mixture of the WBWlacZ strain (wild-type strain marked with lacZ and that allows blue-white screening) and each of the mutants.…”

“…Expression analysis showed that genes required for gluconate, ribose, and arabinose catabolism were induced in the rpoN mutant. These data suggested that specific carbon metabolism genes are negatively regulated by RpoN and that competitive carbon utilization could be an important colonization factor (14).…”

“…Colonies of the WBWlacZ strain appear blue on an X-Gal (5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside) plate in comparison to the wild type and its isogenic mutants that appear white on the plate, thus allowing for a blue-white screen in a competition experiment. The WBWlacZ strain was previously shown to behave identically to the wild type in vitro and in vivo (13,14). Unless stated otherwise, all V. parahaemolyticus strains were grown in lysogeny broth (LB) containing 3% NaCl (LBS) (Fischer Scientific, Pittsburgh, PA) at 37°C with aeration.…”

“…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).…”