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Title:   Author  item
                                            WHITAKER, W.BRIAN - University Of Delawareitem
                                            Richards, Garyitem
                                            BOYD, E. FIDELMA - University Of Delaware
  Submitted to: Infection and Immunity
                                    Publication Type: Peer Reviewed Journal
                                    Publication Acceptance Date: 11/8/2013
                                    Publication  Date: 3/17/2014
                                    Citation: Whitaker, W., Richards, G.P., Boyd, E. 2014. Loss of sigma factor RpoN increases intestinal colonization of vibrio parahaemolyticus in an adult mouse model". Infection and Immunity. 82:544-556.Interpretive Summary: Vibrio parahaemolyticus is a naturally occurring marine bacterium which causes widespread illness among fish and shellfish consumers.  In spite of this fact, the mechanisms by which V. parahaemolyticus colonize the human gut to induce illness are largely unknown.  Vibrios move by means of whip-like flagella which propel the organism through the water or on surfaces.  Under normal conditions, motility is by two types of flagella, a single polar flagellum projecting from the end of the bacterium, and/or lateral flagella projecting from the sides of the bacterium. In this paper we describe the growth and colonization of V. parahaemolyticus in a recently developed mouse model. We also evaluate the effects of several genes known to be involved in V. parahaemolyticus motility.  In the mouse model, three mutant V. parahaemolyticus that were lacking genes for flagella production out-competed the native species within the mouse intestine.  Mutant strains grew faster than the parent strain in media containing various nutritional sources, including a variety of sugars commonly found in intestinal mucus.  Together, these data demonstrate that V. parahaemolyticus strains, which are unable to produce polar flagella, are better suited for intestinal colonization and they reproduce more rapidly.  Energy may be saved by the bacterium by eliminating the need to produce or utilize polar flagella.  Thus, V. parahaemolyticus that are missing the gene for polar flagella appear to utilize that energy to stimulate the colonization of V. parahaemolyticus in the intestinal tract.  This study begins to address the functions of various V. parahaemolyticus genes in vibrio colonization in the human gut.  This research has implications for how various gene mutations in V. parahaemolyticus may enhance or reduce the infectivity of seafood-associated vibrios in humans.
                                Technical Abstract: Vibrio 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 flagella synthesis as well as a wide range of non-flagellar genes.  We constructed a deletion mutation in rpoN (vp2670) in V. parahaemolyticus RIMD2210633 a clinical serogroup O3:K6 isolate and examined the effects in vivo using a streptomycin-treated mouse model of colonization.  Deletion of rpoN rendered V. parahaemolyticus non-motile with reduced biofilm formation, and caused an apparent defect in glutamine synthetase production.  An in vivo competition assay between the rpoN mutant and the wild-type strain showed that the mutant colonized more proficiently and significantly out-competed the wild-type.  Other mutants defective for: polar flagella synthesis (rpoF mutant) and a rpoF/rpoFL double mutant for polar and lateral flagella demonstrated enhanced fitness in vivo compared to the wild-type strain.  These mutants exhibited significantly faster doubling times than wild-type V. parahaemolyticus when grown in mouse intestinal mucin.  Mutants grown on individual components of mucin: gluconate, ribose, and arabinose as sole carbon sources, also exhibited significantly faster doubling times.  Genes in the pathways for the catabolism of these sugars also had significantly higher expression levels in the rpoN mutant than in the wild-type strain.  In conclusion, non-motile V. parahaemolyticus (rpoN, rpoF, and rpoF/rpoFL mutants) are better suited for an in vivo lifestyle, perhaps because they no longer need to expend energy for flagella synthesis or function and may consequently utilize that energy to promote intestinal colonization.


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