Deletion And Characterization Of A Putative Major Type Iv Pilin Protein In Vibrio Parahaemolyticus
Date
2022-05
Authors
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Publisher
University of Delaware
Abstract
Vibrionaceae are a family gram-negative proteobacteria that inhabit marine and estuarine
environments. These bacteria can exist either as free-living organisms or in association with
marine flora and fauna in both parasitic and symbiotic relationships. The model organism in this
study was Vibrio parahaemolyticus, a human pathogen found in marine environments and an
agent of gastrointestinal illness. V. parahaemolyticus forms communal structures known as
biofilms which provide a survival advantage for the bacteria and is an important aspect of
pathogenicity. The formation of a biofilm structure involves the transition from a free-swimming
state to a surface attached state where bacteria aggregate with each other. V. parahaemolyticus
makes use of an appendage called pili to interact with its environment and bring about a wide
variety of functions such as those used in biofilm formation. Bacterial pili are hair-like structures
that emerge from the bacterial membranes and are important for bacterial adhesion, motility, and
twitching among other phenotypes. A subgroup known as Type IV pili (T4P) are designated
based on sequence similarity of the major pilin protein. These pili appendages are dynamic in
nature and the major pili protein subunits are used to quickly form filaments that protrude from
the bacterial surface. The V. parahaemolyticus genome encodes at least four Type IV pili;
TAD1, PilA, MSHA, and TAD2. Characterization of the role of T4P systems PilA and MshA in
biofilm formation has been studied previously. Protein BLAST examination of the V.
parahaemolyticus genome identified an additional putative T4P major pilin subunit mshA2
(VPA0747). The gene VPA0747 appears on the second chromosome of V. parahaemolyticus and
a protein BLAST of the product showed sequence similarity to a MshA1, a T4P major pili
protein in V. parahaemolyticus (99.4% AA similarity). The major pilin protein MshA1 which
VPA0747 is the most similar has been previously shown to be involved with biofilm formation.
In this study we deleted mshA2, VPA0747 in V. parahaemolyticus RIMD 2210633 using the
Gibson assembly protocol to generate an in-frame truncated, nonfunctional mshA2, followed by
allelic exchange. We then characterized this mutant by performing assays such as biofilm,
capsule polysaccharide (CPS), swarming motility, swimming motility, and growth curve
analysis. All assays performed showed wild type phenotypes in the mutant strain demonstrating
no apparent defect for the mshA2 (VPA0747) deletion mutant and indicating that this putative
T4P pili protein is not essential for survival or interactions in the environments that were
examined. Together, these data suggest that MSHA pilus were still being produced in mutants
lacking mshA2. Indicating that the MshA2 protein might not be expressed under the conditions
evaluated or this gene might encode for a minor pilin protein which are not required for the
formation of the MSHA pilus.
Description
Keywords
Proteobacteria, Biofilm, Pili