Transcription Factor σ^B Plays an Important Role in the Production of Extracellular Membrane-Derived Vesicles in <i>Listeria monocytogenes</i>

<div><p>Gram-negative bacteria produce extracellular outer membrane vesicles (OMVs) that interact with host cells. Unlike Gram-negative bacteria, less is known about the production and role of extracellular membrane vesicles (MVs) in Gram-positive bacteria. The food-borne pathogen <i>Listeria monocytogenes</i> can survive under extreme environmental and energy stress conditions and the transcription factor σ^B is involved in this survival ability. Here, we first determined the production of MVs from <i>L. monocytogenes</i> and evaluated whether general stress transcription factor σ^B affected production of MVs in <i>L. monocytogenes. L. monocytogenes</i> secreted MVs during <i>in vitro</i> broth culture. The wild-type strain actively produced MVs approximately nine times more and also produced more intact shapes of MVs than those of the isogenic Δ<i>sigB</i> mutant. A proteomic analysis showed that 130 and 89 MV proteins were identified in the wild-type and Δ<i>sigB</i> mutant strains, respectively. Wild-type strain-derived MVs contained proteins regulated by σ^B such as transporters (OpuCA and OpuCC), stress response (Kat), metabolism (LacD), translation (InfC), and cell division protein (FtsZ). Gene Ontology (GO) enrichment analysis showed that wild-type-derived MV proteins corresponded to several GO terms, including response to stress (heat, acid, and bile resistance) and extracellular polysaccharide biosynthetic process, but not the Δ<i>sigB</i> mutant. Internalin B (InlB) was almost three times more contained in MVs derived from the wild-type strain than in MVs derived from the Δ<i>sigB</i> mutant. Taken together, these results suggest that σ^B plays a pivotal role in the production of MVs and protein profiles contained in MVs. <i>L. monocytogenes</i> MVs may contribute to host infection and survival ability under various stressful conditions. </p> </div

Venn diagram of extracellular membrane vesicle (MV) proteins identified by LC-ESI-MS/MS.

<p>Of the 130 proteins, 46 (35%) were identified only in wild-type <i>L. monocytogenes</i> MVs and of the 89 proteins, five (6%) were identified only in the Δ<i>sigB</i> mutant MVs. Eighty-four MV proteins were identified commonly in the wild-type and Δ<i>sigB</i> mutant of <i>L. monocytogenes</i>.</p

Western blot analysis of Internalin B (InlB) and Listeriolysin O (LLO) in the cell lysate and MVs.

<p>(A) Samples were separated on 10% SDS-PAGE and immunoblotted with anti-InlB and anti-LLO antibodies. CL, <i>L. monocytogenes</i> cell lysate; MVs, membrane-derived vesicles. (B) Band intensities were measured using image analysis software.</p

Extracellular membrane vesicles (MVs) produced by <i>L. monocytogenes</i>.

<p>Transmission electron micrograph of MVs prepared from wild-type (A) and the isogenic ∆<i>sigB</i> mutant of <i>L. monocytogenes</i> (B) cultured in BHI broth. (C) Arrow indicates bilayered structure.</p

Distribution of significant Gene Ontology (GO) terms from extracellular membrane vesicle (MV) proteins that were categorized only in wild-type <i>L. monocytogenes.</i>

<p>Distribution of significant Gene Ontology (GO) terms from extracellular membrane vesicle (MV) proteins that were categorized only in wild-type <i>L. monocytogenes.</i></p

The Elekta XVI-CBCT system and CIRS ATOM phantom.

<p>The Elekta XVI-CBCT system and CIRS ATOM phantom.</p

Line profiles from Fig 8.

<p>A conventional CBCT image is plotted as a solid black line; the TCM images are plotted as dash-dot red lines.</p

Half-fan scanning geometry.

<p>(a) OBI CBCT and (b) XVI CBCT system. The default detector position shifted 14.8 cm using the OBI and 11.5 cm for the XVI in medium-FOV mode.</p

NRMSE and SSIM are calculated for center and off-center slices.

<p>ROIs are defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192933#pone.0192933.g008" target="_blank">Fig 8</a>.</p

Imaging dose to abdominal and pelvic organs from conventional CBCT and TCM scans using a XCAT phantom.

<p>Imaging dose to abdominal and pelvic organs from conventional CBCT and TCM scans using a XCAT phantom.</p