Peptidylarginine Deiminase Inhibitors Reduce Bacterial Membrane Vesicle Release and Sensitize Bacteria to Antibiotic Treatment

ABSTRACTInteraction of microbes with their environment depends on features of the dynamic microbial surface throughout cell growth and division. Surface modifications, whether used to acquire nutrients, defend against other microbes, or resist the pressures of a host immune system, facilitate adaptation to unique surroundings. The release of bioactive membrane vesicles (MVs) from the cell surface is conserved across microbial life, in bacteria, archaea, fungi, and parasites. MV production occurs not onlyin vitrobut alsoin vivoduring infection, underscoring the influence of these surface organelles in microbial physiology and pathogenesis through delivery of enzymes, toxins, communication signals, and antigens recognized by the innate and adaptive immune systems. Derived from a variety of organisms that span kingdoms of life and called by several names (membrane vesicles, outer membrane vesicles [OMVs], exosomes, shedding microvesicles, etc.), the conserved functions and mechanistic strategies of MV release are similar, including the use of ESCRT proteins and ESCRT protein homologues to facilitate these processes in archaea and eukaryotic microbes. Although forms of MV release by different organisms share similar visual, mechanistic, and functional features, there has been little comparison across microbial life. This underappreciated conservation of vesicle release, and the resulting functional impact throughout the tree of life, explored in this review, stresses the importance of vesicle-mediated processes throughout biology.

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Reduced Production of Bacterial Membrane Vesicles Predicts Mortality in ST45/USA600 Methicillin-Resistant Staphylococcus aureus Bacteremia

Antibiotics ◽

10.3390/antibiotics9010002 ◽

2019 ◽

Vol 9 (1) ◽

pp. 2 ◽

Cited By ~ 2

Author(s):

Somrita Dey ◽

Smitha Gudipati ◽

Christopher Giuliano ◽

Marcus J. Zervos ◽

Jonathan M. Monk ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Clinical Microbiology ◽

Methicillin Resistant Staphylococcus Aureus ◽

Membrane Vesicle ◽

Membrane Vesicles ◽

Bacterial Membrane ◽

Staphylococcus Aureus Bacteremia ◽

Immune Biomarkers ◽

Microbial Biomarkers

Immune biomarkers can stratify mortality risk in staphylococcal bacteremia. Microbial biomarkers may provide more consistent signals during early infection. We demonstrate that in ST45/USA600 bacteremia, bacterial membrane vesicle production in vitro predicts clinical mortality (773 vs. 116 RFU, survivors vs. decedents, p < 0.0001). Using a threshold of 301 relative fluorescence units (RFU), the sensitivity and specificity of the membrane vesicles to predict mortality are 78% and 90%, respectively. This platform is facile, scalable and can be integrated into clinical microbiology lab workflows.

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PPAD Activity Promotes Outer Membrane Vesicle Biogenesis and Surface Translocation by Porphyromonas gingivalis

Journal of Bacteriology ◽

10.1128/jb.00343-20 ◽

2020 ◽

Author(s):

Danielle M. Vermilyea ◽

M. Fata Moradali ◽

Hey-Min Kim ◽

Mary E. Davey

Keyword(s):

Outer Membrane ◽

Porphyromonas Gingivalis ◽

Membrane Vesicle ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Virulence Determinants ◽

Peptidylarginine Deiminase ◽

Arginine Metabolism ◽

Key Factor ◽

Surface Translocation

Many bacteria switch between a sessile and a motile mode in response to environmental and host-related signals. Porphyromonas gingivalis, an oral anaerobe implicated in the etiology of chronic periodontal disease, has long been described as a non-motile bacterium. Yet, recent studies have shown that under certain conditions, P. gingivalis is capable of surface translocation. Considering these findings, this work aimed to increase our understanding as to how P. gingivalis transitions between sessile growth and surface migration. Here we show that the peptidylarginine deiminase secreted by P. gingivalis (PPAD), an enzyme previously shown to be upregulated during surface translocation and to constrain biofilm formation, promotes surface translocation. In the absence of PPAD, the production of outer membrane vesicles (OMVs) was drastically reduced. In turn, there was a reduction in gingipain-mediated proteolysis and a reduced zone of hydration around the site of inoculation. RNA-Seq and metabolomics analyses also showed that these changes corresponded to a shift in arginine metabolism. Overall, this study provides new evidence for the functional relevance of PPAD and proteases, as well as the importance of PPAD activity in OMV biogenesis and release. Our findings support the model that citrullination is a critical mechanism during lifestyle transition between surface-attached growth and surface translocation by modulating OMV-mediated proteolysis and arginine metabolism. IMPORTANCE Gram-negative bacteria produce nanosized OMVs that are actively released into their surroundings. The oral anaerobe P. gingivalis is prolific in OMV production and many of the proteins packaged in these vesicles are proteolytic or protein modifying enzymes. This includes key virulence determinants, such as the gingipains and PPAD (a unique peptidylarginine deiminase). Here, we show that PPAD activity (citrullination) is involved in OMV biogenesis. The study reveals an unusual mechanism that allows this bacterium to transform its surroundings. Since OMVs are detected in circulation and in systemic tissues, our study also supports the notion that PPAD activity may be a key factor in the correlation between periodontitis and systemic diseases further supporting PPAD as an important therapeutic target.

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2pB_SS4-1One particle physical property analysis of bacterial membrane vesicle using high-speed AFM

Microscopy ◽

10.1093/jmicro/dfy070 ◽

2018 ◽

Vol 67 (suppl_2) ◽

pp. i23-i23

Author(s):

Yousuke Kikuchi ◽

Nozomu Obana ◽

Masanori Toyofuku ◽

Noriyuki Kodera ◽

Toshio Ando ◽

...

Keyword(s):

High Speed ◽

Membrane Vesicle ◽

Physical Property ◽

Bacterial Membrane ◽

Property Analysis

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Molecular Dissection of Internalization of Porphyromonas gingivalis by Cells using Fluorescent Beads Coated with Bacterial Membrane Vesicle

Cell Structure and Function ◽

10.1247/csf.30.81 ◽

2005 ◽

Vol 30 (2) ◽

pp. 81-91 ◽

Cited By ~ 28

Author(s):

Kayoko Tsuda ◽

Atsuo Amano ◽

Kyohei Umebayashi ◽

Hiroaki Inaba ◽

Ichiro Nakagawa ◽

...

Keyword(s):

Porphyromonas Gingivalis ◽

Membrane Vesicle ◽

Bacterial Membrane ◽

Molecular Dissection ◽

Fluorescent Beads

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Cysteine Depletion Causes Oxidative Stress and Triggers Outer Membrane Vesicle Release by Neisseria meningitidis; Implications for Vaccine Development

PLoS ONE ◽

10.1371/journal.pone.0054314 ◽

2013 ◽

Vol 8 (1) ◽

pp. e54314 ◽

Cited By ~ 33

Author(s):

Bas van de Waterbeemd ◽

Gijsbert Zomer ◽

Jan van den IJssel ◽

Lonneke van Keulen ◽

Michel H. Eppink ◽

...

Keyword(s):

Oxidative Stress ◽

Neisseria Meningitidis ◽

Outer Membrane ◽

Vaccine Development ◽

Membrane Vesicle ◽

Outer Membrane Vesicle ◽

Vesicle Release

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Bacterial membrane vesicle and its diversity

Drug Delivery System ◽

10.2745/dds.36.138 ◽

2021 ◽

Vol 36 (2) ◽

pp. 138-144

Author(s):

Nobuhiko Nomura ◽

Masanori Toyofuku

Keyword(s):

Membrane Vesicle ◽

Bacterial Membrane

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Dynamin-like proteins are essential for vesicle biogenesis in Mycobacterium tuberculosis

10.1101/2020.01.14.906362 ◽

2020 ◽

Cited By ~ 1

Author(s):

Shamba Gupta ◽

Ainhoa Palacios ◽

Atul Khataokar ◽

Brian Weinrick ◽

Jose L. Lavín ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Extracellular Vesicles ◽

Cell Envelope ◽

Membrane Vesicle ◽

Extracellular Vesicle ◽

Dependent Manner ◽

Surface Lipid ◽

Cell Infection ◽

Vesicle Release ◽

Vesicle Biogenesis

ABSTRACTMycobacterium tuberculosis (Mtb) secretes pathogenicity factors and immunologically active molecules via membrane vesicles. However, nothing is known about the mechanisms involved in mycobacterial vesicle biogenesis. This study investigates molecular determinants of membrane vesicle production in Mtb by analyzing Mtb cells under conditions of high vesicle production: iron limitation and VirR restriction. Ultrastructural analysis showed extensive cell envelope restructuring in association with vesicle release that correlated with downregulation of cell surface lipid biosynthesis and peptidoglycan alterations. Comparative transcriptomics showed common upregulation of the iniBAC operon in association with high vesicle production in Mtb cells. Vesicle production analysis demonstrated that the dynamin-like proteins (DLPs) encoded by this operon, IniA and IniC, are necessary for release of EV by Mtb in culture and in infected macrophages. Isoniazid, a first-line antibiotic, used in tuberculosis treatment, was found to stimulate vesicle release in a DLP-dependent manner. Our results provide a new understanding of the function of mycobacterial DLPs and mechanistic insights into vesicle biogenesis. The findings will enable further understanding of the relevance of Mtb-derived extracellular vesicles in the pathogenesis of tuberculosis and may open new avenues for therapeutic research.IMPORTANCEIron is an essential nutrient that promotes survival and growth of M. tuberculosis, the bacterium that causes human tuberculosis (TB). Limited availability of iron, often encountered in the host environment, stimulates M. tuberculosis to secrete membrane-bound extracellular vesicles containing molecules that may help it evade the immune system. Characterizing the bacterial factors and mechanisms involved in the production of mycobacterial vesicles is important for envisioning ways to interfere with this process. Here, we report the discovery of proteins required by M. tuberculosis for vesicle biogenesis in culture and during host cell infection. We also demonstrate a connection between antibiotic response and extracellular vesicle production. The work provides insights into the mechanisms underlying vesicle biogenesis in M. tuberculosis and permits better understanding of the significance of vesicle production to M. tuberculosis-host interactions and antibiotic stress response.

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