scholarly journals Physicochemical Evidence that Francisella FupA and FupB Proteins Are Porins

2020 ◽  
Vol 21 (15) ◽  
pp. 5496
Author(s):  
Claire Siebert ◽  
Corinne Mercier ◽  
Donald K. Martin ◽  
Patricia Renesto ◽  
Beatrice Schaack
Keyword(s):  
Outer Membrane ◽  
Lipid Bilayers ◽  
Lipid Membranes ◽  
Iron Transport ◽  
Molecular Mechanisms ◽  
Protein A ◽  
Membrane Vesicles ◽  
Disodium Salt ◽  
Outer Membrane Vesicles ◽  
Novel Therapeutic Strategies

Responsible for tularemia, Francisella tularensis bacteria are highly infectious Gram-negative, category A bioterrorism agents. The molecular mechanisms for their virulence and resistance to antibiotics remain largely unknown. FupA (Fer Utilization Protein), a protein mediating high-affinity transport of ferrous iron across the outer membrane, is associated with both. Recent studies demonstrated that fupA deletion contributed to lower F. tularensis susceptibility towards fluoroquinolones, by increasing the production of outer membrane vesicles. Although the paralogous FupB protein lacks such activity, iron transport capacity and a role in membrane stability were reported for the FupA/B chimera, a protein found in some F. tularensis strains, including the live vaccine strain (LVS). To investigate the mode of action of these proteins, we purified recombinant FupA, FupB and FupA/B proteins expressed in Escherichia coli and incorporated them into mixed lipid bilayers. We examined the porin-forming activity of the FupA/B proteoliposomes using a fluorescent 8-aminonaphthalene-1,3,6-trisulfonic acid, disodium salt (ANTS) probe. Using electrophysiology on tethered bilayer lipid membranes, we confirmed that the FupA/B fusion protein exhibits pore-forming activity with large ionic conductance, a property shared with both FupA and FupB. This demonstration opens up new avenues for identifying functional genes, and novel therapeutic strategies against F. tularensis infections.

scholarly journals The Mutation of Conservative Asp268 Residue in the Peptidoglycan-Associated Domain of the OmpA Protein Affects Multiple Acinetobacter baumannii Virulence Characteristics

Molecules ◽  
2019 ◽  
Vol 24 (10) ◽  
pp. 1972 ◽  
Author(s):  
Jūratė Skerniškytė ◽  
Emilija Karazijaitė ◽  
Julien Deschamps ◽  
Renatas Krasauskas ◽  
Romain Briandet ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Outer Membrane ◽  
Protein A ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Clinical Strain ◽  
Infection Model ◽  
Terminal Domain ◽  
Ompa Protein

Acinetobacter baumannii is a nosocomial human pathogen of increasing concern due to its multidrug resistance profile. The outer membrane protein A (OmpA) is an abundant bacterial cell surface component involved in A. baumannii pathogenesis. It has been shown that the C-terminal domain of OmpA is located in the periplasm and non-covalently associates with the peptidoglycan layer via two conserved amino acids, thereby anchoring OmpA to the cell wall. Here, we investigated the role of one of the respective residues, D268 in OmpA of A. baumannii clinical strain Ab169, on its virulence characteristics by complementing the ΔompA mutant with the plasmid-borne ompAD268A allele. We show that while restoring the impaired biofilm formation of the ΔompA strain, the Ab169ompAD268A mutant tended to form bacterial filaments, indicating the abnormalities in cell division. Moreover, the Ab169 OmpA D268-mediated association to peptidoglycan was required for the manifestation of twitching motility, desiccation resistance, serum-induced killing, adhesion to epithelial cells and virulence in a nematode infection model, although it was dispensable for the uptake of β-lactam antibiotics by outer membrane vesicles. Overall, the results of this study demonstrate that the OmpA C-terminal domain-mediated association to peptidoglycan is critical for a number of virulent properties displayed by A. baumannii outside and within the host.

scholarly journals LPS Remodeling Triggers Formation of Outer Membrane Vesicles inSalmonella

mBio ◽  
2016 ◽  
Vol 7 (4) ◽  
Author(s):  
Wael Elhenawy ◽  
Michael Bording-Jorgensen ◽  
Ezequiel Valguarnera ◽  
M. Florencia Haurat ◽  
Eytan Wine ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Molecular Mechanisms ◽  
Lipid A ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Mass Spectrometry Analysis ◽  
Membrane Remodeling ◽  
Macrophage Infection ◽  
Spectrometry Analysis

ABSTRACTOuter membrane vesicles (OMV) are proposed to mediate multiple functions during pathogenesis and symbiosis. However, the mechanisms responsible for OMV formation remain poorly understood. It has been shown in eukaryotic membranes that lipids with an inverted-cone shape favor the formation of positive membrane curvatures. Based on these studies, we formulated the hypothesis that lipid A deacylation might impose shape modifications that result in the curvature of the outer membrane (OM) and subsequent OMV formation. We tested the effect of lipid A remodeling on OMV biogenesis employingSalmonella entericaserovar Typhimurium as a model organism. Expression of the lipid A deacylase PagL resulted in increased vesiculation, without inducing an envelope stress response. Mass spectrometry analysis revealed profound differences in the patterns of lipid A in OM and OMV, with accumulation of deacylated lipid A forms exclusively in OMV. OMV biogenesis by intracellular bacteria upon macrophage infection was drastically reduced in apagLmutant strain. We propose a novel mechanism for OMV biogenesis requiring lipid A deacylation in the context of a multifactorial process that involves the orchestrated remodeling of the outer membrane.IMPORTANCEThe role of lipid remodeling in vesiculation is well documented in eukaryotes. Similarly, bacteria produce membrane-derived vesicles; however, the molecular mechanisms underlying their production are yet to be determined. In this work, we investigated the role of outer membrane remodeling in OMV biogenesis inS. Typhimurium. We showed that the expression of the lipid A deacylase PagL results in overvesiculation with deacylated lipid A accumulation exclusively in OMV. AnS. Typhimurium ΔpagLstrain showed a significant reduction in intracellular OMV secretion relative to the wild-type strain. Our results suggest a novel mechanism for OMV biogenesis that involves outer membrane remodeling through lipid A modification. Understanding how OMV are produced by bacteria is important to advance our understanding of the host-pathogen interactions.

Application of cryo-electron microscopy for investigation of Bax-induced pores in apoptosis

2017 ◽  
Vol 6 (1) ◽  
pp. 47-55
Author(s):  
Tomomi Kuwana
Keyword(s):  
Electron Microscopy ◽  
Outer Membrane ◽  
Molecular Mechanisms ◽  
Experimental System ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Mitochondrial Outer Membrane ◽  
Intermembrane Space ◽  
Mitochondrial Outer Membrane Permeabilization ◽  
Cryo Electron Microscopy

AbstractMitochondrial outer membrane permeabilization (MOMP) is a critical step in apoptosis, the molecular mechanisms of which have been a subject of intensive study. This process is important for therapeutic intervention in various diseases, such as cancer. Pro-apoptotic Bax and Bak are functionally redundant and structurally homologous. When activated at the mitochondrial outer membrane, they cause the membrane to permeabilize and release apoptogenic proteins from the intermembrane space. To unravel the molecular mechanisms of this unique and important event, we systematically reduced the experimental system. Simple outer membrane vesicles and liposomes recapitulated many features of MOMP. Although conventional transmission electron microscopy could not detect any membrane changes during MOMP in these vesicles, cryo-electron microscopy successfully revealed Bax-induced delicate pores, owing to its ability to preserve native, hydrated membrane structure. The data are consistent with the idea that Bax is unfolded and embedded in the bilayer and deforms the membrane to form a large pore. Together with the biochemical and structure data in the literature, we now have more comprehensive models of the key function of Bax. We hope that new tools, such as lipid nanodiscs, will give us an atomic-level resolution and finally solve Bax structure in the membrane, where it functions.

scholarly journals Lack of Outer Membrane Protein A Enhances the Release of Outer Membrane Vesicles and Survival ofVibrio choleraeand Suppresses Viability ofAcanthamoeba castellanii

2014 ◽  
Vol 2014 ◽  
pp. 1-6 ◽  
Author(s):  
Soni Priya Valeru ◽  
Salah Shanan ◽  
Haifa Alossimi ◽  
Amir Saeed ◽  
Gunnar Sandström ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Outer Membrane Proteins ◽  
Protein A ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Host Cells ◽  
Wild Type ◽  
Outer Membrane Protein A

Vibrio cholerae, the causative agent of the diarrhoeal disease cholera, survives in aquatic environments. The bacterium has developed a survival strategy to grow and survive insideAcanthamoeba castellanii. It has been shown thatV. choleraeexpresses outer membrane proteins as virulence factors playing a role in the adherence to interacted host cells. This study examined the role of outer membrane protein A (OmpA) and outer membrane vesicles (OMVs) in survival ofV. choleraealone and during its interaction withA. castellanii. The results showed that anOmpAmutant ofV. choleraesurvived longer than wild-typeV. choleraewhen cultivated alone. Cocultivation withA. castellaniienhanced the survival of both bacterial strains andOmpAprotein exhibited no effect on attachment, engulfment, and survival inside the amoebae. However, cocultivation of theOmpAmutant ofV. choleraedecreased the viability ofA. castellaniiand this bacterial strain released more OMVs than wild-typeV. cholerae. Surprisingly, treatment of amoeba cells with OMVs isolated from theOmpAmutant significantly decreased viable counts of the amoeba cells. In conclusion, the results might highlight a regulating rule forOmpAin survival ofV. choleraeand OMVs as a potent virulence factor for this bacterium towards eukaryotes in the environment.

scholarly journals Study on RNA Regulating Iron Transport in Outer Membrane Vesicles of Hypervirulent Klebsiella Pneumoniae

2020 ◽  
Author(s):  
Mao Zhou ◽  
Siyi Wang ◽  
You Lan ◽  
Xin Li ◽  
Xuan Liu ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Outer Membrane ◽  
Iron Transport ◽  
High Throughput Sequencing ◽  
Iron Acquisition ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Transporter Family ◽  
Iron Deficient ◽  
Deficient Medium

Abstract Background: The iron acquisition ability of hypervirulent Klebsiella pneumoniae (hvKP) is an important part of its super virulence mechanism, increasing studies have proved that outer membrane vesicles (OMVs) are involved in the iron acquisition process of bacteria. Thus, we compared the difference in RNA expression in OMVs of hvKP in iron-rich and iron-deficient medium, and explore the possible mechanism of RNA in OMVs involved in hvKP iron acquisition. Results: The results of high-throughput sequencing showed that in iron-deficient medium, there were 239 up-regulated and 89 down-regulated mRNAs in OMVs of hvKP, of which 20 mRNAs related to iron transport was up-regulated, mainly including siderophore synthesis and receptor genes, ATP binding cassette transporter family and iron sulfur cluster. Only two of the differential ncRNAs that regulate these mRNAs are up-regulated, which are lncRNAs.Conclusion: We demonstrated that mRNA and lncRNA in OMVs were directly or indirectly involved in the iron acquisition mechanism of hvKP under iron deficiency environment, which enhanced the adaptive survival ability of hvKP. It provided a basis for further exploring the iron acquisition mechanism of OMVs involved in hvKP.

scholarly journals The peptidoglycan-associated protein NapA plays an important role in the envelope integrity and in the pathogenesis of the lyme disease spirochete

2021 ◽  
Vol 17 (5) ◽  
pp. e1009546
Author(s):  
Marisela M. Davis ◽  
Aaron M. Brock ◽  
Tanner G. DeHart ◽  
Brittany P. Boribong ◽  
Katherine Lee ◽  
...  
Keyword(s):  
Lyme Disease ◽  
Outer Membrane ◽  
Structural Integrity ◽  
Molecular Beacon ◽  
Cell Envelope ◽  
Protein A ◽  
Bacterial Pathogen ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Gram Negative

The bacterial pathogen responsible for causing Lyme disease, Borrelia burgdorferi, is an atypical Gram-negative spirochete that is transmitted to humans via the bite of an infected Ixodes tick. In diderms, peptidoglycan (PG) is sandwiched between the inner and outer membrane of the cell envelope. In many other Gram-negative bacteria, PG is bound by protein(s), which provide both structural integrity and continuity between envelope layers. Here, we present evidence of a peptidoglycan-associated protein (PAP) in B. burgdorferi. Using an unbiased proteomics approach, we identified Neutrophil Attracting Protein A (NapA) as a PAP. Interestingly, NapA is a Dps homologue, which typically functions to bind and protect cellular DNA from damage during times of stress. While B. burgdorferi NapA is known to be involved in the oxidative stress response, it lacks the critical residues necessary for DNA binding. Biochemical and cellular studies demonstrate that NapA is localized to the B. burgdorferi periplasm and is indeed a PAP. Cryo-electron microscopy indicates that mutant bacteria, unable to produce NapA, have structural abnormalities. Defects in cell-wall integrity impact growth rate and cause the napA mutant to be more susceptible to osmotic and PG-specific stresses. NapA-linked PG is secreted in outer membrane vesicles and augments IL-17 production, relative to PG alone. Using microfluidics, we demonstrate that NapA acts as a molecular beacon—exacerbating the pathogenic properties of B. burgdorferi PG. These studies further our understanding of the B. burgdorferi cell envelope, provide critical information that underlies its pathogenesis, and highlight how a highly conserved bacterial protein can evolve mechanistically, while maintaining biological function.

scholarly journals Outer Membrane Vesicle Biosynthesis in Salmonella : Is There More to Gram-Negative Bacteria?

mBio ◽  
2016 ◽  
Vol 7 (4) ◽  
Author(s):  
Joachim Reidl
Keyword(s):  
Outer Membrane ◽  
Molecular Mechanisms ◽  
Membrane Vesicle ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Serovar Typhimurium ◽  
Reported Study

ABSTRACT Recent research has focused on the biological role of outer membrane vesicles (OMVs), which are derived from the outer membranes (OMs) of Gram-negative bacteria, and their potential exploitation as therapeutics. OMVs have been characterized in many ways and functions. Until recently, research focused on hypothetical and empirical models that addressed the molecular mechanisms of OMV biogenesis, such as vesicles bulging from the OM in various ways. The recently reported study by Elhenawy et al. (mBio 7:e00940-16, 2016, http://dx.doi.org/10.1128/mBio.00940-16 ) provided further insights into OMV biogenesis of Salmonella enterica serovar Typhimurium. That study showed that deacylation of lipopolysaccharides (LPS) influences the level of OMV production and, furthermore, determines a sorting of high versus low acylated LPS in OMs and OMVs, respectively. Interestingly, deacylation may inversely correlate with other LPS modifications, suggesting some synergy toward optimized host resistance via best OM compositions for S . Typhimurium.

scholarly journals Outer Membrane Vesicles of Gram-Negative Bacteria: An Outlook on Biogenesis

2021 ◽  
Vol 12 ◽  
Author(s):  
Eric Daniel Avila-Calderón ◽  
María del Socorro Ruiz-Palma ◽  
Ma. Guadalupe Aguilera-Arreola ◽  
Norma Velázquez-Guadarrama ◽  
Enrico A. Ruiz ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Quorum Sensing ◽  
Outer Membrane ◽  
Molecular Mechanisms ◽  
Outer Membrane Proteins ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Cellular Components

Outer membrane vesicles (OMVs) from Gram-negative bacteria were first described more than 50 years ago. However, the molecular mechanisms involved in biogenesis began to be studied only in the last few decades. Presently, the biogenesis and molecular mechanisms for their release are not completely known. This review covers the most recent information on cellular components involved in OMV biogenesis, such as lipoproteins and outer membrane proteins, lipopolysaccharide, phospholipids, quorum-sensing molecules, and flagella.

scholarly journals Comparison of Protection in Rabbits against Host-Adapted and Cultivated Borrelia burgdorferi following Infection-Derived Immunity or Immunization with Outer Membrane Vesicles or Outer Surface Protein A

2000 ◽  
Vol 68 (7) ◽  
pp. 4189-4199 ◽  
Author(s):  
Ellen S. Shang ◽  
Cheryl I. Champion ◽  
Xiao-Yang Wu ◽  
Jonathan T. Skare ◽  
David R. Blanco ◽  
...  
Keyword(s):  
Borrelia Burgdorferi ◽  
Outer Membrane ◽  
Outer Surface ◽  
Outer Membrane Proteins ◽  
Protein A ◽  
Surface Protein ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Outer Surface Protein A ◽  
Outer Surface Protein

ABSTRACT In this study, infection-derived immunity in the rabbit model of Lyme disease was compared to immunity following immunization with purified outer membrane vesicles (OMV) isolated from Borrelia burgdorferi and recombinant outer surface protein A (OspA). Immunization of rabbits with OMV isolated from virulent strain B31 and its avirulent derivative B313 (lacking OspA and DbpA) conferred highly significant protection against intradermal injection with 6 × 104 in vitro-cultivated virulent B. burgdorferi. This is the first demonstration of protective immunogenicity induced by OMV. While immunization with OspA and avirulent B31 OMV provided far less protection against this challenge, rabbits with infection-derived immunity were completely protected. Protection against host-adapted B. burgdorferi was assessed by implantation of skin biopsies taken from rabbit erythema migrans (a uniquely rich source of B. burgdorferi in vertebrate tissue) containing up to 108 spirochetes. While all of the OMV- and OspA-immunized rabbits were fully susceptible to skin and disseminated infection, rabbits with infection-derived immunity were completely protected. Analysis of the antibody responses to outer membrane proteins, including DbpA, OspA, and OspC, suggests that the remarkable protection exhibited by the infection-immune rabbits is due to antibodies directed at antigens unique to or markedly up-regulated in host-adapted B. burgdorferi.

scholarly journals Acinetobacter baumannii Secretes Cytotoxic Outer Membrane Protein A via Outer Membrane Vesicles

PLoS ONE ◽  
2011 ◽  
Vol 6 (2) ◽  
pp. e17027 ◽  
Author(s):  
Jong Sook Jin ◽  
Sang-Oh Kwon ◽  
Dong Chan Moon ◽  
Mamata Gurung ◽  
Jung Hwa Lee ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Acinetobacter Baumannii ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Protein A ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Outer Membrane Protein A
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