scholarly journals Deletion of alpB Gene Influences Outer Membrane Vesicles Biogenesis of Lysobacter sp. XL1

2021 ◽  
Vol 12 ◽  
Author(s):  
Irina V. Kudryakova ◽  
Alexey S. Afoshin ◽  
Tanya V. Ivashina ◽  
Natalia E. Suzina ◽  
Elena A. Leontyevskaya ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Wild Type Strain ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Lower Amount ◽  
Extracellular Milieu ◽  
Allelic Replacement ◽  
Vesicle Biogenesis ◽  
Ultrastructural Characteristics ◽  
Genetic Deletion

Outer membrane vesicles (OMVs) produced by Gram-negative bacteria constitute important factors in defining interactions with the extracellular milieu. Lysobacter sp. XL1 produces OMVs capable of lysing microbial cells due to the presence in their cargo of bacteriolytic protease L5 (AlpB). Although protein L5 has been functionally and biochemically characterized (including aspects of its packing into OMVs), its role in vesicle biogenesis through genetic deletion of alpB had not been studied previously. Here, we have successfully deleted alpB by allelic replacement and show that the alpB deletion mutant produces a significantly lower amount of OMVs that lack bacteriolytic activity and display altered ultrastructural characteristics in relation to the OMVs produced by the wild-type strain. These results confirm that, as previously proposed, protein L5 participates in OMV production through a mechanism that is not yet fully understood.

scholarly journals The role of Zur-regulated lipoprotein A in bacterial morphology, antimicrobial susceptibility, and production of outer membrane vesicles in Acinetobacter baumannii

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Nayeong Kim ◽  
Hyo Jeong Kim ◽  
Man Hwan Oh ◽  
Se Yeon Kim ◽  
Mi Hyun Kim ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Outer Membrane ◽  
Mutant Strain ◽  
Antimicrobial Susceptibility ◽  
Type Strain ◽  
Wild Type Strain ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Wild Type ◽  
Bacterial Morphology

Abstract Background Zinc uptake-regulator (Zur)-regulated lipoprotein A (ZrlA) plays a role in bacterial fitness and overcoming antimicrobial exposure in Acinetobacter baumannii. This study further characterized the zrlA gene and its encoded protein and investigated the roles of the zrlA gene in bacterial morphology, antimicrobial susceptibility, and production of outer membrane vesicles (OMVs) in A. baumannii ATCC 17978. Results In silico and polymerase chain reaction analyses showed that the zrlA gene was conserved among A. baumannii strains with 97–100% sequence homology. Recombinant ZrlA protein exhibited a specific enzymatic activity of D-alanine-D-alanine carboxypeptidase. Wild-type A. baumannii exhibited more morphological heterogeneity than a ΔzrlA mutant strain during stationary phase. The ΔzrlA mutant strain was more susceptible to gentamicin than the wild-type strain. Sizes and protein profiles of OMVs were similar between the wild-type and ΔzrlA mutant strains, but the ΔzrlA mutant strain produced 9.7 times more OMV particles than the wild-type strain. OMVs from the ΔzrlA mutant were more cytotoxic in cultured epithelial cells than OMVs from the wild-type strain. Conclusions The present study demonstrated that A. baumannii ZrlA contributes to bacterial morphogenesis and antimicrobial resistance, but its deletion increases OMV production and OMV-mediated host cell cytotoxicity.

scholarly journals The sensor kinase BfmS controls production of outer membrane vesicles in Acinetobacter baumannii

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Se Yeon Kim ◽  
Mi Hyun Kim ◽  
Seung Il Kim ◽  
Joo Hee Son ◽  
Shukho Kim ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Acinetobacter Baumannii ◽  
Outer Membrane ◽  
Type Strain ◽  
Wild Type Strain ◽  
A549 Cells ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Sensor Kinase ◽  
Wild Type

Abstract Background Acinetobacter baumannii is an important opportunistic pathogen responsible for various nosocomial infections. The BfmRS two-component system plays a role in pathogenesis and antimicrobial resistance of A. baumannii via regulation of bacterial envelope structures. This study investigated the role of the sensor kinase, BfmS, in localization of outer membrane protein A (OmpA) in the outer membrane and production of outer membrane vesicles (OMVs) using wild-type A. baumannii ATCC 17978, ΔbfmS mutant, and bfmS-complemented strains. Results The ΔbfmS mutant showed hypermucoid phenotype in the culture plates, growth retardation under static culture conditions, and reduced susceptibility to aztreonam and colistin compared to the wild-type strain. The ΔbfmS mutant produced less OmpA in the outer membrane but released more OmpA via OMVs than the wild-type strain, even though expression of ompA and its protein production were not different between the two strains. The ΔbfmS mutant produced 2.35 times more OMV particles and 4.46 times more OMV proteins than the wild-type stain. The ΔbfmS mutant OMVs were more cytotoxic towards A549 cells than wild-type strain OMVs. Conclusions The present study demonstrates that BfmS controls production of OMVs in A. baumannii. Moreover, BfmS negatively regulates antimicrobial resistance of A. baumannii and OMV-mediated host cell cytotoxicity. Our results indicate that BfmS negatively controls the pathogenic traits of A. baumannii via cell envelope structures and OMV production.

scholarly journals Proteome Analysis of Outer Membrane Vesicles From a Highly Virulent Strain of Haemophilus parasuis

2021 ◽  
Vol 8 ◽  
Author(s):  
Kunli Zhang ◽  
Pinpin Chu ◽  
Shuai Song ◽  
Dongxia Yang ◽  
Zhibiao Bian ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Virulent Strain ◽  
Bacterial Pathogen ◽  
Proteome Analysis ◽  
Membrane Vesicles ◽  
Subcellular Location ◽  
Outer Membrane Vesicles ◽  
Haemophilus Parasuis ◽  
Extracellular Milieu ◽  
Virulence Potential

Haemophilus parasuis has emerged as an important bacterial pathogen in pig husbandry, as H. parasuis can coinfect pigs with a variety of pathogenic microorganisms and further cause an aggravation of the disease. It is crucial to investigate its pathogenetic mechanism. Gram-negative bacteria naturally secrete outer membrane vesicles (OMVs), and their potent virulence factors play prominent roles that affect the interaction between bacteria and host. Still, the pathogenesis that is associated with the bacterial OMVs has not been well-elucidated. In this study, we investigated the secretion of OMVs from a clinical H. parasuis isolate strain (H45). In addition, we further analyzed the characterization, the comprehensive proteome, and the virulence potential of OMVs. Our data demonstrated that H. parasuis could secrete OMVs into the extracellular milieu during infection. Using liquid chromatography with tandem mass spectrometry (MS/MS) identification and bio-information analysis, we identified 588 different proteins associated with OMVs. Also, we also analyzed the subcellular location and biological function of those proteins. These proteins are mainly involved in immune and iron metabolism. Moreover, we confirmed the pathogenicity of H. parasuis OMVs by observing a strong inflammatory response in J774A.1 and porcine alveolar macrophages. Taken together, our findings suggested that OMVs from H. parasuis were involved in the pathogenesis of this bacterium during infection.

scholarly journals Xanthomonas campestris pv. vesicatoria Secretes Proteases and Xylanases via the Xps Type II Secretion System and Outer Membrane Vesicles

2015 ◽  
Vol 197 (17) ◽  
pp. 2879-2893 ◽  
Author(s):  
Magali Solé ◽  
Felix Scheibner ◽  
Anne-Katrin Hoffmeister ◽  
Nadine Hartmann ◽  
Gerd Hause ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Xanthomonas Campestris ◽  
Pathogenic Bacteria ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Content Type ◽  
Plant Pathogenic Bacteria ◽  
Extracellular Milieu

ABSTRACTMany plant-pathogenic bacteria utilize type II secretion (T2S) systems to secrete degradative enzymes into the extracellular milieu. T2S substrates presumably mediate the degradation of plant cell wall components during the host-pathogen interaction and thus promote bacterial virulence. Previously, the Xps-T2S system fromXanthomonas campestrispv. vesicatoria was shown to contribute to extracellular protease activity and the secretion of a virulence-associated xylanase. The identities and functions of additional T2S substrates fromX. campestrispv. vesicatoria, however, are still unknown. In the present study, the analysis of 25 candidate proteins fromX. campestrispv. vesicatoria led to the identification of two type II secreted predicted xylanases, a putative protease and a lipase which was previously identified as a virulence factor ofX. campestrispv. vesicatoria. Studies with mutant strains revealed that the identified xylanases and the protease contribute to virulence andin plantagrowth ofX. campestrispv. vesicatoria. When analyzed in the related pathogenX. campestrispv. campestris, several T2S substrates fromX. campestrispv. vesicatoria were secreted independently of the T2S systems, presumably because of differences in the T2S substrate specificities of the two pathogens. Furthermore, inX. campestrispv. vesicatoria T2S mutants, secretion of T2S substrates was not completely absent, suggesting the contribution of additional transport systems to protein secretion. In line with this hypothesis, T2S substrates were detected in outer membrane vesicles, which were frequently observed forX. campestrispv. vesicatoria. We, therefore, propose that extracellular virulence-associated enzymes fromX. campestrispv. vesicatoria are targeted to the Xps-T2S system and to outer membrane vesicles.IMPORTANCEThe virulence of plant-pathogenic bacteria often depends on TS2 systems, which secrete degradative enzymes into the extracellular milieu. T2S substrates are being studied in several plant-pathogenic bacteria, includingXanthomonas campestrispv. vesicatoria, which causes bacterial spot disease in tomato and pepper. Here, we show that the T2S system fromX. campestrispv. vesicatoria secretes virulence-associated xylanases, a predicted protease, and a lipase. Secretion assays with the related pathogenX. campestrispv. campestris revealed important differences in the T2S substrate specificities of the two pathogens. Furthermore, electron microscopy showed that T2S substrates fromX. campestrispv. vesicatoria are targeted to outer membrane vesicles (OMVs). Our results, therefore, suggest that OMVs provide an alternative transport route for type II secreted extracellular enzymes.

Electrophysiological Characterization of Transport Across Outer Membrane Channels from Gram-Negative Bacteria in Their Native Environment

2019 ◽  
Author(s):  
Jiajun Wang ◽  
Rémi Terrasse ◽  
Jayesh Arun Bafna ◽  
Lorraine Benier ◽  
Mathias Winterhalter
Keyword(s):  
Outer Membrane ◽  
Lipid Membrane ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Multi Drug Resistance ◽  
Gram Negative Bacteria ◽  
Membrane Channels ◽  
Gram Negative ◽  
Electrophysiological Characterization

Multi-drug resistance in Gram-negative bacteria is often associated with low permeability of the outer membrane. To investigate the role of membrane channels in the uptake of antibiotics, we extract, purify and reconstitute them into artificial planar membranes. To avoid this time-consuming procedure, here we show a robust approach using fusion of native outer membrane vesicles (OMV) into planar lipid bilayer which moreover allows also to some extend the characterization of membrane protein channels in their native environment. Two major membrane channels from <i>Escherichia coli</i>, OmpF and OmpC, were overexpressed from the host and the corresponding OMVs were collected. Each OMV fusion revealed surprisingly single or only few channel activities. The asymmetry of the OMV´s translates after fusion into the lipid membrane with the LPS dominantly present at the side of OMV addition. Compared to conventional reconstitution methods, the channels fused from OMVs containing LPS have similar conductance but a much broader distribution. The addition of Enrofloxacin on the LPS side yields somewhat higher association (<i>k<sub>on</sub></i>) and lower dissociation (<i>k<sub>off</sub></i>) rates compared to LPS-free reconstitution. We conclude that using outer membrane vesicles is a fast and easy approach for functional and structural studies of membrane channels in the native membrane.

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