Carboxy-Terminal Processing Protease Controls Production of Outer Membrane Vesicles and Biofilm in Acinetobacter baumannii

Carboxy-terminal processing protease (Ctp) is a serine protease that controls multiple cellular processes through posttranslational modification of proteins. Acinetobacter baumannii ATCC 17978 ctp mutant, namely MR14, is known to cause cell wall defects and autolysis. The objective of this study was to investigate the role of ctp mutation–driven autolysis in regulating biofilms in A. baumannii and to evaluate the vesiculation caused by cell wall defects. We found that in A. baumannii, Ctp is localized in the cytoplasmic membrane, and loss of Ctp function enhances the biofilm-forming ability of A. baumannii. Quantification of the matrix components revealed that extracellular DNA (eDNA) and proteins were the chief constituents of MR14 biofilm, and the transmission electron microscopy further indicated the presence of numerous dead cells compared with ATCC 17978. The large number of MR14 dead cells is potentially the result of compromised outer membrane integrity, as demonstrated by its high sensitivity to sodium dodecyl sulfate (SDS) and ethylenediaminetetraacetic acid (EDTA). MR14 also exhibited the hypervesiculation phenotype, producing outer-membrane vesicles (OMVs) of large mean size. The MR14 OMVs were more cytotoxic toward A549 cells than ATCC 17978 OMVs. Our overall results indicate that A. baumanniictp negatively controls pathogenic traits through autolysis and OMV biogenesis.

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

BMC Microbiology ◽

10.1186/s12866-019-1679-0 ◽

2019 ◽

Vol 19 (1) ◽

Cited By ~ 3

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.

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

BMC Microbiology ◽

10.1186/s12866-020-02083-0 ◽

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.

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In vitro study of virulence potential of Acinetobacter baumannii outer membrane vesicles

Microbial Pathogenesis ◽

10.1016/j.micpath.2017.08.048 ◽

2017 ◽

Vol 111 ◽

pp. 218-224 ◽

Cited By ~ 12

Author(s):

Chandana Jha ◽

Sujata Ghosh ◽

Vikas Gautam ◽

Pankaj Malhotra ◽

Pallab Ray

Keyword(s):

Acinetobacter Baumannii ◽

Outer Membrane ◽

In Vitro Study ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Vitro Study ◽

Virulence Potential

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

Molecules ◽

10.3390/molecules24101972 ◽

2019 ◽

Vol 24 (10) ◽

pp. 1972 ◽

Cited By ~ 2

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.

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Extracellular DNA and Outer Membrane Vesicles contribute to P. fluorescens SBW25 air-liquid interface biofilms.

10.5194/biofilms9-39 ◽

2020 ◽

Author(s):

Olena Moshynets ◽

Airat Kayumov ◽

Olga Iungin ◽

Svitlana Rymar ◽

Ianina Pokholenko ◽

...

Keyword(s):

Outer Membrane ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Extracellular Dna ◽

Exogenous Dna ◽

Rhizosphere Bacterium ◽

Cellulose Matrix ◽

Dnase Treatment ◽

Biofilm Development ◽

Biofilm Matrix

Outer membrane vesicles (OMVs) and extracellular DNA (eDNA) are important for biofilm formation for many bacteria. OMVs are a perfect transport system to deliver biofilm-related components including eDNA beyond the boundaries of cells, and eDNA itself is an important structural component of biofilms as well as enabling horizontal gene transfer and local adaptation. Both OMVs and eDNA are found in the biofilms produced by the opportunistic human pathogen P. aeruginosa and the plant pathogen P. syringae, but as yet, they have not been reported in the cellulose matrix-based biofilms produced by the related model rhizosphere bacterium Pseudomonas fluorescens SBW25. In this work we have gone back to re-assess the complexity of SBW25 biofilms by looking for evidence of OMVs and eDNA associated with biofilm&#8211;formation. OMVs were first imaged by SEM and LC-MC analysis used to identify 51 biofilm matrix-associated proteins of which 12 were also identified in biofilm OMVs. Interestingly, only 5 proteins were identified in both biofilm matrix and OMV samples, but not in planktonic OMVs, suggesting that these may be biofilm-specific components. &#160; We also observed eDNA by CLSM in both the weak and poorly-attached Viscous Mass (VM) and robust and well-attached Wrinkly Spreader (WS) air-liquid (A-L) interface biofilms produced by wild-type SBW25 and the Wrinkly Spreader mutant. The eDNA fraction could be precipitated from biofilm cell-free supernatant samples which demonstrated that WS biofilms had two-fold&#8211;higher levels than VM biofilms. DNAse treatment effected the development of both types of biofilm and reduced the strength and attachment levels when added to mature VM and WS biofilms. Testing with exogenous DNA suggests that high molecular weight (HMW) DNA is involved in both strength and attachment, perhaps by surface conditioning and interactions with the primary cellulose matrix common to both biofilms. HMW eDNA could be isolated directly from biofilm supernatants whereas two different HMW size fractions could be isolated from OMVs, presumably, from the outer OMV surface because DNAse treatment led to a substantially reduced DNA signal. This suggest that eDNA persistence and degradation in SBW25 biofilms is complex and eDNA fractions may play different roles in biofilm development, protection and adaptation.

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Horizontal Transfer of the OXA-24 Carbapenemase Gene via Outer Membrane Vesicles: a New Mechanism of Dissemination of Carbapenem Resistance Genes in Acinetobacter baumannii

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00929-10 ◽

2011 ◽

Vol 55 (7) ◽

pp. 3084-3090 ◽

Cited By ~ 134

Author(s):

Carlos Rumbo ◽

Esteban Fernández-Moreira ◽

María Merino ◽

Margarita Poza ◽

Jose Antonio Mendez ◽

...

Keyword(s):

Acinetobacter Baumannii ◽

Outer Membrane ◽

Resistance Genes ◽

Carbapenem Resistance ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Content Type ◽

Class D ◽

Carbapenemase Gene

ABSTRACTThe resistance ofAcinetobacter baumanniistrains to carbapenems is a worrying problem in hospital settings. The main mechanism of carbapenem resistance is the expression of β-lactamases (metalloenzymes or class D enzymes). The mechanisms of the dissemination of these genes amongA. baumanniistrains are not fully understood. In this study we used two carbapenem-resistant clinical strains ofA. baumannii(AbH12O-A2 and AbH12O-CU3) expressing the plasmid-borneblaOXA-24gene (plasmids pMMA2 and pMMCU3, respectively) to demonstrate thatA. baumanniireleases outer membrane vesicles (OMVs) duringin vitrogrowth. The use of hybridization studies enabled us to show that these OMVs harbored theblaOXA-24gene. The incubation of these OMVs with the carbapenem-susceptibleA. baumanniiATCC 17978 host strain yielded full resistance to carbapenems. The presence of the original plasmids harboring theblaOXA-24gene was detected in strain ATCC 17978 after the transformation of OMVs. New OMVs harboringblaOXA-24were released byA. baumanniiATCC 17978 after it was transformed with the original OMV-mediated plasmids, indicating the universality of the process. We present the first experimental evidence that clinical isolates ofA. baumanniimay release OMVs as a mechanism of horizontal gene transfer whereby carbapenem resistance genes are delivered to surroundingA. baumanniibacterial isolates.

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Acinetobacter baumannii Extracellular OXA-58 Is Primarily and Selectively Released via Outer Membrane Vesicles after Sec-Dependent Periplasmic Translocation

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01343-15 ◽

2015 ◽

Vol 59 (12) ◽

pp. 7346-7354 ◽

Cited By ~ 21

Author(s):

Yu-Ting Liao ◽

Shu-Chen Kuo ◽

Ming-Hsien Chiang ◽

Yi-Tzu Lee ◽

Wang-Chou Sung ◽

...

Keyword(s):

Acinetobacter Baumannii ◽

Outer Membrane ◽

Signal Peptide ◽

Fluorescent Protein ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Proteinase K ◽

Cell Fractionation ◽

Extracellular Release ◽

Sheltering Effect

ABSTRACTCarbapenem-resistantAcinetobacter baumannii(CRAb) shelter cohabiting carbapenem-susceptible bacteria from carbapenem killing via extracellular release of carbapenem-hydrolyzing class D β-lactamases, including OXA-58. However, the mechanism of the extracellular release of OXA-58 has not been elucidated.In silicoanalysis predicted OXA-58 to be translocated to the periplasm via the Sec system. Using cell fractionation and Western blotting, OXA-58 with the signal peptide and C terminus deleted was not detected in the periplasmic and extracellular fractions. Overexpression of enhanced green fluorescent protein fused to the OXA-58 signal peptide led to its periplasmic translocation but not extracellular release, suggesting that OXA-58 is selectively released. The majority of the extracellular OXA-58 was associated with outer membrane vesicles (OMVs). The OMV-associated OXA-58 was detected only in a strain overexpressing OXA-58. The presence of OXA-58 in OMVs was confirmed by a carbapenem inactivation bioassay, proteomic analysis, and transmission electron microscopy. Imipenem treatment increased OMV formation and caused cell lysis, resulting in an increase in the OMV-associated and OMV-independent release of extracellular OXA-58. OMV-independent OXA-58 hydrolyzed nitrocefin more rapidly than OMV-associated OXA-58 but was more susceptible to proteinase K degradation. Rose bengal, an SecA inhibitor, inhibited the periplasmic translocation and OMV-associated release of OXA-58 and abolished the sheltering effect of CRAb. This study demonstrated that the majority of the extracellular OXA-58 is selectively released via OMVs after Sec-dependent periplasmic translocation. Addition of imipenem increased both OMV-associated and OMV-independent OXA-58, which may have different biological roles. SecA inhibitor could abolish the carbapenem-sheltering effect of CRAb.

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Development of Different Methods for Preparing Acinetobacter baumannii Outer Membrane Vesicles Vaccine: Impact of Preparation Method on Protective Efficacy

Frontiers in Immunology ◽

10.3389/fimmu.2020.01069 ◽

2020 ◽

Vol 11 ◽

Author(s):

Sun Li ◽

Da-Qun Chen ◽

Lu Ji ◽

Si Sun ◽

Zhe Jin ◽

...

Keyword(s):

Acinetobacter Baumannii ◽

Outer Membrane ◽

Preparation Method ◽

Protective Efficacy ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Vaccine Impact

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Toll-Like Receptors 2 and 4 Modulate Pulmonary Inflammation and Host Factors Mediated by Outer Membrane Vesicles Derived from Acinetobacter baumannii

Infection and Immunity ◽

10.1128/iai.00243-19 ◽

2019 ◽

Vol 87 (9) ◽

Cited By ~ 5

Author(s):

Chad R. Marion ◽

Jaewook Lee ◽

Lokesh Sharma ◽

Kyong-Su Park ◽

Changjin Lee ◽

...

Keyword(s):

Acinetobacter Baumannii ◽

Outer Membrane ◽

Pulmonary Inflammation ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Toll Like Receptors ◽

Gram Negative ◽

Content Type ◽

Intranasal Introduction

ABSTRACT Pneumonia due to Gram-negative bacteria is associated with high mortality. Acinetobacter baumannii is a Gram-negative bacterium that is associated with hospital-acquired and ventilator-associated pneumonia. Bacteria have been described to release outer membrane vesicles (OMVs) that are capable of mediating systemic inflammation. The mechanism by which A. baumannii OMVs mediate inflammation is not fully defined. We sought to investigate the roles that Toll-like receptors (TLRs) play in A. baumannii OMV-mediated pulmonary inflammation. We isolated OMVs from A. baumannii cultures and intranasally introduced the OMVs into mice. Intranasal introduction of A. baumannii OMVs mediated pulmonary inflammation, which is associated with neutrophil recruitment and weight loss. In addition, A. baumannii OMVs increased the release of several chemokines and cytokines in the mouse lungs. The proinflammatory responses were partially inhibited in TLR2- and TLR4-deficient mice compared to those of wild-type mice. This study highlights the important roles of TLRs in A. baumannii OMV-induced pulmonary inflammation in vivo.

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