Extracellular vesicles as an alternative copper-secretion mechanism in bacteria

Lima, Steeve; Matinha-Cardoso, Jorge; Giner‐Lamia, Joaquín; Couto, Narciso; Pacheco, Catarina C.; Florencio, Francisco J.; Wright, Phillip C.; Tamagnini, Paula; Oliveira, Paulo

doi:10.1016/j.jhazmat.2022.128594

Cited by 20 publications

(15 citation statements)

References 46 publications

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“…To evaluate the capacity of customizing the protein content of Synechocystis EVs, the super‐folder green fluorescent protein (sfGFP) was used as reporter, as recently described (Lima et al ., 2022). Hence, sfGFP was expressed in Synechocystis cells under the control of the well‐characterized, strong promoter P trc.x.lacO (Ferreira et al ., 2018) and further targeted to the periplasm by the signal peptide of the Synechocystis periplasmic protein FutA2, a substrate of the twin‐arginine translocation (Tat) pathway (Waldron et al ., 2007; Badarau et al ., 2008) (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…To construct the Synechocystis strain expressing the heterologous protein sfGFP and further targeting it to extracellular vesicles (Lima et al, 2022), the synthetic promoter P trc.x.lacO (Ferreira et al, 2018) the signal peptide sequence of the Synechocystis native protein FutA2 (amino acids 1 to 35) as previously used by Polyviou et al (2018), which determines its translocation to the periplasm, and the gene encoding the reporter protein super-folder Green Fluorescent Protein (sfGFP) were amplified by PCR using specific oligonucleotides (Table S8). Assembly of the different DNA fragments was performed by sequential overlap-extension PCR.…”

Section: Engineering Of Synechocystis Heterologous Protein Expression...mentioning

confidence: 99%

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Novel protein carrier system based on cyanobacterial nano‐sized extracellular vesicles for application in fish

Matinha-Cardoso

Coutinho

Lima

et al. 2022

Microbial Biotechnology

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Summary Aquaculture has been one of the fastest‐growing food industry sectors, expanding at the pace of consumers' demands. To promote safe and effective fish growth performance strategies, and to stimulate environmentally friendly solutions to protect fish against disease outbreaks, new approaches are needed to safeguard fish welfare, as well as farmers and consumers interests. Here, we tested the use of cyanobacterial extracellular vesicles (EVs) as a novel nanocarrier system of heterologous proteins for applications in fish. We started by incubating zebrafish larvae with Synechocystis sp. PCC6803 EVs, isolated from selected mutant strains with different cell envelope characteristics. Results show that Synechocystis EVs are biocompatible with fish larvae, regardless of their structural composition, as EVs neither induced fish mortality nor triggered significant inflammatory responses. We establish also that cyanobacteria are amenable to engineering heterologous protein expression and loading into EVs, for which we used the reporter sfGFP. Moreover, upon immersion treatment, we successfully demonstrate that sfGFP‐loaded Synechocystis EVs accumulate in the gastrointestinal tract of zebrafish larvae. This work opens the possibility of using cyanobacterial EVs as a novel biotechnological tool in fish, with prospective applications in carrying proteins/enzymes, for example for modulating their nutritional status or stimulating specific adaptive immune responses.

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Section: Resultsmentioning

confidence: 99%

Section: Engineering Of Synechocystis Heterologous Protein Expression...mentioning

confidence: 99%

Novel protein carrier system based on cyanobacterial nano‐sized extracellular vesicles for application in fish

Matinha-Cardoso

Coutinho

Lima

et al. 2022

Microbial Biotechnology

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“…strain PCC 6803. However, in that study, only a mutant of the TolC outer membrane protein presented hypervesiculation in response to high copper doses and a copper chaperone (CopM) was detected in MVs in association with enhanced copper ( 46 ).…”

Section: Discussionmentioning

confidence: 80%

Bacterial Membrane Vesicles as a Novel Strategy for Extrusion of Antimicrobial Bismuth Drug in Helicobacter pylori

Kumar

Schmitt

Gorgette

et al. 2022

mBio

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“…MV formation has been suggested as one of the Ln uptake systems in the methylotrophic Beijerinckiaceae bacterium RH AL1 ( Wegner et al, 2021 ). MV is also reported to be involved in the excretion of excess copper ( Lima et al, 2022 ), suggesting that MV formation is one of the cellular strategies to excrete excess metals to maintain metal homeostasis. PQQ can bind Ln 3+ ( Lumpe and Daumann, 2019 ) as a possible factor for Ln excretion.…”

Section: Discussionmentioning

confidence: 99%

A Periplasmic Lanthanide Mediator, Lanmodulin, in Methylobacterium aquaticum Strain 22A

Fujitani

Shibata²,

Tani

2022

Front. Microbiol.

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Methylobacterium and Methylorubrum species oxidize methanol via pyrroloquinoline quinone-methanol dehydrogenases (MDHs). MDHs can be classified into two major groups, Ca2+-dependent MDH (MxaF) and lanthanide (Ln3+)-dependent MDH (XoxF), whose expression is regulated by the availability of Ln3+. A set of a siderophore, TonB-dependent receptor, and an ABC transporter that resembles the machinery for iron uptake is involved in the solubilization and transport of Ln3+. The transport of Ln3+ into the cytosol enhances XoxF expression. A unique protein named lanmodulin from Methylorubrum extorquens strain AM1 was identified as a specific Ln3+-binding protein, and its biological function was implicated to be an Ln3+ shuttle in the periplasm. In contrast, it remains unclear how Ln3+ levels in the cells are maintained, because Ln3+ is potentially deleterious to cellular systems due to its strong affinity to phosphate ions. In this study, we investigated the function of a lanmodulin homolog in Methylobacterium aquaticum strain 22A. The expression of a gene encoding lanmodulin (lanM) was induced in response to the presence of La3+. A recombinant LanM underwent conformational change upon La3+ binding. Phenotypic analyses on lanM deletion mutant and overexpressing strains showed that LanM is not necessary for the wild-type and XoxF-dependent mutant’s methylotrophic growth. We found that lanM expression was regulated by MxcQE (a two-component regulator for MxaF) and TonB_Ln (a TonB-dependent receptor for Ln3+). The expression level of mxcQE was altered to be negatively dependent on Ln3+ concentration in ∆lanM, whereas it was constant in the wild type. Furthermore, when exposed to La3+, ∆lanM showed an aggregating phenotype, cell membrane impairment, La deposition in the periplasm evidenced by electron microscopy, differential expression of proteins involved in membrane integrity and phosphate starvation, and possibly lower La content in the membrane vesicle (MV) fractions. Taken together, we concluded that lanmodulin is involved in the complex regulation mechanism of MDHs and homeostasis of cellular Ln levels by facilitating transport and MV-mediated excretion.

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Extracellular vesicles as an alternative copper-secretion mechanism in bacteria

Cited by 20 publications

References 46 publications

Novel protein carrier system based on cyanobacterial nano‐sized extracellular vesicles for application in fish

Novel protein carrier system based on cyanobacterial nano‐sized extracellular vesicles for application in fish

Bacterial Membrane Vesicles as a Novel Strategy for Extrusion of Antimicrobial Bismuth Drug in Helicobacter pylori

A Periplasmic Lanthanide Mediator, Lanmodulin, in Methylobacterium aquaticum Strain 22A

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