Improved Method for Transformation of <i>Vibrio vulnificus</i> by Electroporation

Jayakumar, Jane M.; Shapiro, Orr H.; Almagro‐Moreno, Salvador

doi:10.1002/cpmc.106

Cited by 4 publications

(1 citation statement)

References 25 publications

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“…The construct was introduced into V. vulnificus ENV1 using electroporation ( 58 ). Electrocompetent cells were produced by washing mid-log-phase ENV1 cells with 400 mM sucrose at RT.…”

Section: Methodsmentioning

confidence: 99%

Loss of the Acetate Switch in Vibrio vulnificus Enhances Predation Defense against Tetrahymena pyriformis

Regina

Noorian

Sim

et al. 2022

Appl Environ Microbiol

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Vibrio vulnificus is an opportunistic human pathogen and autochthonous inhabitant of coastal marine environments, where the bacterium is under constant predation by heterotrophic protists or protozoans. As a result of this selection pressure, genetic variants with anti-predation mechanisms are selected for and persist in the environment. Such natural variants may also be pathogenic to animal or human hosts, making it important to understand these defence mechanisms. To identify anti-predator strategies, thirteen V. vulnificus strains of different genotypes isolated from diverse environments were exposed to predation by the ciliated protozoan, Tetrahymena pyriformis , and only strain ENV1 was resistant to predation. Further investigation of the cell-free supernatant showed that ENV1 acidifies the environment by the excretion of organic acids, which is toxic to T. pyriformis . As this predation resistance was dependent on the availability of iron, transcriptomes of V. vulnificus in iron-replete and iron-deplete conditions were compared. This analysis revealed that ENV1 ferments pyruvate and the resultant acetyl-CoA leads to acetate synthesis under aerobic conditions, a hallmark of overflow metabolism. The anaerobic respiration global regulator, arcA , was upregulated when iron was available. An Δ arcA deletion mutant of ENV1 accumulated less acetate and importantly, was sensitive to grazing by T. pyriformis . Based on the transcriptome response and quantification of metabolites, we conclude that ENV1 has adapted to overflow metabolism and has lost a control switch that shifts metabolism from acetate excretion to acetate assimilation, enabling it to excrete acetate continuously. We show that overflow metabolism and the acetate switch contribute to prey-predator interactions. Importance Bacteria in the environment, including Vibrio spp., interact with protozoan predators. To defend against predation, bacteria evolve anti-predator mechanisms ranging from changing morphology, biofilm formation and secretion of toxins or virulence factors. Some of these adaptations may result in strains that are pathogenic to humans. Therefore, it is important to study predator defence strategies of environmental bacteria. V. vulnificus thrives in coastal waters and infects humans. Very little is know about the defence mechanisms V. vulnificus expresses against predation. Here we show that a V. vulnificus strain (ENV1) has rewired the central carbon metabolism enabling the production of excess organic acid that is toxic to the protozoan predator, T. pyriformis . This is a previously unknown mechanism of predation defence that protects against protozoan predators.

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“…The construct was introduced into V. vulnificus ENV1 using electroporation ( 58 ). Electrocompetent cells were produced by washing mid-log-phase ENV1 cells with 400 mM sucrose at RT.…”

Section: Methodsmentioning

confidence: 99%

Loss of the Acetate Switch in Vibrio vulnificus Enhances Predation Defense against Tetrahymena pyriformis

Regina

Noorian

Sim

et al. 2022

Appl Environ Microbiol

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Current approaches to genetic modification of marine bacteria and considerations for improved transformation efficiency

Christi,

Hudson,

Egan

2024

Microbiological Research

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The role of MNK1-mTORC1 pathway in modulating macrophage responses to Vibrio vulnificus infection

Lou,

Xie,

Huang

et al. 2024

Microbiol Spectr

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Vibrio vulnificus ( Vv ) is known to cause life-threatening infections, particularly septicemia. These patients often exhibit elevated levels of pro-inflammatory cytokines. While it is established that mitogen-activated protein kinase (MAPK)-interacting kinase (MNK) contributes to the production of pro-inflammatory cytokines, the role of MNK in macrophages during Vv infection remains unclear. In this study, we investigate the impact of MNK on macrophages. We demonstrate that the inhibition of MNK in J774A.1 cells, when treated with lipopolysaccharide or Vv , resulted in decreased production of tumor necrosis factor alpha and interleukin-6, without affecting their transcription. Interestingly, treatment with MNK inhibitor CGP57380 led to enhanced phosphorylation of MNK1 but decreased phosphorylation of eIF4E. Moreover, MNK1 knockout cells exhibited an increased capacity for phagocytosis and clearance of Vv , with more acidic phagosomes than the parental cells. Notably, CGP57380 did not impact phagocytosis, bacterial clearance, or phagosome acidification in Vv -infected J774A.1 cells. Considering the reported association between MNK and mammalian target of rapamycin complex 1 (mTORC1) activation, we investigated the mTORC1 signaling in MNK1 knockout cells infected with Vv . Our results revealed that attenuation of the mTORC1 signaling in these cells and treatment with the mTORC1 inhibitor rapamycin significantly enhanced bacterial clearance in J774A.1 cells following Vv infection. In summary, our findings suggest that MNK promotes the Vv -induced cytokine production in J774A.1 cells without affecting their transcription levels. MNK1 appears to impair the phagocytosis, bacterial clearance, and phagosome acidification in Vv -infected J774A.1 cells through the MNK1-mTORC1 signaling pathway rather than the MNK1-eIF4E signaling pathway. Our findings highlight the importance of the MNK1-mTORC1 pathway in modulating macrophage responses to Vv infection. IMPORTANCE Mitogen-activated protein kinase (MAPK)-interacting kinase (MNK) plays a role in promoting the production of tumor necrosis factor alpha and interleukin-6 in macrophages during Vibrio vulnificus ( Vv ) infection. Inhibition or knockout of MNK1 in J774A.1 cells resulted in reduced cytokine production without affecting their transcription levels. MNK1 also impairs phagocytosis, bacterial clearance, and phagosome acidification in Vv -infected cells through the MNK1-mammalian target of rapamycin complex 1 (mTORC1) signaling pathway. The findings highlight the importance of the MNK1-mTORC1 pathway in modulating macrophage responses to Vv infection.

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Improved Method for Transformation of Vibrio vulnificus by Electroporation

Cited by 4 publications

References 25 publications

Loss of the Acetate Switch in Vibrio vulnificus Enhances Predation Defense against Tetrahymena pyriformis

Loss of the Acetate Switch in Vibrio vulnificus Enhances Predation Defense against Tetrahymena pyriformis

Current approaches to genetic modification of marine bacteria and considerations for improved transformation efficiency

The role of MNK1-mTORC1 pathway in modulating macrophage responses to Vibrio vulnificus infection

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