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Identification of efficient prokaryotic cell-penetrating peptides with applications in bacterial biotechnology.

ABSTRACT: In bacterial biotechnology, instead of producing functional proteins from plasmids, it is often necessary to deliver functional proteins directly into live cells for genetic manipulation or physiological modification. We constructed a library of cell-penetrating peptides (CPPs) capable of delivering protein cargo into bacteria and developed an efficient delivery method for CPP-conjugated proteins. We screened the library for highly efficient CPPs with no significant cytotoxicity in Escherichia coli and developed a model for predicting the penetration efficiency of a query peptide, enabling the design of new and efficient CPPs. As a proof-of-concept, we used the CPPs for plasmid curing in E. coli and marker gene excision in Methylomonas sp. DH-1. In summary, we demonstrated the utility of CPPs in bacterial engineering. The use of CPPs would facilitate bacterial biotechnology such as genetic engineering, synthetic biology, metabolic engineering, and physiology studies.

SUBMITTER: Lee HM

PROVIDER: S-EPMC7884711 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

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Identification of efficient prokaryotic cell-penetrating peptides with applications in bacterial biotechnology.

Lee Hyang-Mi HM Ren Jun J Tran Kha Mong KM Jeon Byeong-Min BM Park Won-Ung WU Kim Hyunjoo H Lee Kyung Eun KE Oh Yuna Y Choi Myungback M Kim Dae-Sung DS Na Dokyun D

Communications biology 20210215 1

In bacterial biotechnology, instead of producing functional proteins from plasmids, it is often necessary to deliver functional proteins directly into live cells for genetic manipulation or physiological modification. We constructed a library of cell-penetrating peptides (CPPs) capable of delivering protein cargo into bacteria and developed an efficient delivery method for CPP-conjugated proteins. We screened the library for highly efficient CPPs with no significant cytotoxicity in Escherichia c ...[more]

PMID: 33589718

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Project description:Since their identification over twenty-five years ago, the plethora of cell-penetrating peptides (CPP) and their applications has skyrocketed. These 5 to 30 amino acid in length peptides have the unique property of breaching the cell membrane barrier while carrying cargoes larger than themselves into cells in an intact, functional form. CPPs can be conjugated to fluorophores, activatable probes, radioisotopes or contrast agents for imaging tissues, such as tumors. There is no singular mechanism for translocation of CPPs into a cell, and therefore, many CPPs are taken up by a multitude of cell types, creating the challenge of tumor-specific translocation and hindering clinical effectiveness. Varying strategies have been developed to combat this issue and enhance their diagnostic potential by derivatizing CPPs for better targeting by constructing specific cell-activated forms. These methods are currently being used to image integrin-expressing tumors, breast cancer cells, human histiocytic lymphoma and protease-secreting fibrosarcoma cells, to name a few. Additionally, identifying safe, effective therapeutics for malignant tumors has long been an active area of research. CPPs can circumvent many of the complications found in treating cancer with conventional therapeutics by targeted delivery of drugs into tumors, thereby decreasing off-target side effects, a feat not achievable by currently employed conventional chemotherapeutics. Myriad types of chemotherapeutics such as tyrosine kinase inhibitors, antitumor antibodies and nanoparticles can be functionally attached to these peptides, leading to the possibility of delivering established and novel cancer therapeutics directly to tumor tissue. While much research is needed to overcome potential issues with these peptides, they offer a significant advancement over current mechanisms to treat cancer. In this review, we present a brief overview of the research, leading to identification of CPPs with a comprehensive state-of-the-art review on the role of these novel peptides in both cancer diagnostics as well as therapeutics.

Project description:Salmonella infection poses a critical challenge to global public health, and the situation is exacerbated by the increasing prevalence of antibiotic resistance. Bacteriophages (phages) are increasingly being used as antimicrobial agents due to their ability to kill specific bacteria. However, the low cellular uptake of phages has limited their use in treating intracellular bacterial infections. Here, we present a study using engineered phages with cell-penetrating peptides (CPPs) for enhancing the internalization efficiency of phages to inhibit bacterial intracellular infections. Through bioinformatic analysis, we identified a phage-encoded protein harboring an immunoglobulin-like (Ig-like) domain as the potential target for phage display. Using a CRISPR-Cas9-based method, we successfully displayed short peptides on GP94, an Ig-like domain-containing protein, of Salmonella phage selz. We improved phage intracellular uptake in multiple cell types by fusion of various CPPs to GP94. Notably, the phage selzHA-TAT showed promising results in enhancing the intracellular inhibition of Salmonella in different cells. Our research provides a straightforward strategy for displaying CPPs on non-model phages, offering a promising novel and effective therapeutic approach for treating intracellular and drug-resistant bacteria. IMPORTANCE Salmonella infection is a significant threat to global public health, and the increasing prevalence of antibiotic resistance exacerbates the situation. Therefore, finding new and effective ways to combat this pathogen is essential. Phages are natural predators of bacteria and can be used as an alternative to antibiotics to kill specific bacteria, including drug-resistant strains. One significant limitation of using phages as antimicrobial agents is their low cellular uptake, which limits their effectiveness against intracellular bacterial infections. Therefore, finding ways to enhance phage uptake is crucial. Our study provides a straightforward strategy for displaying cell-penetrating peptides on non-model phages, offering a promising novel and effective therapeutic approach for treating intracellular and drug-resistant bacteria. This approach has the potential to address the global challenge of antibiotic resistance and improve public health outcomes.

Dataset Information

Identification of efficient prokaryotic cell-penetrating peptides with applications in bacterial biotechnology.

Publications

Identification of efficient prokaryotic cell-penetrating peptides with applications in bacterial biotechnology.

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