Engineering Components of the Lactobacillus S-Layer for Biotherapeutic Applications

Klotz, Courtney; Barrangou, Rodolphe

doi:10.3389/fmicb.2018.02264

Cited by 21 publications

(5 citation statements)

References 82 publications

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“…Harnessing the endogenous machinery enables efficient genome editing simply by delivering a CRISPR array, together with desired repair templates. The development of such a potent tool has the potential to facilitate the engineering of many valuable bacteria that play critical roles in human health (62,63) and important biological functions in the various habitats Fig. 3.…”

Section: Discussionmentioning

confidence: 99%

Genome editing using the endogenous type I CRISPR-Cas system in Lactobacillus crispatus

Hidalgo-Cantabrana

Goh

Pan

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

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151

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CRISPR-Cas systems are now widely used for genome editing and transcriptional regulation in diverse organisms. The compact and portable nature of class 2 single effector nucleases, such as Cas9 or Cas12, has facilitated directed genome modifications in plants, animals, and microbes. However, most CRISPR-Cas systems belong to the more prevalent class 1 category, which hinges on multiprotein effector complexes. In the present study, we detail how the native type I-E CRISPR-Cas system, with a 5′-AAA-3′ protospacer adjacent motif (PAM) and a 61-nucleotide guide CRISPR RNA (crRNA) can be repurposed for efficient chromosomal targeting and genome editing in Lactobacillus crispatus, an important commensal and beneficial microbe in the vaginal and intestinal tracts. Specifically, we generated diverse mutations encompassing a 643-base pair (bp) deletion (100% efficiency), a stop codon insertion (36%), and a single nucleotide substitution (19%) in the exopolysaccharide priming-glycosyl transferase (p-gtf). Additional genetic targets included a 308-bp deletion (20%) in the prophage DNA packaging Nu1 and a 730-bp insertion of the green fluorescent protein gene downstream of enolase (23%). This approach enables flexible alteration of the formerly genetically recalcitrant species L. crispatus, with potential for probiotic enhancement, biotherapeutic engineering, and mucosal vaccine delivery. These results also provide a framework for repurposing endogenous CRISPR-Cas systems for flexible genome targeting and editing, while expanding the toolbox to include one of the most abundant and diverse systems found in nature.

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Section: Discussionmentioning

confidence: 99%

Genome editing using the endogenous type I CRISPR-Cas system in Lactobacillus crispatus

Hidalgo-Cantabrana

Goh

Pan

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

151

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“…With the exception of the S‐layer proteins whose expression is known to be high and constitutive (Klotz and Barrangou ), the expression of β‐galactosidase and glycolytic enzymes could be affected by changing in the production process parameters and/or media formulation as it was demonstrated for S. thermophilus and Lactococcus lactis (Van den ogaard et al , ; Teusink et al , ).…”

Section: Resultsmentioning

confidence: 99%

Development of omics‐based protocols for the microbiological characterization of multi‐strain formulations marketed as probiotics: the case of VSL#3

Mora

Filardi

Arioli

et al. 2019

Microbial Biotechnology

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Summary The growing commercial interest in multi‐strain formulations marketed as probiotics has not been accompanied by an equal increase in the evaluation of quality levels of these biotechnological products. The multi‐strain product VSL#3 was used as a model to setup a microbiological characterization that could be extended to other formulations with high complexity. Shotgun metagenomics by deep Illumina sequencing was applied to DNA isolated from the commercial VSL#3 product to confirm strains identity safety and composition. Single‐cell analysis was used to evaluate the cell viability, and β‐galactosidase and urease activity have been used as marker to monitor the reproducibility of the production process. Similarly, these lots were characterized in detail by a metaproteomics approach for which a robust protein extraction protocol was combined with advanced mass spectrometry. The results identified over 1600 protein groups belonging to all strains present in the VSL#3 formulation. Of interest, only 3.2 % proteins showed significant differences mainly related to small variations in strain abundance. The protocols developed in this study addressed several quality criteria that are relevant for marketed multi‐strain products and these represent the first efforts to define the quality of complex probiotic formulations such as VSL#3.

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“…bulgaricus have no S-layer proteins ( Hynonen and Palva 2013 ). Similarly, Streptococcus thermophilus also does not express S-layer proteins ( Klotz and Barrangou 2018 ). These reports lead us to speculate that the ligand which binds to rMGL1 is not an S-layer protein.…”

Section: Discussionmentioning

confidence: 99%

Intestinal lamina propria macrophages upregulate interleukin-10 mRNA in response to signals from commensal bacteria recognized by MGL1/CD301a

et al. 2021

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Ligand-induced cellular signaling involved in interleukin 10 (IL-10) production by lamina propria macrophages (LPMs) during their interactions with commensal bacteria is not clearly understood. We previously showed, using mice lacking a C-type lectin MGL1/CD301a, that this molecule on colonic LPMs plays an important role in the induction of IL-10 upon interaction with commensal bacteria, Streptococcus sp. In the present report we show that the physical engagement of MGL1/CD301a on LPMs with in-situ isolated Streptococcus sp. bacteria leads to IL-10 mRNA induction. Spleen tyrosine kinase (Syk), caspase recruitment domain 9 (CARD9), and extracellular signal-regulated kinase (ERK), but not NF-κB pathway, are shown to be indispensable for IL-10 mRNA induction after stimulation with heat-killed Streptococcus sp. Guanidine hydrochloride treatment of Streptococcus sp., which is known to extract bacterial cell surface glycan-rich components, abolished bacterial binding to recombinant MGL1/CD301a. The extract contained materials which bound rMGL1 in ELISA and appeared to induce IL-10 mRNA expression in LPMs in vitro. Lectin blotting showed that the extract contained glycoproteins which are considered as putative ligands for MGL1. Some human commensal Lactobacillus species also induced IL-10 mRNA expression by colonic LPMs in vitro, which depends on the presence of MGL1/CD301a and CARD9. The present results are the first to show that MGL1/CD301a acts as a signal transducer during colonic host–microbe interactions.

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Engineering Components of the Lactobacillus S-Layer for Biotherapeutic Applications

Cited by 21 publications

References 82 publications

Genome editing using the endogenous type I CRISPR-Cas system in Lactobacillus crispatus

Genome editing using the endogenous type I CRISPR-Cas system in Lactobacillus crispatus

Development of omics‐based protocols for the microbiological characterization of multi‐strain formulations marketed as probiotics: the case of VSL#3

Intestinal lamina propria macrophages upregulate interleukin-10 mRNA in response to signals from commensal bacteria recognized by MGL1/CD301a

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