Development of an integration mutagenesis system in<i>Lactobacillus gasseri</i>

Selle, Kurt; Goh, Yong Jun; O’Flaherty, Sarah; Klaenhammer, Todd R.

doi:10.4161/gmic.29101

Cited by 10 publications

(10 citation statements)

References 38 publications

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“…3,32) It is widely accepted that the adhesion and colonization of vaccine delivery vectors play a major role in both non-specific and specific immune stimulation. 16) As early as 1975, Fuller et al found that adhesion to chick crop epithelial cells was observed only with chicken-isolated lactobacillus. 33) In this work, we screened LAB inhabitants from the intestines of the Tibetan chickens, and obtained a new L. salivarius TCMM17 strain, which shows strong adhesive property to CIE cells and can survive in the upper GIT of chickens.…”

Section: Discussionmentioning

confidence: 99%

“…In this context, the chromosomal integration system has been developed using nonreplicative plasmid, which can overcome the safety concerns because of its plasmid-free and antibiotic resistance marker-free properties. [15][16][17] In the present study, an adhesive and safe Lactobacillus salivarius TCMM17 strain (this strain had been deposited in China Center of Industrial Culture Collection (CICC)) was isolated from the intestine mucosa of the Tibetan chicken. Then it was genetically modified to express multi-epitope antigens of infectious bronchitis virus (IBV), which was cell-wall anchored.…”

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confidence: 99%

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Characterization of a new Lactobacillus salivarius strain engineered to express IBV multi-epitope antigens by chromosomal integration

Yang

Wang

et al. 2016

Bioscience, Biotechnology, and Biochemistry

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To obtain adhesive and safe lactic acid bacteria (LAB) strains for expressing heterologous antigens, we screened LAB inhabitants in intestine of Tibetan chickens by analyzing their adhesion and safety properties and the selected LAB was engineered to express heterologous antigen (UTEpi C-A) based on chromosomal integration strategy. We demonstrated that a new Lactobacillu salivarius TCMM17 strain is strongly adhesive to chicken intestinal epithelial cells, contains no endogenous plasmids, is susceptible to tested antimicrobials, and shows no toxicities. In order to examine the potential of TCMM17 strain as heterogenous antigen delivering vehicle, we introduced a UTEpi C-A expression cassette in its chromosome by constructing a non-replicative plasmid (pORI280-UUTEpi C-AD). The recombinant TCMM17 strain (∆TCMM17) stably was found to keep the gene cassette through 50 generations, and successfully displayed EpiC encoded by the cassette on its surface. This work provides a universal platform for development of novel oral vaccines and expression of further antigens of avian pathogens.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Characterization of a new Lactobacillus salivarius strain engineered to express IBV multi-epitope antigens by chromosomal integration

Yang

Wang

et al. 2016

Bioscience, Biotechnology, and Biochemistry

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“…The method has been adapted for numerous Slp studies ranging from reporter integration ( Douglas and Klaenhammer, 2011 ), anchoring/adjuvant assessment ( Kajikawa et al, 2011 ), and targeted antigen delivery systems for disease protection ( Kajikawa et al, 2015 ; O’Flaherty and Klaenhammer, 2016 ). Similar counterselective systems have also been developed in non-S-layer-formers including L. gasseri ATCC 33323 ( Selle et al, 2014 ) and L. casei ATCC 393 ( Song et al, 2014 ), but have yet to be harnessed for Slp analyses. In general, the technique remains a superior approach for characterizing the functional genetics of lactobacilli without the additional pressures required for plasmid maintenance.…”

Section: Slp Engineering Platformsmentioning

confidence: 99%

Engineering Components of the Lactobacillus S-Layer for Biotherapeutic Applications

Klotz

Barrangou

2018

Front. Microbiol.

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Lactic acid bacteria (LAB) are frequently harnessed for the delivery of biomolecules to mucosal tissues. Several species of Lactobacillus are commonly employed for this task, of which a subset are known to possess surface-layers (S-layers). S-layers are two-dimensional crystalline arrays of repeating proteinaceous subunits that form the outermost coating of many prokaryotic cell envelopes. Their periodicity and abundance have made them a target for numerous biotechnological applications. In the following review, we examine the multi-faceted S-layer protein (Slp), and its use in both heterologous protein expression systems and mucosal vaccine delivery frameworks, through its diverse genetic components: the strong native promoter, capable of synthesizing as many as 500 Slp subunits per second; the signal peptide that stimulates robust secretion of recombinant proteins; and the structural domains, which can be harnessed for both cell surface display of foreign peptides or adhesion enhancement of a host bacterium. Although numerous studies have established vaccine platforms based on one or more components of the Lactobacillus S-layer, this area of research still remains largely in its infancy, thus this review is meant to not only highlight past works, but also advocate for the future usage of Slps in biotherapeutic research.

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“…An in-frame deletion of the SrtA-encoding gene, srtA, in L. acidophilus NCFM (LBA1244) or L. gasseri ATCC 33323 (LGAS_0825) was constructed using a uppcounterselective gene replacement system (Goh et al, 2009;Selle et al, 2014). Artemis software (Rutherford et al, 2000) was used to visualize the specific chromosomal region associated with each of the srtA genes, and primers (Table 2) were designed to amplify homologous regions of approximately 600 bp upstream and downstream of the deletion target.…”

Section: Methodsmentioning

confidence: 99%

“…Sortase deletion mutants of L. acidophilus NCFM and L. gasseri ATCC 33323 were generated using upp-based counterselection gene replacement systems (Goh et al, 2009;Selle et al, 2014). The DsrtA mutant of each strain was confirmed by PCR and DNA sequencing using primers flanking the srtA deletion target ( Table 2).…”

Section: Construction Of Sortase-deficient Mutantsmentioning

confidence: 99%

Sortase-deficient lactobacilli: effect on immunomodulation and gut retention

et al. 2015

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Surface proteins of probiotic microbes, including Lactobacillus acidophilus and Lactobacillus gasseri, are believed to promote retention in the gut and mediate host-bacterial communications. Sortase, an enzyme that covalently couples a subset of extracellular proteins containing an LPXTG motif to the cell surface, is of particular interest in characterizing bacterial adherence and communication with the mucosal immune system. A sortase gene, srtA, was identified in L. acidophilus NCFM (LBA1244) and L. gasseri ATCC 33323 (LGAS_0825). Additionally, eight and six intact sortase-dependent proteins were predicted in L. acidophilus and L. gasseri, respectively. Due to the role of sortase in coupling these proteins to the cell wall, DsrtA deletion mutants of L. acidophilus and L. gasseri were created using the upp-based counterselective gene replacement system. Inactivation of sortase did not cause significant alteration in growth or survival in simulated gastrointestinal juices. Meanwhile, both DsrtA mutants showed decreased adhesion to porcine mucin in vitro. Murine dendritic cells exposed to the DsrtA mutant of L. acidophilus or L. gasseri induced lower levels of pro-inflammatory cytokines TNF-a and IL-12, respectively, compared with the parent strains. In vivo co-colonization of the L. acidophilus DsrtA mutant and its parent strain in germ-free 129S6/SvEv mice resulted in a significant one-log reduction of the DsrtA mutant population. Additionally, a similar reduction of the DsrtA mutant was observed in the caecum. This study shows for the first time that sortase-dependent proteins contribute to gut retention of probiotic microbes in the gastrointestinal tract.

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Development of an integration mutagenesis system inLactobacillus gasseri

Cited by 10 publications

References 38 publications

Characterization of a new Lactobacillus salivarius strain engineered to express IBV multi-epitope antigens by chromosomal integration

Characterization of a new Lactobacillus salivarius strain engineered to express IBV multi-epitope antigens by chromosomal integration

Engineering Components of the Lactobacillus S-Layer for Biotherapeutic Applications

Sortase-deficient lactobacilli: effect on immunomodulation and gut retention

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