Engineering Components of the Lactobacillus S-Layer for Biotherapeutic Applications

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

Mariage pour tous dans la presse : itinéraire d’une nomination (2012-2013)

Les apports théoriques de l’analyse du discours praxématique sont au fondement de ce travail qui s’interroge sur les différentes manières de nommer le mariage des couples de personnes de même sexe dans la presse à partir de l’annonce du projet présidentiel en janvier 2012 jusqu’à l’adoption de la loi au Sénat au mois d’avril 2013. L’étude se propose d’analyser le surgissement de la nomination mariage pour tous au sein des tensions mettant aux prises des désignants tels que mariage homosexuel, mariage homo, mariage gay, etc.Theoretical contributions from discourse analysis and praxématique constitute the foundations of this work. Its objective is to question the different ways of naming same sex marriages in the press, from the announcement of the President’s introduction of the bill to its approval by the Senate in April 2013. This study intends to analyze the emergence of the nomination mariage pour tous in the midst of the tensions that opposed designations such as mariage homosexuel, mariage homo, mariage gay, etc.Los aportes teóricos del análisis del discurso y de la praxemática son la base de este trabajo que investiga las diferentes maneras de nombrar el matrimonio de parejas de personas del mismo sexo en la prensa francesa a partir del anuncio del proyecto presidencial del socialismo en enero de 2012 hasta la adopción de la ley en el Senado en abril de 2013. Este estudio se propone analizar el surgimiento de la nominación mariage pour tous en medio de las tensiones que oponen designaciones como mariage homosexuel, mariage homo, mariage gay, etc

Determination of Factors Driving the Genome Editing Field in the CRISPR Era Using Bibliometrics.

Over the past two decades, the discovery of CRISPR-Cas immune systems and the repurposing of their effector nucleases as biotechnological tools have revolutionized genome editing. The corresponding work has been captured by 90,000 authors representing 7,600 affiliations in 126 countries, who have published more than 19,000 papers spanning medicine, agriculture, and biotechnology. Here, we use tech mining and an integrated bibliometric and networks framework to investigate the CRISPR literature over three time periods. The analysis identified seminal papers, leading authors, influential journals, and rising applications and topics interconnected through collaborative networks. A core set of foundational topics gave rise to diverging avenues of research and applications, reflecting a bona fide disruptive emerging technology. This analysis illustrates how bibliometrics can identify key factors, decipher rising trends, and untangle emerging applications and technologies that dynamically shape a morphing field, and provides insights into the trajectory of genome editing

Recombination between phages and CRISPR-cas loci facilitates horizontal gene transfer in staphylococci

This is the author accepted manuscript. The final version is available from Nature Research via the DOI in this record.CRISPR (clustered regularly interspaced short palindromic repeats) loci and their associated (cas) genes encode an adaptive immune system that protects prokaryotes from viral1 and plasmid2 invaders. Following viral (phage) infection, a small fraction of the prokaryotic cells are able to integrate a small sequence of the invader's genome into the CRISPR array1. These sequences, known as spacers, are transcribed and processed into small CRISPR RNA guides3-5 that associate with Cas nucleases to specify a viral target for destruction6-9. Although CRISPR-cas loci are widely distributed throughout microbial genomes and often display hallmarks of horizontal gene transfer10-12, the drivers of CRISPR dissemination remain unclear. Here, we show that spacers can recombine with phage target sequences to mediate a form of specialized transduction of CRISPR elements. Phage targets in phage 85, ΦNM1, ΦNM4 and Φ12 can recombine with spacers in either chromosomal or plasmid-borne CRISPR loci in Staphylococcus, leading to either the transfer of CRISPR-adjacent genes or the propagation of acquired immunity to other bacteria in the population, respectively. Our data demonstrate that spacer sequences not only specify the targets of Cas nucleases but also can promote horizontal gene transfer.Natural Environment Research Council (NERC)Biotechnology & Biological Sciences Research Council (BBSRC)Rita Allen Scholars ProgramNational Institutes of Health (NIH

Occurrence and diversity of CRISPR-Cas systems in the genus bifidobacterium

CRISPR-Cas systems constitute adaptive immune systems for antiviral defense in bacteria. We investigated the occurrence and diversity of CRISPR-Cas systems in 48 Bifidobacterium genomes to gain insights into the diversity and co-evolution of CRISPR-Cas systems within the genus and investigate CRISPR spacer content. We identified the elements necessary for the successful targeting and inference of foreign DNA in select Type II CRISPRCas systems, including the tracrRNA and target PAM sequence. Bifidobacterium species have a very high frequency of CRISPR-Cas occurrence (77%, 37 of 48). We found that many Bifidobacterium species have unusually large and diverse CRISPR-Cas systems that contain spacer sequences showing homology to foreign genetic elements like prophages. A large number of CRISPR spacers in bifidobacteria show perfect homology to prophage sequences harbored in the chromosomes of other species of Bifidobacterium, including some spacers that self-target the chromosome. A correlation was observed between strains that lacked CRISPR-Cas systems and the number of times prophages in that chromosome were targeted by other CRISPR spacers. The presence of prophage-targeting CRISPR spacers and prophage content may shed light on evolutionary processes and strain divergence. Finally, elements of Type II CRISPR-Cas systems, including the tracrRNA and crRNAs, set the stage for the development of genome editing and genetic engineering tools. © 2015 Briner et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.This study was supported by startup funds from North Carolina State University. The authors thank GenProbio srl for financial support of the Laboratory of Probiogenomics. DvS is a member of the Alimentary pharmabiotic Centre funded by Science Foundation Ireland (SFI), through the Irish Government’s National Development Plan (Grant number SFI/12/RC/2273). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer Reviewe

Conserved Genome Organization and Core Transcriptome of the Lactobacillus acidophilus Complex

The Lactobacillus genus encompasses a genetically and functionally diverse group of species, and contains many strains widely formulated in the human food supply chain as probiotics and starter cultures. Within this genetically expansive group, there are several distinct clades that have high levels of homology, one of which is the Lactobacillus acidophilus group. Of the uniting features, small genomes, low GC content, adaptation to dairy environments, and fastidious growth requirements, are some of the most defining characteristics of this group. To better understand what truly links and defines this clade, we sought to characterize the genomic organization and content of the genomes of several members of this group. Through core genome analysis we explored the synteny and intrinsic genetic underpinnings of the L. acidophilus clade, and observed key features related to the evolution and adaptation of these organisms. While genetic content is able to provide a large map of the potential of each organism, it does not always reflect their functionality. Through transcriptomic data we inferred the core transcriptome of the L. acidophilus complex to better define the true metabolic capabilities that unite this clade. Using this approach we have identified seven small ORFs that are both highly conserved and transcribed in diverse members of this clade and could be potential novel small peptide or untranslated RNA regulators. Overall, our results reveal the core features of the L. acidophilus complex and open new avenues for the enhancement and formulation and of next generation probiotics and starter cultures

Functional and comparative genomic analyses of an operon involved in fructooligosaccharide utilization by \u3ci\u3eLactobacillus acidophilus\u3c/i\u3e

Lactobacillus acidophilus is a probiotic organism that displays the ability to use prebiotic compounds such as fructooligosaccharides (FOS), which stimulate the growth of beneficial commensals in the gastrointestinal tract. However, little is known about the mechanisms and genes involved in FOS utilization by Lactobacillus species. Analysis of the L. acidophilus NCFM genome revealed an msm locus composed of a transcriptional regulator of the LacI family, a four-component ATP-binding cassette (ABC) transport system, a fructosidase, and a sucrose phosphorylase. Transcriptional analysis of this operon demonstrated that gene expression was induced by sucrose and FOS but not by glucose or fructose, suggesting some specificity for nonreadily fermentable sugars. Additionally, expression was repressed by glucose but not by fructose, suggesting catabolite repression via two cre-like sequences identified in the promoter–operator region. Insertional inactivation of the genes encoding the ABC transporter substrate-binding protein and the fructosidase reduced the ability of the mutants to grow on FOS. Comparative analysis of gene architecture within this cluster revealed a high degree of synteny with operons in Streptococcus mutans and Streptococcus pneumoniae. However, the association between a fructosidase and an ABC transporter is unusual and may be specific to L. acidophilus. This is a description of a previously undescribed gene locus involved in transport and catabolism of FOS compounds, which can promote competition of beneficial microorganisms in the human gastrointestinal tract

The S-layer Associated Serine Protease Homolog PrtX Impacts Cell Surface-Mediated Microbe-Host Interactions of Lactobacillus acidophilus NCFM

Health-promoting aspects attributed to probiotic microorganisms, including adhesion to intestinal epithelia and modulation of the host mucosal immune system, are mediated by proteins found on the bacterial cell surface. Notably, certain probiotic and commensal bacteria contain a surface (S-) layer as the outermost stratum of the cell wall. S-layers are non-covalently bound semi-porous, crystalline arrays of self-assembling, proteinaceous subunits called S-layer proteins (SLPs). Recent evidence has shown that multiple proteins are non-covalently co-localized within the S-layer, designated S-layer associated proteins (SLAPs). In Lactobacillus acidophilus NCFM, SLP and SLAPs have been implicated in both mucosal immunomodulation and adhesion to the host intestinal epithelium. In this study, a S-layer associated serine protease homolog, PrtX (prtX, lba1578), was deleted from the chromosome of L. acidophilus NCFM. Compared to the parent strain, the PrtX-deficient strain (ΔprtX) demonstrated increased autoaggregation, an altered cellular morphology, and pleiotropic increases in adhesion to mucin and fibronectin, in vitro. Furthermore, ΔprtX demonstrated increased in vitro immune stimulation of IL-6, IL-12, and IL-10 compared to wild-type, when exposed to mouse dendritic cells. Finally, in vivo colonization of germ-free mice with ΔprtX led to an increase in epithelial barrier integrity. The absence of PrtX within the exoproteome of a ΔprtX strain caused morphological changes, resulting in a pleiotropic increase of the organisms’ immunomodulatory properties and interactions with some intestinal epithelial cell components