Molecular characterization of both transesterification reactions of the group II intron circularization pathway.

Group II introns can self-splice from RNA transcripts through branching, hydrolysis and circularization, being released as lariats, linear introns and circles, respectively. In contrast to branching, the circularization pathway is mostly based on assumptions and has been largely overlooked. Here, we address the molecular details of both transesterification reactions of the group II intron circularization pathway in vivo. We show that free E1 is recruited by the intron through base pairing interactions and that released intron circles can generate free E1 by the spliced exon reopening reaction. The first transesterification reaction was found to be induced inaccurately by the 3'OH of the terminal residue of free E1 at the 3' splice site, producing circularization intermediates with heterogeneous 3' ends. Nevertheless, specific terminal 3'OH, selected by a molecular ruler, was shown to precisely attack the 5' splice site and release intron circles with 3'-5' rather than 2'-5' bonds at their circularization junction. Our work supports a circularization model where the recruitment of free E1 and/or displacement of cis-E1 induce a conformational change of the intron active site from the pre-5' to the pre-3' splice site processing conformation, suggesting how circularization might initiate at the 3' instead of the 5' splice site.

Group II Introns Generate Functional Chimeric Relaxase Enzymes with Modified Specificities through Exon Shuffling at Both the RNA and DNA Level.

Group II introns are large self-splicing RNA enzymes with a broad but somewhat irregular phylogenetic distribution. These ancient retromobile elements are the proposed ancestors of approximately half the human genome, including the abundant spliceosomal introns and non-long terminal repeat retrotransposons. In contrast to their eukaryotic derivatives, bacterial group II introns have largely been considered as harmful selfish mobile retroelements that parasitize the genome of their host. As a challenge to this view, we recently uncovered a new intergenic trans-splicing pathway that generates an assortment of mRNA chimeras. The ability of group II introns to combine disparate mRNA fragments was proposed to increase the genetic diversity of the bacterial host by shuffling coding sequences. Here, we show that the Ll.LtrB and Ef.PcfG group II introns from Lactococcus lactis and Enterococcus faecalis respectively can both use the intergenic trans-splicing pathway to catalyze the formation of chimeric relaxase mRNAs and functional proteins. We demonstrated that some of these compound relaxase enzymes yield gain-of-function phenotypes, being significantly more efficient than their precursor wild-type enzymes at supporting bacterial conjugation. We also found that relaxase enzymes with shuffled functional domains are produced in biologically relevant settings under natural expression levels. Finally, we uncovered examples of lactococcal chimeric relaxase genes with junctions exactly at the intron insertion site. Overall, our work demonstrates that the genetic diversity generated by group II introns, at the RNA level by intergenic trans-splicing and at the DNA level by recombination, can yield new functional enzymes with shuffled exons, which can lead to gain-of-function phenotypes.

Bacterial group II introns generate genetic diversity by circularization and trans-splicing from a population of intron-invaded mRNAs.

Group II introns are ancient retroelements that significantly shaped the origin and evolution of contemporary eukaryotic genomes. These self-splicing ribozymes share a common ancestor with the telomerase enzyme, the spliceosome machinery as well as the highly abundant spliceosomal introns and non-LTR retroelements. More than half of the human genome thus consists of various elements that evolved from ancient group II introns, which altogether significantly contribute to key functions and genetic diversity in eukaryotes. Similarly, group II intron-related elements in bacteria such as abortive phage infection (Abi) retroelements, diversity generating retroelements (DGRs) and some CRISPR-Cas systems have evolved to confer important functions to their hosts. In sharp contrast, since bacterial group II introns are scarce, irregularly distributed and frequently spread by lateral transfer, they have mainly been considered as selfish retromobile elements with no beneficial function to their host. Here we unveil a new group II intron function that generates genetic diversity at the RNA level in bacterial cells. We demonstrate that Ll.LtrB, the model group II intron from Lactococcus lactis, recognizes specific sequence motifs within cellular mRNAs by base pairing, and invades them by reverse splicing. Subsequent splicing of ectopically inserted Ll.LtrB, through circularization, induces a novel trans-splicing pathway that generates exon 1-mRNA and mRNA-mRNA intergenic chimeras. Our data also show that recognition of upstream alternative circularization sites on intron-interrupted mRNAs release Ll.LtrB circles harboring mRNA fragments of various lengths at their splice junction. Intergenic trans-splicing and alternative circularization both produce novel group II intron splicing products with potential new functions. Overall, this work describes new splicing pathways in bacteria that generate, similarly to the spliceosome in eukaryotes, genetic diversity at the RNA level while providing additional functional and evolutionary links between group II introns, spliceosomal introns and the spliceosome.

Multifunctional Acidocin 4356 Combats Pseudomonas aeruginosa through Membrane Perturbation and Virulence Attenuation: Experimental Results Confirm Molecular Dynamics Simulation.

A longstanding awareness in generating resistance to common antimicrobial therapies by Gram-negative bacteria has made them a major threat to global health. The application of antimicrobial peptides as a therapeutic agent would be a great opportunity to combat bacterial diseases. Here, we introduce a new antimicrobial peptide (∼8.3 kDa) from probiotic strain Lactobacillus acidophilus ATCC 4356, designated acidocin 4356 (ACD). This multifunctional peptide exerts its anti-infective ability against Pseudomonas aeruginosa through an inhibitory action on virulence factors, bacterial killing, and biofilm degradation. Reliable performance over tough physiological conditions and low hemolytic activity confirmed a new hope for the therapeutic setting. Antibacterial kinetic studies using flow cytometry technique showed that the ACD activity is related to the change in permeability of the membrane. The results obtained from molecular dynamic (MD) simulation were perfectly suited to the experimental data of ACD behavior. The structure-function relationship of this natural compound, along with the results of transmission electron microscopy analysis and MD simulation, confirmed the ability of the ACD aimed at enhancing bacterial membrane perturbation. The peptide was effective in the treatment of P. aeruginosa infection in mouse model. The results support the therapeutic potential of ACD for the treatment of Pseudomonas infections.IMPORTANCE Multidrug-resistant bacteria are a major threat to global health, and the Pseudomonas bacterium with the ability to form biofilms is considered one of the main causative agents of nosocomial infections. Traditional antibiotics have failed because of increased resistance. Thus, finding new biocompatible antibacterial drugs is essential. Antimicrobial peptides are produced by various organisms as a natural defense mechanism against pathogens, inspiring the possible design of the next generation of antibiotics. In this study, a new antimicrobial peptide was isolated from Lactobacillus acidophilus ATCC 4356, counteracting both biofilm and planktonic cells of Pseudomonas aeruginosa A detailed investigation was then conducted concerning the functional mechanism of this peptide by using fluorescence techniques, electron microscopy, and in silico methods. The antibacterial and antibiofilm properties of this peptide may be important in the treatment of Pseudomonas infections.

Circularization pathway of a bacterial group II intron.

Group II introns are large RNA enzymes that can excise as lariats, circles or in a linear form through branching, circularization or hydrolysis, respectively. Branching is by far the main and most studied splicing pathway while circularization was mostly overlooked. We previously showed that removal of the branch point A residue from Ll.LtrB, the group II intron from Lactococcus lactis, exclusively leads to circularization. However, the majority of the released intron circles harbored an additional C residue of unknown origin at the splice junction. Here, we exploited the Ll.LtrB-ΔA mutant to study the circularization pathway of bacterial group II introns in vivo. We demonstrated that the non-encoded C residue, present at the intron circle splice junction, corresponds to the first nt of exon 2. Intron circularization intermediates, harboring the first 2 or 3 nts of exon 2, were found to accumulate showing that branch point removal leads to 3' splice site misrecognition. Traces of properly ligated exons were also detected functionally confirming that a small proportion of Ll.LtrB-ΔA circularizes accurately. Overall, our data provide the first detailed molecular analysis of the group II intron circularization pathway and suggests that circularization is a conserved splicing pathway in bacteria.

The Ll.LtrB intron from Lactococcus lactis excises as circles in vivo: insights into the group II intron circularization pathway.

Group II introns are large ribozymes that require the assistance of intron-encoded or free-standing maturases to splice from their pre-mRNAs in vivo. They mainly splice through the classical branching pathway, being released as RNA lariats. However, group II introns can also splice through secondary pathways like hydrolysis and circularization leading to the release of linear and circular introns, respectively. Here, we assessed in vivo splicing of various constructs of the Ll.LtrB group II intron from the Gram-positive bacterium Lactococcus lactis. The study of excised intron junctions revealed, in addition to branched intron lariats, the presence of perfect end-to-end intron circles and alternatively circularized introns. Removal of the branch point A residue prevented Ll.LtrB excision through the branching pathway but did not hinder intron circle formation. Complete intron RNA circles were found associated with the intron-encoded protein LtrA forming nevertheless inactive RNPs. Traces of double-stranded head-to-tail intron DNA junctions were also detected in L. lactis RNA and nucleic acid extracts. Some intron circles and alternatively circularized introns harbored variable number of non-encoded nucleotides at their splice junction. The presence of mRNA fragments at the splice junction of some intron RNA circles provides insights into the group II intron circularization pathway in bacteria.

Recent horizontal transfer, functional adaptation and dissemination of a bacterial group II intron.

BACKGROUND: Group II introns are catalytically active RNA and mobile retroelements present in certain eukaryotic organelles, bacteria and archaea. These ribozymes self-splice from the pre-mRNA of interrupted genes and reinsert within target DNA sequences by retrohoming and retrotransposition. Evolutionary hypotheses place these retromobile elements at the origin of over half the human genome. Nevertheless, the evolution and dissemination of group II introns was found to be quite difficult to infer. RESULTS: We characterized the functional and evolutionary relationship between the model group II intron from Lactococcus lactis, Ll.LtrB, and Ef.PcfG, a newly discovered intron from a clinical strain of Enterococcus faecalis. Ef.PcfG was found to be homologous to Ll.LtrB and to splice and mobilize in its native environment as well as in L. lactis. Interestingly, Ef.PcfG was shown to splice at the same level as Ll.LtrB but to be significantly less efficient to invade the Ll.LtrB recognition site. We also demonstrated that specific point mutations between the IEPs of both introns correspond to functional adaptations which developed in L. lactis as a response to selective pressure on mobility efficiency independently of splicing. The sequence of all the homologous full-length variants of Ll.LtrB were compared and shown to share a conserved pattern of mutation acquisition. CONCLUSIONS: This work shows that Ll.LtrB and Ef.PcfG are homologous and have a common origin resulting from a recent lateral transfer event followed by further adaptation to the new target site and/or host environment. We hypothesize that Ef.PcfG is the ancestor of Ll.LtrB and was initially acquired by L. lactis, most probably by conjugation, via a single event of horizontal transfer. Strong selective pressure on homing site invasion efficiency then led to the emergence of beneficial point mutations in the IEP, enabling the successful establishment and survival of the group II intron in its novel lactococcal environment. The current colonization state of Ll.LtrB in L. lactis was probably later achieved through recurring episodes of conjugation-based horizontal transfer as well as independent intron mobility events. Overall, our data provide the first evidence of functional adaptation of a group II intron upon invading a new host, offering strong experimental support to the theory that bacterial group II introns, in sharp contrast to their organellar counterparts, behave mostly as mobile elements.

Uncoupling of pro- and anti-inflammatory properties of Staphylococcus aureus.

Staphylococcus aureus is a Gram-positive bacterium that is carried by a quarter of the healthy human population and that can cause severe infections. This pathobiosis has been linked to a balance between Toll-like receptor 2 (TLR2)-dependent pro- and anti-inflammatory responses. The relationship between these two types of responses is unknown. Analysis of 16 nasal isolates of S. aureus showed heterogeneity in their capacity to induce pro- and anti-inflammatory responses, suggesting that these two responses are independent of each other. Uncoupling of these responses was corroborated by selective signaling through phosphoinositol 3-kinase (PI3K)-Akt-mTOR and extracellular signal-regulated kinase (ERK) for the anti-inflammatory response and through p38 for the proinflammatory response. Uncoupling was also observed at the level of phagocytosis and phagosomal processing of S. aureus, which were required solely for the proinflammatory response. Importantly, the anti-inflammatory properties of an S. aureus isolate correlated with its ability to modulate T cell immunity. Our results suggest the presence of anti-inflammatory TLR2 ligands in the staphylococcal cell wall, whose identification may provide templates for novel immunomodulatory drugs.

Contribution of base-pairing interactions between group II intron fragments during trans-splicing in vivo.

Group II introns are mobile genetic elements that self-splice from pre-mRNA transcripts. Some fragmented group II introns found in chloroplastic and mitochondrial genomes are able to assemble and splice in trans. The Ll.LtrB group II intron from the Gram-positive bacterium Lactococcus lactis was shown to splice in trans when fragmented at various locations throughout its structure. Here we used Ll.LtrB to assess the contribution of base-pairing interactions between intron fragments during trans-splicing in vivo. By comparing closely located fragmentation sites, we show that Ll.LtrB trans-splices more efficiently when base-pairing interactions can occur between the two intron fragments. Disruptions and stepwise restorations of specific base-pairing interactions between intron fragments resulted respectively in significant reductions and recoveries of the Ll.LtrB trans-splicing efficiency. Finally, although we confirm that LtrA is an important co-factor for trans-splicing, its overexpression cannot compensate for the reduction in trans-splicing efficiency when the potential base-pairing interactions between intron fragments are disrupted. These findings demonstrate the important contribution of base-pairing interactions for the assembly of group II intron fragments during trans-splicing and rationalizes why such interactions were evolutionarily conserved in natural trans-splicing group II introns.

First unraveling of the hidden and intricate evolutionary history of a bacterial group II intron family.

Bacterial group II introns are large RNA enzymes that self-splice from primary transcripts. Following excision, they can invade various DNA target sites using RNA-based mobility pathways. As fast evolving retromobile elements, which move between genetic loci within and across species, their evolutionary history was proved difficult to study and infer. Here we identified several homologs of Ll.LtrB, the model group II intron from Lactococcus lactis, and traced back their evolutionary relationship through phylogenetic analyses. Our data demonstrate that the Ll.LtrB homologs in Lactococci originate from a single and recent lateral transfer event of Ef.PcfG from Enterococcus faecalis. We also show that these introns disseminated in Lactococci following recurrent episodes of independent mobility events in conjunction with occurrences of lateral transfer. Our phylogenies identified additional lateral transfer events from the environmental clade of the more diverged Lactococci introns to a series of low-GC gram-positive bacterial species including E. faecalis. We also determined that functional intron adaptation occurred early in Lactococci following Ef.PcfG acquisition from E. faecalis and that two of the more diverged Ll.LtrB homologs remain proficient mobile elements despite the significant number of mutations acquired. This study describes the first comprehensive evolutionary history of a bacterial group II intron.

Conjugation as a Highly Sensitive Assay to Study Group II Intron Splicing In Vivo.

Group II introns are noncoding sequences that interrupt genes, and that must be removed or spliced-out at the RNA level during gene expression. Following the transcription of interrupted genes, group II introns self-splice while concurrently ligating their flanking exons to generate mature mRNAs ready for translation. Ll.LtrB, the model group II intron from the gram-positive bacterium Lactococcus lactis, interrupts the gene coding for a relaxase enzyme that initiates the transfer of mobile elements by conjugation. This functional link between group II intron splicing and conjugative transfer enabled us to engineer highly sensitive splicing assays using the native biological context of Ll.LtrB. The splicing efficiency/conjugation assay was developed to determine the splicing competence of various Ll.LtrB mutants, whereas the splicing selection/conjugation assay was established to isolate splicing-proficient variants from a randomly generated bank of mutated introns.

Trans-splicing versatility of the Ll.LtrB group II intron.

Group II introns are found in organelles, bacteria, and archaea. Some harbor an open reading frame (ORF) with reverse transcriptase, maturase, and occasionally endonuclease activities. Group II introns require the assistance of either intron-encoded or free-standing maturases to excise from primary RNA transcripts in vivo. Some ORF-containing group II introns were shown to be mobile retroelements that invade new DNA sites by retrohoming or retrotransposition. Group II introns are also hypothesized to be the ancestors of the spliceosome-dependent nuclear introns and the small nuclear RNAs (snRNAs--U1, U2, U4, U5, and U6) that are part of the spliceosome. The ability of some fragmented group II introns to undergo splicing in trans supports the theory that the snRNAs evolved from portions of group II introns. Here, we developed a Tn5-based genetic screen to explore the trans-splicing potential of the Ll.LtrB group II intron from the Gram-positive bacterium Lactococcus lactis. Proficient trans-splicing variants of Ll.LtrB were selected using a highly sensitive trans-splicing/conjugation screen. We report that numerous fragmentation sites located throughout Ll.LtrB support splicing in trans, showing that this intron is remarkably more tolerant to fragmentation than expected from the fragmentation sites uncovered within natural trans-splicing group II introns. This work unveils the great versatility of group II intron fragments to assemble and accurately trans-splice their flanking exons in vivo.

The circle to lariat ratio of the Ll.LtrB group II intron from Lactococcus lactis is greatly influenced by a variety of biological determinants in vivo.

Bacterial group II introns mostly behave as versatile retromobile genetic elements going through distinct cycles of gain and loss. These large RNA molecules are also ribozymes splicing autocatalytically from their interrupted pre-mRNA transcripts by two different concurrent pathways, branching and circularization. These two splicing pathways were shown to release in bacterial cells significant amounts of branched intron lariats and perfect end-to-end intron circles respectively. On one hand, released intron lariats can invade new sites in RNA and/or DNA by reverse branching while released intron circles are dead end spliced products since they cannot reverse splice through circularization. The presence of two parallel and competing group II intron splicing pathways in bacteria led us to investigate the conditions that influence the overall circle to lariat ratio in vivo. Here we unveil that removing a prominent processing site within the Ll.LtrB group II intron, raising growth temperature of Lactococcus lactis host cells and increasing the expression level of the intron-interrupted gene all increased the relative amount of released intron circles compared to lariats. Strengthening and weakening the base pairing interaction between the intron and its upstream exon respectively increased and decreased the overall levels of released intron circles in comparison to lariats. Host environment was also found to impact the circle to lariat ratio of the Ll.LtrB and Ll.RlxA group II introns from L. lactis and the Ef.PcfG intron from Enterococcus faecalis. Overall, our data show that multiple factors significantly influence the balance between released intron circles and lariats in bacterial cells.

Detection of Group II Intron-Generated Chimeric mRNAs in Bacterial Cells.

Chimeric RNAs are the transcripts composed of exons from two separate genes or transcripts. Although the presence of these joined RNA molecules have mainly been documented in a variety of eukaryotes, we recently demonstrated that the Ll.LtrB group II intron, from the gram-positive bacterium Lactococcus lactis, can generate chimeric mRNAs through a novel intergenic trans-splicing pathway. Here we describe the detailed experimental procedures to detect group II intron-generated mRNA-mRNA chimeras from total RNA extracts using stringent reverse transcription conditions along with a reverse splicing-deficient group II intron as a negative control.

Intranasal Application of Lactococcus lactis W136 Is Safe in Chronic Rhinosinusitis Patients With Previous Sinus Surgery.

Objective: Modulation of the dysbiotic gut microbiome with "healthy" bacteria via a stool transplant or supplementation is increasingly practiced, however this approach has not been explored in the nasal passages. We wished to verify whether Lactococcus lactis W136 (L. lactis W136) bacteria could be safely applied via irrigation to the nasal and sinus passages in individuals with chronic rhinosinusitis (CRS) with previous undergone endoscopic sinus surgery, and whether this was accompanied by bacterial community flora modification. Study Design: Prospective open-label pilot trial of safety and feasibility. Setting: Academic tertiary hospital center. Subjects and Methods: Twenty-four patients with CRS refractory to previous medical and surgical therapy received a 14-day course of BID sinus irrigations containing 1.2 × 109 CFU live L. lactis W136. Patients were monitored for safety using questionnaire, sinus endoscopy, otoscopy, UPSIT-40 smell testing, and endoscopically-obtained conventional sinus culture and a swab for 16S microbiome profiling. Results: All 24 patients receiving at least one treatment successfully completed treatment. L. lactis W136 probiotic treatment was safe, with no major adverse events or new infections. Treatment was associated with improvement in sinus symptoms, QOL, and mucosal scores, which remained improved during the subsequent 14-day observation period. Microbiome changes associated with treatment were limited to an increase of the pathobiont Dolosigranulum pigrum, a bacteria identified as potentially beneficial in the upper airways. Subgroup analysis suggested differences in microbiomes and responses for CRSsNP and CRSwNP phenotypes, but these did not attain significance. Conclusion: Intranasal irrigation of live L. lactis W136 bacteria to patients with refractory chronic rhinosinusitis was safe, and was associated with effects on symptoms, mucosal aspect and microbiome composition. Intranasal bacteria may thus find a role as a treatment strategy for CRS. Clinical Trials Registration: www.ClinicalTrials.gov. identifier: NCT04048174.

Trans-splicing of the Ll.LtrB group II intron in Lactococcus lactis.

The Ll.LtrB intron from the Gram-positive bacterium Lactococcus lactis is one of the most studied bacterial group II introns. Ll.LtrB interrupts the relaxase gene of three L. lactis conjugative elements. The relaxase enzyme recognizes the origin of transfer (oriT ) and initiates the intercellular transfer of its conjugative element. The splicing efficiency of Ll.LtrB from the relaxase transcript thus controls the conjugation level of its host element. Here, we used the level of sex factor conjugation as a read-out for Ll.LtrB splicing efficiency. Using this highly sensitive splicing/conjugation assay (10(7)-fold detection range), we demonstrate that Ll.LtrB can trans-splice in L. lactis when fragmented at various positions such as: three different locations within domain IV, within domain I and within domain III. We also demonstrate that the intron-encoded protein, LtrA, is absolutely required for Ll.LtrB trans-splicing. Characteristic Y-branched trans-spliced introns and ligated exons are detected by RT-PCR from total RNA extracts of cells harbouring fragmented Ll.LtrB. The splicing/conjugation assay we developed constitutes the first model system to study group II intron trans-splicing in vivo. Although only previously observed in bacterial-derived organelles, we demonstrate that assembly and trans-splicing of a fragmented group II intron can take place efficiently in bacterial cells.

Conjugative transfer of the Lactococcus lactis sex factor and pRS01 plasmid to Enterococcus faecalis.

The low G+C gram-positive bacterium Lactococcus lactis harbours two highly similar conjugative elements: an integrative and conjugative element called sex factor and the pRS01 plasmid. Originally, it was believed that the host range of the sex factor was limited to L. lactis subspecies. Here, it is reported that pTRK28 cointegrates of a spectinomycin-marked L. lactis sex factor and of the pRS01 conjugative plasmid can be transferred from L. lactis to Enterococcus faecalis. These results demonstrate the conjugative transfer of these elements to other bacterial species. Furthermore, it is reported that Ll.LtrB, a mobile group II intron carried by both elements, can invade its recognition site upon pRS01 conjugative transfer to E. faecalis.

Retropseudogenes derived from the human Ro/SS-A autoantigen-associated hY RNAs.

We report the characterization in the human genome of 966 pseudogenes derived from the four human Y (hY) RNAs, components of the Ro/SS-A autoantigen. About 95% of the Y RNA pseudogenes are found in corresponding locations on the chimpanzee and human chromosomes. On the contrary, Y pseudogenes in mice are both infrequent and found in different genomic regions. In addition to this rodent/primate discrepancy, the conservation of hY pseudogenes relative to hY genes suggests that they occurred after rodent/primate divergence. Flanking regions of hY pseudogenes contain convincing evidence for involvement of the L1 retrotransposition machinery. Although Alu elements are found in close proximity to most hY pseudogenes, these are not chimeric retrogenes. Point mutations in hY RNA transcripts specifically affecting binding of Ro60 protein likely contributed to their selection for direct trans retrotransposition. This represents a novel requirement for the selection of specific RNAs for their genomic integration by the L1 retrotransposition machinery. Over 40% of the hY pseudogenes are found in intronic regions of protein-coding genes. Considering the functions of proteins known to bind subsets of hY RNAs, hY pseudogenes constitute a new class of L1-dependent non-autonomous retroelements, potentially involved in post-transcriptional regulation of gene expression.

Topical probiotics as a therapeutic alternative for chronic rhinosinusitis: A preclinical proof of concept.

INTRODUCTION: Patients with chronic rhinosinusitis (CRS) have been shown to manifest a high inflammatory phenotype, with a sinus microbiome deficient in gram-positive bacteria. Gram-positive bacteria are capable of downregulating proinflammatory host responses via an interleukin (IL) 10 mediated response and may represent a potential therapeutic alternative for CRS. We wanted to (i) immunoprofile the IL-10 induction capacity of two gram-positive probiotic strains and (ii) verify the tolerance of these strains by the sinus epithelium. METHODS: A peripheral blood mononuclear cell (PBMC) challenge model was used to document probiotic induction of IL-10 and tumor necrosis factor (TNF) alpha responses at various bacterial dilutions. Epithelial cell tolerance was demonstrated by using a primary epithelial cell model derived from patient biopsy specimens (six patients total [three with CRS and three controls]). After an incubation period with either a live or a heat-killed probiotic strain, cell viability was assessed by using light microscopy. RESULTS: Both probiotic strains induced high IL-10 secretion in PBMCs, with differing profiles of TNF alpha production. Microscopic evaluation after probiotic incubation demonstrated intact cell viability for all cell cultures. CONCLUSION: We identified well-tolerated, nonpathogenic, "generally recognized as safe" status gram-positive probiotics with anti-inflammatory properties. Topical probiotics represented a potential novel topical therapeutic strategy for CRS relevant for further clinical evaluation.