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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Isolation and characterization of functional tripartite group II introns using a Tn5-based genetic screen.

BACKGROUND: Group II introns are RNA enzymes that splice themselves from pre-mRNA transcripts. Most bacterial group II introns harbour an open reading frame (ORF), coding for a protein with reverse transcriptase, maturase and occasionally DNA binding and endonuclease activities. Some ORF-containing group II introns were shown to be mobile retroelements that invade new DNA target sites. From an evolutionary perspective, group II introns are 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 important functional elements of the spliceosome machinery. The ability of some group II introns fragmented in two or three pieces to assemble and undergo splicing in trans supports the theory that spliceosomal snRNAs evolved from portions of group II introns. METHODOLOGY/PRINCIPAL FINDINGS: We used a transposon-based genetic screen to explore the ability of the Ll.LtrB group II intron from the Gram-positive bacterium Lactococcus lactis to be fragmented into three pieces in vivo. Trans-splicing tripartite variants of Ll.LtrB were selected using a highly efficient and sensitive trans-splicing/conjugation screen. We report that numerous fragmentation sites located throughout Ll.LtrB support tripartite trans-splicing, showing that this intron is remarkably tolerant to fragmentation. CONCLUSIONS/SIGNIFICANCE: This work unveils the great versatility of group II intron fragments to assemble and accurately trans-splice their flanking exons in vivo. The selected introns represent the first evidence of functional tripartite group II introns in bacteria and provide experimental support for the proposed evolutionary relationship between group II introns and snRNAs.

Oral immunization using live Lactococcus lactis co-expressing LACK and IL-12 protects BALB/c mice against Leishmania major infection.

Leishmaniasis is a parasitic disease affecting over 12 million individuals worldwide. Current treatments are laborious, expensive, cause severe side effects, and emerging drug resistance has been reported. While vaccination is the most cost-effective means to control infectious diseases there is no human vaccine currently available against Leishmania infections. Lactococcus lactis is a non-pathogenic, non-colonizing Gram-positive lactic acid bacterium commonly used in the dairy industry. Recently, L. lactis was used for the expression and delivery of biologically active molecules, such as antigens and cytokines, in mice and humans. In this study, we report the generation of L. lactis(alr-) strains solely expressing the protective Leishmania antigen, LACK, in the cytoplasm, secreted or anchored to the bacterial cell wall or co-expressing mouse IL-12. We show that oral immunization using live L. lactis, secreting both LACK and IL-12 was the only regimen that partially protected BALB/c mice against subsequent Leishmania major challenge. This highlights the importance of temporal and physical proximity of the delivered antigen and adjuvant for optimal immune priming by oral immunization since co-administration of L. lactis strains independently expressing secLACK and secIL-12 did not induce protective immunity. Protected animals displayed a delay in footpad swelling, which correlated with a significant reduction of parasite burden. Immunization with the L. lactis strain secreting both LACK and IL-12 induced an antigen-specific mucosal immune response and a LACK-specific T(H)1 immune response in splenocytes and mesenteric lymph node cells. Further, protection in immunized animals correlated with a strong Leishmania-specific T(H)1 immune response post-challenge, detectable in splenocytes and lymph node cells draining the site of infection. This report demonstrates the use of L. lactis as an oral live vaccine against L. major infection in susceptible BALB/c mice. The vaccine strains generated in this study provide the basis for the development of an inexpensive and safe oral live vaccine against the human parasite Leishmania.

Immunization against Leishmania major infection using LACK- and IL-12-expressing Lactococcus lactis induces delay in footpad swelling.

BACKGROUND: Leishmania is a mammalian parasite affecting over 12 million individuals worldwide. Current treatments are expensive, cause severe side effects, and emerging drug resistance has been reported. Vaccination is the most cost-effective means to control infectious disease but currently there is no vaccine available against Leishmaniasis. Lactococcus lactis is a non-pathogenic, non-colonizing Gram-positive lactic acid bacterium commonly used in the dairy industry. Recently, L. lactis was used to express biologically active molecules including vaccine antigens and cytokines. METHODOLOGY/PRINCIPAL FINDINGS: We report the generation of L. lactis strains expressing the protective Leishmania antigen, LACK, in the cytoplasm, secreted or anchored to the bacterial cell wall. L. lactis was also engineered to secrete biologically active single chain mouse IL-12. Subcutaneous immunization with live L. lactis expressing LACK anchored to the cell wall and L. lactis secreting IL-12 significantly delayed footpad swelling in Leishmania major infected BALB/c mice. The delay in footpad swelling correlated with a significant reduction of parasite burden in immunized animals compared to control groups. Immunization with these two L. lactis strains induced antigen-specific multifunctional T(H)1 CD4(+) and CD8(+) T cells and a systemic LACK-specific T(H)1 immune response. Further, protection in immunized animals correlated with a Leishmania-specific T(H)1 immune response post-challenge. L. lactis secreting mouse IL-12 was essential for directing immune responses to LACK towards a protective T(H)1 response. CONCLUSIONS/SIGNIFICANCE: This report demonstrates the use of L. lactis as a live vaccine against L. major infection in BALB/c mice. The strains generated in this study provide the basis for the development of an inexpensive and safe vaccine against the human parasite Leishmania.

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.

Generation and evaluation of A2-expressing Lactococcus lactis live vaccines against Leishmania donovani in BALB/c mice.

Leishmaniasis is a parasitic disease affecting over 12 million individuals worldwide. As current treatments are insufficient, the development of an effective vaccine is a priority. This study generated and assessed the efficacy of Leishmania vaccines engineered from the non-colonizing, non-pathogenic Gram-positive bacterium Lactococcus lactis. A truncated, codon-optimized version of the A2 antigen from Leishmania donovani was engineered for expression in Lactococcus lactis in three different subcellular compartments: in the cytoplasm, secreted outside the cell or anchored to the cell wall. These three A2-expressing Lactococcus lactis strains were tested for their ability to generate A2-specific immune responses and as live vaccines against visceral Leishmania donovani infection in BALB/c mice. Subcutaneous immunization with live Lactococcus lactis expressing A2 anchored to the cell wall effectively induced high levels of antigen-specific serum antibodies. It was demonstrated that Lactococcus lactis-based vaccines are a feasible approach in the generation of live vaccines against leishmaniasis. The Lactococcus lactis strains generated in this study provide an excellent foundation for further studies on live bacterial vaccines against leishmaniasis and other pathogens.