Mucosal adjuvanticity and mucosal booster effect of colibactin-depleted probiotic Escherichia coli membrane vesicles.

There is a growing interest in development of novel vaccines against respiratory tract infections, due to COVID-19 pandemic. Here, we examined mucosal adjuvanticity and the mucosal booster effect of membrane vesicles (MVs) of a novel probiotic E. coli derivative lacking both flagella and potentially carcinogenic colibactin (ΔflhDΔclbP). ΔflhDΔclbP-derived MVs showed rather strong mucosal adjuvanticity as compared to those of a single flagellar mutant strain (ΔflhD-MVs). In addition, glycoengineered ΔflhDΔclbP-MVs displaying serotype-14 pneumococcal capsular polysaccharide (CPS14+MVs) were well-characterized based on biological and physicochemical parameters. Subcutaneous (SC) and intranasal (IN) booster effects of CPS14+MVs on systemic and mucosal immunity were evaluated in mice that have already been subcutaneously prime-immunized with the same MVs. With a two-dose regimen, an IN boost (SC-IN) elicited stronger IgA responses than homologous prime-boost immunization (SC-SC). With a three-dose regimen, serum IgG levels were comparable among all tested regimens. Homologous immunization (SC-SC-SC) elicited the highest IgM responses among all regimens tested, whereas SC-SC-SC failed to elicit IgA responses in blood and saliva. Furthermore, serum IgA and salivary SIgA levels were increased with an increased number of IN doses administrated. Notably, SC-IN-IN induced not only robust IgG response, but also the highest IgA response in both serum and saliva among the groups. The present findings suggest the potential of a heterologous three-dose administration for building both systemic and mucosal immunity, e.g. an SC-IN-IN vaccine regimen could be beneficial. Another important observation was abundant packaging of colibactin in MVs, suggesting increased applicability of ΔflhDΔclbP-MVs in the context of vaccine safety.

Identification of CgeA as a glycoprotein that anchors polysaccharides to the spore surface in Bacillus subtilis.

The Bacillus subtilis spore is composed of a core, containing chromosomal DNA, surrounded by a cortex layer made of peptidoglycan, and a coat composed of concentric proteinaceous layers. A polysaccharide layer is added to the spore surface, and likely anchored to the crust, the coat outermost layer. However, the identity of the coat protein(s) to which the spore polysaccharides (SPS) are attached is uncertain. First, we showed that the crust proteins CotVWXYZ and CgeA were all contained in the peeled SPS layer obtained from a strain missing CotE, the outer coat morphogenetic protein, suggesting that the SPS is indeed bound to at least one of the spore surface proteins. Second, CgeA is known to be located at the most downstream position in the crust assembly pathway. An analysis of truncated variants of CgeA suggested that its N-terminal half is required for localization to the spore surface, while its C-terminal half is necessary for SPS addition. Third, an amino acid substitution strategy revealed that SPS was anchored at threonine 112 (T112), which constitutes a probable O-glycosylation site on CgeA. Our results indicated that CgeA is a glycoprotein required to initiate SPS assembly and serves as an anchor protein linking the crust and SPS layers.

Visualization and characterization of spore morphogenesis in Paenibacillus polymyxa ATCC39564.

Paenibacillus polymyxa is a spore-forming Gram-positive bacterial species. Both its sporulation process and the spore properties are poorly understood. Here, we investigated sporulation in P. polymyxa ATCC39564. When cultured at 37℃ for 24 h in sporulation medium, more than 80% of the total cells in the culture were spores. Time-lapse imaging revealed that cellular morphological changes during sporulation of P. polymyxa were highly similar to those of B. subtilis. We demonstrated that genetic deletion of spo0A, sigE, sigF, sigG, or sigK, which are highly conserved transcriptional regulators in spore forming bacteria, abolished spore formation. In P. polymyxa, spo0A was required for cell growth in sporulation medium, as well as for the initiation of sporulation. The sigE and sigF mutants formed abnormal multiple asymmetric septa during the early stage of sporulation. The sigG and sigK mutants formed forespores in the sporangium, but they did not become mature. Moreover, fluorescence reporter analysis confirmed compartment-specific gene expression of spoIID and spoVFA in the mother cell and spoIIQ and sspF in the forespore. Transmission electron microscopy imaging revealed that P. polymyxa produces multilayered endospores but lacking a balloon-shaped exosporium. Our results indicate that spore morphogenesis is conserved between P. polymyxa and B. subtilis. However, P. polymyxa genomes lack many homologues encoding spore-coat proteins that are found in B. subtills, suggesting that there are differences in the spore coat composition and surface structure between P. polymyxa and B. subtilis.

Membrane Vesicles Derived From Clostridium botulinum and Related Clostridial Species Induce Innate Immune Responses via MyD88/TRIF Signaling in vitro.

Clostridium botulinum produces botulinum neurotoxin complexes that cause botulism. Previous studies elucidated the molecular pathogenesis of botulinum neurotoxin complexes; however, it currently remains unclear whether other components of the bacterium affect host cells. Recent studies provided insights into the role of bacterial membrane vesicles (MVs) produced by some bacterial species in host immunity and pathology. We herein examined and compared the cellular effects of MVs isolated from four strains of C. botulinum with those of closely related Clostridium sporogenes and two strains of the symbiont Clostridium scindens. MVs derived from all strains induced inflammatory cytokine expression in intestinal epithelial and macrophage cell lines. Cytokine expression was dependent on myeloid differentiation primary response (MyD) 88 and TIR-domain-containing adapter-inducing interferon-ß (TRIF), essential adaptors for toll-like receptors (TLRs), and TLR1/2/4. The inhibition of actin polymerization impeded the uptake of MVs in RAW264.7 cells, however, did not reduce the induction of cytokine expression. On the other hand, the inhibition of dynamin or phosphatidylinositol-3 kinase (PI3K) suppressed the induction of cytokine expression by MVs, suggesting the importance of these factors downstream of TLR signaling. MVs also induced expression of Reg3 family antimicrobial peptides via MyD88/TRIF signaling in primary cultured mouse small intestinal epithelial cells (IECs). The present results indicate that MVs from C. botulinum and related clostridial species induce host innate immune responses.

Autolysis-mediated membrane vesicle formation in Bacillus subtilis.

It is known that Bacillus subtilis releases membrane vesicles (MVs) during the SOS response, which is associated with cell lysis triggered by the PBSX prophage-encoded cell-lytic enzymes XhlAB and XlyA. In this study, we demonstrate that MVs are released under various stress conditions: sucrose fatty acid ester (SFE; surfactant) treatment, cold shock, starvation, and oxygen deficiency. B. subtilis possesses four major host-encoded cell wall-lytic enzymes (autolysins; LytC, LytD, LytE, and LytF). Deletions of the autolysin genes abolished autolysis and the consequent MV production under these stress conditions. In contrast, deletions of xhlAB and xlyA had no effect on autolysis-triggered MV biogenesis, indicating that autolysis is a novel and prophage-independent pathway for MV production in B. subtilis. Moreover, we found that the cell lysis induced by the surfactant treatment was effectively neutralized by the addition of exogenous purified MVs. This result suggests that the MVs can serve as a decoy for the cellular membrane to protect the living cells in the culture from membrane damage by the surfactant. Our results indicate a positive effect of B. subtilis MVs on cell viability and provide new insight into the biological importance of the autolysis phenomenon in B. subtilis.

Extreme C-terminal element of SprA serine integrase is a potential component of the "molecular toggle switch" which controls the recombination and its directionality.

In Bacillus subtilis, a sporulation-related gene, spsM, is disrupted by SPß prophage, but reconstituted during sporulation through SPß excision. The spsM reconstitution is catalyzed by a site-specific DNA recombinase, SprA, and its cognate recombination directionality factor, SprB. SprB interacts with SprA, directing the SprA-mediated recombination reaction from integration to excision; however, the details of the directionality control remains unclear. Here, we demonstrate the importance of the extreme C-terminal region (ECT) of SprA in the DNA recombination and directionality control. We created a series of SprA C-terminal deletants and examined their DNA-binding and recombination activities. Deletions in the ECT caused a loss of integration and excision activity, the magnitudes of which positively correlated with the deletion size. Gel shift study revealed that the loss of the integration activity was attributable to the failure of synaptic complex formation. The excision deficiency was caused by defective interaction with SprB. Moreover, alanine scanning analysis revealed that Phe532 is essential to interact with SprB. SprAF532A , therefore, showed almost no excision activity, while retaining the integration activity. Collectively, these results suggest that the ECT plays the crucial roles in the interaction of SprA with SprB and possibly in the directional control of the recombination.

Biofilms: hot spots of horizontal gene transfer (HGT) in aquatic environments, with a focus on a new HGT mechanism.

Biofilms in water environments are thought to be hot spots for horizontal gene transfer (HGT) of antibiotic resistance genes (ARGs). ARGs can be spread via HGT, though mechanisms are known and have been shown to depend on the environment, bacterial communities and mobile genetic elements. Classically, HGT mechanisms include conjugation, transformation and transduction; more recently, membrane vesicles (MVs) have been reported as DNA reservoirs implicated in interspecies HGT. Here, we review the current knowledge on the HGT mechanisms with a focus on the role of MVs and the methodological innovations in the HGT research.

Compatibility of Site-Specific Recombination Units between Mobile Genetic Elements.

Site-specific recombination (SSR) systems are employed for transfer of mobile genetic elements (MGEs), such as lysogenic phages and integrative conjugative elements (ICEs). SSR between attP/I and attB sites is mediated by an integrase (Int) and a recombination directionality factor (RDF). The genome of Bacillus subtilis 168 contains SPß, an active prophage, skin, a defective prophage, and ICEBs1, an integrative conjugative element. Each of these MGEs harbors the classic SSR unit attL-int-rdf-attR. Here, we demonstrate that these SSR units are all compatible and can substitute for one another. Specifically, when SPß is turned into a defective prophage by deletion of its SSR unit, introduction of the SSR unit of skin or ICE converts it back to an active prophage. We also identified closely related prophages with distinct SSR units that control developmentally regulated gene rearrangements of kamA (L-lysine 2,3-aminomutase). These results suggest that SSR units are interchangeable components of MGEs.

Expansion of the Spore Surface Polysaccharide Layer in Bacillus subtilis by Deletion of Genes Encoding Glycosyltransferases and Glucose Modification Enzymes.

Polysaccharides (PS) decorate the surface of dormant endospores (spores). In the model organism for sporulation, Bacillus subtilis, the composition of the spore PS is not known in detail. Here, we have assessed how PS synthesis enzymes produced during the late stages of sporulation affect spore surface properties. Using four methods, bacterial adhesion to hydrocarbons (BATH) assays, India ink staining, transmission electron microscopy (TEM) with ruthenium red staining, and scanning electron microscopy (SEM), we characterized the contributions of four sporulation gene clusters, spsABCDEFGHIJKL, yfnHGF-yfnED, ytdA-ytcABC, and cgeAB-cgeCDE, on the morphology and properties of the crust, the outermost spore layer. Our results show that all mutations in the sps operon result in the production of spores that are more hydrophobic and lack a visible crust, presumably because of reduced PS deposition, while mutations in cgeD and the yfnH-D cluster noticeably expand the PS layer. In addition, yfnH-D mutant spores exhibit a crust with an unusual weblike morphology. The hydrophobic phenotype from sps mutant spores was partially rescued by a second mutation inactivating any gene in the yfnHGF operon. While spsI, yfnH, and ytdA are paralogous genes, all encoding glucose-1-phosphate nucleotidyltransferases, each paralog appears to contribute in a distinct manner to the spore PS. Our data are consistent with the possibility that each gene cluster is responsible for the production of its own respective deoxyhexose. In summary, we found that disruptions to the PS layer modify spore surface hydrophobicity and that there are multiple saccharide synthesis pathways involved in spore surface properties.IMPORTANCE Many bacteria are characterized by their ability to form highly resistant spores. The dormant spore state allows these species to survive even the harshest treatments with antimicrobial agents. Spore surface properties are particularly relevant because they influence spore dispersal in various habitats from natural to human-made environments. The spore surface in Bacillus subtilis (crust) is composed of a combination of proteins and polysaccharides. By inactivating the enzymes responsible for the synthesis of spore polysaccharides, we can assess how spore surface properties such as hydrophobicity are modulated by the addition of specific carbohydrates. Our findings indicate that several sporulation gene clusters are responsible for the assembly and allocation of surface polysaccharides. Similar mechanisms could be modulating the dispersal of infectious spore-forming bacteria.

Bacillus subtilis YlxR, Which Is Involved in Glucose-Responsive Metabolic Changes, Regulates Expression of tsaD for Protein Quality Control of Pyruvate Dehydrogenase.

Glucose is the most favorable carbon source for many bacteria, which have several glucose-responsive gene networks. Recently, we found that in Bacillus subtilis glucose induces the expression of the extracellular sigma factor genes sigX and sigM through the acetylation of CshA (RNA helicase), which associates with RNA polymerase (RNAP). We performed a transposon mutagenesis screen for mutants with no glucose induction (GI) of sigX-lacZ. While screening for such mutants, we recently found that the GI of sigX/M involves YlxR, a nucleoid-associated protein (NAP) that regulates nearly 400 genes, including metabolic genes. It has been shown that acetylated CshA positively regulates expression of ylxR-containing operon. Here, we report additional mutations in yqfO or tsaD required for the GI of sigX. YqfO contains a universally conserved domain with unknown function. YqfO and YlxR were found to regulate expression of the tsaEBD-containing operon. Mutational analysis using lacZ fusions revealed the adenine-rich cis-element for YlxR. TsaD is a component of the TsaEBD enzyme required for the synthesis of threonylcarbamoyl adenosine (t6A). The t6A modification of tRNA is universal across the three domains of life. Western blot analysis showed that the tsaD mutation in the presence of glucose reduced levels of soluble PdhA, PdhB, and PdhD, which are subunits of the pyruvate dehydrogenase complex (PDHc). This resulted in severely defective PDHc function and thus reduced concentrations of cellular acetyl-CoA, a reaction product of PDHc and plausible source for CshA acetylation. Thus, we discuss a suggested glucose-responsive system (GRS) involving self-reinforcing CshA acetylation. This self-reinforcing pathway may contribute to the maintenance of the acetyl-CoA pool for protein acetylation.

Contributions of crust proteins to spore surface properties in Bacillus subtilis.

Surface properties, such as adhesion and hydrophobicity, constrain dispersal of bacterial spores in the environment. In Bacillus subtilis, these properties are influenced by the outermost layer of the spore, the crust. Previous work has shown that two clusters, cotVWXYZ and cgeAB, encode the protein components of the crust. Here, we characterize the respective roles of these genes in surface properties using Bacterial Adherence to Hydrocarbons assays, negative staining of polysaccharides by India ink and Transmission Electron Microscopy. We showed that inactivation of crust genes caused increases in spore relative hydrophobicity, disrupted the spore polysaccharide layer, and impaired crust structure and attachment to the rest of the coat. We also found that cotO, previously identified for its role in outer coat formation, is necessary for proper encasement of the spore by the crust. In parallel, we conducted fluorescence microscopy experiments to determine the full network of genetic dependencies for subcellular localization of crust proteins. We determined that CotZ is required for the localization of most crust proteins, while CgeA is at the bottom of the genetic interaction hierarchy.

A novel non prophage(-like) gene-intervening element within gerE that is reconstituted during sporulation in Bacillus cereus ATCC10987.

Gene rearrangement is a widely-shared phenomenon in spore forming bacteria, in which prophage(-like) elements interrupting sporulation-specific genes are excised from the host genome to reconstitute the intact gene. Here, we report a novel class of gene-intervening elements, named gin, inserted in the 225 bp gerE-coding region of the B. cereus ATCC10987 genome, which generates a sporulation-specific rearrangement. gin has no phage-related genes and possesses three site-specific recombinase genes; girA, girB, and girC. We demonstrated that the gerE rearrangement occurs at the middle stage of sporulation, in which site-specific DNA recombination took place within the 9 bp consensus sequence flanking the disrupted gerE segments. Deletion analysis of gin uncovered that GirC and an additional factor, GirX, are responsible for gerE reconstitution. Involvement of GirC and GirX in DNA recombination was confirmed by an in vitro recombination assay. These results broaden the definition of the sporulation-specific gene rearrangement phenomenon: gene-intervening elements are not limited to phage DNA but may include non-viral genetic elements that carry a developmentally-regulated site-specific recombination system.

Mechanism of bacterial gene rearrangement: SprA-catalyzed precise DNA recombination and its directionality control by SprB ensure the gene rearrangement and stable expression of spsM during sporulation in Bacillus subtilis.

A sporulation-specific gene, spsM, is disrupted by an active prophage, SPß, in the genome of Bacillus subtilis. SPß excision is required for two critical steps: the onset of the phage lytic cycle and the reconstitution of the spsM-coding frame during sporulation. Our in vitro study demonstrated that SprA, a serine-type integrase, catalyzed integration and excision reactions between attP of SPß and attB within spsM, while SprB, a recombination directionality factor, was necessary only for the excision between attL and attR in the SPß lysogenic chromosome. DNA recombination occurred at the center of the short inverted repeat motif in the unique conserved 16 bp sequence among the att sites (5΄-ACAGATAA/AGCTGTAT-3΄; slash, breakpoint; underlines, inverted repeat), where SprA produced the 3΄-overhanging AA and TT dinucleotides for rejoining the DNA ends through base-pairing. Electrophoretic mobility shift assay showed that SprB promoted synapsis of SprA subunits bound to the two target sites during excision but impaired it during integration. In vivo data demonstrated that sprB expression that lasts until the late stage of sporulation is crucial for stable expression of reconstituted spsM without reintegration of the SPß prophage. These results present a deeper understanding of the mechanism of the prophage-mediated bacterial gene regulatory system.

Developmentally-regulated excision of the SPß prophage reconstitutes a gene required for spore envelope maturation in Bacillus subtilis.

Temperate phages infect bacteria by injecting their DNA into bacterial cells, where it becomes incorporated into the host genome as a prophage. In the genome of Bacillus subtilis 168, an active prophage, SPß, is inserted into a polysaccharide synthesis gene, spsM. Here, we show that a rearrangement occurs during sporulation to reconstitute a functional composite spsM gene by precise excision of SPß from the chromosome. SPß excision requires a putative site-specific recombinase, SprA, and an accessory protein, SprB. A minimized SPß, where all the SPß genes were deleted, except sprA and sprB, retained the SPß excision activity during sporulation, demonstrating that sprA and sprB are necessary and sufficient for the excision. While expression of sprA was observed during vegetative growth, sprB was induced during sporulation and upon mitomycin C treatment, which triggers the phage lytic cycle. We also demonstrated that overexpression of sprB (but not of sprA) resulted in SPß prophage excision without triggering the lytic cycle. These results suggest that sprB is the factor that controls the timing of phage excision. Furthermore, we provide evidence that spsM is essential for the addition of polysaccharides to the spore envelope. The presence of polysaccharides on the spore surface renders the spore hydrophilic in water. This property may be beneficial in allowing spores to disperse in natural environments via water flow. A similar rearrangement occurs in Bacillus amyloliquefaciens FZB42, where a SPß-like element is excised during sporulation to reconstitute a polysaccharide synthesis gene, suggesting that this type of gene rearrangement is common in spore-forming bacteria because it can be spread by phage infection.

Regulated DNA rearrangement during sporulation in Bacillus weihenstephanensis†KBAB4.

Temperate phages can integrate their genomes into a specific region of a host chromosome to produce lysogens (prophage). During genome insertion, prophages may interrupt the gene coding sequence. In Bacillus subtilis, the sigma factor gene sigK is interrupted by a 48 kb prophage-like element. sigK is a composite coding sequence from two partial genes during sporulation. For over two decades, however, no further examples of DNA element-mediated gene reconstitution other than sigK have been identified in spore formers. Here we report that the gene for dipicolinic acid (DPA) synthetase ß subunit spoVFB in B. weihenstephanensis KBAB4 is interrupted by a prophage-like element named vfbin. DPA is synthesized in the mother cell and required for maintaining spore dormancy. We found that spoVFB was a composite coding sequence generated in the mother cell via chromosomal rearrangement that excised vfbin. Furthermore, vfbin caused excision after phage-inducer treatment, but vfbin appeared to be defective as a prophage. We also found various spore-forming bacteria in which sporulation-related genes were disrupted by prophage-like DNA elements. These results demonstrate the first example of a similar mechanism that affects a sporulation gene other than sigK and suggest that this prophage-mediated DNA rearrangement is a common phenomenon in spore-forming bacteria.

Effects of depletion of RNA-binding protein Tex on the expression of toxin genes in Clostridium perfringens.

Tex was originally identified in Bordetella pertussis, where it serves as a transcriptional regulator of toxin genes. However, the Tex of Streptococcus pneumoniae has no regulatory function in the expression of the pneumococcal major toxin pneumolysin. Here, we identified the CPE2168 gene as Tex in Clostridium perfringens, and examined the roles of Tex in toxin gene expression. We found that the deletion mutant for Tex does not affect growth, but the mRNA levels of three hyaluronidase genes (nagH, nagJ, and nagL) and an exo-sialidase (nanJ) were reduced to less than 50% as compared to the parent strain, C. perfringens strain 13. On the other hand, Tex did not affect the expression of proteases, enterotoxins, hemolysins, either of two hyaluronidase genes (nagI and nagK), an exo-sialidase (nanI), or adhesins. Moreover, purified Tex bound to the 5'-portion of target gene mRNAs. Based on these results, we propose that Tex positively regulates the gene expression of a set of toxin genes in C. perfringens.

Stabilization of Clostridium perfringens collagenase mRNA by VR-RNA-dependent cleavage in 5' leader sequence.

The small RNA (sRNA), VR-RNA that is directly regulated by the VirR/VirS two-component system, regulates many genes including toxin genes such as collagenase (colA) and phospholipase C (plc) in Clostridium perfringens. Although the VR-RNA 3' region is sufficient to regulate the colA and plc genes, the molecular mechanism of toxin gene regulation by VR-RNA remains unclear. Here, we found that colA mRNA is cleaved at position -79 and -78 from the A of the first codon (ATG) in the presence of VR-RNA. The processed transcripts were stable compared with longer intact transcripts. On the other hand, colA mRNA was labile in a VR-RNA-deficient strain, and processed transcripts were undetectable. The stability and processing of colA mRNA were restored by transformation of the 3' region of VR-RNA-expression vector. The 3' region of VR-RNA and colA mRNA had significant complementation and interacted in vitro. These results show that VR-RNA base pairs with colA mRNA and induces cleavage in the 5' untranslated region (UTR) of colA mRNA, which leads to the stabilization of colA mRNA and the activation of colA expression.