Role of UDP-N-acetylmuramic acid in the regulation of MurA activity revealed by molecular dynamics simulations.

The peptidoglycan biosynthesis pathway plays a vital role in bacterial cells, and facilitates peptidoglycan layer formation, a fundamental structural component of the bacterial cell wall. The enzymes in this pathway are candidates for antibiotic development, as most do not have mammalian homologues. The UDP-N-acetylglucosamine (UNAG) enolpyruvyl transferase enzyme (MurA) in the peptidoglycan pathway cytoplasmic step is responsible for the phosphoenolpyruvate (PEP)-UNAG catalytic reaction, forming UNAG enolpyruvate and inorganic phosphate. Reportedly, UDP-N-acetylmuramic acid (UNAM) binds tightly to MurA forming a dormant UNAM-PEP-MurA complex and acting as a MurA feedback inhibitor. MurA inhibitors are complex, owing to competitive binding interactions with PEP, UNAM, and UNAG at the MurA active site. We used computational methods to explore UNAM and UNAG binding. UNAM showed stronger hydrogen-bond interactions with the Arg120 and Arg91 residues, which help to stabilize the closed conformation of MurA, than UNAG. Binding free energy calculations using end-point computational methods showed that UNAM has a higher binding affinity than UNAG, when PEP is attached to Cys115. The unbinding process, simulated using τ-random acceleration molecular dynamics, showed that UNAM has a longer relative residence time than UNAG, which is related to several complex dissociation pathways, each with multiple intermediate metastable states. This prevents the loop from opening and exposing the Arg120 residue to accommodate UNAG and potential new ligands. Moreover, we demonstrate the importance of Cys115-linked PEP in closed-state loop stabilization. We provide a basis for evaluating novel UNAM analogues as potential MurA inhibitors. PUBLIC SIGNIFICANCE: MurA is a critical enzyme involved in bacterial cell wall biosynthesis and is involved in antibiotic resistance development. UNAM can remain in the target protein's active site for an extended time compared to its natural substrate, UNAG. The prolonged interaction of this highly stable complex known as the 'dormant complex' comprises UNAM-PEP-MurA and offers insights into antibiotic development, providing potential options against drug-resistant bacteria and advancing our understanding of microbial biology.

Lacticaseibacillus rhamnosus CRL1505 Peptidoglycan Modulates the Inflammation-Coagulation Response Triggered by Poly(I:C) in the Respiratory Tract.

Lacticaseibacillus rhamnosus CRL1505 beneficially modulates the inflammation-coagulation response during respiratory viral infections. This study evaluated the capacity of the peptidoglycan obtained from the CRL1505 strain (PG-Lr1505) to modulate the immuno-coagulative response triggered by the viral pathogen-associated molecular pattern poly(I:C) in the respiratory tract. Adult BALB/c mice were nasally treated with PG-Lr1505 for two days. Treated and untreated control mice were then nasally challenged with poly(I:C). Mice received three doses of poly(I:C) with a 24 h rest period between each administration. The immuno-coagulative response was studied after the last administration of poly(I:C). The challenge with poly(I:C) significantly increased blood and respiratory pro-inflammatory mediators, decreased prothrombin activity (PT), and increased von Willebrand factor (vWF) levels in plasma. Furthermore, tissue factor (TF), tissue factor pathway inhibitor (TFPI), and thrombomodulin (TM) expressions were increased in the lungs. PG-Lr1505-treated mice showed significant modulation of hemostatic parameters in plasma (PT in %, Control = 71.3 ± 3.8, PG-Lr1505 = 94.0 ± 4.0, p < 0.01) and lungs. Moreover, PG-Lr1505-treated mice demonstrated reduced TF in F4/80 cells from lungs, higher pro-inflammatory mediators, and increased IL-10 compared to poly(I:C) control mice (IL-10 in pg/mL, Control = 379.1 ± 12.1, PG-Lr1505 = 483.9 ± 11.3, p < 0.0001). These changes induced by PG-Lr1505 correlated with a significant reduction in lung tissue damage. Complementary in vitro studies using Raw 264.7 cells confirmed the beneficial effect of PG-Lr1505 on poly(I:C)-induced inflammation, since increased IL-10 expression, as well as reduced damage, production of inflammatory mediators, and hemostatic parameter expressions were observed. In addition, protease-activated receptor-1 (PAR1) activation in lungs and Raw 264.7 cells was observed after TLR3 stimulation, which was differentially modulated by PG-Lr1505. The peptidoglycan from L. rhamnosus CRL1505 is able to regulate inflammation, the procoagulant state, and PAR1 activation in mice and macrophages in the context of the activation of TLR3 signaling pathways, contributing to a beneficial modulation of inflammation-hemostasis crosstalk.

Arthrobacter vasquezii sp. nov., isolated from a soil sample from Union Glacier, Antarctica.

A Gram-stain-positive, catalase-positive, non-motile bacteria, with a rod-coccus cycle (designated as EH-1B-1T) was isolated from a soil sample from Union Glacier in Ellsworth Mountains, Antarctica. Strain EH-1B-1T had an optimal growth temperature of 28 °C and grew at pH 7-10. The major cellular fatty acids were anteiso-C15 : 0, iso-C15 : 0, C16 : 0 and anteiso-C17 : 0. The G+C content based on the whole genome sequence was 63.1 mol%. Strain EH-1B-1T was most closely related to members of the genus Arthrobacter, namely Arthrobacter subterraneus and Arthrobacter tumbae. The strain grew on tryptic soy agar, Reasoner's 2A agar, lysogeny broth agar and nutrient agar. The average nucleotide identity and digital DNA-DNA hybridization values between strain EH-1B-1T and its closest reference type strains ranged from 78 to 88 % and from 20.9 to 36.3 %, respectively. Based on phenotypic, chemotypic and genotypic evidence, it is proposed that strain EH-1B-1T represents a novel species of Arthrobacter, for which the name Arthrobacter vasquezii sp. nov. is proposed, with strain EH-1B-1T (RGM 3386T=LMG 32961T) as the type strain.

Architecture and genomic arrangement of the MurE-MurF bacterial cell wall biosynthesis complex.

Peptidoglycan (PG) is a central component of the bacterial cell wall, and the disruption of its biosynthetic pathway has been a successful antibacterial strategy for decades. PG biosynthesis is initiated in the cytoplasm through sequential reactions catalyzed by Mur enzymes that have been suggested to associate into a multimembered complex. This idea is supported by the observation that in many eubacteria, mur genes are present in a single operon within the well conserved dcw cluster, and in some cases, pairs of mur genes are fused to encode a single, chimeric polypeptide. We performed a vast genomic analysis using >140 bacterial genomes and mapped Mur chimeras in numerous phyla, with Proteobacteria carrying the highest number. MurE-MurF, the most prevalent chimera, exists in forms that are either directly associated or separated by a linker. The crystal structure of the MurE-MurF chimera from Bordetella pertussis reveals a head-to-tail, elongated architecture supported by an interconnecting hydrophobic patch that stabilizes the positions of the two proteins. Fluorescence polarization assays reveal that MurE-MurF interacts with other Mur ligases via its central domains with KDs in the high nanomolar range, backing the existence of a Mur complex in the cytoplasm. These data support the idea of stronger evolutionary constraints on gene order when encoded proteins are intended for association, establish a link between Mur ligase interaction, complex assembly and genome evolution, and shed light on regulatory mechanisms of protein expression and stability in pathways of critical importance for bacterial survival.

Identification and characterization of a novel lytic peptidoglycan transglycosylase (MltC) in Shigella dysenteriae.

Shigellosis remains a worldwide health problem due to the lack of vaccines and the emergence of antibiotic-resistant strains. Shigella (S.) dysenteriae has rigid peptidoglycan (PG), and its tight regulation of biosynthesis and remodeling is essential for bacterial integrity. Lytic transglycosylases are highly conserved PG autolysins in bacteria that play essential roles in bacterial growth. However, their precise functions are obscure. We aimed to identify, clone, and express MltC, a unique autolysin in Escherichia (E.) coli C41 strain. The purification of recombinant MltC protein was performed using affinity chromatography and size-exclusion chromatography methods. The PG enzymatic activity of MltC was investigated using Zymogram and Fluorescein isothiocyanate (FITC)-labeled PG assays. Also, we aimed to detect its localization in bacterial fractions (cytoplasm and membrane) by western blot using specific polyclonal anti-MltC antibodies and its probable partners using immunoprecipitation and mass spectrometry applications. Purified MltC showed autolysin activity. Native MltC showed various locations in S. dysenteriae cells during different growth phases. In the Lag and early stationary phases, MltC was not found in cytoplasm and membrane fractions. However, it was detected in cytoplasm and membrane fractions during the exponential phase. In the late stationary phase, MltC was expressed in the membrane fraction only. Different candidate protein partners of MltC were identified that could be essential for bacterial growth and pathogenicity. This is the first study to suggest that MltC is indeed autolysin and could be a new drug target for the treatment of shigellosis by understanding its biological functions.

DNA aptamers selection for Staphylococcus aureus cells by SELEX and Cell-SELEX.

BACKGROUND: Staphylococcus aureus is the most common bacteria found in skin, soft tissues, bone, and bone prostheses infections. The aim of this study was to select DNA aptamers for S. aureus to be applied in the diagnosis of bacteria. METHODS AND RESULTS: We used SELEX (Systematic Evolution of Ligands by EXponencial Enrichment) for peptidoglycan followed by cell-SELEX with S. aureus cells as target. Four sequences showed significantly higher binding to S. aureus distinguishing it from the control cells of other significant microbial species: Escherichia coli, Candida albicans, Streptococcus pyogenes and Streptococcus pneumoniae. In particular, ApSA1 (Kd = 62.7 ± 5.6 nM) and ApSA3 (Kd = 43.3 ± 3.0 nM) sequences combined high affinity and specificity for S. aureus, considering all microorganisms tested. CONCLUSIONS: Our results demonstrated that these aptamers were able to identify peptidoglycan in the S. aureus surface and have great potential for use in the development of radiopharmaceuticals capable to identify S. aureus infectious foci, as well as in other aptamer-based methodologies for bacteria diagnosis.

MES SV40 Cells Are Sensitive to Lipopolysaccharide, Peptidoglycan, and Poly I:C Expressing IL-36 Cytokines.

Mesangial cells (MC) maintain the architecture and cellular communication and indirectly join in the glomerular filtration rate for the correct functioning of the glomerulus. Consequently, these cells are activated constantly in response to changes in the intraglomerular environment due to a metabolic imbalance or infection. IL-36, a member of the IL-1 family, is a cytokine that initiates and maintains inflammation in different tissues in acute and chronic pathologies, including the skin, lungs, and intestines. In the kidney, IL-36 has been described in the development of tubulointerstitial lesions, the production of an inflammatory environment, and is associated with metabolic and mesangioproliferative disorders. The participation of IL-36 in functional dysregulation and the consequent generation of the inflammatory environment by MCs in the presence of microbial stimulation is not yet elucidated. In this work, the MES SV40 cell cultures were stimulated with classical pathogen-associated molecular patterns (PAMPs), mimicking an infection by negative and positive bacteria as well as a viral infection. Lipopolysaccharide (LPS), peptidoglycan (PGN) microbial wall components, and a viral mimic poly I:C were used, and the mRNA and protein expression of the IL-36 members were assessed. We observed a differential and dose-dependent IL-36 mRNA and protein expression under LPS, PGN, and poly I:C stimulation. IL-36ß was only found when the cells were treated with LPS, while IL-36α and IL-36γ were favored by PGN and poly I:C stimulation. We suggest that the microbial components participate in the activation of MCs, leading them to the production of IL-36, in which a specific member may participate in the origin and maintenance of inflammation in the glomerular environment that is associated with infections.

Valproic acid restricts mast cell activation by Listeria monocytogenes.

Mast cells (MC) play a central role in the early containment of bacterial infections, such as that caused by Listeria monocytogenes (L.m). The mechanisms of MC activation induced by L.m infection are well known, so it is possible to evaluate whether they are susceptible to targeting and modulation by different drugs. Recent evidence indicates that valproic acid (VPA) inhibits the immune response which favors L.m pathogenesis in vivo. Herein, we examined the immunomodulatory effect of VPA on L.m-mediated MC activation. To this end, bone marrow-derived mast cells (BMMC) were pre-incubated with VPA and then stimulated with L.m. We found that VPA reduced MC degranulation and cytokine release induced by L.m. MC activation during L.m infection relies on Toll-Like Receptor 2 (TLR2) engagement, however VPA treatment did not affect MC TLR2 cell surface expression. Moreover, VPA was able to decrease MC activation by the classic TLR2 ligands, peptidoglycan and lipopeptide Pam3CSK4. VPA also reduced cytokine production in response to Listeriolysin O (LLO), which activates MC by a TLR2-independent mechanism. In addition, VPA decreased the activation of critical events on MC signaling cascades, such as the increase on intracellular Ca2+ and phosphorylation of p38, ERK1/2 and -p65 subunit of NF-κB. Altogether, our data demonstrate that VPA affects key cell signaling events that regulate MC activation following L.m infection. These results indicate that VPA can modulate the functional activity of different immune cells that participate in the control of L.m infection.

Characterization of Secondary Structure and Thermal Stability by Biophysical Methods of the D-alanyl,D-alanine Ligase B Protein from Escherichia coli.

BACKGROUND: Peptidoglycan (PG) is a key structural component of the bacterial cell wall and interruption of its biosynthesis is a validated target for antimicrobials. Of the enzymes involved in PG biosynthesis, D-alanyl,D-alanine ligase B (DdlB) is responsible for the condensation of two alanines, forming D-Ala-D-Ala, which is required for subsequent extracellular transpeptidase crosslinking of the mature peptidoglycan polymer. OBJECTIVE: We aimed at the biophysical characterization of recombinant Escherichia coli DdlB (EcDdlB), considering parameters of melting temperature (Tm), calorimetry and Van't Hoff enthalpy changes of denaturation ( ΔHUcal and ΔHUvH ), as well as characterization of elements of secondary structure at three different pHs. METHODS: DdlB was overexpressed in E. coli BL21 and purified by affinity chromatography. Thermal stability and structural characteristics of the purified enzyme were analyzed by circular dichroism (CD), differential scanning calorimetry and fluorescence spectroscopy. RESULTS: The stability of EcDdlB increased with proximity to its pI of 5.0, reaching the maximum at pH 5.4 with Tm and ΔHUvH U of 52.68 ºC and 484 kJ.mol-1, respectively. Deconvolutions of the CD spectra at 20 ºC showed a majority percentage of α-helix at pH 5.4 and 9.4, whereas for pH 7.4, an equal contribution of ß-structures and α-helices was calculated. Thermal denaturation process of EcDdlB proved to be irreversible with an increase in ß-structures that can contribute to the formation of protein aggregates. CONCLUSION: Such results will be useful for energy minimization of structural models aimed at virtual screening simulations, providing useful information in the search for drugs that inhibit peptidoglycan synthesis.

Biochemical characterization of LysVpKK5 endolysin from a marine vibriophage.

Endolysins have been proposed as a potential antibacterial alternative for aquaculture, especially against Vibrio; the bacterial-agents that most frequently cause disease. Although multiple marine vibriophages have been characterized to date, research on vibriophage endolysins is recent. In this study, biochemical characterization of LysVpKK5 endolysin encoded by Vibrio parahaemolyticus-infecting VpKK5 phage was performed. In silico analysis revealed that LysVpKK5 possesses a conserved amidase_2 domain with a zinc-binding motif of high structural similarity to T7 lysozyme (RMSD = 0.107 Å). Contrary to expectations, the activity was inhibited with Zn2+ and was improved with other divalent cations, especially Ca2+. It showed optimal muralytic activity at pH 10, and curiously, no lytic activity at pH ≤ 7 was recorded. As for the thermal stability test, the optimal activity was recorded at 30 °C; the higher residual activity was recorded at 4 °C, and was lost at ≥ 50 °C. On the other hand, increasing NaCl concentrations reduced the activity gradually; the optimal activity was recorded at 50 mM NaCl. On the other hand, the enzymatic activity at 0.5 M NaCl was approx 30% and of approx 50% in seawater. LysVpKK5 endolysin exhibited a higher activity on V. parahaemolyticus ATCC-17802 strain, in comparison with AHPND + strains.

MagC is a NplC/P60-like member of the α-2-macroglobulin Mag complex of Pseudomonas aeruginosa that interacts with peptidoglycan.

Bacterial α-2 macroglobulins (A2Ms) structurally resemble the large spectrum protease inhibitors of the eukaryotic immune system. In Pseudomonas aeruginosa, MagD acts as an A2M and is expressed within a six-gene operon encoding the MagA-F proteins. In this work, we employ isothermal calorimetry (ITC), analytical ultracentrifugation (AUC), and X-ray crystallography to investigate the function of MagC and show that MagC associates with the macroglobulin complex and with the peptidoglycan (PG). However, the catalytic residues of MagC display an inactive conformation that could suggest that it binds to PG but does not degrade it. We hypothesize that MagC could serve as an anchor between the MagD macroglobulin and the PG and could provide stabilization and/or regulation for the entire complex.

Peptidoglycans modulating the interaction of a bactericide compound with lipids at the air-water interface.

A known monoterpene, named γ-terpineol, was incorporated in mixed Langmuir monolayers composed of dipalmitoyl-phosphoethanolamine (DPPE) and peptidoglycans as a model of microbial membranes. Surface pressure and surface potential isotherms, dynamical surface rheology, Brewster angle microscopy (BAM), and infrared spectroscopy were employed to characterize the compound-membrane interactions. The compound expanded the monolayers denoting repulsive interactions. At 30 mN/m, the monolayer presented lower viscoelastic and in-plane elasticity parameters and an increased all-trans/gauche conformers ratio for the alkyl chains, confirming molecular order. The morphology of the monolayer was analyzed by BAM, which revealed a heterogeneous distribution of γ-terpineol along the mixed monolayer, which tends to segregate. In conclusion, the compound changes the thermodynamic, electric, rheological, morphological, and structural properties of the peptidoglycan-DPPE monolayer, which may be essential to understand, at the molecular level, the action of bioactives in selected membrane models.

Distribution and preservation of the components of the engulfment. What is beyond representative genomes?

Engulfment requires the coordinated, targeted synthesis and degradation of peptidoglycan at the leading edge of the engulfing membrane to allow the mother cell to completely engulf the forespore. Proteins such as the DMP and Q:AH complexes in Bacillus subtilis are essential for engulfment, as are a set of accessory proteins including GerM and SpoIIB, among others. Experimental and bioinformatic studies of these proteins in bacteria distinct from Bacillus subtilis indicate that fundamental differences exist regarding the organization and mechanisms used to successfully perform engulfment. As a consequence, the distribution and prevalence of the proteins involved in engulfment and other proteins that participate in different sporulation stages have been studied using bioinformatic approaches. These works are based on the prediction of orthologs in the genomes of representative Firmicutes and have been helpful in tracing hypotheses about the origin and evolution of sporulation genes, some of which have been postulated as sporulation signatures. To date, an extensive study of these signatures outside of the representative Firmicutes is not available. Here, we asked whether phyletic profiles of proteins involved in engulfment can be used as signatures able to describe the sporulation phenotype. We tested this hypothesis in a set of 954 Firmicutes, finding preserved phyletic profiles defining signatures at the genus level. Finally, a phylogenetic reconstruction based on non-redundant phyletic profiles at the family level shows the non-monophyletic origin of these proteins due to gain/loss events along the phylum Firmicutes.

Production of IL-1ß, IL-6 and CXCL8 by endometrium of crossbred heifers stimulated with various pathogen-associated molecular patterns.

Uterine bacterial infections are common during the post-partum period of dairy herds and, apparently, incidences in crossbred cattle are less than in Holsteins. The aims of this study were (I) to evaluate production of interleukin 1-ß (IL-1ß), interleukin-6 (IL-6) and chemokine CXCL8 using endometrial explants from Bos indicus crossbred heifers at diestrous, stimulated by various pathogen-associated molecular patterns (PAMP), and (II) assess production of these cytokines by lipopolysaccharide-stimulated endometrial explants from heifers when samples were collected at different stages of estrous cycle. In the first experiment, endometrial explants from heifers at diestrous were stimulated by ten-fold serial dilutions of lipopolysaccharide (LPS), triacylated lipopeptide (PAM3) or peptidoglycan (PGN). In the second experiment, endometrial explants collected at different stages of the estrous cycle were treated with LPS. Concentrations of IL-1ß, IL-6 and CXCL8 were quantified in supernatant. There was a marked (P < 0.05) production of IL-1ß, IL-6, and CXCL8 in response to LPS treatment. There was also production of IL-1ß (P < 0.05) in response to PGN treatment. Explant samples collected at different stages of the estrous cycle responded to LPS treatment with production of IL-1ß and IL-6, but with no differences (P > 0.05) between stages of estrous cycle. In conclusion, endometrial samples of crossbred Zebu-based heifers collected during diestrous produced IL-1ß, IL-6 and CXCL8 in response to LPS and IL-1ß in response to PGN. The cytokine production in response to LPS, however, was not affected by the stage of the estrous cycle in Bos indicus crossbred heifers.

Targeting the Achilles' Heel of Bacteria: Different Mechanisms To Break Down the Peptidoglycan Cell Wall during Bacterial Warfare.

Bacteria commonly live in dense polymicrobial communities and compete for scarce resources. Consequently, they employ a diverse array of mechanisms to harm, inhibit, and kill their competitors. The cell wall is essential for bacterial survival by providing mechanical strength to resist osmotic stress. Because peptidoglycan is the major component of the cell wall and its synthesis is a complex multistep pathway that requires the coordinate action of several enzymes, it provides a target for rival bacteria, which have developed a large arsenal of antibacterial molecules to attack the peptidoglycan of competitors. These molecules include antibiotics, bacteriocins, and contact-dependent effectors that are either secreted into the medium or directly translocated into a target cell. In this minireview, we summarize the diversity of these molecules and highlight distinct mechanisms to disrupt the peptidoglycan, giving special attention to molecules that are known or have the potential to be used during interbacterial competitions.

Cleavage of proteoglycans, plasma proteins and the platelet-derived growth factor receptor in the hemorrhagic process induced by snake venom metalloproteinases.

Envenoming by viperid snakes results in a complex pattern of tissue damage, including hemorrhage, which in severe cases may lead to permanent sequelae. Snake venom metalloproteinases (SVMPs) are main players in this pathogenesis, acting synergistically upon different mammalian proteomes. Hemorrhagic Factor 3 (HF3), a P-III class SVMP from Bothrops jararaca, induces severe local hemorrhage at pmol doses in a murine model. Our hypothesis is that in a complex scenario of tissue damage, HF3 triggers proteolytic cascades by acting on a partially known substrate repertoire. Here, we focused on the hypothesis that different proteoglycans, plasma proteins, and the platelet derived growth factor receptor (PDGFR) could be involved in the HF3-induced hemorrhagic process. In surface plasmon resonance assays, various proteoglycans were demonstrated to interact with HF3, and their incubation with HF3 showed degradation or limited proteolysis. Likewise, Western blot analysis showed in vivo degradation of biglycan, decorin, glypican, lumican and syndecan in the HF3-induced hemorrhagic process. Moreover, antithrombin III, complement components C3 and C4, factor II and plasminogen were cleaved in vitro by HF3. Notably, HF3 cleaved PDGFR (alpha and beta) and PDGF in vitro, while both receptor forms were detected as cleaved in vivo in the hemorrhagic process induced by HF3. These findings outline the multifactorial character of SVMP-induced tissue damage, including the transient activation of tissue proteinases, and underscore for the first time that endothelial glycocalyx proteoglycans and PDGFR are targets of SVMPs in the disruption of microvasculature integrity and generation of hemorrhage.

The Role of Alveolar Macrophages in the Improved Protection against Respiratory Syncytial Virus and Pneumococcal Superinfection Induced by the Peptidoglycan of Lactobacillus rhamnosus CRL1505.

The nasal priming with nonviable Lactobacillus rhamnosus CRL1505 (NV1505) or its purified peptidoglycan (PG1505) differentially modulates the respiratory innate immune response in infant mice, improving their resistance to primary respiratory syncytial virus (RSV) infection and secondary pneumococcal pneumonia. In association with the protection against RSV-pneumococcal superinfection, it was found that NV1505 or PG1505 significantly enhance the numbers of CD11c+SiglecF+ alveolar macrophages (AMs) producing interferon (IFN)-ß. In this work, we aimed to further advance in the characterization of the beneficial effects of NV1505 and PG1505 in the context of a respiratory superinfection by evaluating whether their immunomodulatory properties are dependent on AM functions. Macrophage depletion experiments and a detailed study of their production of cytokines and antiviral factors clearly demonstrated the key role of this immune cell population in the improvement of both the reduction of pathogens loads and the protection against lung tissue damage induced by the immunobiotic CRL1505 strain. Studies at basal conditions during primary RSV or S. pneumoniae infections, as well as during secondary pneumococcal pneumonia, brought the following five notable findings regarding the immunomodulatory effects of NV1505 and PG1505: (a) AMs play a key role in the beneficial modulation of the respiratory innate immune response and protection against RSV infection, (b) AMs are necessary for improved protection against primary and secondary pneumococcal pneumonia, (c) the generation of activated/trained AMs would be essential for the enhanced protection against respiratory pathogens, (d) other immune and nonimmune cell populations in the respiratory tract may contribute to the protection against bacterial and viral infections, and (e) the immunomodulatory properties of NV1505 and PG1505 are strain-specific. These findings significantly improve our knowledge about the immunological mechanisms involved in the modulation of respiratory immunity induced by beneficial microbes.

A Family of T6SS Antibacterial Effectors Related to l,d-Transpeptidases Targets the Peptidoglycan.

Type VI secretion systems (T6SSs) are nanomachines used by bacteria to inject toxic effectors into competitors. The identity and mechanism of many effectors remain unknown. We characterized a Salmonella T6SS antibacterial effector called Tlde1 that is toxic in target-cell periplasm and is neutralized by its cognate immunity protein (Tldi1). Microscopy analysis reveals that cells expressing Tlde1 stop dividing and lose cell envelope integrity. Bioinformatic analysis uncovers similarities between Tlde1 and the catalytic domain of l,d-transpeptidases. Point mutations on conserved catalytic residues abrogate toxicity. Biochemical assays reveal that Tlde1 displays both l,d-carboxypeptidase activity by cleaving peptidoglycan tetrapeptides between meso-diaminopimelic acid3 and d-alanine4 and l,d-transpeptidase exchange activity by replacing d-alanine4 by a non-canonical d-amino acid. Phylogenetic analysis shows that Tlde1 homologs constitute a family of T6SS-associated effectors broadly distributed among Proteobacteria. This work expands our current knowledge about bacterial effectors used in interbacterial competition and reveals a different mechanism of bacterial antagonism.

Arthrobacter ulcerisalmonis sp. nov., isolated from an ulcer of a farmed Atlantic salmon (Salmo salar), and emended description of the genus Arthrobacter sensu lato.

A Gram-stain positive, pleomorphic, oxidase-negative, non-motile isolate from the ulcer of a farmed Atlantic salmon (Salmo salar), designated strain T11bT, was subjected to a comprehensive taxonomic investigation. A comparative analysis of the 16S rRNA gene sequence showed highest similarities to the type strains of Pseudarthrobacter siccitolerans (98.1 %) and Arthrobacter methylotrophus and Pseudarthrobacter phenanthrenivorans (both 98.0 %). The highest ANI value observed between the assembled genome of T11bT and the publicly available Pseudarthrobacter and Arthrobacter type strain genomes were 81.15 and 80.99 %, respectively. The major respiratory quinone was menaquinone MK-9(H2). The polyamine pattern contained predominantly spermidine. The polar lipid profile consisted of the major lipids diphosphatidylglycerol, phosphatidylglycerol, monogalactosyl-diacylglycerol and dimannosylglyceride. Minor amouts of trimannosyldiacylglycerol and phosphatidylinositol were also detected. The peptidoglycan was of the type A3α l-Lys-l-Ser-l-Thr-l-Ala (A11.23). In the fatty acid profile, anteiso and iso branched fatty acids predominated (anteiso C15 : 0, iso C16 : 0, anteiso C17 : 0). Moderate to low DNA-DNA similarities, physiological traits as well as unique traits in the fatty acid pattern distinguished strain T11bT from the next related species. All these data point to the fact that strain T11bT represents a novel species of the genus Arthrobacter for which we propose the name Arthrobacter ulcerisalmonis sp. nov. The type strain is T11bT (=CIP 111621T=CCM 8854T=LMG 30632T=DSM 107127T).

Bacillus natronophilus sp. nov., an alkaliphilic bacterium isolated from a soda lake.

An alkaliphilic, moderately halophilic, heterotrophic, rod-shaped, spore-forming bacterium (M30T) was isolated from a sediment sample collected from a soda lake (Lake Magadi, Tanzania). Strain M30T was strictly aerobic, catalase-positive, oxidase-negative and non-motile. Growth occurred at 12-43 °C (optimum, 25-30 °C), at pH 8.0-12 (optimum, pH 9.5-10) and at salinities of 0.5-15 % (w/v) NaCl (optimum 5 %). It utilized various sugars and organic acids as sole carbon sources and was positive for amylase, cellulase, gelatinase, protease and xylanase activities. The cell-wall peptidoglycan contained meso-diaminopimelic acid and the polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, one unidentified lipid and one unidentified phospholipid. The DNA G+C content was 48.9 mol%. The predominant menaquinone was MK-7 and the major fatty acids (>10 %) comprised anteiso-C15 : 0, iso-C15 : 0, and anteiso-C17 : 0. Phylogenetic analysis based on 16S rRNA gene sequence affiliated M30T to the genus Bacillus and showed the highest similarities to Bacillus populi FJAT-45347T (96.4 %) and Bacillus aurantiacus K1-5T (96.2 %). Based on the data from the current polyphasic study, M30T represents a novel species of the genus Bacillus, for which the name Bacillus natronophilus sp. nov. is proposed. The type strain is M30T (=JCM 32118T=CGMCC 1.16739T=MCC 3010T).