Cytosolic Sensing of Intracellular Staphylococcus aureus by Mast Cells Elicits a Type I IFN Response That Enhances Cell-Autonomous Immunity.

Strategically located at mucosal sites, mast cells are instrumental in sensing invading pathogens and modulating the quality of the ensuing immune responses depending on the nature of the infecting microbe. It is believed that mast cells produce type I IFN (IFN-I) in response to viruses, but not to bacterial infections, because of the incapacity of bacterial pathogens to internalize within mast cells, where signaling cascades leading to IFN-I production are generated. However, we have previously reported that, in contrast with other bacterial pathogens, Staphylococcus aureus can internalize into mast cells and therefore could trigger a unique response. In this study, we have investigated the molecular cross-talk between internalized S. aureus and the human mast cells HMC-1 using a dual RNA sequencing approach. We found that a proportion of internalized S. aureus underwent profound transcriptional reprogramming within HMC-1 cells to adapt to the nutrients and stress encountered in the intracellular environment and remained viable. HMC-1 cells, in turn, recognized intracellular S. aureus via cGMP-AMP synthase-STING-TANK-binding kinase 1 signaling pathway, leading to the production of IFN-I. Bacterial internalization and viability were crucial for IFN-I induction because inhibition of S. aureus internalization or infection with heat-killed bacteria completely prevented the production of IFN-I by HMC-1 cells. Feeding back in an autocrine manner in S. aureus-harboring HMC-1 cells and in a paracrine manner in noninfected neighboring HMC-1 cells, IFN-I promoted a cell-autonomous antimicrobial state by inducing the transcription of IFN-I-stimulated genes. This study provides unprecedented evidence of the capacity of mast cells to produce IFN-I in response to a bacterial pathogen.

Aurantibacillus circumpalustris gen. nov., sp. nov., the first characterized representative of the Bacteroidota candidate family env.OPS 17 and proposal of Aurantibacillaceae fam. nov.

16S rRNA sequence types associated with the candidate family env.OPS 17 have been reported from various environments, but no representatives have been characterized and validly named. Bacteria of env.OPS 17 are affiliated with the order Sphingobacteriales and were first detected more than two decades ago in the vicinity of a thermal spring in Yellowstone National Park. Strain Swamp196T, isolated from the soil surrounding a swamp in Northern Germany, is the first characterized representative of candidate family env.OPS 17. Cells of strain Swamp196T are rod-shaped, non-motile, non-spore-forming, non-capsulated and stain Gram-negative. Colonies are small and orange-coloured. The strain is mesophilic and grows under aerobic or microaerophilic conditions. It grows chemo-organotrophically over a narrow range of pH and exclusively on proteinaceous substrates. The major cellular fatty acids are iso-C15 : 0, iso-C15 : 1 ω10c, C18 : 1 ω9c and C16 : 1 ω7c and the major polar lipids are two unidentified aminophospholipids, one unidentified aminolipid and one unidentified lipid. The predominant respiratory quinone is MK-7. The DNA G+C content of genomic DNA is 35.5 mol%. Strain Swamp196T is related to Pedobacter cryophilus AR-3-17T, Arcticibacter pallidicorallinus Hh36T and Pedobacter daechungensis Dae 13T with 16S rRNA gene sequence similarity of 84.1, 83.8 and 83.5 %, respectively. Based on our phenotypic, genomic and phylogenetic analysis, we propose the novel species Aurantibacillus circumpalustris sp. nov (type strain Swamp196T=DSM 105849T=CECT 30420T) of the novel genus Aurantibacillus gen. nov. and the novel family Aurantibacillaceae fam. nov.

Stratiformator vulcanicus gen. nov., sp. nov., a marine member of the family Planctomycetaceae isolated from a red biofilm in the Tyrrhenian Sea close to the volcanic island Panarea.

A novel planctomycetal strain, designated Pan189T, was isolated from biofilm material sampled close to Panarea Island in the Tyrrhenian Sea. Cells of strain Pan189T are round grain rice-shaped, form pink colonies and display typical planctomycetal characteristics including asymmetric cell division through polar budding and presence of crateriform structures. Cells bear a stalk opposite to the division pole and fimbriae cover the cell surface. Strain Pan189T has a mesophilic (optimum at 24 °C) and neutrophilic (optimum at pH 7.5) growth profile, is aerobic and heterotrophic. Under laboratory-scale cultivation conditions, it reached a generation time of 102 h (µmax = 0.0068 h-1), which places the strain among the slowest growing members of the phylum Planctomycetota characterized so far. The genome size of the strain is with 5.23 Mb at the lower limit among the family Planctomycetaceae (5.1-8.9 Mb). Phylogenetically, the strain represents a novel genus and species in the family Planctomycetaceae, order Planctomycetales, class Planctomycetia. We propose the name Stratiformator vulcanicus gen. nov., sp. nov. for the novel taxon, that is represented by the type strain Pan189T (= DSM 101711 T = CECT 30699 T).

Symbiosis between nanohaloarchaeon and haloarchaeon is based on utilization of different polysaccharides.

Nano-sized archaeota, with their small genomes and limited metabolic capabilities, are known to associate with other microbes, thereby compensating for their own auxotrophies. These diminutive and yet ubiquitous organisms thrive in hypersaline habitats that they share with haloarchaea. Here, we reveal the genetic and physiological nature of a nanohaloarchaeon-haloarchaeon association, with both microbes obtained from a solar saltern and reproducibly cultivated together in vitro. The nanohaloarchaeon Candidatus Nanohalobium constans LC1Nh is an aerotolerant, sugar-fermenting anaerobe, lacking key anabolic machinery and respiratory complexes. The nanohaloarchaeon cells are found physically connected to the chitinolytic haloarchaeon Halomicrobium sp. LC1Hm. Our experiments revealed that this haloarchaeon can hydrolyze chitin outside the cell (to produce the monosaccharide N-acetylglucosamine), using this beta-glucan to obtain carbon and energy for growth. However, LC1Hm could not metabolize either glycogen or starch (both alpha-glucans) or other polysaccharides tested. Remarkably, the nanohaloarchaeon's ability to hydrolyze glycogen and starch to glucose enabled growth of Halomicrobium sp. LC1Hm in the absence of a chitin. These findings indicated that the nanohaloarchaeon-haloarchaeon association is both mutualistic and symbiotic; in this case, each microbe relies on its partner's ability to degrade different polysaccharides. This suggests, in turn, that other nano-sized archaeota may also be beneficial for their hosts. Given that availability of carbon substrates can vary both spatially and temporarily, the susceptibility of Halomicrobium to colonization by Ca Nanohalobium can be interpreted as a strategy to maximize the long-term fitness of the host.

New insights into the energy metabolism and taxonomy of Deferribacteres revealed by the characterization of a new isolate from a hypersaline microbial mat.

Strain L21-Ace-BEST , isolated from a lithifying cyanobacterial mat, could be assigned to a novel species and genus within the class Deferribacteres. It is an important model organism for the study of anaerobic acetate degradation under hypersaline conditions. The metabolism of strain L21-Ace-BEST was characterized by biochemical studies, comparative genome analyses, and the evaluation of gene expression patterns. The central metabolic pathway is the citric acid cycle, which is mainly controlled by the enzyme succinyl-CoA:acetate-CoA transferase. The potential use of a reversed oxidative citric acid cycle to fix CO2 has been revealed through genome analysis. However, no autotrophic growth was detected in this strain, whereas sulfide and H2 can be used mixotrophically. Preferred electron acceptors for the anaerobic oxidation of acetate are nitrate, fumarate and dimethyl sulfoxide, while oxygen can be utilized only under microoxic conditions. Aerotolerant growth by fermentation was observed at higher oxygen concentrations. The redox cycling of sulfur/sulfide enables the generation of reducing power for the assimilation of acetate during growth and could prevent the over-reduction of cells in stationary phase. Extracellular electron transfer appears to be an essential component of the respiratory metabolism in this clade of Deferribacteres and may be involved in the reduction of nitrite to ammonium.

Cellular adaptation of Clostridioides difficile to high salinity encompasses a compatible solute-responsive change in cell morphology.

Infections by the pathogenic gut bacterium Clostridioides difficile cause severe diarrhoeas up to a toxic megacolon and are currently among the major causes of lethal bacterial infections. Successful bacterial propagation in the gut is strongly associated with the adaptation to changing nutrition-caused environmental conditions; e.g. environmental salt stresses. Concentrations of 350 mM NaCl, the prevailing salinity in the colon, led to significantly reduced growth of C. difficile. Metabolomics of salt-stressed bacteria revealed a major reduction of the central energy generation pathways, including the Stickland-fermentation reactions. No obvious synthesis of compatible solutes was observed up to 24 h of growth. The ensuing limited tolerance to high salinity and absence of compatible solute synthesis might result from an evolutionary adaptation to the exclusive life of C. difficile in the mammalian gut. Addition of the compatible solutes carnitine, glycine-betaine, γ-butyrobetaine, crotonobetaine, homobetaine, proline-betaine and dimethylsulfoniopropionate restored growth (choline and proline failed) under conditions of high salinity. A bioinformatically identified OpuF-type ABC-transporter imported most of the used compatible solutes. A long-term adaptation after 48 h included a shift of the Stickland fermentation-based energy metabolism from the utilization to the accumulation of l-proline and resulted in restored growth. Surprisingly, salt stress resulted in the formation of coccoid C. difficile cells instead of the typical rod-shaped cells, a process reverted by the addition of several compatible solutes. Hence, compatible solute import via OpuF is the major immediate adaptation strategy of C. difficile to high salinity-incurred cellular stress.

Salmonella Typhimurium discreet-invasion of the murine gut absorptive epithelium.

Salmonella enterica serovar Typhimurium (S.Tm) infections of cultured cell lines have given rise to the ruffle model for epithelial cell invasion. According to this model, the Type-Three-Secretion-System-1 (TTSS-1) effectors SopB, SopE and SopE2 drive an explosive actin nucleation cascade, resulting in large lamellipodia- and filopodia-containing ruffles and cooperative S.Tm uptake. However, cell line experiments poorly recapitulate many of the cell and tissue features encountered in the host's gut mucosa. Here, we employed bacterial genetics and multiple imaging modalities to compare S.Tm invasion of cultured epithelial cell lines and the gut absorptive epithelium in vivo in mice. In contrast to the prevailing ruffle-model, we find that absorptive epithelial cell entry in the mouse gut occurs through "discreet-invasion". This distinct entry mode requires the conserved TTSS-1 effector SipA, involves modest elongation of local microvilli in the absence of expansive ruffles, and does not favor cooperative invasion. Discreet-invasion preferentially targets apicolateral hot spots at cell-cell junctions and shows strong dependence on local cell neighborhood. This proof-of-principle evidence challenges the current model for how S.Tm can enter gut absorptive epithelial cells in their intact in vivo context.

Characterisation of new anti-O157 bacteriophages of bovine origin representing three genera.

Shiga-toxin-producing Escherichia coli (STEC) strains of the serogroup O157 are foodborne pathogens associated with severe clinical disease. As antibiotics are counter-indicated for treatment of these infections, they represent prime candidates for targeted application of bacteriophages to reduce infection burden. In this study, we characterised lytic bacteriophages representing three phage genera for activity against E. coli O157 strains. The phages vb_EcoM_bov9_1 (Tequatrovirus), vb_EcoM_bov11CS3 (Vequintavirus), and vb_EcoS_bov25_1D (Dhillonvirus) showed effective lysis of enterohaemorrhagic E. coli EHEC O157:H7 strains, while also exhibiting activity against other strains of the O157 serogroup, as well as of the 'big six' (STEC) serogroups, albeit with lower efficiency. They had a burst size of 293, 127 and 18 per cell and a latent period of 35, 5 and 30 min, respectively. In situ challenge experiments using the O157 Sakai strain on minced beef showed a reduction by 2-3-fold when treated with phages at a 0.1 MOI (multiplicity of infection), and approximately 1 log reduction when exposed to MOI values of 10 and 100. A cocktail of the phages, applied at 10 × and 100 × MOI showed 2 to 3 log reduction when samples were treated at room temperature, and all treatments at 37 °C with 100 × MOI resulted in a 5 to 6 log reduction in cell count. Our results indicate that the phages vb_EcoM_bov9_1 and vb_EcoM_bov11CS3, which have higher burst sizes, are promising candidates for biocontrol experiments aimed at the eradication of E. coli O157 strains in animals or foodstuff.

Blastococcus tunisiensis sp. nov., isolated from limestone collected in Tunisia.

A new actinobacterium strain, designated BMG 823T, was isolated from a limestone sample collected in Tunisia. Its taxonomic position was scrutinized using a polyphasic approach. Colonies of strain BMG 823T were pink orange-coloured, regular and had a moist surface. Cells are Gram-stain-positive, catalase-negative and oxidase-negative. The strain grew at pH 5.5-9, 10-40 °C and in presence of up to 4 % NaCl (w/v). Chemotaxonomically, strain BMG 823T was characterized by cell-wall type III containing meso-diaminopimelic acid as diamino acid, glucose, ribose and rhamnose as whole-cell sugars, MK-9(H4) as predominant menaquinone, and phosphatidylcholine, diphosphadidylglycerol, phosphatidethanolamine, phosphatidylcholine, phosphatidylinositol, unidentified glycolipid, unidentified aminophospholipids and unidentified glycophospholipid as major polar lipids. The fatty acid profile consisted of iso-C16 : 0 and iso-C17 : 1 ω9. Phylogenetic trees based on 16S rRNA gene and genome sequences placed strain BMG 823T within the genus Blastococcus and separated it from all type strains of validly published species. Comparison of 16S rRNA gene sequence similarity, digital DNA-DNA hybridization and average nucleotide identity indicated that strain BMG 823T was most closely related to Blastococcus litoris DSM 106127T and Blastococcus colisei BMG 822T with pairwise values well below the species differentiation thresholds. The distinct phenotypic and genotypic features of strain BMG 823T (=DSM 46838T=CECT 8881T) within the genus Blastococcus warrant its recognition as the type strain for the new species for which we propose the name Blastococcus tunisiensis sp. nov.

Mucisphaera calidilacus gen. nov., sp. nov., a novel planctomycete of the class Phycisphaerae isolated in the shallow sea hydrothermal system of the Lipari Islands.

For extending the current collection of axenic cultures of planctomycetes, we describe in this study the isolation and characterisation of strain Pan265T obtained from a red biofilm in the hydrothermal vent system close to the Lipari Islands in the Tyrrhenian Sea, north of Sicily, Italy. The strain forms light pink colonies on solid medium and grows as a viscous colloid in liquid culture, likely as the result of formation of a dense extracellular matrix observed during electron microscopy. Cells of the novel isolate are spherical, motile and divide by binary fission. Strain Pan265T is mesophilic (temperature optimum 30-33 °C), neutrophilic (pH optimum 7.0-8.0), aerobic and heterotrophic. The strain has a genome size of 3.49 Mb and a DNA G + C content of 63.9%. Phylogenetically, the strain belongs to the family Phycisphaeraceae, order Phycisphaerales, class Phycisphaerae. Our polyphasic analysis supports the delineation of strain Pan265T from the known genera in this family. Therefore, we conclude to assign strain Pan265T to a novel species within a novel genus, for which we propose the name Mucisphaera calidilacus gen. nov., sp. nov. The novel species is the type species of the novel genus and is represented by strain Pan265T (= DSM 100697T = CECT 30425T) as type strain.

Analysis of bacterial communities in a municipal duck pond during a phytoplankton bloom and isolation of Anatilimnocola aggregata gen. nov., sp. nov., Lacipirellula limnantheis sp. nov. and Urbifossiella limnaea gen. nov., sp. nov. belonging to the phylum Planctomycetes.

Waterbodies such as lakes and ponds are fragile environments affected by human influences. Suitable conditions can result in massive growth of phototrophs, commonly referred to as phytoplankton blooms. Such events benefit heterotrophic bacteria able to use compounds secreted by phototrophs or their biomass as major nutrient source. One example of such bacteria are Planctomycetes, which are abundant on the surfaces of marine macroscopic phototrophs; however, less data are available on their ecological roles in limnic environments. In this study, we followed a cultivation-independent deep sequencing approach to study the bacterial community composition during a cyanobacterial bloom event in a municipal duck pond. In addition to cyanobacteria, which caused the bloom event, members of the phylum Planctomycetes were significantly enriched in the cyanobacteria-attached fraction compared to the free-living fraction. Separate datasets based on isolated DNA and RNA point towards considerable differences in the abundance and activity of planctomycetal families, indicating different activity peaks of these families during the cyanobacterial bloom. Motivated by the finding that the sampling location harbours untapped bacterial diversity, we included a complementary cultivation-dependent approach and isolated and characterized three novel limnic strains belonging to the phylum Planctomycetes.

The Lytic Siphophage vB_StyS-LmqsSP1 Reduces the Number of Salmonella enterica Serovar Typhimurium Isolates on Chicken Skin.

Phage-based biocontrol of bacteria is considered a natural approach to combat foodborne pathogens. Salmonella spp. are notifiable and highly prevalent pathogens that cause foodborne diseases worldwide. In this study, six bacteriophages were isolated and further characterized that infect food-derived Salmonella isolates from different meat sources. The siphovirus VB_StyS-LmqsSP1, which was isolated from a cow's nasal swab, was further subjected to in-depth characterization. Phage-host interaction investigations in liquid medium showed that vB_StyS-LmqsSP1 can suppress the growth of Salmonella species isolates at 37°C for 10 h and significantly reduce the bacterial titer at 4°C. A reduction of 1.4 to 3 log units was observed in investigations with two food-derived Salmonella isolates and one reference strain under cooling conditions using multiplicities of infection (MOIs) of 104 and 105. Phage application on chicken skin resulted in a reduction of about 2 log units in the tested Salmonella isolates from the first 3 h throughout a 1-week experiment at cooling temperature and with an MOI of 105. The one-step growth curve analysis using vB_StyS-LmqsSP1 demonstrated a 60-min latent period and a burst size of 50 to 61 PFU/infected cell for all tested hosts. Furthermore, the genome of the phage was determined to be free from genes causing undesired effects. Based on the phenotypic and genotypic properties, LmqsSP1 was assigned as a promising candidate for biocontrol of Salmonella enterica serovar Typhimurium in food. IMPORTANCE Salmonella enterica is one of the major global causes of foodborne enteritis in humans. The use of chemical sanitizers for reducing bacterial pathogens in the food chain can result in the spread of bacterial resistance. Targeted and clean-label intervention strategies can reduce Salmonella contamination in food. The significance of our research demonstrates the suitability of a bacteriophage (vB_StyS-LmqsSP1) for biocontrol of Salmonella enterica serovar Typhimurium on poultry due to its lytic efficacy under conditions prevalent in food production environments.

Hook length of the bacterial flagellum is optimized for maximal stability of the flagellar bundle.

Most bacteria swim in liquid environments by rotating one or several flagella. The long external filament of the flagellum is connected to a membrane-embedded basal body by a flexible universal joint, the hook, which allows the transmission of motor torque to the filament. The length of the hook is controlled on a nanometer scale by a sophisticated molecular ruler mechanism. However, why its length is stringently controlled has remained elusive. We engineered and studied a diverse set of hook-length variants of Salmonella enterica. Measurements of plate-assay motility, single-cell swimming speed, and directional persistence in quasi-2D and population-averaged swimming speed and body angular velocity in 3D revealed that the motility performance is optimal around the wild-type hook length. We conclude that too-short hooks may be too stiff to function as a junction and too-long hooks may buckle and create instability in the flagellar bundle. Accordingly, peritrichously flagellated bacteria move most efficiently as the distance travelled per body rotation is maximal and body wobbling is minimized. Thus, our results suggest that the molecular ruler mechanism evolved to control flagellar hook growth to the optimal length consistent with efficient bundle formation. The hook-length control mechanism is therefore a prime example of how bacteria evolved elegant but robust mechanisms to maximize their fitness under specific environmental constraints.

Mitochondria Are a Subset of Extracellular Vesicles Released by Activated Monocytes and Induce Type I IFN and TNF Responses in Endothelial Cells.

RATIONALE: Extracellular vesicles, including microvesicles, are increasingly recognized as important mediators in cardiovascular disease. The cargo and surface proteins they carry are considered to define their biological activity, including their inflammatory properties. Monocyte to endothelial cell signaling is a prerequisite for the propagation of inflammatory responses. However, the contribution of microvesicles in this process is poorly understood. OBJECTIVE: To elucidate the mechanisms by which microvesicles derived from activated monocytic cells exert inflammatory effects on endothelial cells. METHODS AND RESULTS: LPS (lipopolysaccharide)-stimulated monocytic cells release free mitochondria and microvesicles with mitochondrial content as demonstrated by flow cytometry, quantitative polymerase chain reaction, Western Blot, and transmission electron microscopy. Using RNAseq analysis and quantitative reverse transcription-polymerase chain reaction, we demonstrated that both mitochondria directly isolated from and microvesicles released by LPS-activated monocytic cells, as well as circulating microvesicles isolated from volunteers receiving low-dose LPS-injections, induce type I IFN (interferon), and TNF (tumor necrosis factor) responses in endothelial cells. Depletion of free mitochondria significantly reduced the ability of these microvesicles to induce type I IFN and TNF-dependent genes. We identified mitochondria-associated TNFα and RNA from stressed mitochondria as major inducers of these responses. Finally, we demonstrated that the proinflammatory potential of microvesicles and directly isolated mitochondria were drastically reduced when they were derived from monocytic cells with nonrespiring mitochondria or monocytic cells cultured in the presence of pyruvate or the mitochondrial reactive oxygen species scavenger MitoTEMPO. CONCLUSIONS: Mitochondria and mitochondria embedded in microvesicles constitute a major subset of extracellular vesicles released by activated monocytes, and their proinflammatory activity on endothelial cells is determined by the activation status of their parental cells. Thus, mitochondria may represent critical intercellular mediators in cardiovascular disease and other inflammatory settings associated with type I IFN and TNF signaling.

Kibdelosporangium persicum sp. nov., a new member of the Actinomycetes from a hot desert in Iran.

Isolate 4NS15T was isolated from a neglected arid habitat in Kerman, Iran. The strain showed 16S rRNA gene sequence similarity values of 98.9 % to the type strains of Kibdelosporangium aridum subsp. aridum, Kibdelosporangium phytohabitans and Kibdelosporangium philippinense and 98.6 % to the type strain K. aridum subsp. largum, respectively. Genome-based phylogenetic analysis revealed that isolate 4NS15T is closely related to Kibdelosporangium aridum subsp. aridum DSM 43828T. The digital DNA-DNA hybridization value between the genome sequences of 4NS15T and strain DSM 43828T is 29.8 %. Strain 4NS15T produces long chains of spores without a sporangium-like structure which can be distinguished from other Kibdelosporangium species. Isolate 4NS15T has a genome size of 10.35 Mbp with a G+C content of 68.1 mol%. Whole-cell hydrolysates of isolate 4NS15T are rich in meso-diaminopimelic acid and cell-wall sugars such as arabinose, galactose, glucose and ribose. Major fatty acids (>10 %) are C16 : 0, iso-C16 : 0 and iso-C15 : 0. The phospholipid profile contains diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylhydroxyethanolamine, aminolipid and glycoaminolipid. The predominant menaquinone is MK-9(H4). Based on its phenotypic and genotypic characteristics, isolate 4NS15T (NCCB 100701=CIP 111705=DSM 110728) merits recognition as representing a novel species of the genus Kibdelosporangium, for which the name Kibdelosporangium persicum sp. nov. is proposed.

Streptomonospora litoralis sp. nov., a halophilic thiopeptides producer isolated from sand collected at Cuxhaven beach.

Strain M2T was isolated from the beach of Cuxhaven, Wadden Sea, Germany, in course of a program to attain new producers of bioactive natural products. Strain M2T produces litoralimycin and sulfomycin-type thiopeptides. Bioinformatic analysis revealed a potential biosynthetic gene cluster encoding for the M2T thiopeptides. The strain is Gram-stain-positive, rod shaped, non-motile, spore forming, showing a yellow colony color and forms extensively branched substrate mycelium and aerial hyphae. Inferred from the 16S rRNA gene phylogeny strain M2T affiliates with the genus Streptomonospora. It shows 96.6% 16S rRNA gene sequence similarity to the type species Streptomonospora salina DSM 44593 T and forms a distinct branch with Streptomonospora sediminis DSM 45723 T with 97.0% 16S rRNA gene sequence similarity. Genome-based phylogenetic analysis revealed that M2T is closely related to Streptomonospora alba YIM 90003 T with a digital DNA-DNA hybridisation (dDDH) value of 26.6%. The predominant menaquinones of M2T are MK-10(H6), MK-10(H8), and MK-11(H6) (> 10%). Major cellular fatty acids are iso-C16:0, anteiso C17:0 and C18:0 10-methyl. The polar lipid profile consisted of diphosphatidylglycerol phosphatidyl glycerol, phosphatidylinositol, phosphatidylcholine, phosphatidylethanolamine, three glycolipids, two unknown phospholipids, and two unknown lipids. The genome size of type strain M2T is 5,878,427 bp with 72.1 mol % G + C content. Based on the results obtained from phylogenetic and chemotaxonomic studies, strain M2T (= DSM 106425 T = NCCB 100650 T) is considered to represent a novel species within the genus Streptomonospora for which the name Streptomonospora litoralis sp. nov. is proposed.

Streptomyces bathyalis sp. nov., an actinobacterium isolated from the sponge in a deep sea.

A novel actinobacterium, designated ASO4wetT, was isolated from the unidentified sponge (SO4) in the deep sea collected of the North Atlantic Ocean. Study of 16S rRNA gene sequences indicated that strain ASO4wetT is a member of the genus Streptomyces and showed the closest similarities to Streptomyces karpasiensis K413T (98.87 %), Streptomyces glycovorans YIM M 10366T (98.38 %), and Streptomyces abyssalis YIM M 10400T (97.53 %). Strain ASO4wetT contained MK-9(H8) as the predominant menaquinone and the major fatty acids are iso-C16:0, anteiso-C15:0, and iso-C15:0. Polyphasic taxonomy was carried out between strain ASO4wetT and its phylogenetically closely related Streptomyces strains, which further elucidated their relatedness and revealed that strain ASO4wetT could be distinguished from currently known Streptomyces species. Strain ASO4wetT clearly represents a novel species in genus Streptomyces. We propose the name Streptomyces bathyalis sp. nov., with the type strain ASO4wetT (= DSM 106605T = NCCB 100657T). Analysis of the whole-genome sequence of S. bathyalis revealed that genome size is 7,377,472 bp with 6332 coding sequences.

Cortactin Is Required for Efficient FAK, Src and Abl Tyrosine Kinase Activation and Phosphorylation of Helicobacter pylori CagA.

Cortactin is a well-known regulatory protein of the host actin cytoskeleton and represents an attractive target of microbial pathogens like Helicobacter pylori. H. pylori manipulates cortactin's phosphorylation status by type-IV secretion-dependent injection of its virulence protein CagA. Multiple host tyrosine kinases, like FAK, Src, and Abl, are activated during infection, but the pathway(s) involved is (are) not yet fully established. Among them, Src and Abl target CagA and stimulate tyrosine phosphorylation of the latter at its EPIYA-motifs. To investigate the role of cortactin in more detail, we generated a CRISPR/Cas9 knockout of cortactin in AGS gastric epithelial cells. Surprisingly, we found that FAK, Src, and Abl kinase activities were dramatically downregulated associated with widely diminished CagA phosphorylation in cortactin knockout cells compared to the parental control. Together, we report here a yet unrecognized cortactin-dependent signaling pathway involving FAK, Src, and Abl activation, and controlling efficient phosphorylation of injected CagA during infection. Thus, the cortactin status could serve as a potential new biomarker of gastric cancer development.

Adenosine Triphosphate Neutralizes Pneumolysin-Induced Neutrophil Activation.

BACKGROUND: In tissue infections, adenosine triphosphate (ATP) is released into extracellular space and contributes to purinergic chemotaxis. Neutrophils are important players in bacterial clearance and are recruited to the site of tissue infections. Pneumococcal infections can lead to uncontrolled hyperinflammation of the tissue along with substantial tissue damage through excessive neutrophil activation and uncontrolled granule release. We aimed to investigate the role of ATP in neutrophil response to pneumococcal infections. METHODS: Primary human neutrophils were exposed to the pneumococcal strain TIGR4 and its pneumolysin-deficient mutant or directly to different concentrations of recombinant pneumolysin. Neutrophil activation was assessed by measurement of secreted azurophilic granule protein resistin and profiling of the secretome, using mass spectrometry. RESULTS: Pneumococci are potent inducers of neutrophil degranulation. Pneumolysin was identified as a major trigger of neutrophil activation. This process is partially lysis independent and inhibited by ATP. Pneumolysin and ATP interact with each other in the extracellular space leading to reduced neutrophil activation. Proteome analyses of the neutrophil secretome confirmed that ATP inhibits pneumolysin-dependent neutrophil activation. CONCLUSIONS: Our findings suggest that despite its cytolytic activity, pneumolysin serves as a potent neutrophil activating factor. Extracellular ATP mitigates pneumolysin-induced neutrophil activation.

The immunogenic potential of bacterial flagella for Salmonella-mediated tumor therapy.

Genetically engineered Salmonella Typhimurium are potent vectors for prophylactic and therapeutic measures against pathogens as well as cancer. This is based on the potent adjuvanticity that supports strong immune responses. The physiology of Salmonella is well understood. It simplifies engineering of both enhanced immune-stimulatory properties as well as safety features, thus, resulting in an appropriate balance between attenuation and efficacy for clinical applications. A major virulence factor of Salmonella is the flagellum. It is also a strong pathogen-associated molecular pattern recognized by extracellular and intracellular receptors of immune cells of the host. At the same time, it represents a serious metabolic burden. Accordingly, the bacteria evolved tight regulatory mechanisms that control flagella synthesis in vivo. Here, we systematically investigated the immunogenicity and adjuvant properties of various flagella mutants of Salmonella in vitro and in a mouse cancer model in vivo. We found that mutants lacking the flagellum-specific ATPase FliHIJ or the inner membrane ring FliF displayed the greatest stimulatory capacity and strongest antitumor effects, while remaining safe in vivo. Scanning electron microscopy revealed the presence of outer membrane vesicles in the ΔfliF and ΔfliHIJ mutants. Finally, the combination of the ΔfliF and ΔfliHIJ mutations with our previously described attenuated and immunogenic background strain SF102 displayed strong efficacy against the highly resistant cancer cell line RenCa. We thus conclude that manipulating flagella biosynthesis has great potential for the construction of highly efficacious and versatile Salmonella vector strains.