Campylobacter jejuni Surface-Bound Protease HtrA, but Not the Secreted Protease nor Protease in Shed Membrane Vesicles, Disrupts Epithelial Cell-to-Cell Junctions.

Fundamental functions of the intestinal epithelium include the digestion of food, absorption of nutrients, and its ability to act as the first barrier against intruding microbes. Campylobacter jejuni is a major zoonotic pathogen accounting for a substantial portion of bacterial foodborne illnesses. The germ colonizes the intestines of birds and is mainly transmitted to humans through the consumption of contaminated poultry meat. In the human gastrointestinal tract, the bacterium triggers campylobacteriosis that can progress to serious secondary disorders, including reactive arthritis, inflammatory bowel disease and Guillain-Barré syndrome. We recently discovered that C. jejuni serine protease HtrA disrupts intestinal epithelial barrier functions via cleavage of the tight and adherens junction components occludin, claudin-8 and E-cadherin. However, it is unknown whether epithelial damage is mediated by the secreted soluble enzyme, by HtrA contained in shed outer-membrane vesicles (OMVs) or by another mechanism that has yet to be identified. In the present study, we investigated whether soluble recombinant HtrA and/or purified OMVs induce junctional damage to polarized intestinal epithelial cells compared to live C. jejuni bacteria. By using electron and confocal immunofluorescence microscopy, we show that HtrA-expressing C. jejuni bacteria trigger efficient junctional cell damage, but not soluble purified HtrA or HtrA-containing OMVs, not even at high concentrations far exceeding physiological levels. Instead, we found that only bacteria with active protein biosynthesis effectively cleave junctional proteins, which is followed by paracellular transmigration of C. jejuni through the epithelial cell layer. These findings shed new light on the pathogenic activities of HtrA and virulence strategies of C. jejuni.

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).

A single-nucleotide polymorphism in Helicobacter pylori promotes gastric cancer development.

Single-nucleotide polymorphisms (SNPs) in various human genes are key factors in carcinogenesis. However, whether SNPs in bacterial pathogens are similarly crucial in cancer development is unknown. Here, we analyzed 1,043 genomes of the stomach pathogen Helicobacter pylori and pinpointed a SNP in the serine protease HtrA (position serine/leucine 171) that significantly correlates with gastric cancer. Our functional studies reveal that the 171S-to-171L mutation triggers HtrA trimer formation and enhances proteolytic activity and cleavage of epithelial junction proteins occludin and tumor-suppressor E-cadherin. 171L-type HtrA, but not 171S-HtrA-possessing H. pylori, inflicts severe epithelial damage, enhances injection of oncoprotein CagA into epithelial cells, increases NF-κB-mediated inflammation and cell proliferation through nuclear accumulation of ß-catenin, and promotes host DNA double-strand breaks, collectively triggering malignant changes. These findings highlight the 171S/L HtrA mutation as a unique bacterial cancer-associated SNP and as a potential biomarker for risk predictions in H. pylori infections.

Functional diversity of nanohaloarchaea within xylan-degrading consortia.

Extremely halophilic representatives of the phylum Candidatus Nanohaloarchaeota (members of the DPANN superphyla) are obligately associated with extremely halophilic archaea of the phylum Halobacteriota (according to the GTDB taxonomy). Using culture-independent molecular techniques, their presence in various hypersaline ecosystems around the world has been confirmed over the past decade. However, the vast majority of nanohaloarchaea remain uncultivated, and thus their metabolic capabilities and ecophysiology are currently poorly understood. Using the (meta)genomic, transcriptomic, and DNA methylome platforms, the metabolism and functional prediction of the ecophysiology of two novel extremely halophilic symbiotic nanohaloarchaea (Ca. Nanohalococcus occultus and Ca. Nanohalovita haloferacivicina) stably cultivated in the laboratory as members of a xylose-degrading binary culture with a haloarchaeal host, Haloferax lucentense, was determined. Like all known DPANN superphylum nanoorganisms, these new sugar-fermenting nanohaloarchaea lack many fundamental biosynthetic repertoires, making them exclusively dependent on their respective host for survival. In addition, given the cultivability of the new nanohaloarchaea, we managed to discover many unique features in these new organisms that have never been observed in nano-sized archaea both within the phylum Ca. Nanohaloarchaeota and the entire superphylum DPANN. This includes the analysis of the expression of organism-specific non-coding regulatory (nc)RNAs (with an elucidation of their 2D-secondary structures) as well as profiling of DNA methylation. While some ncRNA molecules have been predicted with high confidence as RNAs of an archaeal signal recognition particle involved in delaying protein translation, others resemble the structure of ribosome-associated ncRNAs, although none belong to any known family. Moreover, the new nanohaloarchaea have very complex cellular defense mechanisms. In addition to the defense mechanism provided by the type II restriction-modification system, consisting of Dcm-like DNA methyltransferase and Mrr restriction endonuclease, Ca. Nanohalococcus encodes an active type I-D CRISPR/Cas system, containing 77 spacers divided into two loci. Despite their diminutive genomes and as part of their host interaction mechanism, the genomes of new nanohaloarchaea do encode giant surface proteins, and one of them (9,409 amino acids long) is the largest protein of any sequenced nanohaloarchaea and the largest protein ever discovered in cultivated archaea.

Nanohaloarchaea as beneficiaries of xylan degradation by haloarchaea.

Climate change, desertification, salinisation of soils and the changing hydrology of the Earth are creating or modifying microbial habitats at all scales including the oceans, saline groundwaters and brine lakes. In environments that are saline or hypersaline, the biodegradation of recalcitrant plant and animal polysaccharides can be inhibited by salt-induced microbial stress and/or by limitation of the metabolic capabilities of halophilic microbes. We recently demonstrated that the chitinolytic haloarchaeon Halomicrobium can serve as the host for an ectosymbiont, nanohaloarchaeon 'Candidatus Nanohalobium constans'. Here, we consider whether nanohaloarchaea can benefit from the haloarchaea-mediated degradation of xylan, a major hemicellulose component of wood. Using samples of natural evaporitic brines and anthropogenic solar salterns, we describe genome-inferred trophic relations in two extremely halophilic xylan-degrading three-member consortia. We succeeded in genome assembly and closure for all members of both xylan-degrading cultures and elucidated the respective food chains within these consortia. We provide evidence that ectosymbiontic nanohaloarchaea is an active ecophysiological component of extremely halophilic xylan-degrading communities (although by proxy) in hypersaline environments. In each consortium, nanohaloarchaea occur as ectosymbionts of Haloferax, which in turn act as scavenger of oligosaccharides produced by xylan-hydrolysing Halorhabdus. We further obtained and characterised the nanohaloarchaea-host associations using microscopy, multi-omics and cultivation approaches. The current study also doubled culturable nanohaloarchaeal symbionts and demonstrated that these enigmatic nano-sized archaea can be readily isolated in binary co-cultures using an appropriate enrichment strategy. We discuss the implications of xylan degradation by halophiles in biotechnology and for the United Nation's Sustainable Development Goals.

Characterization of Temperate LPS-Binding Bordetella avium Phages That Lack Superinfection Immunity.

Bordetella avium causes a highly infectious upper respiratory tract disease in turkeys and other poultry with high economic losses. Considering the antimicrobial resistance crisis, bacteriophages (phages) may be an alternative approach for treating bacterial infections such as bordetellosis. Here, we describe seven B. avium phages, isolated from drinking water and feces from chicken and turkey farms. They showed strong bacteriolytic activity with a broad host range and used lipopolysaccharides (LPS) as a host receptor for their adsorption. All phages are myoviruses based on their structure observed by transmission electron microscopy. Genome sequence analyses revealed genome assembly sizes ranging from 39,087 to 43,144 bp. Their permutated genomes were organized colinearly, with a conserved module order, and were packed according to a predicted headful packing strategy. Notably, they contained genes encoding putative markers of lysogeny, indicative of temperate phages, despite their lytic phenotype. Further investigation revealed that the phages could indeed undergo a lysogenic life cycle with varying frequency. However, the lysogenic bacteria were still susceptible to superinfection with the same phages. This lack of stable superinfection immunity after lysogenization appears to be a characteristic feature of B. avium phages, which is favorable in terms of a potential therapeutic use of phages for the treatment of avian bordetellosis. IMPORTANCE To maintain the effectiveness of antibiotics over the long term, alternatives to treat infectious diseases are urgently needed. Therefore, phages have recently come back into focus as they can specifically infect and lyse bacteria and are naturally occurring. However, there is little information on phages that can infect pathogenic bacteria from animals, such as the causative agent of bordetellosis of poultry, B. avium. Therefore, in this study, B. avium phages were isolated and comprehensively characterized, including whole-genome analysis. Although phenotypically the phages were thought to undergo a lytic cycle, we demonstrated that they undergo a lysogenic phase, but that infection does not confer stable host superinfection immunity. These findings provide important information that could be relevant for potential biocontrol of avian bordetellosis by using phage therapy.

Staphylococcus aureus binding to Seraph® 100 Microbind® Affinity Filter: Effects of surface protein expression and treatment duration.

INTRODUCTION: Extracorporeal blood purification systems represent a promising alternative for treatment of blood stream infections with multiresistant bacteria. OBJECTIVES: The aim of this study was to analyse the binding activity of S. aureus to Seraph affinity filters based on heparin coated beads and to identify effectors influencing this binding activity. RESULTS: To test the binding activity, we used gfp-expressing S. aureus Newman strains inoculated either in 0.9% NaCl or in blood plasma and determined the number of unbound bacteria by FACS analyses after passing through Seraph affinity filters. The binding activity of S. aureus was clearly impaired in human plasma: while a percent removal of 42% was observed in 0.9% NaCl (p-value 0.0472) using Seraph mini columns, a percent removal of only 10% was achieved in human plasma (p-value 0.0934). The different composition of surface proteins in S. aureus caused by the loss of SarA, SigB, Lgt, and SaeS had no significant influence on its binding activity. In a clinically relevant approach using the Seraph® 100 Microbind® Affinity Filter and 1000 ml of human blood plasma from four different donors, the duration of treatment was shown to have a critical effect on the rate of bacterial reduction. Within the first four hours, the number of bacteria decreased continuously and the reduction in bacteria reached statistical significance after two hours of treatment (percentage reduction 64%, p-value 0.01165). The final reduction after four hours of treatment was close to 90% and is dependent on donor. The capacity of Seraph® 100 for S. aureus in human plasma was approximately 5 x 108 cells. CONCLUSIONS: The Seraph affinity filter, based on heparin-coated beads, is a highly efficient method for reducing S. aureus in human blood plasma, with efficiency dependent on blood plasma composition and treatment duration.

Genotype-phenotype correlations within the Geodermatophilaceae.

The integration of genomic information into microbial systematics along with physiological and chemotaxonomic parameters provides for a reliable classification of prokaryotes. In silico analysis of chemotaxonomic traits is now being introduced to replace characteristics traditionally determined in the laboratory with the dual goal of both increasing the speed of the description of taxa and the accuracy and consistency of taxonomic reports. Genomics has already successfully been applied in the taxonomic rearrangement of Geodermatophilaceae (Actinomycetota) but in the light of new genomic data the taxonomy of the family needs to be revisited. In conjunction with the taxonomic characterisation of four strains phylogenetically located within the family, we conducted a phylogenetic analysis of the whole proteomes of the sequenced type strains and established genotype-phenotype correlations for traits related to chemotaxonomy, cell morphology and metabolism. Results indicated that the four isolates under study represent four novel species within the genus Blastococcus. Additionally, the genera Blastococcus, Geodermatophilus and Modestobacter were shown to be paraphyletic. Consequently, the new genera Trujillonella, Pleomorpha and Goekera were proposed within the Geodermatophilaceae and Blastococcus endophyticus was reclassified as Trujillonella endophytica comb. nov., Geodermatophilus daqingensis as Pleomorpha daqingensis comb. nov. and Modestobacter deserti as Goekera deserti comb. nov. Accordingly, we also proposed emended descriptions of Blastococcus aggregatus, Blastococcus jejuensis, Blastococcus saxobsidens and Blastococcus xanthilyniticus. In silico chemotaxonomic results were overall consistent with wet-lab results. Even though in silico discriminatory levels varied depending on the respective chemotaxonomic trait, this approach is promising for effectively replacing and/or complementing chemotaxonomic analyses at taxonomic ranks above the species level. Finally, interesting but previously overlooked insights regarding morphology and ecology were revealed by the presence of a repertoire of genes related to flagellum synthesis, chemotaxis, spore production and pilus assembly in all representatives of the family. A rich carbon metabolism including four different CO2 fixation pathways and a battery of enzymes able to degrade complex carbohydrates were also identified in Blastococcus genomes.

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.

Patatin-like phospholipase CapV in Escherichia coli - morphological and physiological effects of one amino acid substitution.

In rod-shaped bacteria, morphological plasticity occurs in response to stress, which blocks cell division to promote filamentation. We demonstrate here that overexpression of the patatin-like phospholipase variant CapVQ329R, but not CapV, causes pronounced sulA-independent pyridoxine-inhibited cell filamentation in the Escherichia coli K-12-derivative MG1655 associated with restriction of flagella production and swimming motility. Conserved amino acids in canonical patatin-like phospholipase A motifs, but not the nucleophilic serine, are required to mediate CapVQ329R phenotypes. Furthermore, CapVQ329R production substantially alters the lipidome and colony morphotype including rdar biofilm formation with modulation of the production of the biofilm activator CsgD, and affects additional bacterial traits such as the efficiency of phage infection and antimicrobial susceptibility. Moreover, genetically diverse commensal and pathogenic E. coli strains and Salmonella typhimurium responded with cell filamentation and modulation in colony morphotype formation to CapVQ329R expression. In conclusion, this work identifies the CapV variant CapVQ329R as a pleiotropic regulator, emphasizes a scaffold function for patatin-like phospholipases, and highlights the impact of the substitution of a single conserved amino acid for protein functionality and alteration of host physiology.

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.

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.

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.

Campylobacter Bacteriophage Cocktail Design Based on an Advanced Selection Scheme.

Campylobacteriosis is a worldwide-occurring disease and has been the most commonly reported zoonotic gastrointestinal infection in the European Union in recent years. The development of successful phage-based intervention strategies will require a better understanding of phage-bacteria interactions to facilitate advances in phage cocktail design. Therefore, this study aimed to investigate the effects of newly isolated group II and group III phages and their combinations on current Campylobacter field strains. A continuous workflow for host range and efficiency of plating (EOP) value determination was combined with a qPCR-based phage group identification and a liquid-based planktonic killing assay (PKA). An advanced analysis scheme allowed us to evaluate phage cocktails by their efficacy in inhibiting bacterial population growth and the resulting phage concentrations. The results of this study indicate that data obtained from PKAs are more accurate than host range data based on plaque formation (EOP). Planktonic killing assays with Campylobacter appear to be a useful tool for a straightforward cocktail design. Results show that a group II phage vB_CcM-LmqsCP218-2c2 and group III phage vB_CjM-LmqsCP1-1 mixture would be most promising for practical applications against Campylobacter coli and Campylobacter jejuni.

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.

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.

The group A Streptococcus interleukin-8 protease SpyCEP promotes bacterial intracellular survival by evasion of autophagy.

Autophagy serves an innate immune function in defending the host against invading bacteria, including group A Streptococcus (GAS). Autophagy is regulated by numerous host proteins, including the endogenous negative regulator calpain, a cytosolic protease. Globally disseminated serotype M1T1 GAS strains associated with high invasive disease potential express numerous virulence factors and resist autophagic clearance. Upon in vitro infection of human epithelial cell lines with representative wild-type GAS M1T1 strain 5448 (M1.5448), we observed increased calpain activation linked to a specific GAS virulence factor, the IL-8 protease SpyCEP. Calpain activation inhibited autophagy and decreased capture of cytosolic GAS in autophagosomes. In contrast, the serotype M6 GAS strain JRS4 (M6.JRS4), which is highly susceptible to host autophagy-mediated killing, expresses low levels of SpyCEP and does not activate calpain. Overexpression of SpyCEP in M6.JRS4 stimulated calpain activation, inhibited autophagy and significantly decreased bacterial capture in autophagosomes. These paired loss- and gain-of-function studies reveal a novel role for the bacterial protease SpyCEP in enabling GAS M1 evasion of autophagy and host innate immune clearance.

Corrigendum: Genotype-phenotype correlations within the Geodermatophilaceae.

[This corrects the article DOI: 10.3389/fmicb.2022.975365.].

Novel protocol for the isolation of highly purified neonatal murine microglia and astrocytes.

BACKGROUND: The crosstalk and reactivity of the cell type glia, especially microglia and astrocytes, have progressively gathered research attention in understanding proper brain function regulated by the innate immune response. Therefore, methods to isolate highly viable and pure glia for the analysis on a cell-specific level are indispensable. NEW METHOD: We modified previously established techniques: Animal numbers were reduced by multiple microglial harvests from the same mixed glial culture, thereby maximizing microglial yields following the principles of the 3Rs (replacement, reduction, and refinement). We optimized Magnetic-activated cell sorting (MACS®) of microglia and astrocytes by applying cultivated primary glial cell suspensions instead of directly sorting dissociated single cell suspension. RESULTS: We generated highly viable and pure microglia and astrocytes derived from a single mixed culture with a purity of ~99%, as confirmed by FACS analysis. Field emission scanning electron microscopy (FESEM) demonstrated integrity of the MACS-purified glial cells. Tumor necrosis factor (TNF) and Interleukin-10 (IL-10) ELISA confirmed pro- and anti-inflammatory responses to be functional in purified glia, but significantly weakened compared to non-purified cells, further highlighting the importance of cellular crosstalk for proper immune activation. COMPARISON WITH EXISTING METHOD(S): Unlike previous studies that either isolated a single type of glia or displayed a substantial proportion of contamination with other cell types, we achieved isolation of both microglia and astrocytes at an increased purity (99-100%). CONCLUSIONS: We have created an optimized protocol for the efficient purification of both primary microglia and astrocytes. Our results clearly demonstrate the importance of purity in glial cell cultivation in order to examine immune responses, which particularly holds true for astrocytes. We propose the novel protocol as a tool to investigate the cell type-specific crosstalk between microglia and astrocytes in the frame of CNS diseases.