Incomplete IgG avidity maturation after seasonal coronavirus infections.

In classical viral infections, the avidity of immunoglobulin G (IgG) is low during acute infection and high a few months later. As recently reported, SARS-CoV-2 infections are not following this scheme, but they are rather characterized by incomplete avidity maturation. This study was performed to clarify whether infection with seasonal coronaviruses also leads to incomplete avidity maturation. The avidity of IgG toward the nucleoprotein (NP) of the seasonal coronaviruses 229E, NL63, OC43, HKU1 and of SARS-CoV-2 was determined in the sera from 88 healthy, SARS-CoV-2-negative subjects and in the sera from 70 COVID-19 outpatients, using the recomLine SARS-CoV-2 assay with recombinant antigens. In the sera from SARS-CoV-2-negative subjects, incomplete avidity maturation (persistent low and intermediate avidity indices) was the lowest for infections with the alpha-coronaviruses 229E (33.3%) and NL63 (61.3%), and the highest for the beta-coronaviruses OC43 (77.5%) and HKU1 (71.4%). In the sera from COVID-19 patients, the degree of incomplete avidity maturation of IgG toward NP of 223E, OC43, and HKU1 was not significantly different from that found in SARS-CoV-2-negative subjects, but a significant increase in avidity was observed for IgG toward NP of NL63. Though there was no cross-reaction between SARS-CoV-2 and seasonal coronaviruses, higher concentrations of IgG directed toward seasonal coronaviruses seemed to indirectly increase avidity maturation of IgG directed toward SARS-CoV-2. Our data show that incomplete IgG avidity maturation represents a characteristic consequence of coronavirus infections. This raises problems for the serological differentiation between acute and past infections and may be important for the biology of coronaviruses.

Heterozygous truncating variants in SUFU cause congenital ocular motor apraxia.

PURPOSE: This study aimed to delineate the genetic basis of congenital ocular motor apraxia (COMA) in patients not otherwise classifiable. METHODS: We compiled clinical and neuroimaging data of individuals from six unrelated families with distinct clinical features of COMA who do not share common diagnostic characteristics of Joubert syndrome or other known genetic conditions associated with COMA. We used exome sequencing to identify pathogenic variants and functional studies in patient-derived fibroblasts. RESULTS: In 15 individuals, we detected familial as well as de novo heterozygous truncating causative variants in the Suppressor of Fused (SUFU) gene, a negative regulator of the Hedgehog (HH) signaling pathway. Functional studies showed no differences in cilia occurrence, morphology, or localization of ciliary proteins, such as smoothened. However, analysis of expression of HH signaling target genes detected a significant increase in the general signaling activity in COMA patient-derived fibroblasts compared with control cells. We observed higher basal HH signaling activity resulting in increased basal expression levels of GLI1, GLI2, GLI3, and Patched1. Neuroimaging revealed subtle cerebellar changes, but no full-blown molar tooth sign. CONCLUSION: Taken together, our data imply that the clinical phenotype associated with heterozygous truncating germline variants in SUFU is a forme fruste of Joubert syndrome.

Vaccination versus infection with SARS-CoV-2: Establishment of a high avidity IgG response versus incomplete avidity maturation.

Avidity is defined as the binding strength of immunoglobulin G (IgG) toward its target epitope. Avidity is directly related to affinity, as both processes are determined by the best fit of IgG to epitopes. We confirm and extend data on incomplete avidity maturation of IgG toward severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleoprotein (NP), spike protein-1 (S1), and its receptor-binding domain (RBD) in coronavirus disease 2019 (COVID-19) patients. In SARS-CoV-2-infected individuals, an initial rise in avidity maturation was ending abruptly, leading to IgG of persistently low or intermediate avidity. Incomplete avidity maturation might facilitate secondary SARS-CoV-2 infections and thus prevent the establishment of herd immunity. Incomplete avidity maturation after infection with SARS-CoV-2 (with only 11.8% of cases showing finally IgG of high avidity, that is, an avidity index > 0.6) was contrasted by regular and rapid establishment of high avidity in SARS-CoV-2 naïve individuals after two vaccination steps with the BioNTech messenger RNA (mRNA) Vaccine (78% of cases with high avidity). One vaccination step was not sufficient for induction of complete avidity maturation in vaccinated SARS-CoV-2 naïve individuals, as it induced high avidity only in 2.9% of cases within 3 weeks. However, one vaccination step was sufficient to induce high avidity in individuals with previous SARS-CoV-2 infection.

Displacement of the transcription factor B reader domain during transcription initiation.

Transcription initiation by archaeal RNA polymerase (RNAP) and eukaryotic RNAP II requires the general transcription factor (TF) B/ IIB. Structural analyses of eukaryotic transcription initiation complexes locate the B-reader domain of TFIIB in close proximity to the active site of RNAP II. Here, we present the first crosslinking mapping data that describe the dynamic transitions of an archaeal TFB to provide evidence for structural rearrangements within the transcription complex during transition from initiation to early elongation phase of transcription. Using a highly specific UV-inducible crosslinking system based on the unnatural amino acid para-benzoyl-phenylalanine allowed us to analyze contacts of the Pyrococcus furiosus TFB B-reader domain with site-specific radiolabeled DNA templates in preinitiation and initially transcribing complexes. Crosslink reactions at different initiation steps demonstrate interactions of TFB with DNA at registers +6 to +14, and reduced contacts at +15, with structural transitions of the B-reader domain detected at register +10. Our data suggest that the B-reader domain of TFB interacts with nascent RNA at register +6 and +8 and it is displaced from the transcribed-strand during the transition from +9 to +10, followed by the collapse of the transcription bubble and release of TFB from register +15 onwards.

Molecular architecture of the N-type ATPase rotor ring from Burkholderia pseudomallei.

The genome of the highly infectious bacterium Burkholderia pseudomallei harbors an atp operon that encodes an N-type rotary ATPase, in addition to an operon for a regular F-type rotary ATPase. The molecular architecture of N-type ATPases is unknown and their biochemical properties and cellular functions are largely unexplored. We studied the B. pseudomallei N1No-type ATPase and investigated the structure and ion specificity of its membrane-embedded c-ring rotor by single-particle electron cryo-microscopy. Of several amphiphilic compounds tested for solubilizing the complex, the choice of the low-density, low-CMC detergent LDAO was optimal in terms of map quality and resolution. The cryoEM map of the c-ring at 6.1 Å resolution reveals a heptadecameric oligomer with a molecular mass of ~141 kDa. Biochemical measurements indicate that the c17 ring is H+ specific, demonstrating that the ATPase is proton-coupled. The c17 ring stoichiometry results in a very high ion-to-ATP ratio of 5.7. We propose that this N-ATPase is a highly efficient proton pump that helps these melioidosis-causing bacteria to survive in the hostile, acidic environment of phagosomes.

TFE and Spt4/5 open and close the RNA polymerase clamp during the transcription cycle.

Transcription is an intrinsically dynamic process and requires the coordinated interplay of RNA polymerases (RNAPs) with nucleic acids and transcription factors. Classical structural biology techniques have revealed detailed snapshots of a subset of conformational states of the RNAP as they exist in crystals. A detailed view of the conformational space sampled by the RNAP and the molecular mechanisms of the basal transcription factors E (TFE) and Spt4/5 through conformational constraints has remained elusive. We monitored the conformational changes of the flexible clamp of the RNAP by combining a fluorescently labeled recombinant 12-subunit RNAP system with single-molecule FRET measurements. We measured and compared the distances across the DNA binding channel of the archaeal RNAP. Our results show that the transition of the closed to the open initiation complex, which occurs concomitant with DNA melting, is coordinated with an opening of the RNAP clamp that is stimulated by TFE. We show that the clamp in elongation complexes is modulated by the nontemplate strand and by the processivity factor Spt4/5, both of which stimulate transcription processivity. Taken together, our results reveal an intricate network of interactions within transcription complexes between RNAP, transcription factors, and nucleic acids that allosterically modulate the RNAP during the transcription cycle.

Hippocampal synaptic connectivity in phenylketonuria.

In humans, lack of phenylalanine hydroxylase (Pah) activity results in phenylketonuria (PKU), which is associated with the development of severe mental retardation after birth. The underlying mechanisms, however, are poorly understood. Mutations of the Pah gene in Pah(enu2)/c57bl6 mice result in elevated levels of phenylalanine in serum similar to those in humans suffering from PKU. In our study, long-term potentiation (LTP) and paired-pulse facilitation, measured at CA3-CA1 Schaffer collateral synapses, were impaired in acute hippocampal slices of Pah(enu2)/c57bl6 mice. In addition, we found reduced expression of presynaptic proteins, such as synaptophysin and the synaptosomal-associated protein 25 (SNAP-25), and enhanced expression of postsynaptic marker proteins, such as synaptopodin and spinophilin. Stereological counting of spine synapses at the ultrastructural level revealed higher synaptic density in the hippocampus, commencing at 3 weeks and persisting up to 12 weeks after birth. Consistent effects were seen in response to phenylalanine treatment in cultures of dissociated hippocampal neurones. Most importantly, in the hippocampus of Pah(enu2)/c57bl6 mice, we found a significant reduction in microglia activity. Reorganization of hippocampal circuitry after birth, namely synaptic pruning, relies on elimination of weak synapses by activated microglia in response to neuronal activity. Hence, our data strongly suggest that reduced microglial activity in response to impaired synaptic transmission affects physiological postnatal remodelling of synapses in the hippocampus and may trigger the development of mental retardation in PKU patients after birth.

Large T antigen variants of human polyomaviruses 9 and 12 and seroreactivity against their N terminus.

The tumour antigens (TAgs) of mammalian polyomaviruses (PyVs) are key proteins responsible for modulating the host cell cycle and are involved in virus replication as well as cell transformation and tumour formation. Here we aimed to identify mRNA sequences of known and novel TAgs encoded by the recently discovered human polyomaviruses 9 and 12 (HPyV9 and HPyV12) in cell culture. Synthetic viral genomes were transfected into human and animal cell lines. Gene expression occurred in most cell lines, as measured by quantitative PCR of cDNA copies of mRNA encoding major structural protein VP1. Large TAg- and small TAg-encoding mRNAs were detected in all cell lines, and additional spliced mRNAs were identified encoding TAg variants of 145 aa (HPyV9) and 84 aa (HPyV12). Using as antigens in ELISA the N-terminal 78 aa common to all respective TAg variants of HPyV9 and HPyV12, seroreactivity of 100 healthy blood donors, 54 patients with malignant diseases of the gastrointestinal tract (GIT) and 32 patients with non-malignant diseases of the GIT was analysed. For comparison, the corresponding TAg N termini of BK PyV (BKPyV) and Merkel cell PyV (MCPyV) were included. Frequent reactivity against HPyV9, HPyV12 and BKPyV TAgs, but not MCPyV TAg, was observed in all tested groups. This indicates expression activity of the early region of three human PyVs in healthy and diseased subjects.

A new type of Na(+)-driven ATP synthase membrane rotor with a two-carboxylate ion-coupling motif.

The anaerobic bacterium Fusobacterium nucleatum uses glutamate decarboxylation to generate a transmembrane gradient of Na⁺. Here, we demonstrate that this ion-motive force is directly coupled to ATP synthesis, via an F1F0-ATP synthase with a novel Na⁺ recognition motif, shared by other human pathogens. Molecular modeling and free-energy simulations of the rotary element of the enzyme, the c-ring, indicate Na⁺ specificity in physiological settings. Consistently, activity measurements showed Na⁺ stimulation of the enzyme, either membrane-embedded or isolated, and ATP synthesis was sensitive to the Na⁺ ionophore monensin. Furthermore, Na⁺ has a protective effect against inhibitors targeting the ion-binding sites, both in the complete ATP synthase and the isolated c-ring. Definitive evidence of Na⁺ coupling is provided by two identical crystal structures of the c11 ring, solved by X-ray crystallography at 2.2 and 2.6 Šresolution, at pH 5.3 and 8.7, respectively. Na⁺ ions occupy all binding sites, each coordinated by four amino acids and a water molecule. Intriguingly, two carboxylates instead of one mediate ion binding. Simulations and experiments demonstrate that this motif implies that a proton is concurrently bound to all sites, although Na⁺ alone drives the rotary mechanism. The structure thus reveals a new mode of ion coupling in ATP synthases and provides a basis for drug-design efforts against this opportunistic pathogen.

Fluorescently labeled recombinant RNAP system to probe archaeal transcription initiation.

The transcriptional apparatus is one of the most complex cellular machineries and in order to fully appreciate the behavior of these protein-nucleic acid assemblies one has to understand the molecular details of the system. In addition to classical biochemical and structural studies, fluorescence-based techniques turned out as an important--and sometimes the critical--tool to obtain information about the molecular mechanisms of transcription. Fluorescence is not only a multi-modal parameter that can report on molecular interactions, environment and oligomerization status. Measured on the single-molecule level it also informs about the heterogeneity of the system and gives access to distances and distance changes in the molecular relevant nanometer regime. A pre-requisite for fluorescence-based measurements is the site-specific incorporation of one or multiple fluorescent dyes. In this respect, the archaeal transcription system is ideally suited as it is available in a fully recombinant form and thus allows for site-specific modification via sophisticated labeling schemes. The application of fluorescence based approaches to the archaeal transcription apparatus changed our understanding of the molecular mechanisms and dynamics that drive archaeal transcription and unraveled the architecture of transcriptional complexes not amenable to structural interrogation.

Eukaryotic and archaeal TBP and TFB/TF(II)B follow different promoter DNA bending pathways.

During transcription initiation, the promoter DNA is recognized and bent by the basal transcription factor TATA-binding protein (TBP). Subsequent association of transcription factor B (TFB) with the TBP-DNA complex is followed by the recruitment of the ribonucleic acid polymerase resulting in the formation of the pre-initiation complex. TBP and TFB/TF(II)B are highly conserved in structure and function among the eukaryotic-archaeal domain but intriguingly have to operate under vastly different conditions. Employing single-pair fluorescence resonance energy transfer, we monitored DNA bending by eukaryotic and archaeal TBPs in the absence and presence of TFB in real-time. We observed that the lifetime of the TBP-DNA interaction differs significantly between the archaeal and eukaryotic system. We show that the eukaryotic DNA-TBP interaction is characterized by a linear, stepwise bending mechanism with an intermediate state distinguished by a distinct bending angle. TF(II)B specifically stabilizes the fully bent TBP-promoter DNA complex and we identify this step as a regulatory checkpoint. In contrast, the archaeal TBP-DNA interaction is extremely dynamic and TBP from the archaeal organism Sulfolobus acidocaldarius strictly requires TFB for DNA bending. Thus, we demonstrate that transcription initiation follows diverse pathways on the way to the formation of the pre-initiation complex.

Early and late promoters of BK polyomavirus, Merkel cell polyomavirus, Trichodysplasia spinulosa-associated polyomavirus and human polyomavirus 12 are among the strongest of all known human polyomaviruses in 10 different cell lines.

Recently, 11 new human polyomaviruses (HPyVs) have been isolated and named KI, WU, Merkel cell polyomavirus (MCPyV), HPyV6, -7, -9, -10 and -12, Trichodysplasia spinulosa-associated polyomavirus (TSPyV), STLPyV and NJPyV-2013. Little is known about cell tropism of the novel HPyVs, and cell cultures allowing virus propagation are lacking. Because viral tropism partially depends on the interaction of cellular transcription factors with the viral promoter, we monitored the promoter activity of all known HPyVs. Therefore, we compared the relative early and late promoter activity of the BK polyomavirus (BKPyV) (WW strain) with the corresponding activities of the other HPyVs in 10 different cell lines derived from brain, colon, kidney, liver, lung, the oral cavity and skin. Our results show that the BKPyV, MCPyV, TSPyV and HPyV12 early promoters displayed the strongest activity in most cell lines tested, while the remaining HPyV had relative low early promoter activity. HPyV12 showed the highest late promoter activity of all HPyVs in most cell lines, but also the BKPyV, MCPyV and TSPyV late promoters belonged to the stronger ones among HPyVs. The HPyVs with weak early promoter activity had in general also weak late promoter activity, except for HPyV10 whose late promoter was relatively strong in six of the 10 cell lines. A 20 bp deletion in the promoter of an HPyV12 variant significantly affected both early and late promoter activity in most cell lines. In conclusion, our findings suggest which cell lines may be suitable for virus propagation and may give an indication of the cell tropism of the HPyVs.

Ubiquitin-specific protease-like 1 (USPL1) is a SUMO isopeptidase with essential, non-catalytic functions.

Isopeptidases are essential regulators of protein ubiquitination and sumoylation. However, only two families of SUMO isopeptidases are at present known. Here, we report an activity-based search with the suicide inhibitor haemagglutinin (HA)-SUMO-vinylmethylester that led to the identification of a surprising new SUMO protease, ubiquitin-specific protease-like 1 (USPL1). Indeed, USPL1 neither binds nor cleaves ubiquitin, but is a potent SUMO isopeptidase both in vitro and in cells. C13orf22l--an essential but distant zebrafish homologue of USPL1--also acts on SUMO, indicating functional conservation. We have identified invariant USPL1 residues required for SUMO binding and cleavage. USPL1 is a low-abundance protein that colocalizes with coilin in Cajal bodies. Its depletion does not affect global sumoylation, but causes striking coilin mislocalization and impairs cell proliferation, functions that are not dependent on USPL1 catalytic activity. Thus, USPL1 represents a third type of SUMO protease, with essential functions in Cajal body biology.

A starting point for fluorescence-based single-molecule measurements in biomolecular research.

Single-molecule fluorescence techniques are ideally suited to provide information about the structure-function-dynamics relationship of a biomolecule as static and dynamic heterogeneity can be easily detected. However, what type of single-molecule fluorescence technique is suited for which kind of biological question and what are the obstacles on the way to a successful single-molecule microscopy experiment? In this review, we provide practical insights into fluorescence-based single-molecule experiments aiming for scientists who wish to take their experiments to the single-molecule level. We especially focus on fluorescence resonance energy transfer (FRET) experiments as these are a widely employed tool for the investigation of biomolecular mechanisms. We will guide the reader through the most critical steps that determine the success and quality of diffusion-based confocal and immobilization-based total internal reflection fluorescence microscopy. We discuss the specific chemical and photophysical requirements that make fluorescent dyes suitable for single-molecule fluorescence experiments. Most importantly, we review recently emerged photoprotection systems as well as passivation and immobilization strategies that enable the observation of fluorescently labeled molecules under biocompatible conditions. Moreover, we discuss how the optical single-molecule toolkit has been extended in recent years to capture the physiological complexity of a cell making it even more relevant for biological research.

Acetate augmentation boosts the ethanol production rate and specificity by Clostridium ljungdahlii during gas fermentation with pure carbon monoxide.

Recycling waste gases from industry is vital for the transition toward a circular economy. The model microbe Clostridium ljungdahlii reduces carbon from syngas and primarily produces acetate and ethanol. Here, a gas fermentation experiment is presented in chemostats with C. ljungdahlii and pure carbon monoxide (CO) as feedstock while entirely omitting yeast extract. A maximum ethanol production rate of 0.07 ± 0.01 g L-1 h-1 and a maximum average ethanol/acetate ratio of 1.41 ± 0.39 was observed under steady-state conditions. This confirmed that CO as the sole feedstock pushes the metabolism toward more reduced fermentation products. This effect was even more pronounced when 15 mM sodium acetate was added to the feed medium. An ethanol production rate of 0.23 ± 0.01 g L-1 h-1 was achieved, representing an increase of more than 240%. This increase was accompanied by an increase in cell density and selectivity toward ethanol, with a maximum average ethanol/acetate ratio of 92.96 ± 28.39. Oxygen contaminations voided this effect, although the cultures were still able to maintain a stable biomass concentration and ethanol production rate. These findings highlight the potential of CO-fermentation with acetate augmentation and the importance of preventing oxygen contaminations.

Novel, non-colonizing, single-strain live biotherapeutic product ADS024 protects against Clostridioides difficile infection challenge in vivo.

BACKGROUND: The Centers for Disease Control and Prevention estimate that Clostridioides difficile (C. difficile) causes half a million infections (CDI) annually and is a major cause of total infectious disease death in the United States, causing inflammation of the colon and potentially deadly diarrhea. We recently reported the isolation of ADS024, a Bacillus velezensis (B. velezensis) strain, which demonstrated direct in vitro bactericidal activity against C. difficile, with minimal collateral impact on other members of the gut microbiota. In this study, we hypothesized that in vitro activities of ADS024 will translate in vivo to protect against CDI challenge in mouse models. AIM: To investigate the in vivo efficacy of B. velezensis ADS024 in protecting against CDI challenge in mouse models. METHODS: To mimic disruption of the gut microbiota, the mice were exposed to vancomycin prior to dosing with ADS024. For the mouse single-dose study, the recovery of ADS024 was assessed via microbiological analysis of intestinal and fecal samples at 4 h, 8 h, and 24 h after a single oral dose of 5 × 108 colony-forming units (CFU)/mouse of freshly grown ADS024. The single-dose study in miniature swine included groups that had been pre-dosed with vancomycin and that had been exposed to a dose range of ADS024, and a group that was not pre-dosed with vancomycin and received a single dose of ADS024. The ADS024 colonies [assessed by quantitative polymerase chain reaction (qPCR) using ADS024-specific primers] were counted on agar plates. For the 28-d miniature swine study, qPCR was used to measure ADS024 levels from fecal samples after oral administration of ADS024 capsules containing 5 × 109 CFU for 28 consecutive days, followed by MiSeq compositional sequencing and bioinformatic analyses to measure the impact of ADS024 on microbiota. Two studies were performed to determine the efficacy of ADS024 in a mouse model of CDI: Study 1 to determine the effects of fresh ADS024 culture and ADS024 spore preparations on the clinical manifestations of CDI in mice, and Study 2 to compare the efficacy of single daily doses vs dosing 3 times per day with fresh ADS024. C. difficile challenge was performed 24 h after the start of ADS024 exposure. To model the human distal colon, an anerobic fecal fermentation system was used. MiSeq compositional sequencing and bioinformatic analyses were performed to measure microbiota diversity changes following ADS024 treatment. To assess the potential of ADS024 to be a source of antibiotic resistance, its susceptibility to 18 different antibiotics was tested. RESULTS: In a mouse model of CDI challenge, single daily doses of ADS024 were as efficacious as multiple daily doses in protecting against subsequent challenge by C. difficile pathogen-induced disease. ADS024 showed no evidence of colonization based on the observation that the ADS024 colonies were not recovered 24 h after single doses in mice or 72 h after single doses in miniature swine. In a 28-d repeat-dose study in miniature swine, ADS024 was not detected in fecal samples using plating and qPCR methods. Phylogenetic analysis performed in the human distal colon model showed that ADS024 had a selective impact on the healthy human colonic microbiota, similarly to the in vivo studies performed in miniature swine. Safety assessments indicated that ADS024 was susceptible to all the antibiotics tested, while in silico testing revealed a low potential for off-target activity or virulence and antibiotic-resistance mechanisms. CONCLUSION: Our findings, demonstrating in vivo efficacy of ADS024 in protecting against CDI challenge in mouse models, support the use of ADS024 in preventing recurrent CDI following standard antibiotic treatment.

The role of virome in the gastrointestinal tract and beyond.

The human gut virome is comprised of diverse commensal and pathogenic viruses. The colonization by these viruses begins right after birth through vaginal delivery, then continues through breastfeeding, and broader environmental exposure. Their constant interaction with their bacterial hosts in the body shapes not only our microbiomes but us. In addition, these viruses interact with the immune cells, trigger a broad range of immune responses, and influence different metabolic pathways. Besides its key role in regulating the human gut homeostasis, the intestinal virome contributes to disease development in distant organs, both directly and indirectly. In this review, we will describe the changes in the gut virome through life, health, and disease, followed by discussing the interactions between the virome, the microbiome, and the human host as well as providing an overview of their contribution to gut disease and disease of distant organs.

Discovery of Highly Active Derivatives of Daptomycin by Assessing the Effect of Amino Acid Substitutions at Positions 8 and 11 on a Daptomycin Analogue.

Daptomycin is an important antibiotic used for treating serious infections caused by Gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci. Establishing structure-activity relationships of daptomycin is important for developing new daptomycin-based antibiotics with expanded clinical applications and for tackling the ever-increasing problem of antimicrobial resistance. Toward this end, Dap-K6-E12-W13, an active analogue of daptomycin in which the uncommon amino acids in daptomycin are replaced with their common counterparts, was used as a model system for studying the effect of amino acid variation at positions 8 and 11 on in vitro biological activity against a model organism, Bacillus subtilis, and calcium-dependent insertion into model membranes. None of the new peptides were more active than Dap-K6-E12-W13; however, substitution at positions 8 and/or 11 with cationic residues resulted in little or no loss of activity, and some of these analogues were able to insert into model membranes at lower calcium ion concentrations than the parent peptide. Incorporation of these cationic residues into positions 8 and/or 11 of daptomycin itself yielded some derivatives that exhibited lower minimum inhibitory concentrations than daptomycin against B. subtilis 1046 as well as comparable and sometimes superior activity against clinical isolates of MRSA.

Identification of ADS024, a newly characterized strain of Bacillus velezensis with direct Clostridiodes difficile killing and toxin degradation bio-activities.

Clostridioides difficile infection (CDI) remains a significant health threat worldwide. C. difficile is an opportunistic, toxigenic pathogen that takes advantage of a disrupted gut microbiome to grow and produce signs and symptoms ranging from diarrhea to pseudomembranous colitis. Antibiotics used to treat C. difficile infection are usually broad spectrum and can further disrupt the commensal gut microbiota, leaving patients susceptible to recurrent C. difficile infection. There is a growing need for therapeutic options that can continue to inhibit the outgrowth of C. difficile after antibiotic treatment is completed. Treatments that degrade C. difficile toxins while having minimal collateral impact on gut bacteria are also needed to prevent recurrence. Therapeutic bacteria capable of producing a range of antimicrobial compounds, proteases, and other bioactive metabolites represent a potentially powerful tool for preventing CDI recurrence following resolution of symptoms. Here, we describe the identification and initial characterization of ADS024 (formerly ART24), a novel therapeutic bacterium that can kill C. difficile in vitro with limited impact on other commensal bacteria. In addition to directly killing C. difficile, ADS024 also produces proteases capable of degrading C. difficile toxins, the drivers of symptoms associated with most cases of CDI. ADS024 is in clinical development for the prevention of CDI recurrence as a single-strain live biotherapeutic product, and this initial data set supports further studies aimed at evaluating ADS024 in future human clinical trials.

BTEX biodegradation is linked to bacterial community assembly patterns in contaminated groundwater ecosystem.

The control of degrader populations and the stochasticity and certainty of the microbial community in contaminated groundwater are not well-understood. In this study, a long-term contaminated groundwater ecosystem was selected to investigate the impact of BTEX on microbial communities and how microbial communities respond to BTEX pollution. 16S rRNA gene sequencing and metagenomic sequencing provided insights on microbial community assemblage patterns and their role in BTEX cleaning. The operational taxonomy units (OTUs) in the contaminated groundwater ecosystem were clustered distinguishably between the Plume and the Deeper Zone (lower contaminated zone). ßNTI analysis revealed that the assembly strategies of abundant and rare OTU subcommunities preferred deterministic processes. Redundancy Analysis (RDA) and mantel testing indicated that benzene, toluene, ethylbenzene, and xylenes (BTEX) strongly drove the abundant OTU subcommunity, while the rare OTU subcommunity was only weakly affected. Deltaproteobacteria, the most dominant degrading microorganism, contains the complete degradation genes in the plume layer. In summary, our finding revealed that BTEX was the major factor in shaping the microbial community structure, and functional bacteria contribute greatly to water cleaning. Investigating the pattern of microbial community assembly will provide insights into the ecological controls of contaminant degradation in groundwater.