Intracellular delivery of nuclear localization sequence peptide mitigates COVID-19 by inhibiting nuclear transport of inflammation-associated transcription factors.

The novel severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), responsible for coronavirus disease 2019 (COVID-19), can trigger dysregulated immune responses known as the cytokine release syndrome (CRS), leading to severe organ dysfunction and respiratory distress. Our study focuses on developing an improved cell-permeable nuclear import inhibitor (iCP-NI), capable of blocking the nuclear transport of inflammation-associated transcription factors, specifically nuclear factor kappa B (NF-κB). By fusing advanced macromolecule transduction domains and nuclear localization sequences from human NF-κB, iCP-NI selectively interacts with importin α5, effectively reducing the expression of proinflammatory cytokines. In mouse models mimic SARS-CoV-2-induced pneumonitis, iCP-NI treatment demonstrated a significant decrease in mortality rates by suppressing proinflammatory cytokine production and immune cell infiltration in the lungs. Similarly, in hamsters infected with SARS-CoV-2, iCP-NI effectively protected the lung from inflammatory damage by reducing tumor necrosis factor-α, interleukin-6 (IL-6), and IL-17 levels. These promising results highlight the potential of iCP-NI as a therapeutic approach for COVID-19-related lung complications and other inflammatory lung diseases.

Genome-wide association study of signature genetic alterations among pseudomonas aeruginosa cystic fibrosis isolates.

Pseudomonas aeruginosa (PA) is an opportunistic pathogen that causes diverse human infections including chronic airway infection in patients with cystic fibrosis (CF). Comparing the genomes of CF and non-CF PA isolates has great potential to identify the genetic basis of pathogenicity. To gain a deeper understanding of PA adaptation in CF airways, we performed a genome-wide association study (GWAS) on 1,001 PA genomes. Genetic variations identified among CF isolates were categorized into (i) alterations in protein-coding regions, either large- or small-scale, and (ii) polymorphic variation in intergenic regions. We introduced each CF-associated genetic alteration into the genome of PAO1, a prototype PA strain, and validated the outcomes experimentally. Loci readily mutated among CF isolates included genes encoding a probable sulfatase, a probable TonB-dependent receptor (PA2332~PA2336), L-cystine transporter (YecS, PA0313), and a probable transcriptional regulator (PA5438). A promoter region of a heme/hemoglobin uptake outer membrane receptor (PhuR, PA4710) was also different between the CF and non-CF isolate groups. Our analysis highlights ways in which the PA genome evolves to survive and persist within the context of chronic CF infection.

Chemical inhibitors of the conserved bacterial transcriptional regulator DksA1 suppressed quorum sensing-mediated virulence of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen whose virulence is dependent on quorum sensing (QS). DksA1, an RNA polymerase-binding transcriptional regulator, plays a role in determining a number of phenotypes, including QS-mediated virulence. We therefore envisioned that DksA1 inhibitors may help to control P. aeruginosa infection. Here, we screened a library of 6970 chemical compounds and identified two compounds (henceforth termed Dkstatins) that specifically suppressed DksA1 activity. Treatment with these two compounds also substantially decreased the production of elastase and pyocyanin, dominant virulence determinants of P. aeruginosa, and protected murine hosts from lethal infection from a prototype strain of P. aeruginosa, PAO1. The Dkstatins also suppressed production of homoserine lactone (HSL)-based autoinducers that activate P. aeruginosa QS. The level of 3-oxo-C12-HSL produced by Dkstatin-treated wildtype PAO1 closely resembled that of the ΔdksA1 mutant. RNA-Seq analysis showed that transcription levels of QS- and virulence-associated genes were markedly reduced in Dkstatin-treated PAO1 cells, indicating that Dkstatin-mediated suppression occurs at the transcriptional level. Importantly, Dkstatins increased the antibiotic susceptibilities of PAO1, particularly to protein synthesis inhibitors, such as tobramycin and tetracycline. Co-immunoprecipitation assays demonstrated that these Dkstatins interfered with DksA1 binding to the ß subunit of RNA polymerase, pointing to a potential mechanism of action. Collectively, our results illustrate that inhibition of P. aeruginosa QS may be achieved via DksA1 inhibitors and that Dkstatins may serve as potential lead compounds to control infection.

Transcriptome analysis reveals that the RNA polymerase-binding protein DksA1 has pleiotropic functions in Pseudomonas aeruginosa.

The stringent response (SR) is a highly conserved stress response in bacteria. It is composed of two factors, (i) a nucleotide alarmone, guanosine tetra- and pentaphosphate ((p)ppGpp), and (ii) an RNA polymerase-binding protein, DksA, that regulates various phenotypes, including bacterial virulence. The clinically significant opportunistic bacterial pathogen Pseudomonas aeruginosa possesses two genes, dksA1 and dksA2, that encode DksA proteins. It remains elusive, however, which of these two genes plays a more important role in SR regulation. In this work, we compared genome-wide, RNA-Seq-based transcriptome profiles of ΔdksA1, ΔdksA2, and ΔdksA1ΔdksA2 mutants to globally assess the effects of these gene deletions on transcript levels coupled with phenotypic analyses. The ΔdksA1 mutant exhibited substantial defects in a wide range of phenotypes, including quorum sensing (QS), anaerobiosis, and motility, whereas the ΔdksA2 mutant exhibited no significant phenotypic changes, suggesting that the dksA2 gene may not have an essential function in P. aeruginosa under the conditions used here. Of note, the ΔdksA1 mutants displayed substantially increased transcription of genes involved in polyamine biosynthesis, and we also detected increased polyamine levels in these mutants. Because SAM is a shared precursor for the production of both QS autoinducers and polyamines, these findings suggest that DksA1 deficiency skews the flow of SAM toward polyamine production rather than to QS signaling. Together, our results indicate that DksA1, but not DksA2, controls many important phenotypes in P. aeruginosa We conclude that DksA1 may represent a potential target whose inhibition may help manage recalcitrant P. aeruginosa infections.

Microbiome and mycobiome interaction in house dust mites and impact on airway cells.

BACKGROUND: Major allergen sources Dermatophagoides farinae, Dermatophagoides pteronyssinus and Tyrophagus putrescentiae have been reported to have distinct microbiomes. The purpose of this study was to investigate the effect of each mite on airway epithelial cells as a model of airway allergic disease. METHODS: Transcriptomic analysis (RNA-seq) of an airway epithelial cell line (BEAS-2B) was performed to compare gene expression patterns after treatment with extracts of three mite species (D. farinae, D. pteronyssinus and T. putrescentiae). In addition, mycobiome deep sequencing of mites was employed to identify fungal species that interact with the microbiomes of the mites. RESULTS: Immune responses to bacteria were enriched only in the D. farinae-treated group as this species harboured larger numbers of bacteria than the other mites, and the high level of LPS in D. farinae caused proinflammatory cytokine production in airway epithelial cells. In addition, antibiotic metabolism pathways were enriched in D. pteronyssinus-treated cells but not in D. farinae -treated cells. Subsequent experiments revealed that D. pteronyssinus had a high fungal load that inhibited bacterial survival in this mite species. CONCLUSION: The large amount of bacteria in D. farinae causes airway epithelial cells to produce more allergy-related cytokines than D. pteronyssinus, since fungi present in D. pteronyssinus suppress the abundance of mite-associated bacteria.

The Effect of Formula-based Nutritional Treatment on Colitis in a Murine Model.

BACKGROUND: Exclusive enteral nutrition (EEN) induces remission in pediatric Crohn's disease (CD). The exact mechanism of EEN therapy in CD is unknown, but alteration of the intestinal microflora after EEN is thought to affect mucosal healing. To determine the link between EEN therapy and therapeutic efficacy in CD, we established a murine model of dextran sulfate sodium (DSS)-induced colitis and applied EEN therapy. METHODS: Eight-week-old C57BL/6 mice were administered DSS for 4 days to induce colitis, and either normal chow (NC) or EEN was administered for the following 4 days. The mice were grouped according to the feeding pattern after DSS administration: DSS/NC and DSS/EEN groups. The clinical course was confirmed via daily observation of the weight and stool. Fecal samples were collected and 16sRNA sequencing was used. The mice were sacrificed to confirm colonic histopathology. RESULTS: Weight reduction and increase in disease activity were observed as the day progressed for 4 days after DSS administration. There was significant weight recovery and improvement in disease activity in the EEN group compared to that in the NC group. Verrucomicrobia and Proteobacteria abundances tended to increase and Bacteroidetes abundance decreased in the EEN group. In the EEN group, significant changes in the ß-diversity of the microbiota were observed. In the analysis of microbiome species, abundances of Akkermansia muciniphila, Clostridium cocleatum, mucin-degrading bacteria, Flintibacter butyricus, and Parabacteroides goldsteinii, which are beneficial microbiota, were significantly increased in the EEN group compared to those in the NC group. More abundant mucins were confirmed in the colonic histopathology of the EEN group. These microbial and histopathological differences suggested that EEN might improve colitis symptoms in a murine colitis model by promoting mucin recycling and subsequently inducing the healing effect of the gut barrier. CONCLUSION: EEN showed clinical efficacy in a murine model of colitis. Based on the increase in mucin-degrading bacteria and the pathological increase in mucin production after EEN administration, it can be observed that mucin plays an important role in the therapeutic effect of EEN.

IL-17C Protects Nasal Epithelium from Pseudomonas aeruginosa Infection.

IL-17 family cytokines are directly involved in host immune responses and the critical mediators for host defense against infection or inflammation. IL-17C is highly expressed in respiratory epithelium and is induced after acute bacterial lung infection. However, the definite function of IL-17C induced by Pseudomonas aeruginosa (PAO1 strain) is not fully understood, and our study was designed to demonstrate IL-17C-induced immune response against PAO1 infection in nasal epithelium. Passage-2 normal human nasal epithelial (NHNE) cells were infected with PAO1 and the relationship between IL-17C-related immune responses and the iron absorption of PAO1, depending on inoculation of recombinant human IL-17C (rhIL-17C), was assessed by measuring the siderophore activity of PAO1. Microarray data showed that IL-17C expression increased 34.7 times at 8 hours postinfection (hpi) in NHNE cells, and IL-17C mRNA levels increased until 48 hpi. The PAO1 colonies significantly increased from 8 hpi in NHNE cells, and siderophore activity of PAO1 was enhanced in the supernatants of PAO1-infected NHNE cells. Interestingly, PAO1 colonies were reduced in PAO1-infected NHNE cells treated with rhIL-17C, and supernatants from NHNE cells treated with rhIL-17C also exhibited decreased PAO1 colonies. We found that the siderophore activity of PAO1 was significantly reduced in the supernatants of NHNE cells treated with rhIL-17C where LCN2 expression was highly elevated. Our findings indicate that IL-17C mediates an antibacterial effect against PAO1 by inhibiting siderophore activity in nasal epithelium. We propose that IL-17C might be an efficient mediator to suppress PAO1 infection through disturbing iron absorption of PAO1 in nasal epithelium.

Guanosine tetra- and pentaphosphate increase antibiotic tolerance by reducing reactive oxygen species production in Vibrio cholerae.

The pathogen Vibrio cholerae is the causative agent of cholera. Emergence of antibiotic-resistant V. cholerae strains is increasing, but the underlying mechanisms remain unclear. Herein, we report that the stringent response regulator and stress alarmone guanosine tetra- and pentaphosphate ((p)ppGpp) significantly contributes to antibiotic tolerance in V. cholerae We found that N16961, a pandemic V. cholerae strain, and its isogenic (p)ppGpp-overexpressing mutant ΔrelAΔspoT are both more antibiotic-resistant than (p)ppGpp0 (ΔrelAΔrelVΔspoT) and ΔdksA mutants, which cannot produce or utilize (p)ppGpp, respectively. We also found that additional disruption of the aconitase B-encoding and tricarboxylic acid (TCA) cycle gene acnB in the (p)ppGpp0 mutant increases its antibiotic tolerance. Moreover, expression of TCA cycle genes, including acnB, was increased in (p)ppGpp0, but not in the antibiotic-resistant ΔrelAΔspoT mutant, suggesting that (p)ppGpp suppresses TCA cycle activity, thereby entailing antibiotic resistance. Importantly, when grown anaerobically or incubated with an iron chelator, the (p)ppGpp0 mutant became antibiotic-tolerant, suggesting that reactive oxygen species (ROS) are involved in antibiotic-mediated bacterial killing. Consistent with that hypothesis, tetracycline treatment markedly increased ROS production in the antibiotic-susceptible mutants. Interestingly, expression of the Fe(III) ABC transporter substrate-binding protein FbpA was increased 10-fold in (p)ppGpp0, and fbpA gene deletion restored viability of tetracycline-exposed (p)ppGpp0 cells. Of note, FbpA expression was repressed in the (p)ppGpp-accumulating mutant, resulting in a reduction of intracellular free iron, required for the ROS-generating Fenton reaction. Our results indicate that (p)ppGpp-mediated suppression of central metabolism and iron uptake reduces antibiotic-induced oxidative stress in V. cholerae.

Molecular Determinants of the Thickened Matrix in a Dual-Species Pseudomonas aeruginosa and Enterococcus faecalis Biofilm.

Biofilms are microbial communities that inhabit various surfaces and are surrounded by extracellular matrices (ECMs). Clinical microbiologists have shown that the majority of chronic infections are caused by biofilms, following the introduction of the first biofilm infection model by J. W. Costerton and colleagues (J. Lam, R. Chan, K. Lam, and J. W. Costerton, Infect Immun 28:546-556, 1980). However, treatments for chronic biofilm infections are still limited to surgical removal of the infected sites. Pseudomonas aeruginosa and Enterococcus faecalis are two frequently identified bacterial species in biofilm infections; nevertheless, the interactions between these two species, especially during biofilm growth, are not clearly understood. In this study, we observed phenotypic changes in a dual-species biofilm of P. aeruginosa and E. faecalis, including a dramatic increase in biofilm matrix thickness. For clear elucidation of the spatial distribution of the dual-species biofilm, P. aeruginosa and E. faecalis were labeled with red and green fluorescence, respectively. E. faecalis was located at the lower part of the dual-species biofilm, while P. aeruginosa developed a structured biofilm on the upper part. Mutants with altered exopolysaccharide (EPS) productions were constructed in order to determine the molecular basis for the synergistic effect of the dual-species biofilm. Increased biofilm matrix thickness was associated with EPSs, not extracellular DNA. In particular, Pel and Psl contributed to interspecies and intraspecies interactions, respectively, in the dual-species P. aeruginosa and E. faecalis biofilm. Accordingly, targeting Pel and Psl might be an effective part of eradicating P. aeruginosa polymicrobial biofilms.IMPORTANCE Chronic infection is a serious problem in the medical field. Scientists have observed that chronic infections are closely associated with biofilms, and the vast majority of infection-causing biofilms are polymicrobial. Many studies have reported that microbes in polymicrobial biofilms interact with each other and that the bacterial interactions result in elevated virulence, in terms of factors, such as infectivity and antibiotic resistance. Pseudomonas aeruginosa and Enterococcus faecalis are frequently isolated pathogens in chronic biofilm infections. Nevertheless, while both bacteria are known to be agents of numerous nosocomial infections and can cause serious diseases, interactions between the bacteria in biofilms have rarely been examined. In this investigation, we aimed to characterize P. aeruginosa and E. faecalis dual-species biofilms and to determine the molecular factors that cause synergistic effects, especially on the matrix thickening of the biofilm. We suspect that our findings will contribute to the development of more efficient methods for eradicating polymicrobial biofilm infections.

(p)ppGpp, a Small Nucleotide Regulator, Directs the Metabolic Fate of Glucose in Vibrio cholerae.

When V. cholerae encounters nutritional stress, it activates (p)ppGpp-mediated stringent response. The genes relA and relV are involved in the production of (p)ppGpp, whereas the spoT gene encodes an enzyme that hydrolyzes it. Herein, we show that the bacterial capability to produce (p)ppGpp plays an essential role in glucose metabolism. The V. cholerae mutants defective in (p)ppGpp production (i.e. ΔrelAΔrelV and ΔrelAΔrelVΔspoT mutants) lost their viability because of uncontrolled production of organic acids, when grown with extra glucose. In contrast, the ΔrelAΔspoT mutant, a (p)ppGpp overproducer strain, exhibited better growth in the presence of the same glucose concentration. An RNA sequencing analysis demonstrated that transcriptions of genes consisting of an operon for acetoin biosynthesis were markedly elevated in N16961, a seventh pandemic O1 strain, but not in its (p)ppGpp(0) mutant during glucose-stimulated growth. Transposon insertion in acetoin biosynthesis gene cluster resulted in glucose-induced loss of viability of the ΔrelAΔspoT mutant, further suggesting the crucial role of acetoin production in balanced growth under glucose-rich environments. Additional deletion of the aphA gene, encoding a negative regulator for acetoin production, failed to rescue the (p)ppGpp(0) mutant from the defective glucose-mediated growth, suggesting that (p)ppGpp-mediated acetoin production occurs independent of the presence of AphA. Overall, our results reveal that (p)ppGpp, in addition to its well known role as a stringent response mediator, positively regulates acetoin production that contributes to the successful glucose metabolism and consequently the proliferation of V. cholerae cells under a glucose-rich environment, a condition that may mimic the human intestine.

Cholera toxin production during anaerobic trimethylamine N-oxide respiration is mediated by stringent response in Vibrio cholerae.

As a facultative anaerobe, Vibrio cholerae can grow by anaerobic respiration. Production of cholera toxin (CT), a major virulence factor of V. cholerae, is highly promoted during anaerobic growth using trimethylamine N-oxide (TMAO) as an alternative electron acceptor. Here, we investigated the molecular mechanisms of TMAO-stimulated CT production and uncovered the crucial involvement of stringent response in this process. V. cholerae 7th pandemic strain N16961 produced a significantly elevated level of ppGpp, the bacterial stringent response alarmone, during anaerobic TMAO respiration. Bacterial viability was impaired, and DNA replication was also affected under the same growth condition, further suggesting that stringent response is induced. A ΔrelA ΔspoT ppGpp overproducer strain produced an enhanced level of CT, whereas anaerobic growth via TMAO respiration was severely inhibited. In contrast, a ppGpp-null strain (ΔrelA ΔspoT ΔrelV) grew substantially better, but produced no CT, suggesting that CT production and bacterial growth are inversely regulated in response to ppGpp accumulation. Bacterial capability to produce CT was completely lost when the dksA gene, which encodes a protein that works cooperatively with ppGpp, was deleted. In the ΔdksA mutant, stringent response growth inhibition was alleviated, further supporting the inverse regulation of CT production and anaerobic growth. In vivo virulence of ΔrelA ΔspoT ΔrelV or ΔdksA mutants was significantly attenuated. The ΔrelA ΔspoT mutant maintained virulence when infected with exogenous TMAO despite its defective growth. Together, our results reveal that stringent response is activated under TMAO-stimulated anaerobic growth, and it regulates CT production in a growth-dependent manner in V. cholerae.

A drug-repositioning screening identifies pentetic acid as a potential therapeutic agent for suppressing the elastase-mediated virulence of Pseudomonas aeruginosa.

Pseudomonas aeruginosa, a Gram-negative bacterium of clinical significance, produces elastase as a predominant exoprotease. Here, we screened a library of chemical compounds currently used for human medication and identified diethylene triamine penta-acetic acid (DTPA, pentetic acid) as an agent that suppresses the production of elastase. Elastase activity found in the prototype P. aeruginosa strain PAO1 was significantly decreased when grown with a concentration as low as 20 µM DTPA. Supplementation with Zn(2+) or Mn(2+) ions restored the suppressive effect of DTPA, suggesting that the DTPA-mediated decrease in elastase activity is associated with ion-chelating activity. In DTPA-treated PAO1 cells, transcription of the elastase-encoding lasB gene and levels of the Pseudomonas quinolone signal (PQS), a molecule that mediates P. aeruginosa quorum sensing (QS), were significantly downregulated, reflecting the potential involvement of the PQS QS system in DTPA-mediated elastase suppression. Biofilm formation was also decreased by DTPA treatment. When A549 alveolar type II-like adenocarcinoma cells were infected with PAO1 cells in the presence of DTPA, A549 cell viability was substantially increased. Furthermore, the intranasal delivery of DTPA to PAO1-infected mice alleviated the pathogenic effects of PAO1 cells in the animals. Together, our results revealed a novel function for a known molecule that may help treat P. aeruginosa airway infection.

Airway epithelial CD47 plays a critical role in inducing influenza virus-mediated bacterial super-infection.

Respiratory viral infection increases host susceptibility to secondary bacterial infections, yet the precise dynamics within airway epithelia remain elusive. Here, we elucidate the pivotal role of CD47 in the airway epithelium during bacterial super-infection. We demonstrated that upon influenza virus infection, CD47 expression was upregulated and localized on the apical surface of ciliated cells within primary human nasal or bronchial epithelial cells. This induced CD47 exposure provided attachment sites for Staphylococcus aureus, thereby compromising the epithelial barrier integrity. Through bacterial adhesion assays and in vitro pull-down assays, we identified fibronectin-binding proteins (FnBP) of S. aureus as a key component that binds to CD47. Furthermore, we found that ciliated cell-specific CD47 deficiency or neutralizing antibody-mediated CD47 inactivation enhanced in vivo survival rates. These findings suggest that interfering with the interaction between airway epithelial CD47 and pathogenic bacterial FnBP holds promise for alleviating the adverse effects of super-infection.

Activation of cholera toxin production by anaerobic respiration of trimethylamine N-oxide in Vibrio cholerae.

Vibrio cholerae is a gram-negative bacterium that causes cholera. Although the pathogenesis caused by this deadly pathogen takes place in the intestine, commonly thought to be anaerobic, anaerobiosis-induced virulence regulations are not fully elucidated. Anerobic growth of the V. cholerae strain, N16961, was promoted when trimethylamine N-oxide (TMAO) was used as an alternative electron acceptor. Strikingly, cholera toxin (CT) production was markedly induced during anaerobic TMAO respiration. N16961 mutants unable to metabolize TMAO were incapable of producing CT, suggesting a mechanistic link between anaerobic TMAO respiration and CT production. TMAO reductase is transported to the periplasm via the twin arginine transport (TAT) system. A similar defect in both anaerobic TMAO respiration and CT production was also observed in a N16961 TAT mutant. In contrast, the abilities to grow on TMAO and to produce CT were not affected in a mutant of the general secretion pathway. This suggests that V. cholerae may utilize the TAT system to secrete CT during TMAO respiration. During anaerobic growth with TMAO, N16961 cells exhibit green fluorescence when stained with 2',7'-dichlorofluorescein diacetate, a specific dye for reactive oxygen species (ROS). Furthermore, CT production was decreased in the presence of an ROS scavenger suggesting a positive role of ROS in regulating CT production. When TMAO was co-administered to infant mice infected with N16961, the mice exhibited more severe pathogenic symptoms. Together, our results reveal a novel anaerobic growth condition that stimulates V. cholerae to produce its major virulence factor.

Dissemination of metallo-ß-lactamase-producing Pseudomonas aeruginosa of sequence type 235 in Asian countries.

OBJECTIVES: To investigate the epidemiological traits of metallo-ß-lactamase (MBL)-producing Pseudomonas aeruginosa (MPPA) clinical isolates collected by the Asian Network for Surveillance of Resistant Pathogens (ANSORP). METHODS: A total of 16 MPPA clinical isolates were collected from six Asian countries in 2000 to 2009 by ANSORP. The MBL gene was detected by PCR amplification. The genetic organization of the class 1 integron carrying the MBL gene cassette was investigated by PCR mapping and sequencing. Southern blotting, repetitive sequence-based PCR and multilocus sequence typing (MLST) experiments were performed to characterize the isolates. RESULTS: PCR and sequencing experiments detected the blaVIM-2 (n = 12), blaVIM-3 (n = 1), blaIMP-6 (n = 2) and blaIMP-26 (n = 1) genes. The MBL genes were located on the chromosome in all isolates except one. Furthermore, all the MBL genes were located in a class 1 integron. All the MPPA isolates from Malaysia, Thailand, Sri Lanka and Korea were identified as sequence type (ST) 235 by MLST. Three VIM-2-producing isolates from India were identified as ST773, and one isolate harbouring VIM-3 from Taiwan was identified as ST298. CONCLUSIONS: P. aeruginosa ST235 might play a role in dissemination of MBL genes in Asian countries.

Functional screening of a metagenomic library reveals operons responsible for enhanced intestinal colonization by gut commensal microbes.

Evidence suggests that gut microbes colonize the mammalian intestine through propagation as an adhesive microbial community. A bacterial artificial chromosome (BAC) library of murine bowel microbiota DNA in the surrogate host Escherichia coli DH10B was screened for enhanced adherence capability. Two out of 5,472 DH10B clones, 10G6 and 25G1, exhibited enhanced capabilities to adhere to inanimate surfaces in functional screens. DNA segments inserted into the 10G6 and 25G1 clones were 52 and 41 kb and included 47 and 41 protein-coding open reading frames (ORFs), respectively. DNA sequence alignments, tetranucleotide frequency, and codon usage analysis strongly suggest that these two DNA fragments are derived from species belonging to the genus Bacteroides. Consistent with this finding, a large portion of the predicted gene products were highly homologous to those of Bacteroides spp. Transposon mutagenesis and subsequent experiments that involved heterologous expression identified two operons associated with enhanced adherence. E. coli strains transformed with the 10a or 25b operon adhered to the surface of intestinal epithelium and colonized the mouse intestine more vigorously than did the control strain. This study has revealed the genetic determinants of unknown commensals (probably resembling Bacteroides species) that enhance the ability of the bacteria to colonize the murine bowel.

Gut commensal Kineothrix alysoides mitigates liver dysfunction by restoring lipid metabolism and gut microbial balance.

Metabolic dysfunction-associated steatotic liver disease (MASLD), previously known as Non-Alcoholic Fatty Liver Disease, is a widespread liver condition characterized by excessive fat buildup in hepatocytes without significant alcohol consumption. Manipulation of the gut microbiome has been considered to prevent and improve the occurrence and progression of MASLD, particularly through the gut-liver axis. This study aimed to investigate the correlation between the gut microbiome and liver function and determine whether the gut microbiome can ameliorate MASLD. We comparatively analyzed the gut microbiome composition between mice fed normal chow and those fed a high-fat diet and observed that the abundance of Kineothrix alysoides decreased in the high-fat group. Further analysis showed that treatment with K. alysoides in the high-fat diet group led to decreased weight loss, and MASLD attenuation. Importantly, K. alysoides treatment attenuated MASLD in mice fed a high-fat, high-fructose diet (HFHF), which can cause advanced liver damage. Furthermore, administration of K. alysoides altered the gut microbial composition in the HFHF diet group and improved MASLD. Overall, these findings demonstrate the potential of K. alysoides in restoring gut health and facilitating lipid metabolism to prevent and treat MASLD.

How Deep Learning in Antiviral Molecular Profiling Identified Anti-SARS-CoV-2 Inhibitors.

The integration of artificial intelligence (AI) into drug discovery has markedly advanced the search for effective therapeutics. In our study, we employed a comprehensive computational-experimental approach to identify potential anti-SARS-CoV-2 compounds. We developed a predictive model to assess the activities of compounds based on their structural features. This model screened a library of approximately 700,000 compounds, culminating in the selection of the top 100 candidates for experimental validation. In vitro assays on human intestinal epithelial cells (Caco-2) revealed that 19 of these compounds exhibited inhibitory activity. Notably, eight compounds demonstrated dose-dependent activity in Vero cell lines, with half-maximal effective concentration (EC50) values ranging from 1 µM to 7 µM. Furthermore, we utilized a clustering approach to pinpoint potential nucleoside analog inhibitors, leading to the discovery of two promising candidates: azathioprine and its metabolite, thioinosinic acid. Both compounds showed in vitro activity against SARS-CoV-2, with thioinosinic acid also significantly reducing viral loads in mouse lungs. These findings underscore the utility of AI in accelerating drug discovery processes.

A Moonlighting Protein Secreted by a Nasal Microbiome Fortifies the Innate Host Defense Against Bacterial and Viral Infections.

Evidence suggests that the human respiratory tract, as with the gastrointestinal tract, has evolved to its current state in association with commensal microbes. However, little is known about how the airway microbiome affects the development of airway immune system. Here, we uncover a previously unidentified mode of interaction between host airway immunity and a unique strain (AIT01) of Staphylococcus epidermidis, a predominant species of the nasal microbiome. Intranasal administration of AIT01 increased the population of neutrophils and monocytes in mouse lungs. The recruitment of these immune cells resulted in the protection of the murine host against infection by Pseudomonas aeruginosa, a pathogenic bacterium. Interestingly, an AIT01-secreted protein identified as GAPDH, a well-known bacterial moonlighting protein, mediated this protective effect. Intranasal delivery of the purified GAPDH conferred significant resistance against other Gram-negative pathogens (Klebsiella pneumoniae and Acinetobacter baumannii) and influenza A virus. Our findings demonstrate the potential of a native nasal microbe and its secretory protein to enhance innate immune defense against airway infections. These results offer a promising preventive measure, particularly relevant in the context of global pandemics.

Vitamin B12-mediated restoration of defective anaerobic growth leads to reduced biofilm formation in Pseudomonas aeruginosa.

Pseudomonas aeruginosa undergoes cell elongation and forms robust biofilms during anaerobic respiratory growth using nitrate (NO(3)(-)) as an alternative electron acceptor. Understanding the mechanism of cell shape change induced upon anaerobiosis is crucial to the development of effective treatments against P. aeruginosa biofilm infection. Here, we uncovered the molecular basis of anaerobiosis-triggered cell elongation and identified vitamin B(12) to be a molecule that can reinstate defective anaerobic growth of P. aeruginosa. The ratio of total cellular DNA content to protein content was significantly decreased in the PAO1 strain grown under anaerobic conditions, indicating that DNA replication is impaired during anaerobic growth. Anaerobic growth of PAO1 reached a higher cell density in the presence of vitamin B(12), an essential coenzyme of class II ribonucleotide reductase. In addition, cell morphology returned to a normal rod shape and transcription of stress-response genes was downregulated under the same anaerobic growth conditions. These results suggest that vitamin B(12), the production of which was suppressed during anaerobic growth, can restore cellular machineries for DNA replication and therefore facilitate better anaerobic growth of P. aeruginosa with normal cell division. Importantly, biofilm formation was substantially decreased when grown with vitamin B(12), further demonstrating that anaerobiosis-induced cell elongation is responsible for robust biofilm formation. Taken together, our data reveal mechanistic details of a morphological change that naturally occurs during anaerobic growth of P. aeruginosa and illustrates the ability of vitamin B(12) to modulate the biofilm-forming capacity of P. aeruginosa under such condition.