Genome-Directed Discovery of Bicyclic Cinnamoyl-Containing Nonribosomal Peptides with Anticoronaviral Activity from Streptomyces griseus.

Two novel cinnamoyl-containing nonribosomal peptides (CCNPs) grisgenomycin A and B were identified in Streptomyces griseus NBRC 13350 (CGMCC 4.5718) and ATCC 12475, through genome mining using conserved adjacent LuxR family regulators as probes and activators. Notably, grisgenomycins represent a new group of bicyclic decapeptides featuring an unprecedented C-C bond between the tryptophan carbocycle and the cinnamoyl group. A plausible biosynthetic pathway for grisgenomycins was deduced by a bioinformatics analysis. Grisgenomycins exhibited activity against human coronaviruses at the micromolar level.

Predominance of Escherichia-Shigella in Gut Microbiome and Its Potential Correlation with Elevated Level of Plasma Tumor Necrosis Factor Alpha in Patients with Tuberculous Meningitis.

Tuberculous meningitis (TBM), the most lethal and disabling form of tuberculosis (TB), may be related to gut microbiota composition, warranting further study. Here we systematically compared gut microbiota compositions and blood cytokine profiles of TBM patients, pulmonary TB patients, and healthy controls. Notably, the significant gut microbiota dysbiosis observed in TBM patients was associated with markedly high proportions of Escherichia-Shigella species as well as increased blood levels of tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6). Next, we obtained a fecal bacterial isolate from a TBM patient and administered it via oral gavage to mice in order to develop a murine gut microbiota dysbiosis model for use in exploring mechanisms underlying the observed relationship between gut microbial dysbiosis and TBM. Thereafter, cells of commensal Escherichia coli (E. coli) were isolated and administered to model mice by gavage and then mice were inoculated with Mycobacterium tuberculosis (M. tuberculosis). Subsequently, these mice exhibited increased blood TNF-α levels accompanied by downregulated expression of tight junction protein claudin-5, increased brain tissue bacterial burden, and elevated central nervous system inflammation relative to corresponding indicators in controls administered PBS by gavage. Thus, our results demonstrated that a signature dysbiotic gut microbiome profile containing a high proportion of E. coli was potentially associated with an increased circulating TNF-α level in TBM patients. Collectively, these results suggest that modulation of dysbiotic gut microbiota holds promise as a new strategy for preventing or alleviating TBM. IMPORTANCE As the most severe form of tuberculosis, the pathogenesis of tuberculous meningitis (TBM) is still unclear. Gut microbiota dysbiosis plays an important role in a variety of central nervous system diseases. However, the relationship between gut microbiota and TBM has not been identified. In our study, significant dysbiosis in gut microbiota composition with a high proportion of E. coli and increased levels of TNF-α in plasma was noted in TBM patients. A commensal E. coli was isolated and shown to increase the plasma level of TNF-α and downregulate brain tight junction protein claudin-5 in the murine model. Gavage administration of E. coli aggravated the bacterial burden and increased the inflammatory responses in the central nervous system after M. tuberculosis infection. Dysbiosis of gut microbiota may be a promising therapeutic target and biomarker for TBM prevention or treatment.

Significant difference in Th1/Th2 paradigm induced by tuberculosis-specific antigens between IGRA-positive and IGRA-negative patients.

False negative interferon-γ release assay (IGRA) results constitute the major dilemma for the diagnosis of tuberculosis (TB) infections. Herein, we conducted a cohort study to compare the host immunological response to TB-specific antigens between active TB patients with positive and negative IGRA results and control groups. A total of 274 laboratory-confirmed TB patients were included in our analysis, consisting of 221 were IGRA positive and 53 were IGRA negative. Patients with the elderly were identified as an independent risk factor for negative IGRA results. In addition, the elevated level of IL-4 and the decreased levels of IFN-γ, IL-2, IL-6, IL-1ß, and IL-12 in IGRA negative TB relative to IGRA positive TB group, demonstrating a significant difference in Th1/Th2 paradigm between two groups. The IFN-γ&IL-2 based assay could correctly identify 247 out of 307 MTB-infected individuals [271 TB patients and 36 individuals with latent TB infection (LTBI)], demonstrating a sensitivity of 80.5%. Then the IFN-γ and IL-4 were applied to distinguish healthy control and IGRA-negative group. When using the stepwise algorithm, the sensitivity for detecting Mycobacterium tuberculosis (MTB) infections was significantly increased from 80.5% to 89.6%. Additionally, patients with negative IGRA results had a conversion to culture-negative status longer than those with positive IGRA results. In conclusion, a stepwise algorithm outperforms IGRA assays to accurately identify MTB infections by the combination IFN-γ, IL-2, and IL-4. Further study is needed to evaluate the accuracy of our diagnostic algorithm in the LTBI population.

Increased Tuberculosis Incidence Due to Immunotherapy Based on PD-1 and PD-L1 Blockade: A Systematic Review and Meta-Analysis.

Objectives: In this study, we conducted a systematic review to determine tuberculosis (TB) incidence due to immunotherapy with programmed cell death protein-1 (PD-1)/PD ligand (PD-L1) blockade in cancer patients. Methods: We searched PubMed, Cochrance Library, Excerpt Medica Database (Embase), ClinicalTrials.gov, Chinese BioMedical Literature Database (CBM), China National Knowledge Infrastructure Database (CNKI), Wanfang and China Science and Technology Journal Database to identify studies between January 1, 2000 and April 30, 2021, on the reports of TB cases in patients treated with PD-1/PD-L1 blockade. Methodological quality of eligible studies was assessed, and random-effect model meta-analysis was performed to generate the pooled incidence estimate of TB cases in patients undergoing PD-1/PD-L1 therapy. Results: We initially identified 745 records, of which 27 studies ultimately met the inclusion criteria and were included in our meta-analysis. A total of 35 TB cases occurred among patients treated with PD-1/PD-L1 blockade. Nivolumab (51.4%) was the most frequently used PD-1/PD-L1 blockade for cancer treatment. In addition, pulmonary TB was the most common form of tuberculosis seen in 77.1% cases. Clinical outcomes were recorded in 18 patients, of whom 77.8% were cured or achieved remission, and 22.2% were died of TB. Pooled analysis determined that the TB rate in this population was 2,000 cases per 100,000 persons, and the estimated rate for TB associated with PD-1/PD-L1 blockade was 35 times higher than that in the general population. Conclusion: To conclude, our results demonstrate that the clinical use of PD-1/PD-L1 inhibitors significantly increases risk of TB reactivation. An extremely high mortality rate due to TB disease is noted in the patients with PD-1/PD-L1 blockade.

Clinical isolates of Mycobacterium tuberculosis with different genotypes exhibit distinct host macrophage responses in vitro.

Introduction. Mycobacterium tuberculosis (M.tb), the causative agent of tuberculosis, can survive as an intracellular parasite after entering macrophages via phagocytosis. M.tb strains are genotypically distinct and engage in diverse pathogen-host interactions, with different host immune responses triggered by different M.tb strains. Importantly, differences in intracellular accumulation and triggering of host macrophage responses during early infection stages are key determinants that shape the final outcomes of host innate immune responses to different M.tb strains.Hypothesis/Gap Statement. Clinical M.tb strains with different genotypes elicit different host innate immune responses in vitro.Aim. This work aimed to compare host innate immune responses elicited by genotypically diverse, clinically derived M.tb strains in vitro.Methodology. RAW264.7 cells were infected with three lineage 2 and lineage 4 clinically derived M.tb strains and strain H37Rv. Strains were evaluated for differences in intracellular growth, induction of macrophage apoptosis, and induction of expression of proinflammatory cytokines and associated pattern recognition receptors.Results. Highly variable cytokine profiles were observed subsequent to RAW264.7 cell infection with the different strains. The Beijing genotype strain, a modern Beijing strain belonging to lineage 2, induced milder host proinflammatory responses and less apoptosis and exhibited greater intracellular growth as compared to the other strains. Moreover, mRNA expression levels of iNOS in Beijing and MANU2 genotype strains exceeded corresponding levels obtained for the T1 genotype strain. Meanwhile, mRNA expression levels of toll-like receptor (TLR)-encoding genes TLR2 and TLR7 in macrophages infected with the Beijing genotype strain were higher than corresponding levels observed in MANU2 genotype strain-infected macrophages.Conclusion. The higher intracellular survival rate and lower level of host cell apoptosis associated with macrophage infection with the Beijing genotype strain indicated greater virulence of this strain relative to that of the other strains. Furthermore, in vitro immune responses induced by the Beijing genotype strain were unique in that this strain induced a weaker inflammatory response than was induced by T1 or MANU2 genotype strains. Nevertheless, additional evidence is needed to confirm that Beijing genotype strains possess greater virulence than strains with other genotypes.

Exploring novel herbicidin analogues by transcriptional regulator overexpression and MS/MS molecular networking.

BACKGROUND: Herbicidin F has an undecose tricyclic furano-pyrano-pyran structure with post-decorations. It was detected from Streptomyces mobaraensis US-43 fermentation broth as a trace component by HPLC-MS analysis. As herbicidins exhibit herbicidal, antibacterial, antifungal and antiparasitic activities, we are attracted to explore more analogues for further development. RESULTS: The genome of S. mobaraensis US-43 was sequenced and a herbicidin biosynthetic gene cluster (hcd) was localized. The cluster contains structural genes, one transporter and three potential transcription regulatory genes. Overexpression of the three regulators respectively showed that only hcdR2 overexpression significantly improved the production of herbicidin F, and obviously increased the transcripts of 7 structural genes as well as the transporter gene. After performing homology searches using BLASTP in the GenBank database, 14 hcd-like clusters were found with a cluster-situated hcdR2 homologue. These HcdR2 orthologues showed overall structural similarity, especially in the C-terminal DNA binding domain. Based on bioinformatics analysis, a 21-bp consensus binding motif of HcdR2 was detected within 30 promoter regions in these genome-mined clusters. EMSA results verified that HcdR2 bound to the predicted consensus sequence. Additionally, we employed molecular networking to explore novel herbicidin analogues in hcdR2 overexpression strain. As a result, ten herbicidin analogues including six new compounds were identified based on MS/MS fragments. Herbicidin O was further purified and confirmed by 1H NMR spectrum. CONCLUSIONS: A herbicidin biosynthetic gene cluster (hcd) was identified in S. mobaraensis US-43. HcdR2, a member of LuxR family, was identified as the pathway-specific positive regulator, and the production of herbicidin F was dramatically increased by overexpression of hcdR2. Combined with molecular networking, ten herbicidin congeners including six novel herbicidin analogues were picked out from the secondary metabolites of hcdR2 overexpression strain. The orthologues of herbicidin F pathway-specific regulator HcdR2 were present in most of the genome-mined homologous biosynthetic gene clusters, which possessed at least one consensus binding motif with LuxR family characteristic. These results indicated that the combination of overexpression of hcdR2 orthologous regulator and molecular networking might be an effective way to exploit the "cryptic" herbicidin-related biosynthetic gene clusters for discovery of novel herbicidin analogues.

Preliminary study on the virulence of XDR-TB: low virulence owing to less cytokine expression through the TLR 2 and TLR4 pathways in BLAB/C mice.

Extensively drug-resistant tuberculosis (XDR-TB) imposes a new threat to the world. In this study, XDR-TB and drug-sensitive TB (DS-TB) strains with similar genotypes were investigated. The results showed that BLAB/C mice infected with XDR-TB had reduced Toll-like receptor (TLR) 2 and TLR4 expression in lung tissues than DS-TB mice at 2 weeks post-infection. Mice infected with XDR-TB showed lower levels of TNF-α, IFN-γ, IL-4 and IL-10 in the sera than those infected with DS-TB. Moreover, mice infected with XDR-TB survived longer period and had less severe alveolar damage and smaller granulomas than those infected with DS-TB. These results were further confirmed in THP-1 cells. Using the flow cytometry and qRT-PCR methods, we found that XDR-TB stimulated lower TLR2 and TLR4 expression. Based on the above results, we cautiously inferred that low virulence of XDR-TB owed to the less cytokine expression through the TLR 2 and 4 pathways. The effect of XDR-TB and DS-TB can be further tested in TLR2 or TLR4 knock-out mice and TLRs-silenced THP-1 cells.

[Influences of Mycobacterium tuberculosis on the levels of human acute monocytic leukemia cell line THP-1 apoptosis and death].

OBJECTIVE: To explore the influences of Mycobacterium tuberculosis on the levels of human acute monocytic leukemia cell line THP-1 apoptosis and death. METHODS: Human acute monocytic leukemia cell line THP-1 were infected with Mycobacterium tuberculosis strains H37Ra, H37Rv, or Beijing genotype (BJTB), respectively, to construct the infection models. Cell apoptosis was detected using flow cytometry. The distribution of the apoptotic proteins was detected using immunofluorescent staining assays. The cells late apoptosis was detected using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining assays. The change of cell death was determined by Tyrpan blue staining assays. RESULTS: THP-1 apoptosis was induced by Mycobacterium tuberculosis strains H37Ra, H37Rv, and BJTB. H37Ra strongly induced THP-1 apoptosis, H37Rv weakly induced THP-1 apoptosis, and BJTB induced THP-1 apoptosis at the lowest level among these three Mycobacterium tuberculosis strains. On the contrary, BJTB strongly induced THP-1 death, H37Rv weakly induced THP-1 death, and H37Ra induced THP-1 death at the lowest level. CONCLUSIONS: Mycobacterial strains with different virulence induce different levels of apoptosis and death of THP-1 cells. Compared with highly virulent strains, attenuated strains induce more apoptosis and less death.