Transcriptomic Analysis of Mycobacterial Infected Macrophages Reveals a High MOI Specific Type I IFN Signaling.

Macrophage (MΦ) infection models are important tools for studying host-mycobacterial interactions. Although the multiplicity of infection (MOI) is an important experimental variable, the selection of MOI in mycobacterial infection experiments is largely empirical, without reference to solid experimental data. To provide relevant data, we used RNA-seq to analyze the gene expression profiles of MΦs 4 or 24 h after infection with Mycobacterium marinum (M. m) at MOIs ranging from 0.1 to 50. Analysis of differentially expressed genes (DEGs) showed that different MOIs are linked to distinct transcriptomic changes and only 10% of DEGs were shared by MΦ infected at all MOIs. KEGG pathway enrichment analysis revealed that type I interferon (IFN)-related pathways were inoculant dose-dependent and enriched only at high MOIs, whereas TNF pathways were inoculant dose-independent and enriched at all MOIs. Protein-protein interaction (PPI) network alignment showed that different MOIs had distinct key node genes. By fluorescence-activated cell sorting and follow-up RT-PCR analysis, we could separate infected MΦs from uninfected MΦs and found phagocytosis of mycobacteria to be the determinant factor for type I IFN production. The distinct transcriptional regulation of RAW264.7 MΦ genes at different MOIs was also seen with Mycobacterium tuberculosis (M.tb) infections and primary MΦ infection models. In summary, transcriptional profiling of mycobacterial infected MΦs revealed that different MOIs activate distinct immune pathways and the type I IFN pathway is activated only at high MOIs. This study should provide guidance for selecting the MOI most appropriate for different research questions.

Association of HTR1A Gene Polymorphisms with Efficacy and Plasma Concentrations of Atypical Antipsychotics in the Treatment of Male Patients with Schizophrenia.

Purpose: We investigate the association of HTR1A rs10042486 and rs6295 with efficacy and plasma concentrations of atypical antipsychotics in the treatment of male patients with schizophrenia. Patients and Methods: A total of 140 male patients diagnosed with schizophrenia who were treated with any single atypical antipsychotic between May 2020 and May 2022 were retrospectively included. Clinical symptoms were assessed using Positive and Negative Syndrome Scale (PANSS). All SNPs were typed using Agena Bioscience MassARRAY DNA mass spectrometry. Plasma concentrations of antipsychotics at week 3, 6 and 12 after treatment commence were analyzed using mass spectrometry. Results: For efficacy of atypical antipsychotics, we observed no significant difference between HTR1A rs10042486, rs6295 and positive symptom improvement, where the patients with heterozygous mutant at the rs10042486 and rs6295 locus were superior to those with wild-type or homozygous mutant genotypes on negative symptom improvement, especially at 12 weeks of follow-up when the difference between genotypes at the rs6295 locus have statistical significance (P = 0.037). For plasma concentration, we found that quetiapine plasma concentrations were significantly lower in patients with mutation-heterozygous types than in wild-type and homozygous mutation genotypes at week 6. In contrast, higher plasma concentrations were found for mutant heterozygous than wild genotypes in the risperidone monotherapy analysis, and the difference among genotypes at the rs6295 locus was statistically significant at 6 weeks of follow-up. Conclusion: The assessment of the correlation of genetic polymorphisms of HTR1A rs6295 and rs10042486 in male patients with schizophrenia with the monitoring of therapeutic drug concentrations and therapeutic efficacy provides a constructive foundation for the clinical individualization of antipsychotics, such as quetiapine and risperidone, which is important in selecting the dose of the medication and improving the improvement of negative symptoms.

Mycobacterial CpsA activates type I IFN signaling in macrophages via cGAS-mediated pathway.

Type I interferon (IFN) production is crucial in tuberculosis pathogenesis, yet the bacterial factors initiating this process are incompletely understood. CpsA, protein of Mycobacterium marinum and Mycobacterium tuberculosis, plays a key role in maintaining bacterial virulence and inhibiting host cell LC3-associated phagocytosis. By utilizing CpsA full deletion mutant studies, we re-verified its essential role in infection-induced pathology and revealed its new role in type I IFN expression. CpsA deficiency hindered IFN production in infected macrophages in vitro as well as zebrafish and mice in vivo. This effect was linked to the cGAS-TBK1-IRF3 pathway, as evidenced by decreased TBK1 and IRF3 phosphorylation in CpsA-deficient bacterial strain-infected macrophages. Moreover, we further show that CpsA deficiency cause decreased cytosolic DNA levels, correlating with impaired phagosomal membrane rupture. Our findings reveal a new function of mycobacterial CpsA in type I IFN production and offer insight into the molecular mechanisms underlying mycobacterial infection pathology.

Clinical performance of nucleotide MALDI-TOF-MS in the rapid diagnosis of pulmonary tuberculosis and drug resistance.

OBJECTIVE: To evaluate the application value of nucleotide matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) technology in the rapid diagnosis of pulmonary tuberculosis (PTB) and its drug resistance. METHODS: From February 2021 to January 2022, respiratory specimens from 214 suspected PTB patients at the First Hospital of Quanzhou were collected. Nucleotide MALDI-TOF-MS and BACTEC MGIT 960 culture methods were used for the detection of Mycobacterium tuberculosis (MTB) and drug resistance to anti-tuberculosis drugs. RESULTS: Compared with culture method, nucleotide MALDI-TOF-MS technology had a sensitivity, specificity, and accuracy of 92.2%, 74.1%, and 82.7%, respectively, for the detection of MTB in respiratory specimens. With clinical diagnosis as the reference standard, the sensitivity and accuracy of nucleotide MALDI-TOF-MS were 82.5% and 86.0%, respectively, which were higher than those of the culture method (69.2% and 78.0%, respectively). The specificity of nucleotide MALDI-TOF-MS was 93.0%, which was slightly lower than that of culture method (95.8%). As for drug resistance, the results of nucleotide MALDI-TOF-MS exhibited good consistence with culture methods for rifampin, isoniazid, ethambutol, and streptomycin. CONCLUSION: Nucleotide MALDI-TOF-MS detection has a good clinical performance for rapid detection of MTB and drug sensitivity to rifampin, isoniazid, ethambutol, and streptomycin directly on respiratory specimens.

Modern Beijing sublineage of Mycobacterium tuberculosis shift macrophage into a hyperinflammatory status.

The high prevalence of the modern Beijing sublineage of Mycobacterium tuberculosis may be related to increased virulence, although the responsible mechanisms remain poorly understood. We previously described enhanced triacylglycerol accumulation in modern Beijing strains. Here we show that modern Beijing strains grow faster in vitro and trigger a vigorous immune response and pronounced macrophage infiltration. Transcriptomic analysis of bone marrow derived macrophages infected with modern Beijing lineage strains revealed a significant enrichment of infection, cholesterol homeostasis and amino acid metabolic pathways. The upregulation of proinflammatory / bactericidal cytokines was confirmed by RT-PCR analysis, which is also in consistent with the reduced bacterial burden in modern strains infected macrophages. These results suggest that modern Beijing strains elicit a hyperinflammatory response which might indicate a stronger virulence and contribute to their extensive global prevalence.

Mycobacterium tuberculosis strains of the modern Beijing sublineage excessively accumulate triacylglycerols in vitro.

Mycobacterium tuberculosis (Mtb) strains of modern Beijing sublineage appear to be more transmissible and cause more severe disease than strains of other sublineages, but the responsible pathogenic mechanisms remain unclear. We previously identified genetic changes that are specific for the modern Beijing sublineage, and here we characterize the lipidome and transcriptome differences between modern and ancient Beijing sublineages. We report that modern Beijing strains accumulated 2.89 (95%CI: 2.05-3.73) times more triacylglycerol (TAG) than ancient Beijing strains in vitro. We also observed that modern Beijing strains had a 2.64-fold (95%CI: 1.29-4.00) upregulation of tgs2 (annotated as TAG synthetase 2), whose role in TAG accumulation was further confirmed in Mycobacterium marinum (Mm). Because TAG serves as a crucial carbon source and reservoir of free fatty acids, the results suggest that the excessive accumulation of TAG might fuel the growth of modern Beijing strains after infection and lead to rapid development of disease.

Diverse effects of mycobacterial proline-proline-glutamic acid proteins upon interaction with host macrophages.

The proline-proline-glutamic acid (PPE) family proteins are abundant only in pathogenic Mycobacteria, but their general functions are far from unveiled. To investigate their roles in how Mycobacterium tuberculosis (Mtb) resists killing by the host, 25 PPE recombinant Mycobacterium smegmatis strains that overexpress Mtb PPE proteins were constructed. During phagocytosis, a similar amount of intracellular bacteria was observed at 2 h post-infection (hpi) for 24 PPE recombinants, while a 50% reduction of entrance was observed for the PPE29 recombinant. In addition, we found that 20 ppe genes significantly influenced the survival of mycobacteria within macrophage cells. Mycobacterial survival was promoted by overexpression of 18 of these genes and inhibited by the other two. Highest survival was observed for the PPE27 recombinant. We also measured the levels of proinflammatory cytokines tumor necrosis factor-alpha and interleukin-6 secreted by macrophages. The overall effects varied among the different PPE recombinants. Moreover, we also found that various PPE recombinants exhibited increased resistance against oxidative, acidic and sodium dodecyl sulfate stresses that could be encountered in vivo. Together, our results indicate that PPE proteins play distinct roles in mycobacterial survival in macrophages. The findings described here broaden our understanding of mycobacterial pathogenicity.

PPE38 Protein of Mycobacterium tuberculosis Inhibits Macrophage MHC Class I Expression and Dampens CD8+ T Cell Responses.

Suppression of CD8+ T cell activation is a critical mechanism used by Mycobacterium tuberculosis (MTB) to escape protective host immune responses. PPE38 belongs to the unique PPE family of MTB and in our previous study, PPE38 protein was speculated to participate in manipulating macrophage MHC class I pathway. To test this hypothesis, the function of mycobacterial PPE38 protein was assessed here using macrophage and mouse infection models. Decreased amount of MHC class I was observed on the surface of macrophages infected with PPE38-expressing mycobacteria. The transcript of genes encoding MHC class I was also inhibited by PPE38. After infection of C57BL/6 mice with Mycobacterium smegmatis expressing PPE38 (Msmeg-PPE38), decreased number of CD8+ T cells was found in spleen, liver, and lungs through immunohistochemical analysis, comparing to the control strain harboring empty vector (Msmeg-V). Consistently, flow cytometry assay showed that fewer effector/memory CD8+ T cells (CD44highCD62Llow) were activated in spleen from Msmeg-PPE38 infected mice. Moreover, Msmeg-PPE38 confers a growth advantage over Msmeg-V in C57BL/6 mice, indicating an effect of PPE38 to favor mycobacterial persistence in vivo. Overall, this study shows a unique biological function of PPE38 protein to facilitate mycobacteria to escape host immunity, and provides hints for TB vaccine development.

The FBPase Encoding Gene glpX Is Required for Gluconeogenesis, Bacterial Proliferation and Division In Vivo of Mycobacterium marinum.

Lipids have been identified as important carbon sources for Mycobacterium tuberculosis (Mtb) to utilize in vivo. Thus gluconeogenesis bears a key role for Mtb to survive and replicate in host. A rate-limiting enzyme of gluconeogenesis, fructose 1, 6-bisphosphatase (FBPase) is encoded by the gene glpX. The functions of glpX were studied in M. marinum, a closely related species to Mtb. The glpX deletion strain (ΔglpX) displayed altered gluconeogenesis, attenuated virulence, and altered bacterial proliferation. Metabolic profiles indicate an accumulation of the FBPase substrate, fructose 1, 6-bisphosphate (FBP) and altered gluconeogenic flux when ΔglpX is cultivated in a gluconeogenic carbon substrate, acetate. In both macrophages and zebrafish, the proliferation of ΔglpX was halted, resulting in dramatically attenuated virulence. Intracellular ΔglpX exhibited an elongated morphology, which was also observed when ΔglpX was grown in a gluconeogenic carbon source. This elongated morphology is also supported by the observation of unseparated multi-nucleoid cell, indicating that a complete mycobacterial division in vivo is correlated with intact gluconeogenesis. Together, our results indicate that glpX has essential functions in gluconeogenesis, and plays an indispensable role in bacterial proliferation in vivo and virulence of M. marinum.