Comparative analysis of genomic characteristics and immune response between Mycobacterium tuberculosis strains cultured continuously for 25 years and H37Rv.

Tuberculosis (TB) continues to pose a significant global health challenge, emphasizing the critical need for effective preventive measures. Although many studies have tried to develop new attenuated vaccines, there is no effective TB vaccine. In this study, we report a novel attenuated Mycobacterium tuberculosis (M. tb) strain, CHVAC-25, cultured continuously for 25 years in the laboratory. CHVAC-25 exhibited significantly reduced virulence compared to both the virulent H37Rv strain in C57BL/6J and severe combined immunodeficiency disease mice. The comparative genomic analysis identified 93 potential absent genomic segments and 65 single nucleotide polymorphic sites across 47 coding genes. Notably, the deletion mutation of ppsC (Rv2933) involved in phthiocerol dimycocerosate synthesis likely contributes to CHVAC-25 virulence attenuation. Furthermore, the comparative analysis of immune responses between H37Rv- and CHVAC-25-infected macrophages showed that CHVAC-25 triggered a robust upregulation of 173 genes, particularly cytokines crucial for combating M. tb infection. Additionally, the survival of CHVAC-25 was significantly reduced compared to H37Rv in macrophages. These findings reiterate the possibility of obtaining attenuated M. tb strains through prolonged laboratory cultivation, echoing the initial conception of H37Ra nearly a century ago. Additionally, the similarity of CHVAC-25 to genotypes associated with attenuated M. tb vaccine positions it as a promising candidate for TB vaccine development.

Quantitative analysis of the lysine acetylome reveals the role of SIRT3-mediated HSP60 deacetylation in suppressing intracellular Mycobacterium tuberculosis survival.

Protein acetylation and deacetylation are key epigenetic modifications that regulate the initiation and development of several diseases. In the context of infection with Mycobacterium tuberculosis (M. tb), these processes are essential for host-pathogen interactions and immune responses. However, the specific effects of acetylation and deacetylation on cellular functions during M. tb infection are not fully understood. This study employed Tandem Mass Tag (TMT) labeling for quantitative proteomic profiling to examine the acetylproteome (acetylome) profiles of noninfected and M. tb-infected macrophages. We identified 715 acetylated peptides from 1,072 proteins and quantified 544 lysine acetylation sites (Kac) in 402 proteins in noninfected and M. tb-infected macrophages. Our research revealed a link between acetylation events and metabolic changes during M. tb infection. Notably, the deacetylation of heat shock protein 60 (HSP60), a key chaperone protein, was significantly associated with this process. Specifically, the deacetylation of HSP60 at K96 by sirtuin3 (SIRT3) enhances macrophage apoptosis, leading to the elimination of intracellular M. tb. These findings underscore the pivotal role of the SIRT3-HSP60 axis in the host immune response to M. tb. This study offers a new perspective on host protein acetylation and suggests that targeting host-directed therapies could be a promising approach for tuberculosis immunotherapy. IMPORTANCE: Protein acetylation is crucial for the onset, development, and outcome of tuberculosis (TB). Our study comprehensively investigated the dynamics of lysine acetylation during M. tb infection, shedding light on the intricate host-pathogen interactions that underlie the pathogenesis of tuberculosis. Using an advanced quantitative lysine proteomics approach, different profiles of acetylation sites and proteins in macrophages infected with M. tb were identified. Functional enrichment and protein-protein network analyses revealed significant associations between acetylated proteins and key cellular pathways, highlighting their critical role in the host response to M. tb infection. Furthermore, the deacetylation of HSP60 and its influence on macrophage-mediated clearance of M. tb underscore the functional significance of acetylation in tuberculosis pathogenesis. In conclusion, this study provides valuable insights into the regulatory mechanisms governing host immune responses to M. tb infection and offers promising avenues for developing novel therapeutic interventions against TB.

The role of Mycobacterium tuberculosis acetyltransferase and protein acetylation modifications in tuberculosis.

Tuberculosis (TB) is a widespread infectious disease caused by Mycobacterium tuberculosis (M. tb), which has been a significant burden for a long time. Post-translational modifications (PTMs) are essential for protein function in both eukaryotic and prokaryotic cells. This review focuses on the contribution of protein acetylation to the function of M. tb and its infected macrophages. The acetylation of M. tb proteins plays a critical role in virulence, drug resistance, regulation of metabolism, and host anti-TB immune response. Similarly, the PTMs of host proteins induced by M. tb are crucial for the development, treatment, and prevention of diseases. Host protein acetylation induced by M. tb is significant in regulating host immunity against TB, which substantially affects the disease's development. The review summarizes the functions and mechanisms of M. tb acetyltransferase in virulence and drug resistance. It also discusses the role and mechanism of M. tb in regulating host protein acetylation and immune response regulation. Furthermore, the current scenario of isoniazid usage in M. tb therapy treatment is examined. Overall, this review provides valuable information that can serve as a preliminary basis for studying pathogenic research, developing new drugs, exploring in-depth drug resistance mechanisms, and providing precise treatment for TB.

Retrospective Analysis of 28 Cases of Tuberculosis in Pregnant Women in China.

While tuberculosis (TB) in pregnant women is reported globally, clinical data is unavailable in China. To describe clinical features and identify difficulties in the diagnosis of pregnancy-related TB, we performed a retrospective study of 28 TB inpatients at Beijing Chest Hospital. The results were presented in terms of interquartile range (IQR) for age, and medians and percentages with respect to the categorical variables. One patient (3.6%) was immediately diagnosed; for 27 patients (96.4%), the median interval from the initial onset of symptoms to diagnosis was five weeks. Eight cases (28.6%) were microbiologically confirmed. 22 (78.6%) were pulmonary TB (PTB), while six (21.4%) were extrapulmonary TB (EPTB). In addition, eight (28.6%) were miliary TB and six (21.4%) were cerebral TB. 27 (96.4%) were cured and one (3.6%) died. 15 neonates were identified, nine of which were healthy. Two were small for the gestational age (SGA) and one was a stillbirth. Three had neonatal TB, one of which died. Nine were legal abortions and four were spontaneous abortions. Indeed, there was a substantial delay in the diagnosis of TB in the pregnant women and a high incidence of both miliary and cerebral TB was evident. With timely treatment, prognosis is positive.

Application of the CRISPRi system to repress sepF expression in Mycobacterium smegmatis.

Despite technical advances in introducing genomic deletions and modulating gene expression, direct inactivation of essential genes in mycobacteria remains difficult. In this study, we described clustered regularly interspaced short palindromic repeat interference (CRISPRi) technology to repress the expression of sepF (MSMEG_4219) based on nuclease-deficient CRISPR-associated protein 9 (Cas9) and small guide RNA (sgRNA) specific to the target sequence in Mycobacterium smegmatis. Using this CRISPRi approach, we achieved the repression of sepF by up to 98% in M. smegmatis without off-target effects. The depleted Msm_sepF strains resulted in growth and morphology changes including elongated, filamentous and branched bacterial cells, but the levels of the interacting partners ftsZ and murG were not modified in M. smegmatis. The sepF gene was proven to be an essential gene in M. smegmatis. This study provided an improved and detailed technical procedure for the application of CRISPRi technology in mycobacteria, and this approach was demonstrated to be a simple and efficient tool for regulating the expression of essential genes in M. smegmatis.

Case Report: Primary Tuberculosis of the Bilateral Breast.

Primary breast tuberculosis is rare. We report a case of bilateral primary breast tuberculosis. The patient received incisional drainage and debridement of both breasts. Histopathology of the breast tissues revealed chronic granulomatous inflammation and positive acid-fast stain. The patient received antitubercular therapy for 18 months, and she achieved complete resolution.

IL-21 inhibits IL-17A-producing γδ T-cell response after infection with Bacillus Calmette-Guérin via induction of apoptosis.

Innate γδ T cells expressing Vγ6 produce IL-17A at an early stage following infection with Mycobacterium bovis Bacillus Calmette-Guérin (BCG). In this study, we used IL-21 receptor knockout (IL-21R KO) mice and IL-21-producing recombinant BCG mice (rBCG-Ag85B-IL-21) to examine the role of IL-21 in the regulation of IL-17A-producing innate γδ T-cell response following BCG infection. IL-17A-producing Vγ6+ γδ T cells increased in the peritoneal cavity of IL-21R KO mice more than in wild type mice after BCG infection. In contrast, the number of IL-17A-producing Vγ6+ γδ T cells was significantly lower after inoculation with rBCG-Ag85B-IL-21 compared with control rBCG-Ag85B. Notably, exogenous IL-21 selectively induced apoptosis of IL-17A-producing Vγ6+ γδ T cells via Bim. Thus, these results suggest that IL-21 acts as a potent inhibitor of a IL-17A-producing γδ T-cell subset during BCG infection.

New polymorphs of 9-nitro-camptothecin prepared using a supercritical anti-solvent process.

Recrystallization and micronization of 9-nitro-camptothecin (9-NC) has been investigated using the supercritical anti-solvent (SAS) technology in this study. Five operating factors, i.e., the type of organic solvent, the concentration of 9-NC in the solution, the flow rate of 9-NC solution, the precipitation pressure and the temperature, were optimized using a selected OA16 (4(5)) orthogonal array design and a series of characterizations were performed for all samples. The results showed that the processed 9-NC particles exhibited smaller particle size and narrower particle size distribution as compared with 9-NC raw material (Form I), and the optimum micronization conditions for preparing 9-NC with minimum particle size were determined by variance analysis, where the solvent plays the most important role in the formation and transformation of polymorphs. Three new polymorphic forms (Form II, III and IV) of 9-NC, which present different physicochemical properties, were generated after the SAS process. The predicted structures of the 9-NC crystals, which were consistent with the experiments, were performed from their experimental XRD data by the direct space approach using the Reflex module of Materials Studio. Meanwhile, the optimal sample (Form III) was proved to have higher cytotoxicity against the cancer cells, which suggested the therapeutic efficacy of 9-NC is polymorph-dependent.

Proteomic analysis reveals significant elevation of heat shock protein 70 in patients with chronic heart failure due to arrhythmogenic right ventricular cardiomyopathy.

As proteins are the ultimate biological determinants of phenotype of disease, we screened altered proteins associated with heart failure due to arrhythmogenic right ventricular cardiomyopathy (ARVC) to identify biomarkers potential for rapid diagnosis of heart failure. By 2-dimensional gel electrophoresis and mass spectrometry, we identified five commonly altered proteins with more than 1.5 fold changes in eight ARVC failing hearts using eight non-failing hearts as reference. Noticeably, one of the altered proteins, heat shock protein 70 (HSP70), was increased by 1.64 fold in ARVC failing hearts compared with non-failing hearts. The increase of cardiac HSP70 was further validated by Western blot, immunochemistry, and enzyme-linked immunosorbent assay (ELISA) in failing hearts due to not only ARVC, but also dilated (DCM, n = 18) and ischemic cardiomyopathy (ICM, n = 8). Serum HSP70 was also observed to be significantly increased in heart failure patients derived from the three forms of cardiomyopathies. In addition, we observed hypoxia/serum depletion stimulation induced significantly elevation of intracellular and extracellular HSP70 in cultured neonatal rat cardiomyocytes. For the first time to our knowledge, we revealed and clearly demonstrated significant up-regulation of cardiac and serum HSP70 in ARVC heart failure patients. Our results indicate that elevated HSP70 is the common feature of heart failure due to ARVC, DCM, and ICM, which suggests that HSP70 may be used as a biomarker for the presence of heart failure due to cardiomyopathies of different etiologies and may hold diagnostic/prognostic potential in clinical practice.

Upregulated expression of cardiac ankyrin repeat protein in human failing hearts due to arrhythmogenic right ventricular cardiomyopathy.

AIMS: Expression of cardiac ankyrin repeat protein (CARP) is augmented in heart failure due to dilated or ischaemic cardiomyopathy. It is unclear whether CARP is upregulated in heart failure due to arrhythmogenic right ventricular cardiomyopathy (ARVC). In the present study, we investigated the expression pattern of CARP and the correlation between CARP and the well-known heart failure marker pro-atrial natriuretic peptide (proANP) in ARVC failing hearts. METHODS AND RESULTS: Gene microarray analysis demonstrated increased CARP expression in ARVC failing hearts compared with non-failing control hearts, which was further validated by real-time RT-PCR, western blot, and ELISA at the mRNA and protein levels. Fractionation experiments revealed that the upregulation of CARP expression is restricted to the nuclei of residual cardiac cells in ARVC failing hearts. Regression analysis showed a positive correlation between CARP and proANP in ARVC failing hearts. CONCLUSION: Augmented CARP expression may be a common molecular event in failing hearts regardless of cardiomyopathic aetiology. The upregulation of nuclear CARP expression and positive correlation between cardiac CARP and proANP suggests that CARP may be used as a genetic marker existing in the nuclei in contrast to proANP existing in the cytosol of cardiac cells in heart failure patients.

Apolipoprotein D as a novel marker in human end-stage heart failure: a preliminary study.

Apolipoprotein D (Apo D) is reported to be in close association with developing and mature blood vessels, and involved in enhanced smooth muscle cell migration after injury. This study was designed to clarify the expression pattern of Apo D and the possibility of Apo D as a new marker in human end-stage heart failure. Individual RNA samples obtained from independent left ventricular tissue of six heart failure patients derived from cardiomyopathies of different aetiologies during cardiac transplantation and six non-failing control subjects were hybridized to the gene microarray containing, in total, 35 000 well-characterized Homo sapiens genes. Apo D was one of the highly expressed genes (3.3-fold upregulated) detected by microarray, which was further confirmed by quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR) (5.88-fold upregulated) in failing hearts compared with non-failing hearts. Both Western blotting and immunohistochemistry analyses also demonstrated the higher levels of Apo D protein in failing hearts. Importantly, we observed elevated levels of plasma Apo D in heart failure patients compared with non-failing control subjects. We demonstrated, for the first time to our knowledge, that Apo D was highly expressed in the mRNA and protein levels in human failing hearts compared with non-failing hearts. Furthermore, our finding of elevated plasma Apo D levels in patients with heart failure provides clues that Apo D may act not only as a cardiac molecular marker but also as a circulating biomarker in patients with heart failure.

[Establishment of an assay for PrP(Sc) detection based on streptomycin precipitation].

To establish a new Western blotting assay for PrP(Sc) detection, we optimized the Western blotting assay with a precipitation procedure of streptomycin sulfate. After digestion with PK, 10% scrapie infected hamster brain homogenates were incubated with 60 mmol/L streptomycin and the precipitated PrP(Sc) was recovered by centrifugation. The enrichment of PrP(Sc) by streptomycin sulfate precipitation was evaluated using Western blotting assay. The results showed streptomycin could bind to PK-treated PrP(Sc), forming high molecular masses, but not influence the glycosylated patterns on SDS-PAGE. Western blot assay revealed that the detective sensitivity of the streptomycin-precipitation PrP(Sc) was remarkably improved. As a sensitive, specific, rapid and flexible protocol for PrP(Sc), the protocol in this study has the potential utility, alone or combined with other techniques, for the detection of low level PrP(Sc) in the specimens from central nerve system, or from peripheral organs or body fluids.

Human tau protein forms complex with PrP and some GSS- and fCJD-related PrP mutants possess stronger binding activities with tau in vitro.

Microtubule associated protein tau is considered to play roles in some types of human transmissible spongiform encephalopathies (TSE). In this study, the full-length and several truncated human tau proteins were expressed from E. coli and purified. Using GST pull down, co-immunoprecipitation assay and tau-coated ELISA, the molecular interaction between tau protein and PrP was confirmed in the context of the full-length human tau. The N terminus (amino acids 1-91) and tandem repeats region (amino acids 186-283) of tau protein were responsible for the interaction with PrP. The octapeptide repeats within PrP directly affected the binding activity of PrP with tau. GSS-related mutant PrP102L and fCJD- related mutants with two and seven extra octarepeats showed more active binding capacity with tau than wild-type PrP. The molecular interactions between PrP and tau protein highlight a potential role of tau in the biological function of PrP and the pathogenesis of TSE.

[Preliminary analyses for influence of mutant PrPs with different number of octapeptide].

OBJECTIVE: The present study was conducted to understand the effects of PrP in different octapeptide repeats on proliferation of HeLa cells. METHODS AND RESULTS: Mutant PrPs with octapeptide repeat insertion were transiently expressed in HeLa cells and their results of MTT assay showed stronger cytotoxic effect on the proliferation of cells than wild-type PrP. Annexin V/PI assay also demonstrated that the expression of mutant PrPs was much easier to induce apoptosis than wild-type in HeLa cells. The percentage of both early and late stage apoptosis in mutant groups were significantly higher than that of wild type. CONCLUSION: These data suggest that the expression of mutant PrPs associated with familial CJD is much easier to induce apoptosis in cultured cells than expression of wild type PrP.

[Generation and identification of phage engineering antibodies library against hamster prion protein].

OBJECTIVE: Generation and Identification of Phage Engineering Antibodies Library against Hamster Prion Protein. METHODS: Fab antibodies were identified and confirmed. BALB/c mice were immuned with PrP proteins. After the third immunization, the total RNA was extracted from the mice spleens. The genes of heavy Fd Fragments and light chain of antibodies amplified by a series of specific primers of human IgG Fab fragment were cloned into phagemid vector pComb3. RESULTS: The combinatorial Fab library were constructed successfully and the cloning efficiencies both of light chain and Fd fragments were about near 10(6). The Fab library were panned by four cycles and screened with purified haPrP23-231 antigen on microtiter plates. 12 mAbs were isolated after four cycles of panning, five of which were sequenced and resulted sequence data were analyzed by alignment with GenBank immunoglobulin genes. Two strain of new heavy and light chain genes of Fab antibodies were identified and confirmed. CONCLUSION: The research in this article will provide foundation for study of diagnosis and therapy of prion.

Generation of genetic engineering monoclonal antibodies against prion protein.

Two strains of Fab monoclonal antibodies (mAbs) against prion protein, designated as IV-66 and IV-78, were selected from the phage display libraries. The gene sequences encoding the light kappa chain and heavy Fd chain of IV-78 were inserted into a baculovirus expression cassette vector for mouse IgG expression. Western blot, Dot-ELISA and immunoprecipitation confirmed that these Fab and IgG mAbs reacted well with the recombinant hamster and human PrP proteins expressed in prokaryotic and in mammalian cells and PrP(Sc) from scrapie-infected hamsters. It demonstrates that mAbs against prion protein are successfully generated by phage-display technique.

Preparation of monoclonal antibodies against prion proteins with full-length hamster PrP.

OBJECTIVE: To prepare the PrP specific monoclonal antibodies (mAbs) that can be used for the detection of mammalian prions and study of pathogenesis of prion diseases. METHODS: Several BALB/c mice were immunized with recombinant hamster prion protein (HaPrP). Three hybridoma cell lines designated as B7, B9, and B10, secreting monoclonal antibodies against HaPrP, were established by hybridoma technique. The mAbs reactivities were evaluated with ELISA, Western blot, and immunohistochemistry. RESULTS: The mAbs produced by these cell lines reacted well with different recombinant hamster PrP proteins. Western blot analyses showed that mAbs B7 and B9 reacted with PrPSc from the scrapie-infected animals after proteinase K digestion with three glycosylated forms. The mAbs exhibited cross-reactivity with various PrPC from several other mammalian species, including humans and cattles. Immunohistochemistry assays confirmed that mAbs B7 and B9 could recognize not only extracellular but also intracellular PrPsSc. CONCLUSION: The mAbs of prion protein are successfully generated by hybridoma technique and can be applied for the diagnosis of prion associated diseases.