Language：English   Publishing type：Research paper (scientific journal)  

Tuberculosis remains a large health threat, despite the availability of the tuberculosis vaccine, BCG. As BCG efficacy gradually decreases from adolescence, BCG-Prime and antigen-booster may be an efficient strategy to confer vaccine efficacy. Mycobacterial DNA-binding protein 1 (MDP1, namely Rv2986c, hupB or HU) is a major Mycobacterium tuberculosis protein that induces vaccine-efficacy by co-administration with CpG DNA. To produce MDP1 for booster-vaccine use, we have created recombinant MDP1 produced in both Escherichia coli (eMDP1) and Mycolicibacterium smegmatis (mMDP1), an avirulent rapid-growing mycobacteria. We tested their immunogenicity by checking interferon (IFN)-gamma production by stimulated peripheral blood cells derived from BCG-vaccinated individuals. Similar to native M. tuberculosis MDP1, we observed that most lysin resides in the C-terminal half of mMDP1 are highly methylated. In contrast, eMDP1 had less post-translational modifications and IFN-gamma stimulation. mMDP1 stimulated the highest amount of IFN-gamma production among the examined native M. tuberculosis proteins including immunodominant MPT32 and Antigen 85 complex. MDP1-mediated IFN-gamma production was more strongly enhanced when combined with a new type of CpG DNA G9.1 than any other tested CpG DNAs. Taken together, these results suggest that the combination of mMDP1 and G9.1 possess high potential use for human booster vaccine against tuberculosis.

DOI： 10.1038/s41598-024-58836-8

PubMed

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Publishing type：Research paper (scientific journal)   Publisher：Wiley  

Abstract

Vaccination is an important factor in public health. The recombinant bacillus Calmette Guérin (rBCG) vaccine, which expresses foreign antigens, is expected to be a superior vaccine against infectious diseases. Here, we report a new recombination platform in which the BCG Tokyo strain is transformed with nucleotide sequences encoding foreign protein fused with the MPB70 immunogenic protein precursor. By RNA‐sequencing, mpb70 was found to be the most transcribed among all known genes of BCG Tokyo. Small oligopeptide, namely, polyhistidine tag, was able to be expressed in and secreted from rBCG through a process in which polyhistidine tag fused with intact MPB70 were transcribed by an mpb70 promoter. This methodology was applied to develop an rBCG expressing the receptor binding domain (RBD) of severe acute respiratory syndrome coronavirus 2. Immunoblotting images and mass spectrometry data showed that RBD was also secreted from rBCG. Sera from mice vaccinated with the rBCG showed a tendency of weak neutralizing capacity. The secretion was retained even after a freeze‐drying process. The freeze‐dried rBCG was administered to and recovered from mice. Recovered rBCG kept secreting RBD. Collectively, our recombination platform offers stable secretion of foreign antigens and can be applied to the development of practical rBCGs.

DOI： 10.1111/1348-0421.13116

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Publishing type：Research paper (scientific journal)   Publisher：Oxford University Press (OUP)  

Abstract

Mycobacteria are the major human pathogens with the capacity to become dormant persisters. Mycobacterial DNA-binding protein 1 (MDP1), an abundant histone-like protein in dormant mycobacteria, induces dormancy phenotypes, e.g. chromosome compaction and growth suppression. For these functions, the polycationic intrinsically disordered region (IDR) is essential. However, the disordered property of IDR stands in the way of clarifying the molecular mechanism. Here we clarified the molecular and structural mechanism of DNA compaction by MDP1. Using high-speed atomic force microscopy, we observed that monomeric MDP1 bundles two adjacent DNA duplexes side-by-side via IDR. Combined with coarse-grained molecular dynamics simulation, we revealed the novel dynamic DNA cross-linking model of MDP1 in which a stretched IDR cross-links two DNA duplexes like double-sided tape. IDR is able to hijack HU function, resulting in the induction of strong mycobacterial growth arrest. This IDR-mediated reversible DNA cross-linking is a reasonable model for MDP1 suppression of the genomic function in the resuscitable non-replicating dormant mycobacteria.

DOI： 10.1093/nar/gkad1149

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Language：English   Publishing type：Research paper (scientific journal)  

<jats:title>Abstract</jats:title><jats:p>Survival of the live attenuated Bacillus Calmette-Guérin (BCG) vaccine amidst harsh host environments is key for BCG effectiveness as it allows continuous immune response induction and protection against tuberculosis. Mycobacterial DNA binding protein 1 (MDP1), a nucleoid associated protein, is essential in BCG. However, there is limited knowledge on the extent of MDP1 gene regulation and how this influences BCG survival. Here, we demonstrate that MDP1 conditional knockdown (cKD) BCG grows slower than vector control in vitro<jats:italic>,</jats:italic> and dies faster upon exposure to antibiotics (bedaquiline) and oxidative stress (H<jats:sub>2</jats:sub>O<jats:sub>2</jats:sub> and menadione). MDP1-cKD BCG also exhibited low infectivity and survival in THP-1 macrophages and mice indicating possible susceptibility to host mediated stress. Consequently, low in vivo survival resulted in reduced cytokine (IFN-gamma and TNF-alpha) production by splenocytes. Temporal transcriptome profiling showed more upregulated (81–240) than downregulated (5–175) genes in response to MDP1 suppression. Pathway analysis showed suppression of biosynthetic pathways that coincide with low in vitro growth. Notable was the deferential expression of genes involved in stress response (<jats:italic>sigI</jats:italic>), maintenance of DNA integrity (<jats:italic>mutT1</jats:italic>), REDOX balance (<jats:italic>WhiB3</jats:italic>), and host interactions (<jats:italic>PE/PE_PGRS</jats:italic>). Thus, this study shows MDP1’s importance in BCG survival and highlights MDP1-dependent gene regulation suggesting its role in growth and stress adaptation.</jats:p>

DOI： 10.1038/s41598-023-40941-9

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Language：English   Publishing type：Research paper (scientific journal)  

<jats:title>Abstract</jats:title><jats:p>Accurate point-of-care testing (POCT) is critical for managing tuberculosis (TB). However, current antibody-based diagnosis shows low specificity and sensitivity. To find proper antigen candidates for TB diagnosis by antibodies, we assessed IgGs responsiveness to <jats:italic>Mycobacterium tuberculosis</jats:italic> proteins in pulmonary TB (PTB) patients. We employed major secreted proteins, such as Rv1860, Ag85C, PstS1, Rv2878c, Ag85B, and Rv1926c that were directly purified from <jats:italic>M. tuberculosis</jats:italic>. In the first screening, we found that IgG levels were significantly elevated in PTB patients only against Rv1860, PstS1, and Ag85B among tested antigens. However, recombinant PstS1 and Ag85B from <jats:italic>Escherichia coli (E. coli)</jats:italic> couldn’t distinguish PTB patients and healthy controls (HC). Recombinant Rv1860 was not checked due to its little expression. Then, the 59 confirmed PTB patients from Soetomo General Academic Hospital, Surabaya, Indonesia, and 102 HC were tested to Rv1860 and Ag85B only due to the low yield of the PstS1 from <jats:italic>M. tuberculosis</jats:italic>. The ROC analysis using native Ag85B and Rv1860 showed an acceptable area under curve for diagnosis, which is 0.812 (95% CI 0.734–0.890, <jats:italic>p</jats:italic> &lt; 0.0001) and 0.821 (95% CI 0.752–0.890, <jats:italic>p</jats:italic> &lt; 0.0001). This study indicates that taking consideration of native protein structure is key in developing TB’s POCT by antibody-based diagnosis.</jats:p>

DOI： 10.1038/s41598-023-39436-4

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Language：English   Presentation type：Poster presentation  

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Presentation type：Poster presentation  

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Presentation type：Poster presentation  

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Presentation type：Oral presentation (general)  

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Type：Academic society, research group, etc. 

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