BACKGROUND AND OBJECTIVE

Nontuberculous mycobacteria (NTM) lung disease appears like tuberculosis infection but is resistant to primary anti-tuberculosis drugs. Hence, patients whose sputum sample tests positive for acid-fast bacilli (AFB) and bacterial culture for several times should be assessed for colonization or infection with NTM in a damaged lung secondary to TB. In such cases, though drug-resistant TB may be adequately treated, treatment may need to be directed towards the NTM as well. In NTM therapy, the duration and choice of treatment agent is based upon the specific organism and disease extent. This study used one-step multiplex PCR (mPCR) assay for rapid differentiation of solid cultures in Ogawa medium as Mycobacterium tuberculosis (MTB) and/or NTM.

A total of 80 stocked isolates obtained from the Lung Center of the Philippines from January to December 2018 were screened for NTM in terms of growth in Ogawa medium, acid fastness, and MPT64 TB antigen test result. These were from sputum specimens of multidrug-resistant tuberculosis (MDR-TB) patients. DNA was extracted from cultures (n=55) grown in Ogawa medium and one-step mPCR was performed to identify NTM to the species level.

Out of 80 samples screened, a total of 55 isolates were identified as NTM. One-step mPCR identified 12.73% (7/55) as M. abscessus, 34.55% (19/55) as M. massiliense, 1.82% (1/55) as M. kansasii, and 50.91% (28/55) were identified only up to genus Mycobacteria spp. Neither M. avium complex nor M. intracellulare was identified among the samples tested.

One-step mPCR was able to identify isolates as MTB or NTM coinciding with the initial screening using MPT64 TB antigen test. Multiplex PCR has given a more specific identificati on to the species level. The use of mPCR in identifying MTB and clinically significant NTM’s is suitable for the adequate treatment of mycobacterial infection.

Early secreted antigenic target of 6 kDa protein (ESAT-6) is the major virulence factor of Mycobacterium tuberculosis (MTB), which can resist the clearance of MTB in bodies by inhibiting macrophage phagocytosis and autophagy reaction, thus impeding the immune defense function of the body against MTB infection. In addition, ESAT-6-induced apoptosis of macrophage and massive necrosis of innate immune cells can foster MTB proliferation and colonization, leading to systemic MTB infection. Moreover, ESAT-6 hampers the protective immune response of Th1 cells, reducing the secretion of pro-inflammatory cytokines and contributing to immune dysfunction, thus accelerating the course of MTB infection. During the process, the high immunogenicity of ESAT-6 can be leveraged as a dominant antigen in the development of new TB vaccines, making it a promising candidate with broad prospects for further development.
Humans ; Mycobacterium tuberculosis ; Vaccines ; Cytokines ; Apoptosis ; Autophagy ; Sepsis

Humans ; Retrospective Studies ; Bronchiectasis ; Mycobacterium avium Complex ; Syndrome ; Nocardia Infections/drug therapy* ; Nocardia ; Mycobacterium avium-intracellulare Infection

Humans ; Isoniazid/pharmacology* ; Mycobacterium tuberculosis/genetics* ; Rifampin/pharmacology* ; Antitubercular Agents/pharmacology* ; Mutation ; Microbial Sensitivity Tests ; Tuberculosis, Multidrug-Resistant/microbiology* ; Drug Resistance, Multiple, Bacterial/genetics* ; Bacterial Proteins/genetics*

As a branched chain amino acid, L-valine is widely used in the medicine and feed sectors. In this study, a microbial cell factory for efficient production of L-valine was constructed by combining various metabolic engineering strategies. First, precursor supply for L-valine biosynthesis was enhanced by strengthening the glycolysis pathway and weakening the metabolic pathway of by-products. Subsequently, the key enzyme in the L-valine synthesis pathway, acetylhydroxylate synthase, was engineered by site-directed mutation to relieve the feedback inhibition of the engineered strain. Moreover, promoter engineering was used to optimize the gene expression level of key enzymes in L-valine biosynthetic pathway. Furthermore, cofactor engineering was adopted to change the cofactor preference of acetohydroxyacid isomeroreductase and branched-chain amino acid aminotransferase from NADPH to NADH. The engineered strain C. glutamicum K020 showed a significant increase in L-valine titer, yield and productivity in 5 L fed-batch bioreactor, up to 110 g/L, 0.51 g/g and 2.29 g/(L‧h), respectively.
Valine ; Corynebacterium glutamicum/genetics* ; Metabolic Engineering ; Amino Acids, Branched-Chain ; Bioreactors

L-glutamic acid is the world's largest bulk amino acid product that is widely used in the food, pharmaceutical and chemical industries. Using Corynebacterium glutamicum G01 as the starting strain, the fermentation by-product alanine content was firstly reduced by knocking out the gene encoding alanine aminotransferase (alaT), a major by-product related to alanine synthesis. Secondly, since the α-ketoglutarate node carbon flow plays an important role in glutamate synthesis, the ribosome-binding site (RBS) sequence optimization was used to reduce the activity of α-ketoglutarate dehydrogenase and enhance the glutamate anabolic flow. The endogenous conversion of α-ketoglutarate to glutamate was also enhanced by screening different glutamate dehydrogenase. Subsequently, the glutamate transporter was rationally desgined to improve the glutamate efflux capacity. Finally, the fermentation conditions of the strain constructed using the above strategy were optimized in 5 L fermenters by a gradient temperature increase combined with a batch replenishment strategy. The glutamic acid production reached (135.33±4.68) g/L, which was 41.2% higher than that of the original strain (96.53±2.32) g/L. The yield was 55.8%, which was 11.6% higher than that of the original strain (44.2%). The combined strategy improved the titer and the yield of glutamic acid, which provides a reference for the metabolic modification of glutamic acid producing strains.
Glutamic Acid ; Corynebacterium glutamicum/genetics* ; Ketoglutaric Acids ; Metabolic Engineering ; Alanine

Polyhydroxyalkanoate depolymerase (PHAD) can be used for the degradation and recovery of polyhydroxyalkanoate (PHA). In order to develop a PHAD with good stability under high temperature, PHAD from Thermomonospora umbrina (TumPHAD) was heterelogously expressed in Escherichia coli BL21(DE3). At the same time, a mutant A190C/V240C with enhanced stability was obtained via rational design of disulfide bonds. Characterization of enzymatic properties showed that the mutant A190C/V240C had an optimum temperature of 60 ℃, which was 20 ℃ higher than that of the wild type. The half-life at 50 ℃ was 7 hours, at 50 ℃ which was 21 times longer than that of the wild type. The mutant A190C/V240C was used for the degradation of polyhydroxybutyrate (PHB), one of the typical PHA. At 50 ℃, the degradation rate of PHB being treated for 2 hours and 12 hours was 2.1 times and 3.8 times higher than that of the wild type, respectively. The TumPHAD mutant A190C/V240C obtained in this study shows tolerance to high temperature resistance, good thermal stability and strong PHB degradation ability, which may facilitate the degradation and recovery of PHB.
Thermomonospora ; Actinomycetales ; Escherichia coli/genetics* ; Polyhydroxyalkanoates

Mycolic acids (MAs), i.e. 2-alkyl, 3-hydroxy long-chain fatty acids, are the hallmark of the cell envelope of Mycobacterium tuberculosis and are related with antibiotic resistance and host immune escape. Nowadays, they've become hot target of new anti-tuberculosis drugs. There are two main methods to detect MAs, ¹⁴C metabolic labeling thin-layer chromatography (TLC) and liquid chromatograph mass spectrometer (LC-MS). However, the user qualification of ¹⁴C or the lack of standards for LC-MS hampered the easy use of this method. TLC is a common way to analyze chemical substance and can be used to analyze MAs. In this study, we used tetrabutylammonium hydroxide and methyl iodide to hydrolyze and formylate MAs from mycobacterium cell wall. Subsequently, we used diethyl ether to extract methyl mycolate. By this method, we can easily extract and analyze MA in regular biological labs. The results demonstrated that this method could be used to compare MAs of different mycobacterium in different growth phases, MAs of mycobacteria treated by anti-tuberculosis drugs or MAs of mycobacterium mutants. Therefore, we can use this method as an initial validation for the changes of MAs in researches such as new drug screening without using radioisotope or when the standards are not available.
Mycolic Acids/metabolism* ; Chromatography, Thin Layer ; Mycobacterium tuberculosis ; Fatty Acids ; Antitubercular Agents/pharmacology*

To prepare a lipid nanoparticle (LNP)-based subunit vaccine of Mycobacterium tuberculosis (Mtb) antigen EsxV and study its immunological characteristics, the LNP containing EsxV and c-di-AMP (EsxV: C: L) was prepared by thin film dispersion method, and its encapsulation rate, LNP morphology, particle size, surface charge and polyphase dispersion index were measured. BALB/c mice were immunized with EsxV: C: L by nasal drops. The levels of serum and mucosal antibodies, transcription and secretion of cytokines in lung and spleen, and the proportion of T cell subsets were detected after immunization. EsxV: C: L LNPs were obtained with uniform size and they were spherical and negatively charged. Compared with EsxV: C immunization, EsxV: C: L mucosal inoculation induced increased sIgA level in respiratory tract mucosa. Levels of IL-2 secreted from spleen and ratios of memory T cells and tissue-resident T cells in mice were also elevated. In conclusion, EsxV: C: L could induce stronger mucosal immunity and memory T cell immune responses, which may provide better protection against Mtb infection.
Animals ; Mice ; Mycobacterium tuberculosis ; Antigens, Bacterial ; Immunization ; Nanoparticles ; Vaccines, Subunit ; Mice, Inbred BALB C

Cyclodipeptide (CDP) composed of two amino acids is the simplest cyclic peptide. These two amino acids form a typical diketopiperazine (DKP) ring by linking each other with peptide bonds. This characteristic stable ring skeleton is the foundation of CDP to display extensive and excellent bioactivities, which is beneficial for CDPs' pharmaceutical research and development. The natural CDP products are well isolated from actinomycetes. These bacteria can synthesize DKP backbones with nonribosomal peptide synthetase (NRPS) or cyclodipeptide synthase (CDPS). Moreover, actinomycetes could produce a variety of CDPs through different enzymatic modification. The presence of these abundant and diversified catalysis indicates that actinomycetes are promising microbial resource for exploring CDPs. This review summarized the pathways for DKP backbones biosynthesis and their post-modification mechanism in actinomycetes. The aim of this review was to accelerate the genome mining of CDPs and their isolation, purification and structure identification, and to facilitate revealing the biosynthesis mechanism of novel CDPs as well as their synthetic biology design.
Actinobacteria/metabolism* ; Actinomyces/metabolism* ; Biological Products/metabolism* ; Bacteria/metabolism* ; Diketopiperazines/metabolism* ; Amino Acids