• Journal of Microbiology and Biotechnology
• /
• v.30 no.12
• /
• pp.1819-1826
• /
• 2020

Increasing evidence suggests a potential role of microbial colonization in the inception of chronic airway diseases. However, it is not clear whether the lung and gut microbiome dysbiosis is coincidental or a result of mutual interaction. In this study, we investigated the airway microbiome in interleukin 13 (IL-13)-rich lung environment and related alterations of the gut microbiome. IL-13-overexpressing transgenic (TG) mice presented enhanced eosinophilic inflammatory responses and mucus production, together with airway hyperresponsiveness and subepithelial fibrosis. While bronchoalveolar lavage fluid and cecum samples obtained from 10-week-old IL-13 TG mice and their C57BL/6 wild-type (WT) littermates showed no significant differences in alpha diversity of lung and gut microbiome, they presented altered beta diversity in both lung and gut microbiota in the IL-13 TG mice compared to the WT mice. Lung-specific IL-13 overexpression also altered the composition of the gut as well as the lung microbiome. In particular, IL-13 TG mice showed an increased proportion of Proteobacteria and Cyanobacteria and a decreased amount of Bacteroidetes in the lungs, and depletion of Firmicutes and Proteobacteria in the gut. The patterns of polymicrobial interaction within the lung microbiota were different between WT and IL-13 TG mice. For instance, in IL-13 TG mice, lung Mesorhizobium significantly affected the alpha diversity of both lung and gut microbiomes. In summary, chronic asthma-like pathologic changes can alter the lung microbiota and affect the gut microbiome. These findings suggest that the lung-gut microbial axis might actually work in asthma.

• Journal of Microbiology and Biotechnology
• /
• v.31 no.5
• /
• pp.667-675
• /
• 2021

Streptococcus agalactiae is an important bacterial pathogen and causative agent of diseases including neonatal sepsis and meningitis, as well as infections in healthy adults and pregnant women. Although antibiotic treatments effectively relieve symptoms, the emergence and transmission of multidrug-resistant strains indicate the need for an effective immunotherapy. Effector T helper (Th) 17 cells are a relatively newly discovered subpopulation of helper CD4⁺ T lymphocytes, and which, by expressing interleukin (IL)-17A, play crucial roles in host defenses against a variety of pathogens, including bacteria and viruses. However, whether S. agalactiae infection can induce the differentiation of CD4⁺ T cells into Th17 cells, and whether IL-17A can play an effective role against S. agalactiae infections, are still unclear. In this study, we analyzed the responses of CD4⁺ T cells and their defensive effects after S. agalactiae infection. The results showed that S. agalactiae infection induces not only the formation of Th1 cells expressing interferon (IFN)-γ, but also the differentiation of mouse splenic CD4⁺ T cells into Th17 cells, which highly express IL-17A. In addition, the bacterial load of S. agalactiae was significantly increased and decreased in organs as determined by antibody neutralization and IL-17A addition experiments, respectively. The results confirmed that IL-17A is required by the host to defend against S. agalactiae and that it plays an important role in effectively eliminating S. agalactiae. Our findings therefore prompt us to adopt effective methods to regulate the expression of IL-17A as a potent strategy for the prevention and treatment of S. agalactiae infection.

• Journal of Microbiology and Biotechnology
• /
• v.31 no.2
• /
• pp.207-216
• /
• 2021

Supplement of high-protein food plays an important role in improving the symptoms of malnutrition and the immune capacity of the body, but the association of high-protein diet and gut microbiota remained unaddressed. Here, we systematically analyzed the internal organs and gut microbiota in C57(WT) or PD-1H-depleted (KO) mice (T cells were activated) fed with pupae or feed for six weeks. We observed that the body weight gain in the mice fed with pupae increased less significantly than that of the feed group, while the villi and small intestine lengths in the pupa group were reduced compared with that of mice given feed. However, the average body weight of the KO mice increased compared with that of the WT mice fed with pupae or feed. Pupae increased the concentration of blood glucose in WT, but not in KO mice. Moreover, in the feed group, there was no difference in the weight of the internal organs between the WT and KO mice, but in the pupae-fed group, liver weight was decreased and spleen weight was increased compared with that of KO mice. The amounts/plural/amounts of Melainabacteria, Chloroflexi, and Armatimonadetes were specifically upregulated by pupae, and this upregulation was weakened or eliminated by PD-1H depletion. Some bacteria with high abundance in the feed-fed KO mice, such as Deferribacteres, Melainabacteria, Acidobacteria, Bacteroidetes, Spirochaetes and Verrucomicrobia, were decreased in pupae-fed KO mice, and Proteobacteria and Deinococcus were specifically enriched in pupae-fed KO mice. Bacteroidetes, Firmicutes and Akkermansia were associated with weight loss in the pupae-fed group while Lachnospiraceae and Anaerobiospirillum were related glucose metabolism and energy consumption. Based on high-throughput sequencing, we discovered that some gut bacteria specifically regulated the metabolism of a high-protein diet, and PD-1H deficiency improved life quality and sustained blood glucose. Moreover, PD-1H responses to high-protein diet through modulating the type and quantity of gut bacteria. These findings provide evidence about the association among gut microbiota, T cell activation (for PD-1H depletion) and high-protein diet metabolism, have important theoretical significance for nutrition and health research.

• Journal of Microbiology and Biotechnology
• /
• v.31 no.2
• /
• pp.259-271
• /
• 2021

Many bacteria metabolize aromatic compounds via catechol as a catabolic intermediate, and possess multiple genes or clusters encoding catechol-cleavage enzymes. The presence of multiple isozyme-encoding genes is a widespread phenomenon that seems to give the carrying strains a selective advantage in the natural environment over those with only a single copy. In the naphthalene-degrading strain Pseudomonas putida ND6, catechol can be converted into intermediates of the tricarboxylic acid cycle via either the ortho- or meta-cleavage pathways. In this study, we demonstrated that the catechol ortho-cleavage pathway genes (catBICIAI and catBIICIIAII) on the chromosome play an important role. The catI and catII operons are co-transcribed, whereas catAI and catAII are under independent transcriptional regulation. We examined the binding of regulatory proteins to promoters. In the presence of cis-cis-muconate, a well-studied inducer of the cat gene cluster, CatRI and CatRII occupy an additional downstream site, designated as the activation binding site. Notably, CatRI binds to both the catI and catII promoters with high affinity, while CatRII binds weakly. This is likely caused by a T to G mutation in the G/T-N11-A motif. Specifically, we found that CatRI and CatRII regulate catBICIAI and catBIICIIAII in a cooperative manner, which provides new insights into naphthalene degradation.

• Journal of Microbiology and Biotechnology
• /
• v.32 no.2
• /
• pp.187-194
• /
• 2022

Two α-ʟ-arabinofuranosidases (BfdABF1 and BfdABF3) and a β-ᴅ-xylosidase (BfdXYL2) genes were cloned from Bifidobacterium dentium ATCC 27679, and functionally expressed in E. coli BL21(DE3). BfdABF1 showed the highest activity in 50 mM sodium acetate buffer at pH 5.0 and 25℃. This exo-enzyme could hydrolyze p-nitrophenyl arabinofuranoside, arabino-oligosaccharides (AOS), arabinoxylo-oligosaccharides (AXOS) such as 3²-α-ʟ-arabinofuranosyl-xylobiose (A³X), and 2³-α-ʟ-arabinofuranosyl-xylotriose (A²XX), whereas hardly hydrolyzed polymeric substrates such as debranched arabinan and arabinoxylans. BfdABF1 is a typical exo-ABF with the higher specific activity on the oligomeric substrates than the polymers. It prefers to α-(1,2)-ʟ-arabinofuranosidic linkages compared to α-(1,3)-linkages. Especially, BfdABF1 could slowly hydrolyze 2³,3³-di-α-ʟ-arabinofuranosyl-xylotriose (A²⁺³XX). Meanwhile, BfdABF3 showed the highest activity in sodium acetate at pH 6.0 and 50℃, and it has the exclusively high activities on AXOS such as A³X and A²XX. BfdABF3 mainly catalyzes the removal of ʟ-arabinose side chains from various AXOS. BfdXYL2 exhibited the highest activity in sodium citrate at pH 5.0 and 55℃, and it specifically hydrolyzed p-nitrophenyl xylopyranoside and xylo-oligosaccharides (XOS). Also, BfdXYL2 could slowly hydrolyze AOS and AXOS such as A³X. Based on the detailed hydrolytic modes of action of three exo-hydrolases (BfdABF1, BfdABF3, and BfdXYL2) from Bf. dentium, their probable roles in the hemiceullose-utilization system of Bf. dentium are proposed in the present study. These intracellular exo-hydrolases can synergistically produce ʟ-arabinose and ᴅ-xylose from various AOS, XOS, and AXOS.

• Journal of Microbiology and Biotechnology
• /
• v.31 no.11
• /
• pp.1583-1590
• /
• 2021

Studies have demonstrated that PE_PGRS45 is constitutively expressed under various environmental conditions (such as nutrient depletion, hypoxia, and low pH) of the in vitro growth conditions examined, indicating that PE_PGRS45 protein is critical to the basic functions of Mycobacterium tuberculosis. However, there are few reports about the biochemical function and pathogenic mechanism of PE_PGRS45 protein. The fact that this M. tuberculosis gene is not easily expressed in E. coli may be mainly due to the high content of G+C and the use of unique codons. Fusion tags are indispensable tools used to improve the soluble expression of recombinant proteins and accelerate the characterization of protein structure and function. In the present study, His6, Trx, and His6-MBP were used as fusion tags, but only MBP-PE_PGRS45 was expressed solubly. The purification using His6-MBP tag-specific binding to the Ni column was easy to separate after the tag cleavage. We used the purified PE_PGRS45 to immunize New Zealand rabbits and obtained anti-PE_PGRS45 serum. We found that the titer of polyclonal antibodies against PE_PGR45 was higher than 1:256000. The result shows that purified PE_PGRS45 can induce New Zealand rabbits to produce high-titer antibodies. In conclusion, the recombinant protein PE_PGRS45 was successfully expressed in E. coli and specific antiserum was prepared, which will be followed by further evaluation of these specific antigens to develop highly sensitive and specific diagnostic tests for tuberculosis.

• Journal of Microbiology and Biotechnology
• /
• v.32 no.3
• /
• pp.294-301
• /
• 2022

In our greenhouse experiment, soil heat treatment groups (50, 80, and 121℃) significantly promoted growth and disease suppression of Panax notoginseng in consecutively cultivated soil (CCS) samples (p < 0.01), and 80℃ worked better than 50℃ and 121℃ (p < 0.01). Furthermore, we found that heat treatment at 80℃ changes the microbial diversity in CCS, and the inhibition ratios of culturable microorganisms, such as fungi and actinomycetes, were nearly 100%. However, the heat-tolerant bacterial community was preserved. The 16S rRNA gene and internal transcribed spacer (ITS) sequencing analyses indicated that the soil heat treatment had a greater effect on the Chao1 index and Shannon's diversity index of bacteria than fungi, and the relative abundances of Firmicutes and Proteobacteria were significantly higher than without heating (80 and 121℃, p < 0.05). Soil probiotic bacteria, such as Bacillus (67%), Sporosarcina (9%), Paenibacillus (6%), Paenisporosarcina (6%), and Cohnella (4%), remained in the soil after the 80℃ and 121℃ heat treatments. Although steam increased the relative abundances of most of the heat-tolerant microbes before sowing, richness and diversity gradually recovered to the level of CCS, regardless of fungi or bacteria, after replanting. Thus, we added heat-tolerant microbes (such as Bacillus) after steaming, which reduced the relative abundance of pathogens, recruited antagonistic bacteria, and provided a long-term protective effect compared to the steaming and Bacillus alone (p < 0.05). Taken together, the current study provides novel insight into sustainable agriculture in a consecutively cultivated system.

• Journal of Microbiology and Biotechnology
• /
• v.31 no.11
• /
• pp.1501-1507
• /
• 2021

Lagerstroemia ovalifolia Teijsm. & Binn. (LO) (crape myrtle) has reportedly been used as traditional herbal medicine (THM) in Java, Indonesia. Our previous study revealed that the LO leaf extract (LOLE) exerted anti-inflammatory effects on lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Based on this finding, the current study aimed to evaluate the protective effects of LOLE in a mouse model of LPS-induced acute lung injury (ALI). The results showed that treatment with LPS enhanced the inflammatory cell influx into the lungs and increased the number of macrophages and the secretion of the inflammatory cytokines in the bronchoalveolar lavage fluid (BALF) of mice. However, these effects were notably abrogated with LOLE pretreatment. Furthermore, the increase of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and monocyte chemoattractant protein-1 (MCP-1) expression in the lung tissues of mice with ALI was also reversed by LOLE. In addition, LOLE significantly suppressed the LPS-induced activation of the MAPK/NF-κB signaling pathway and led to heme oxygenase-1 (HO-1) induction in the lungs. Additionally, in vitro experiments showed that LOLE enhanced the expression of HO-1 in RAW264.7 macrophages. The aforementioned findings collectively indicate that LOLE exerts an ameliorative effect on inflammatory response in the airway of ALI mice.

• Journal of Microbiology and Biotechnology
• /
• v.32 no.4
• /
• pp.405-418
• /
• 2022

Simotang oral liquid (SMT) is a traditional Chinese medicine (TCM) consisting of four natural plants and is used to alleviate gastrointestinal side effects after chemotherapy and functional dyspepsia (FD). However, the mechanism by which SMT helps cure these gastrointestinal diseases is still unknown. Here, we discovered that SMT could alleviate gastrointestinal side effects after chemotherapy by altering gut microbiota. C57BL/6J mice were treated with cisplatin (DDP) and SMT, and biological samples were collected. Pathological changes in the small intestine were observed, and the intestinal injury score was assessed. The expression levels of the inflammatory factors IL-1β and IL-6 and the adhesive factors Occludin and ZO-1 in mouse blood or small intestine tissue were also detected. Moreover, the gut microbiota was analyzed by high-throughput sequencing of 16S rRNA amplicons. SMT was found to effectively reduce gastrointestinal mucositis after DDP injection, which lowered inflammation and tightened the intestinal epithelial cells. Gut microbiota analysis showed that the abundance of the anti-inflammatory microbiota was downregulated and that the inflammatory microbiota was upregulated in DDP-treated mice. SMT upregulated anti-inflammatory and anticancer microbiota abundance, while the inflammatory microbiota was downregulated. An antibiotic cocktail (ABX) was also used to delete mice gut microbiota to test the importance of gut microbiota, and we found that SMT could not alleviate gastrointestinal mucositis after DDP injection, showing that gut microbiota might be an important mediator of SMT treatment. Our study provides evidence that SMT might moderate gastrointestinal mucositis after chemotherapy by altering gut microbiota.

• Journal of Microbiology and Biotechnology
• /
• v.32 no.4
• /
• pp.531-540
• /
• 2022

Due to the high incidence of malignant melanoma, the establishment of in vitro models that recapitulate the tumor microenvironment is of great biological and clinical importance for tumor treatment and drug research. In this study, 3D printing technology was used to prepare GelMA/PEGDA composite scaffolds that mimic the microenvironment of human malignant melanoma cell (A375) growth and construct in vitro melanoma micro-models. The GelMA/PEGDA hybrid scaffold was tested by the mechanical property, cell live/dead assay, cell proliferation assay, cytoskeleton staining and drug loading assay. The growth of tumor cells in two- and three-dimensional culture systems and the anti-cancer effect of luteolin were evaluated using the live/dead staining method and the Cell Counting Kit-8 (CCK-8) method. The results showed a high aggregation of tumor cells on the 3D scaffold, which was suitable for long-term culture. Cytoskeleton staining and immunofluorescent protein staining were used to evaluate the degree of differentiation of tumor cells under 2D and 3D culture systems. The results indicated that 3D bioprinted scaffolds were more suitable for tumor cell expansion and differentiation, and the tumor cells were more aggressive. In addition, luteolin was time- and dose-dependent on tumor cells, and tumor cells in the 3D culture system were more resistant to the drug.