Immune-Enhancing Effect of Submerged Culture of Ceriporia lacerata Mycelia on Cyclophosphamide-Induced Immunosuppressed Mice and the Underlying Mechanisms in Macrophages

The white-rot fungi Ceriporia lacerata is used in bioremediation, such as lignocellulose degradation, in nature. Submerged cultures and extracts of C. lacerata mycelia (CLM) have been reported to contain various active ingredients, including β-glucan and extracellular polysaccharides, and to exert anti-diabetogenic properties in mice and cell lines. However, the immunostimulatory effects have not yet been reported. This study aimed to identify the immunomodulatory effects, and underlying mechanisms thereof, of submerged cultures of CLM using RAW264.7 macrophages and cyclophosphamide (CTX)-induced immunosuppression in mice. Compared to CTX-induced immunosuppressed mice, the spleen and thymus indexes in mice orally administered CLM were significantly increased; body weight loss was alleviated; and natural killer (NK) cytotoxicity, lymphocyte proliferation, and cytokine (tumor necrosis factor [TNF]-α, interferon [IFN]-γ, and interleukin [IL]-2) production were elevated in the serum. In RAW264.7 macrophages, treatment with CLM induced phagocytic activity, increased the production of nitric oxide (NO), and promoted mRNA expression of the immunomodulatory cytokines TNF-α, IFN-γ, IL-1β, IL-6, IL-10, and IL-12. In addition, CLM increased the inducible NO synthase (iNOS) concentration in macrophages, similar to lipopolysaccharide (LPS) stimulation. Mechanistic studies showed that CLM induced the activation of the NF-κB, PI3k/Akt, ERK1/2, and JNK1/2 pathways. Moreover, the phosphorylation of NF-κB and IκB induced by CLM in RAW264.7 cells was suppressed by specific MAPKs and PI3K inhibitors. Further experiments with a TLR4 inhibitor demonstrated that the production of TNF-α, IL-1β, and IL-6 induced by CLM was decreased after TLR4 was blocked. Overall, CLM protected against CTX-induced adverse reactions by enhancing humoral and cellular immune functions, and has potential as an immunomodulatory agent.

T6SS Accessory Proteins, Including DUF2169 Domain-Containing Protein and Pentapeptide Repeats Protein, Contribute to Bacterial Virulence in T6SS Group_5 of Burkholderia glumae BGR1

Burkholderia glumae are bacteria pathogenic to rice plants that cause a disease called bacterial panicle blight (BPB) in rice panicles. BPB, induced by B. glumae, causes enormous economic losses to the rice agricultural industry. B. glumae also causes bacterial disease in other crops because it has various virulence factors, such as toxins, proteases, lipases, extracellular polysaccharides, bacterial motility, and bacterial secretion systems. In particular, B. glumae BGR1 harbors type VI secretion system (T6SS) with functionally distinct roles: the prokaryotic targeting system and the eukaryotic targeting system. The functional activity of T6SS requires 13 core components and T6SS accessory proteins, such as adapters containing DUF2169, DUF4123, and DUF1795 domains. There are two genes, bglu_1g23320 and bglu_2g07420, encoding the DUF2169 domain-containing protein in the genome of B. glumae BGR1. bglu_2g07420 belongs to the gene cluster of T6SS group_5 in B. glumae BGR1, whereas bglu_1g23320 does not belong to any T6SS gene cluster in B. glumae BGR1. T6SS group_5 of B. glumae BGR1 is involved in bacterial virulence in rice plants. The DUF2169 domain-containing protein with a single domain can function by itself; however, Δu1g23320 showed no attenuated virulence in rice plants. In contrast, Δu2g07420DUF2169 and Δu2g07420PPR did exhibit attenuated virulence in rice plants. These results suggest that the pentapeptide repeats region of the C-terminal additional domain, as well as the DUF2169 domain, is required for complete functioning of the DUF2169 domain-containing protein encoded by bglu_2g07420. bglu_2g07410, which encodes the pentapeptide repeats protein, composed of only the pentapeptide repeats region, is located downstream of bglu_2g07420. Δu2g07410 also shows attenuated virulence in rice plants. This finding suggests that the pentapeptide repeats protein, encoded by bglu_2g07410, is involved in bacterial virulence. This study is the first report that the DUF2169 domain-containing protein and pentapeptide repeats protein are involved in bacterial virulence to the rice plants as T6SS accessory proteins, encoded in the gene cluster of the T6SS group_5.

The Microbial Diversity and Biofilm-Forming Characteristic of Two Traditional Tibetan Kefir Grains

In this study, a high-throughput sequencing technique was used to analyze bacterial and fungal diversity of two traditional Tibetan kefir grains from Linzhi (K1) and Naqu (K2) regions. Comparative bioinformatic analyses indicated that Lactobacillus kefiranofaciens, L. kefiri and Kluyveromyces marxianus were the main dominant strains in K1 and K2. In order to research the relationship of the growth of kefir grains, the biofilm and the extracellular polysaccharides (EPS) produced by microorganisms, the proliferation rate of kefir grains, the yield and chemical structure of EPS and the optimal days for biofilm formation were determined. The results showed that the growth rate, the yield of EPS and the biofilm formation ability of K1 were higher than K2, and the optimal day of their biofilm formation was the same in 10th day. Additionally, the live cells, dead cells and EPS in biofilm formation of K1 and K2 were observed by fluorescence microscope to clarify the formation process of kefir grains. To determine the influence of microbial interactions on biofilm and the formation of kefir grains, the essential role of microbial quorum sensing needs further attention.

Acetylation of glucosyltransferases regulates Streptococcus mutans biofilm formation and virulence

Lysine acetylation is a frequently occurring post-translational modification (PTM), emerging as an important metabolic regulatory mechanism in prokaryotes. This process is achieved enzymatically by the protein acetyltransferase (KAT) to specifically transfer the acetyl group, or non-enzymatically by direct intermediates (acetyl phosphate or acetyl-CoA). Although lysine acetylation modification of glucosyltransferases (Gtfs), the important virulence factor in Streptococcus mutans, was reported in our previous study, the KAT has not been identified. Here, we believe that the KAT ActG can acetylate Gtfs in the enzymatic mechanism. By overexpressing 15 KATs in S. mutans, the synthesized water-insoluble extracellular polysaccharides (EPS) and biofilm biomass were measured, and KAT (actG) was identified. The in-frame deletion mutant of actG was constructed to validate the function of actG. The results showed that actG could negatively regulate the water-insoluble EPS synthesis and biofilm formation. We used mass spectrometry (MS) to identify GtfB and GtfC as the possible substrates of ActG. This was also demonstrated by in vitro acetylation assays, indicating that ActG could increase the acetylation levels of GtfB and GtfC enzymatically and decrease their activities. We further found that the expression level of actG in part explained the virulence differences in clinically isolated strains. Moreover, overexpression of actG in S. mutans attenuated its cariogenicity in the rat caries model. Taken together, our study demonstrated that the KAT ActG could induce the acetylation of GtfB and GtfC enzymatically in S. mutans, providing insights into the function of lysine acetylation in bacterial virulence and pathogenicity.

The vicK gene of Streptococcus mutans mediates its cariogenicity via exopolysaccharides metabolism

Streptococcus mutans (S. mutans) is generally regarded as a major contributor to dental caries because of its ability to synthesize extracellular polysaccharides (EPS) that aid in the formation of plaque biofilm. The VicRKX system of S. mutans plays an important role in biofilm formation. The aim of this study was to investigate the effects of vicK gene on specific characteristics of EPS in S. mutans biofilm. We constructed single-species biofilms formed by different mutants of vicK gene. Production and distribution of EPS were detected through atomic force microscopy, scanning electron microscopy and confocal laser scanning microscopy. Microcosmic structures of EPS were analyzed by gel permeation chromatography and gas chromatography-mass spectrometry. Cariogenicity of the vicK mutant was assessed in a specific pathogen-free rat model. Transcriptional levels of cariogenicity-associated genes were confirmed by quantitative real-time polymerase chain reaction. The results showed that deletion of vicK gene suppressed biofilm formation as well as EPS production, and EPS were synthesized mostly around the cells. Molecular weight and monosaccharide components underwent evident alterations. Biofilms formed in vivo were sparse and contributed a decreased degree of caries. Moreover, expressional levels of genes related to EPS synthesis were down-regulated, except for gtfB. Our report demonstrates that vicK gene enhances biofilm formation and subsequent caries development. And this may due to its regulations on EPS metabolism, like synthesis or microcosmic features of EPS. This study suggests that vicK gene and EPS can be considered as promising targets to modulate dental caries.

Capsules and Extracellular Polysaccharides in Escherichia coli and Salmonella

Escherichia coli and Salmonella isolates produce a range of different polysaccharide structures that play important roles in their biology. E. coli isolates often possess capsular polysaccharides (K antigens), which form a surface structural layer. These possess a wide range of repeat-unit structures.

Discovery of Novel Dihydrolipoamide S-Succinyltransferase Inhibitors Based on Fragment Virtual Screening

A series of new oxadiazole sulfone derivatives containing an amide moiety was synthesized based on fragment virtual screening to screen high-efficiency antibacterial agents for rice bacterial diseases. All target compounds showed greater bactericidal activity than commercial bactericides. 3-(4-fluorophenyl)-N-((5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)methyl)acrylamide (10) showed excellent antibacterial activity against Xanthomonas oryzae pv. oryzae and Xanthomonas oryzae pv. oryzicola, with EC50 values of 0.36 and 0.53 mg/L, respectively, which were superior to thiodiazole copper (113.38 and 131.54 mg/L) and bismerthiazol (83.07 and 105.90 mg/L). The protective activity of compound 10 against rice bacterial leaf blight and rice bacterial leaf streak was 43.2% and 53.6%, respectively, which was superior to that of JHXJZ (34.1% and 26.4%) and thiodiazole copper (33.0% and 30.2%). The curative activity of compound 10 against rice bacterial leaf blight and rice bacterial leaf streak was 44.5% and 51.7%, respectively, which was superior to that of JHXJZ (32.6% and 24.4%) and thiodiazole copper (27.1% and 28.6%). Moreover, compound 10 might inhibit the growth of Xanthomonas oryzae pv. oryzae and Xanthomonas oryzae pv. oryzicola by affecting the extracellular polysaccharides, destroying cell membranes, and inhibiting the enzyme activity of dihydrolipoamide S-succinyltransferase.