Search

Abstract Macrophages play a pivotal role in the inflammatory response to the zoonotic pathogen E. coli, responsible for causing enteric infections. While considerable research has been conducted to comprehend the pathogenesis of this disease, scant attention devoted to host-derived H2S. Herein, we reported that E. coli infection enhanced the expression of CSE in macrophages, accompanied by a significantly increased inflammatory response. This process may be mediated by the involvement of excessive autophagy. Inhibition of AMPK or autophagy with pharmacological inhibitors could alleviate the inflammation. Additionally, cell model showed that the mRNA expression of classic inflammatory factors (Il-1β, Il-6), macrophage polarization markers (iNOS, Arg1) and ROS production was significantly down-regulated after employing CSE specific inhibitor PAG. And PAG is capable of inhibiting excessive autophagy through the LKB1-AMPK-ULK1 axis. Interestingly, exogenous H2S could suppress inflammation response. Our study emphasizes the importance of CSE in regulating the macrophage-mediated response to E. coli. Increased CSE in macrophages leads to excessive inflammation, which should be considered a new target for drug development to treat intestinal infection.

Abstract Foamy macrophages containing prominent cytoplasmic lipid droplets (LDs) are found in a variety of infectious diseases. However, their role in Streptococcus uberis-induced mastitis is unknown. Herein, we report that S. uberis infection enhances the fatty acid synthesis pathway in macrophages, resulting in a sharp increase in LD levels, accompanied by a significantly enhanced inflammatory response. This process is mediated by the involvement of fatty acid binding protein 4 (FABP4), a subtype of the fatty acid-binding protein family that plays critical roles in metabolism and inflammation. In addition, FABP4 siRNA inhibitor cell models showed that the deposition of LDs decreased, and the mRNA expression of Tnf, Il1b and Il6 was significantly downregulated after gene silencing. As a result, the bacterial load in macrophages increased. Taken together, these data demonstrate that macrophage LD formation is a host-driven component of the immune response to S. uberis. FABP4 contributes to promoting inflammation via LDs, which should be considered a new target for drug development to treat infections.

ABSTRACT Klebsiella pneumoniae is an opportunistic pathogen that causes serious infections in humans and animals. However, the availability of epidemiological information on clinical mastitis due to K. pneumoniae is limited. To acquire new information regarding K. pneumoniae mastitis, data were mined about K. pneumoniae strains on dairy cattle farms (farms A to H) in 7 Chinese provinces in 2021. Hypermucoviscous strains of K. pneumoniae were obtained by the string test. MICs of antimicrobial agents were determined via the broth microdilution method. Ten antimicrobial resistance genes and virulence genes were identified by PCR. The prevalence of K. pneumoniae was 35.91% (65/181), and 100% of the bacteria were sensitive to enrofloxacin. Nine antimicrobial resistance genes and virulence genes were identified and compared among farms. The hypermucoviscous phenotype was present in 94.44% of isolates from farm B, which may be a function of the rmpA virulence gene. Based on these data, the multidrug-resistant strains SD-14 and HB-21 were chosen and sequenced. Genotypes were assayed for K. pneumoniae isolates from different countries and different hosts using multilocus sequence typing (MLST). Ninety-four sequence types (STs) were found, and 6 STs present a risk for spreading in specific regions. Interestingly, ST43 was observed in bovine isolates for the first time. Our study partially reveals the current distribution characteristics of bovine K. pneumoniae in China and may provide a theoretical basis for the prevention and treatment of bovine K. pneumoniae mastitis. IMPORTANCE K. pneumonia is ubiquitous in nature and infects a wide range of hosts, including animals, and humans. It is one of the leading inducements of clinical mastitis (CM) in dairy cows, a prevalent and costly disease that is predominantly associated with bacterial infection. In general, CM caused by Gram-negative bacteria is more difficult to cure than that associated with Gram-positive pathogens, with an average cost per case of 211.03 U.S. dollars (USD) for Gram-negative bacterial infections compared with 133.73 USD for Gram-positive bacterial CM cases. After Escherichia coli, K. pneumoniae is the second most common Gram-negative cause of bovine CM, but it is the most detrimental in terms of decreased milk yield, discarded milk, treatment costs, death, and culling. In view of the economic implications of K. pneumoniae infection in dairy farming, research into population structure and antibiotic resistance is particularly important.

Here, we reported a Ginkgo endophyte, Aspergillus sp. Gbtc 2, isolated from the root tissue. Its flavonoid biosynthesis pathway was reconstructed, the effect of phenylalanine on the production of flavonoids was explored, and the flavonoid metabolites were identified with the high-resolution Liquid chromatography–mass spectrometry (LC–MS). Some essential genes were annotated to form the upstream of the complete biosynthesis pathway, indicating that Aspergillus sp. Gbtc 2 has the ability to synthesize the C6–C3–C6 flavonoid monomers. HPLC results showed that adding an appropriate amount of phenylalanine could promote the production of flavonoids by Aspergillus Gbtc 2. LC–MS results depicted a significant difference in many flavonoids between intracellularly and extracellularly. Most of the flavonoids gathered in the cell contained glycosylation groups, while almost all components with multiple hydroxyls showed much higher concentrations extracellularly than intracellularly; they likely have different biological functions. A variety of these substances can be mapped back to the pathway pattern of flavonoid biosynthesis and prove the ability of flavonoid production once again. This study expanded the information on flavonoid biosynthesis in Aspergillus and provided a solid theoretical basis for developing the fungi into genetically engineered strains undertaking flavonoid industrialized production.

Altitude affects plant growth and metabolism, but the effect of altitude on plant endophytic microorganisms is still unclear. In this study, we selected 16 Ginkgo biloba trees to study the response of leaves’ endophytes to flavonoids and altitude (from 530 m to 1,310 m). HPLC results showed that flavonoids in Ginkgo biloba leaves increased by more than 150% with attitude rising from 530 m to 1,310 m, which revealed a positive correlation with altitude. Ginkgo biloba might regulate the increased flavonoids in leaves to resist the increasing light intensity. 16S rDNA sequencing results showed that the endophytic bacterial communities of Ginkgo biloba at different altitudes significantly differed. Ginkgo leaf endophytes’ alpha diversity decreased with increasing flavonoids content and altitude. The increased flavonoids might increase the environmental pressure on endophytes and affect the endophytic community in Ginkgo biloba leaves. The bacterial network in Ginkgo biloba leaves became more complex with increasing altitude, which might be one of the strategies of leaf endophytes to cope with increasing flavonoids. Metagenomes results predicted with PICRUSt showed that the abundance of flavonoid biosynthesis and photosynthesis genes were significantly decreased with the increase of flavonoid contents. High flavonoid content in leaves appeared to inhibit microbial flavonoid synthesis. Our findings indicate that altitude can modulate microbial community structure through regulating plant metabolites, which is important to uncovering the interaction of microbes, host and the environment.

Flavonoids are crucial in physiological and pharmaceutical processes, especially the treatment of cancer and the prevention of cardiovascular and cerebrovascular diseases. Flavonoid-producing plants and fungi have been extensively reported, but bacteria have been much less investigated as a source of flavonoid production. Deinococcus sp. 43, a spherical flavonoid-producing bacteria from the Ginkgo rhizosphere, was reported in this study. First, the whole genome of Deinococcus sp. 43 was sequenced and a series of flavonoid anabolic genes were annotated. Simultaneously, High Performance Liquid Chromatography (HPLC) results showed that Deinococcus sp. 43 was capable of producing flavonoids, with a maximum quercetin output of 2.9 mg/L. Moreover, the relative expression of key genes involved in flavonoid synthesis was determined to test the completeness of the flavonoid anabolic pathway. The results of LC-MS analysis demonstrated that the flavonoids produced by Deinococcus sp. 43 were significantly different between intracellular and extracellular environments. The concentration of multiple glycosylated flavonoids was substantially higher in extracellular than intracellular environments, while the majority of flavonoids obtained in intracellular environments were hydroxylated multiple times. Lastly, the flavonoid biosynthetic pathway of Deinococcus sp. 43 was constructed based on the genomic analysis and the detected flavonoids. In conclusion, this study represents the first comprehensive characterization of the flavonoid-producing pathway of Deinococcus. The findings demonstrate that the strain has excellent potential as a genetically engineered strain for the industrial production of flavonoids.

The response of soil bacterial communities from farmland ecosystems to cadmium (Cd) pollution, in which a steep concentration gradient of more than 100 mg/kg has naturally formed, has not previously been fully reported. In this study, a field investigation was conducted in a typical severe Cd-polluted farmland ecosystem, and the bacterial community response to the steep Cd gradient was analyzed. The results showed that Cd concentration sharply decreased from 159.2 mg/kg to 4.18 mg/kg among four sampling sites alongside an irrigation canal over a distance of 150 m. Bacterial diversity and richness were significantly lower in highly polluted sites, and random forest analysis indicated that Cd gradient played a decisive role in reducing alpha diversity. Redundancy analysis (RDA) and co-occurrence network indicated that the synergistic effects of pH, Cd, and phosphorus were the main drivers shaping community structure. The functional results predicted by BugBase suggested that the bacterial community may adapt to the harsh environment by recruiting Cd-resistant microbes and improving oxidative stress tolerance of the whole community. Cd-resistant microorganisms such as Burkholderia, Bradyrhizobium, and Sulfurifustis, which directly or indirectly participate in diminishing oxidative damage of Cd, may play essential roles in maintaining community stability and might be potential bacterial resources for the bioremediation of Cd pollution.

Plant-specialized secondary metabolites have ecological functions in mediating interactions between plants and their entophytes. In this study, high-throughput gene sequencing was used to analyze the composition and abundance of bacteria from Ginkgo leaves at five different sampling times. The results indicated that the bacterial community structure varied during leaf developmental stage. Bacterial diversity was observed to be the highest at T2 stage and the lowest at T1 stage. Proteobacteria, Firmicutes, Actinobacteria, Chloroflexi, Cyanobacteria, and Bacteroidetes were found as the dominant phyla. The major genera also showed consistency across sampling times, but there was a significant variation in their abundance, such as Bacillus, Lysinibacillus, and Staphylococcus. Significant correlations were observed between endophytic bacteria and flavonoids. Especially, Staphylococcus showed a significant positive correlation with quercetin, and changes in the abundance of Staphylococcus also showed a strong correlation with flavonoid content. In order to determine the effect of flavonoids on endophytic bacteria of Ginkgo leaves, an extracorporeal culture of related strains (a strain of Staphylococcus and a strain of Deinococcus) was performed, and it was found that the effect of flavonoids on them remained consistent. The predicted result of Tax4Fun2 revealed that flavonoids might lead to a lower abundance of endophytic microorganisms, which further proved the correlation between bacterial communities and flavonoids. This study provided the first insight into the bacterial community composition during the development of Ginkgo leaves and the correlation between the endophytic bacteria and flavonoids.

Ginkgo biloba is a pharmaceutical resource for terpenes and flavonoids. However, few insights discussed endophytes’ role in Ginkgo, and whether genetic exchange happens between Ginkgo and endophytes remains unclear. Herein, functional gene profiles and repetitive sequences were analyzed to focus on these issues. A total of 25 endophyte strains were isolated from the Ginkgo root and distributed in 16 genera of 6 phyla. Significant morphological diversities lead to the diversity in the COG functional classification. KEGG mapping revealed that endophytic bacteria and fungi potentially synthesize chalcone, while endophytic fungi might also promote flavonoid derivatization. Both bacteria and fungi may facilitate the lignin synthesis. Aspergillus sp. Gbtc_1 exhibited the feasibility of regulating alcohols to lignans. Although Ginkgo and the endophytes have not observed the critical levopimaradiene synthase in ginkgolides synthesis, the upstream pathways of terpenoid precursors are likely intact. The MVK genes in Ginkgo may have alternative non-homologous copies or be compensated by endophytes in long-term symbiosis. Cellulomonas sp. Gbtc_1 became the only bacteria to harbor both MEP and MVA pathways. Endophytes may perform the mutual transformation of IPP and DMAPP in the root. Ginkgo and bacteria may lead to the synthesis and derivatization of the carotenoid pathway. The isoquinoline alkaloid biosynthesis seemed lost in the Ginkgo root community, but L-dopa is more probably converted into dopamine as an essential signal-transduction substance. So, endophytes may participate in the secondary metabolism of the Ginkgo in a shared or complementary manner. Moreover, a few endophytic sequences predicted as Ty3/Gypsy and Ty1/Copia superfamilies exhibited extremely high similarity to those of Ginkgo. CDSs in such endophytic LTR-RT sequences were also highly homologous to one Ginkgo CDS. Therefore, LTR-RTs may be a rare unit flowing between the Ginkgo host and endophytes to exchange genetic information. Collectively, this research effectively expanded the insight on the symbiotic relationship between the Ginkgo host and the endophytes in the root.

Streptococcus uberis (S. uberis) is an important pathogen causing mastitis, which causes continuous inflammation and dysfunction of mammary glands and leads to enormous economic losses. Most research on infection continues to be microbial metabolism-centric, and many overlook the fact that pathogens require energy from host. Mouse is a common animal model for studying bovine mastitis. In this perspective, we uncover metabolic reprogramming during host immune responses is associated with infection-driven inflammation, particularly when caused by intracellular bacteria. Taurine, a metabolic regulator, has been shown to effectively ameliorate metabolic diseases. We evaluated the role of taurine in the metabolic regulation of S. uberis-induced mastitis. Metabolic profiling indicates that S. uberis exposure triggers inflammation and metabolic dysfunction of mammary glands and mammary epithelial cells (the main functional cells in mammary glands). Challenge with S. uberis upregulates glycolysis and oxidative phosphorylation in MECs. Pretreatment with taurine restores metabolic homeostasis, reverses metabolic dysfunction by decrease of lipid, amino acid and especially energy disturbance in the infectious context, and alleviates excessive inflammatory responses. These outcomes depend on taurine-mediated activation of the AMPK–mTOR pathway, which inhibits the over activation of inflammatory responses and alleviates cellular damage. Thus, metabolic homeostasis is essential for reducing inflammation. Metabolic modulation can be used as a prophylactic strategy against mastitis.

Xenobiotics are generally known as man-made refractory organic pollutants widely distributed in various environments. For exploring the bioremediation possibility of xenobiotics, two novel xenobiotics-degrading strains affiliated with Azonexaceae were isolated. We report here the phylogenetics, genome, and geo-distribution of a novel and ubiquitous Azonexaceae species that primarily joins in the cometabolic process of some xenobiotics in natural communities. Strains s22 and t15 could be proposed as a novel species within Dechloromonas based on genomic and multi-phylogenetic analysis. Pan-genome analysis showed that the 63 core genes in Dechloromonas include genes for dozens of metabolisms such as nitrogen fixation protein (nifU), nitrogen regulatory protein (glnK), dCTP deaminase, C4-dicarboxylate transporter, and fructose-bisphosphate aldolase. Strains s22 and t15 have the ability to metabolize nitrogen, including nitrogen fixation, NirS-dependent denitrification, and dissimilatory nitrate reduction. Moreover, the novel species possesses the EnvZ-OmpR two-component system for controlling osmotic stress and QseC-QseB system for quorum sensing to rapidly sense environmental changes. It is intriguing that this new species has a series of genes for the biodegradation of some xenobiotics such as azathioprine, 6-Mercaptopurine, trinitrotoluene, chloroalkane, and chloroalkene. Specifically, glutathione S-transferase (GST) and 4-oxalocrotonate tautomerase (praC) in this novel species play important roles in the detoxification metabolism of some xenobiotics like dioxin, trichloroethene, chloroacetyl chloride, benzo[a]pyrene, and aflatoxin B1. Using data from GenBank, DDBJ and EMBL databases, we also demonstrated that members of this novel species were found globally in plants (e.g. rice), guts (e.g. insect), pristine and contaminated regions. Given these data, Dechloromonas sp. strains s22 and t15 take part in the biodegradation of some xenobiotics through key enzymes.

Endophytes are highly associated with plant growth and health. Exploring the variation of bacterial communities in different plant niches is essential for understanding microbe-plant interactions. In this study, high-throughput gene sequencing was used to analyze the composition and abundance of bacteria from the rhizospheric soil and different parts of the Macleaya cordata. The results indicated that the bacterial community structure varied widely among compartments. Bacterial diversity was observed to be the highest in the rhizospheric soil and the lowest in fruits. Proteobacteria, Actinobacteria, and Bacteroidetes were found as the dominant phyla. The genera Sphingomonas (∼47.77%) and Methylobacterium (∼45.25%) dominated in fruits and leaves, respectively. High-performance liquid chromatography (HPLC) was employed to measure the alkaloid content of different plant parts. Significant correlations were observed between endophytic bacteria and alkaloids. Especially, Sphingomonas showed a significant positive correlation with sanguinarine and chelerythrine. All four alkaloids were negatively correlated with the microbiota of stems. The predicted result of PICRUST2 revealed that the synthesis of plant alkaloids might lead to a higher abundance of endophytic microorganisms with genes related to alkaloid synthesis, further demonstrated the correlation between bacterial communities and alkaloids. This study provided the first insight into the bacterial community composition in different parts of Macleaya cordata and the correlation between the endophytic bacteria and alkaloids.

Ginkgo leaves are always resources for flavonoids pharmaceutical industry. However, the effect of the elevation and tree age changes on flavonoid biosynthesis have not been detailly explored in Ginkgo leaves. In addition, whether these environmental pressures have similar effects on the biosynthesis of other non-flavonoids polyphenolics in phenylpropanoid biosynthesis is not known at present. In this research, de novo transcriptome sequencing of Ginkgo leaves was performed coupled with ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry analyses to obtain a comprehensive understanding of the influence of elevation and tree age on phenylpropanoid biosynthesis. A total of 557,659,530 clean reads were assembled into 188,155 unigenes, of which 135,102 (71.80%) were successfully annotated in seven public databases. The putative DFRs, LARs, and ANRs were significantly up-regulated with the increase of elevation in young Ginkgo tree leaves. The relative concentration of flavonoid derivatives with high parent ion intensity was likely to imply that the elevation increase promoted the biosynthesis of flavonoids. Complex gene variations involved in flavonoid biosynthesis were observed with the tree age increase. However, flavonoid derivatives analysis predicted that the rise of tree age was more likely to be detrimental to the flavonoids manufacture. Otherwise, multiple genes implicated in the synthesis of hydroxycinnamates, lignin, and lignan exhibited fluctuations with the elevation increase. Significantly up-regulated CADs and down-regulated PRDs potentially led to the accumulation of p-Coumaryl alcohol, one of the lignin monomers, and might inhibit further lignification. Overall, the putative DFRs seemed to show more considerable variability toward these stress, and appeared to be the main regulatory point in the flavonoid biosynthesis. Light enhancement caused by elevation increase may be the main reason for flavonoids accumulation. Flavonoid biosynthesis exhibited a greater degree of perturbation than that of hydroxycinnamates, lignins and lignans, potentially suggesting that flavonoid biosynthesis might be more susceptible than other branch pathways involved in phenylpropanoid biosynthesis. This research effectively expanded the functional genomic library and provide new insights into phenylpropanoid biosynthesis in Ginkgo.