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High lead (Pb) levels in agricultural soil and wastewater threaten ecosystems and organism health. Microbial remediation is a cost-effective, efficient, and eco-friendly alternative to traditional physical or chemical methods for Pb remediation. Previous research indicates that micro-organisms employ various strategies to combat Pb pollution, including biosorption, bioprecipitation, biomineralization, and bioaccumulation. This study delves into recent advancements in Pb-remediation techniques utilizing bacteria, fungi, and microalgae, elucidating their detoxification pathways and the factors that influence Pb removal through specific case studies. It investigates how bacteria immobilize Pb by generating nanoparticles that convert dissolved lead (Pb-II) into less harmful forms to mitigate its adverse impacts. Furthermore, the current review explores the molecular-level mechanisms and genetic engineering techniques through which microbes develop resistance to Pb. We outline the challenges and potential avenues for research in microbial remediation of Pb-polluted habitats, exploring the interplay between Pb and micro-organisms and their potential in Pb removal.

Bartonellae are considered to be emerging opportunistic pathogens. The bacteria are transmitted by blood-sucking arthropods, and their hosts are a wide range of mammals including humans. After a protective barrier breach in mammals, Bartonella colonizes endothelial cells (ECs), enters the bloodstream, and infects erythrocytes. Current research primarily focuses on investigating the interaction between Bartonella and ECs and erythrocytes, with recent attention also paid to immune-related aspects. Various molecules related to Bartonella’s pathogenicity have been identified. The present review aims to provide a comprehensive overview of the newly described molecular and immune responses associated with Bartonella’s pathogenicity.

Antimicrobial resistance (AMR) is a global concern threatening public health. Developing novel antibiotics is one of the effective strategies to tackle AMR. Serine/threonine kinases (STKs) have been recently shown to play critical roles in the physiology and pathogenesis of several important bacterial pathogens which are regarded as a promising antimicrobial drug target. We previously reported the roles of STK in the regulation of bacterial cell division, metabolism, and pathogenesis in Streptococcus suis, an important zoonotic bacterial pathogen. In this study, we firstly identified the Thr167 and Ser175 residues in the activation loop of S. suis STK (ssSTK) as the kinase autophosphorylation sites. Phenotyping results demonstrated that the autophosphorylation deficient strain resembled the stk deletion strain showing essentiality for bacterial growth in minimal medium, abnormal morphology, and decreased virulence when compared with the wild-type S. suis SC19 strain. Based on these findings, we established an ssSTK inhibitor screening approach by measuring the growth of S. suis in a minimal medium and testing the autophosphorylation inhibition by measuring the consumption of ATP in an enzymatic reaction by ssSTK. A series of inhibitors against ssSTK are identified from a commercial kinase inhibitors library, including Staurosporine, K252a, AT9283, and APY29. These inhibitors showed antimicrobial activity in vitro. Moreover, by using Galleria mellonella larvae infection assay, compound APY29 displayed in vivo efficacy against S. suis infection. Additionally, it was predicted by molecular docking that these inhibitors could interact with ssSTK. Collectively, our data illustrated the essential roles of ssSTK autophosphorylation in the physiology and pathogenicity of S. suis and consider these inhibitors as promising antimicrobial lead compounds.

Excessive application of the herbicide chlorimuron-ethyl (CE) severely harms subsequent crops and poses severe risks to environmental health. Therefore, methods for efficiently decreasing and eliminating CE residues are urgently needed. Microbial consortia show potential for bioremediation due to their strong metabolic complementarity and synthesis. In this study, a microbial consortium entitled L1 was enriched from soil contaminated with CE by a “top-down” synthetic biology strategy. The consortium could degrade 98.04% of 100 mg L−1 CE within 6 days. We characterized it from the samples at four time points during the degradation process and a sample without degradation activity via metagenome and 16S rDNA sequencing. The results revealed 39 genera in consortium L1, among which Methyloversatilis (34.31%), Starkeya (28.60%), and Pseudoxanthomonas (7.01%) showed relatively high abundances. Temporal succession and the loss of degradability did not alter the diversity and community composition of L1 but changed the community structure. Taxon-functional contribution analysis predicted that glutathione transferase [EC 2.5.1.18], urease [EC 3.5.1.5], and allophanate hydrolase [EC 3.5.1.54] are relevant for the degradation of CE and that Methyloversatilis, Pseudoxanthomonas, Methylopila, Hyphomicrobium, Stenotrophomonas, and Sphingomonas were the main degrading genera. The degradation pathway of CE by L1 may involve cleavage of the CE carbamide bridge to produce 2-amino-4-chloro-6-methoxypyrimidine and ethyl o-sulfonamide benzoate. The results of network analysis indicated close interactions, cross-feeding, and co-metabolic relationships between strains in the consortium, and most of the above six degrading genera were keystone taxa in the network. Additionally, the degradation of CE by L1 required not only “functional bacteria” with degradation capacity but also “auxiliary bacteria” without degradation capacity but that indirectly facilitate/inhibit the degradation process; however, the abundance of “auxiliary bacteria” should be controlled in an appropriate range. These findings improve the understanding of the synergistic effects of degrading bacterial consortia, which will provide insight for isolating degrading bacterial resources and constructing artificial efficient bacterial consortia. Furthermore, our results provide a new route for pollution control and biodegradation of sulfonylurea herbicides.

SARS-CoV-2 and its variants, such as the Omicron continue to threaten public health. The virus recognizes the host cell by attaching its Spike (S) receptor-binding domain (RBD) to the host receptor, ACE2. Therefore, RBD is a primary target for neutralizing antibodies and vaccines. Here, we report the isolation and biological and structural characterization of a single-chain antibody (nanobody) from RBD-immunized alpaca. The nanobody, named DL28, binds to RBD tightly with a KD of 1.56 nM and neutralizes the original SARS-CoV-2 strain with an IC50 of 0.41 μg mL−1. Neutralization assays with a panel of variants of concern (VOCs) reveal its wide-spectrum activity with IC50 values ranging from 0.35 to 1.66 μg mL−1 for the Alpha/Beta/Gamma/Delta and an IC50 of 0.66 μg mL−1 for the currently prevalent Omicron. Competition binding assays show that DL28 blocks ACE2-binding. However, structural characterizations and mutagenesis suggest that unlike most antibodies, the blockage by DL28 does not involve direct competition or steric hindrance. Rather, DL28 may use a “conformation competition” mechanism where it excludes ACE2 by keeping an RBD loop in a conformation incompatible with ACE2-binding.

A tigecycline-resistant Acinetobacter pittii clinical strain from pleural fluid carrying a blaNDM–1 gene and a novel blaOXA gene, blaOXA–1045, was isolated and characterized. The AP2044 strain acquired two copies of the blaNDM–1 gene and six antibiotic resistance genes (ARGs) from other pathogens. According to the whole-genome investigation, the GC ratios of ARGs (50–60%) were greater than those of the chromosomal backbone (39.46%), indicating that ARGs were horizontally transferred. OXA-1045 belonged to the OXA-213 subfamily and the amino acid sequence of OXA-1045 showed 89% similarity to the amino acid sequences of OXA-213. Then, blaOXA–1045 and blaOXA–213 were cloned and the minimum inhibitory concentrations (MICs) of β-lactams in the transformants were determined using the broth microdilution method. OXA-1045 was able to confer a reduced susceptibility to piperacillin and piperacillin-tazobactam compared to OXA-213. AP2044 strain exhibited low pathogenicity in Galleria mellonella infection models. The observation of condensed biofilm using the crystal violet staining method and scanning electron microscopy (SEM) suggested that the AP2044 strain was a weak biofilm producer. Quantitative reverse transcription-PCR (qRT-PCR) was used to detect the expression of resistance-nodulation-cell division (RND) efflux pump-related genes. The transcription level of adeB and adeJ genes increased significantly and was correlated with tigecycline resistance. Therefore, our genomic and phenotypic investigations revealed that the AP2044 strain had significant genome plasticity and natural transformation potential, and the emergence of antibiotic resistance in these unusual bacteria should be a concern for future investigations.

A linalool/polycaprolactone (LL/PCL) antibacterial film was prepared by using a coaxial electrospinning process, and its physical and chemical properties were characterized. The antibacterial film was formed into an active antibacterial gasket, and its effect on salmon preservation was analyzed. The results show that the LL/PCL nanofiber membrane had a well-developed microstructure, and the fiber surface was smooth and uniform. The diameter of the fibers in the PCL membrane without the core material (linalool) was 113.92 ± 23.74 nm. In contrast, the diameter of the coaxial nanofiber membrane with linalool increased, and the diameter of the LL/PCL membranes with 20% and 40% linalool was 220.62 ± 44.01 and 232.22 ± 56.27 nm, respectively. The hydrophobicity and water vapor permeability were enhanced, whereas the tensile strength and elongation at break decreased slightly, while the thermal stability did not differ significantly with the incorporation of linalool. Analysis of the sustained release of linalool showed that the LL/PCL coaxial fiber membranes could release linalool into the reaction system for a long time. The LL/PCL nanofiber film was used to create an antibacterial active gasket for salmon preservation experiments. Sensory evaluation and analyses of the total bacterial count, total volatile basic nitrogen (TVB-N), thiobarbituric acid reactive substances (TBARS), pH, texture (hardness, elasticity, chewiness, and viscoelasticity), water distribution change, and aroma using an electronic nose were used to determine the quality of salmon. It was found that food-grade tinfoil and the PCL gasket had no significant effect on the freshness of salmon, while the active antibacterial gasket samples containing linalool could decrease the rate of decay salmon and effectively prolong the shelf-life of salmon by releasing linalool.

Pseudomonas fluorescens is a common specific spoilage organism (SSO) of aquatic products. The spoilage ability of SSO can be regulated by the quorum sensing (QS) system. However, the QS system in P. fluorescens and their relationship with the spoilage potential have not been systematically analyzed. In the present study, the complete genome of P. fluorescens PF08 isolated from spoilage turbot was sequenced. The identification of key genes that involved in the QS, enzyme synthesis, sulfur, and amino acid metabolism explained the spoilage potential of P. fluorescens PF08. Results of quantitative real-time PCR revealed the key role of the P. fluorescens PF08 QS system in regulating the transcription of spoilage-related genes and its sensitivity to environmental stress. These findings provide insight into the spoilage features of P. fluorescens PF08 from a genomic perspective. The knowledge may be valuable in the development of new strategies for the targeted inhibition of aquatic product spoilage based on QS interference.

The Coronavirus disease 2019 (COVID-19) pandemic presents an unprecedented public health crisis worldwide. Although several vaccines are available, the global supply of vaccines, particularly within developing countries, is inadequate, and this necessitates a need for the development of less expensive, accessible vaccine options. To this end, here, we used the Escherichia coli expression system to produce a recombinant fusion protein comprising the receptor binding domain (RBD) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; residues 319–541) and the fragment A domain of Cross-Reacting Material 197 (CRM197); hereafter, CRMA-RBD. We show that this CRMA-RBD fusion protein has excellent physicochemical properties and strong reactivity with COVID-19 convalescent sera and representative neutralizing antibodies (nAbs). Furthermore, compared with the use of a traditional aluminum adjuvant, we find that combining the CRMA-RBD protein with a nitrogen bisphosphonate-modified zinc-aluminum hybrid adjuvant (FH-002C-Ac) leads to stronger humoral immune responses in mice, with 4-log neutralizing antibody titers. Overall, our study highlights the value of this E. coli-expressed fusion protein as an alternative vaccine candidate strategy against COVID-19.

There is little known of immunologic factors leading to the occurrence of occult HBV infection (OBI). Specific cellular immune response to hepatitis B virus (HBV) core/pol peptides was compared between blood donor populations, including 37 OBIs, 53 chronic HBV infections (CHB), 47 resolved infections, and 56 non-infected controls, respectively. The rate of CD4+/CD8+ T cell proliferation in OBI or CHB carriers was higher than in HBV resolved and non-infected individuals (P < 0.05). The intensity of IFN-γ-secretion T-cell response of OBI carriers was highest, followed by CHB and resolved infections, and non-infected individuals (P < 0.05). The frequency of intracellular IFN-γ and IL-17A CD4+/CD8+ and IL-21 CD4+ T-cell responses was significantly higher in resolved infections than in OBI or CHB carriers (P < 0.05), while the level of extracellular IL-17A of peripheral blood mononuclear cells (PBMCs) was higher in OBI and CHB carriers than in resolved infections (P < 0.01). The frequency of intracellular IL-10 CD4+ T-cell response in CHB, OBI, and resolved infections was higher than in HBV non-infected individuals (P < 0.01). Intracellular IL-10 CD8+ T cell and extracellular IL-10 T-cell responses were higher in CHB than in OBI (P = 0.012) or HBV resolved infections (P < 0.01). In conclusion, the higher level of effective T-cell response with IFN-γ, IL-17A, and IL-21 contributes to resolved infection outcome, while higher levels of suppressive T-cell response with IL-10 result in HBV chronicity. OBI is an intermediary status between HBV resolved and chronic infections, in which IL-21 effector and IL-10 suppressor T-cell responses play an important role in directing the outcome of HBV infection.

BackgroundDengue poses a large burden on the public health systems worldwide. severe dengue (SD) could lead to more serious clinical symptoms and even death. This study aimed to identify the cause of SD in a clinical trial during the dengue outbreak in Xishuangbanna in 2019, and could provide new insights into the pathogenic mechanisms of SD.MethodsMosquito-borne viral (DENV, JEV, and CHIKV) infections were identified. The epidemiological factors and clinical symptoms of inpatients in Xishuangbanna were recorded. The IgG and IgM levels in the serum of dengue inpatients were evaluated, and secondary infections were identified. Then, the structural proteins (C/PrM/E) were sequenced and compared with those of the same type of DENV in the same area as before, and their structures were predicted by the SWISS-MODEL (expasy.org). The full-length viral genomes were sequenced and aligned with representative strains by BioEidt or MEGA 5.0.ResultsIn this outbreak, the clinical symptoms were more serious in SD. The proportion of SD inpatients of male and Han nationality was larger than that of dengue fever (DF) inpatients (p < 0.05). DENV-2 infection was the majority in DF, with 45 inpatients. However, DENV-1 infection was the most common SD, with 54 inpatients. There were 3 DENV-3-positive inpatients in the DF group and 6 ZIKV-positive inpatients in the SD group. A secondary infection accounted for 76.47% (78 cases) of SD inpatients, but secondary infections were only in 20% (17 cases) of DF inpatients. In the three-dimensional structure of protein analysis, the C/PrM/E of DENV-1 and DENV-2 showed more stability than previous epidemic strains, while DENV-3 in 2019 showed a looser spatial structure. After a complete genome sequencing and analysis, all six DENV-2 strains belonged to cosmopolitan, five of which clustered into one branch. The GC/AT of the five strains decreased from 2014 to 2018. Compared with DF strains, SD strains had no mutations of commonness.ConclusionsSD may related to secondary heteromorphic dengue in Xishuangbanna in 2019. The coinfection of ZIKV could be another related factor for SD. The currently datas were very limited and only suggestive.

Trichinellosis caused by Trichinella spiralis is a worldwide food-borne parasitic zoonosis. Several approaches have been performed to control T. spiralis infection, including veterinary vaccines, which contribute to improving animal health and increasing public health by preventing the transmission of trichinellosis from animals to humans. In the past several decades, many vaccine studies have been performed in effort to control T. spiralis infection by reducing the muscle larvae and adult worms burden. Various candidate antigens, selected from excretory-secretory (ES) products and different functional proteins involved in the process of establishing infection have been investigated in rodent or swine models to explore their protective effect against T. spiralis infection. Moreover, different types of vaccines have been developed to improve the protective effect against T. spiralis infection in rodent or swine models, such as live attenuated vaccines, natural antigen vaccines, recombinant protein vaccines, DNA vaccines, and synthesized epitope vaccines. However, few studies of T. spiralis vaccines have been performed in pigs, and future research should focus on exploring the protective effect of different types of vaccines in swine models. Here, we present an overview of the strategies for the development of effective T. spiralis vaccines and summarize the factors of influencing the effectiveness of vaccines. We also discuss several propositions in improving the effectiveness of vaccines and may provide a route map for future T. spiralis vaccines development.

Studies have shown that the cholesterol-lowering medicine statins alter the gut microbiome, induce chronic metabolic inflammation, and disrupt glycemic homeostasis. In this study, we aimed to investigate whether effects of atorvastatin (Ator) on gut microbiome and metabolic inflammation could be causally correlated. Mice at 8-week age were fed with high-fat diet (HFD) or HFD with Ator (HFD+Ator) for 16 weeks. 16S rRNA sequencing of stool and RNA sequencing of colon tissue were employed to analyze the intestinal alterations that could be induced by Ator. A human colon carcinoma cell line (Caco2) was used for in vitro experiments on barrier function. Compared to HFD, HFD+Ator induced more weight gain, impaired glucose tolerance, and led to gut microbiota dysbiosis, such as suppressing Akkermansia muciniphila in mice. The expressions of tight junction (TJ) proteins were attenuated in the colon, and the serum LPS-binding-protein (LBP) level was elevated in HFD+Ator mice, so as to transcriptionally activate the intestinal nuclear factor-k-gene binding (NF-κB) signaling pathway. Consistently, Ator impaired the barrier function of Caco2, and treatment of supernatant of A. Muciniphila culture could decrease the intestinal permeability and recover the attenuated expression of TJ proteins induced by Ator. In conclusion, long-term use of Ator with HFD may alter gut microbiota, induce intestinal barrier dysfunction, and hence promote chronic inflammation that contributes to disrupted glycemic homeostasis.

Since December 2019, a novel coronavirus (SARS-CoV-2) has resulted in a global pandemic of coronavirus disease (COVID-19). Although viral nucleic acid test (NAT) has been applied predominantly to detect SARS-CoV-2 RNA for confirmation diagnosis of COVID-19, an urgent need for alternative, rapid, and sensitive immunoassays is required for primary screening of virus. In this study, we developed a smartphone-based nanozyme-linked immunosorbent assay (SP-NLISA) for detecting the specific nucleocapsid phosphoprotein (NP) of SARS-CoV-2 in 37 serum samples from 20 COVID-19 patients who were diagnosed by NAT previously. By using SP-NLISA, 28/37 (75.7%) serum samples were detected for NP antigens and no cross-reactivity with blood donors’ control samples collected from different areas of China. In a control assay using the conventional enzyme-linked immunosorbent assay (ELISA), only 7/37 (18.91%) serum samples were detected for NP antigens and no cross-reactivity with control samples. SP-NLISA could be used for rapid detection of SARS-CoV-2 NP antigen in primary screening of SARS-CoV-2 infected individuals.

An efficient bioflocculant-producing strain, Raoultella ornithinolytica 160-1, was identified by 16S rRNA and mass spectrometry analyses. Rapid production of bioflocculant EPS-160 was obtained with 10.01 g/(L⋅d) after optimized by response surface methodology. With the aid of Al(III), more than 90% flocculation activity of EPS-160 at 8 mg/L dosage was achieved in 5 min. Thus, this novel Al(III) dependent bioflocculant was used in combined with chemical coagulants AlCl3 to remove kaolin suspensions and wastewater treatment. The results indicated that the addition of EPS-160 in aggregation system not only largely improved the flocculation ability than the individual use of chemical flocculant (over 30 percent), but also overcome the decrease of flocculation activity due to the overdose of AlCl3 and maintained the optimum dosage of AlCl3 in a wide range (11–23 mg/L). The zeta potentials and EPS-160 structure indicated that both charge neutralization and bridging were the flocculation mechanism with kaolin. During the wastewater treatment, this composite flocculants consisted of EPS-160 and AlCl3 also had great performance for turbidity elimination. Moreover, with the properties of high flocculation activity, hyperthermal stability, pH tolerance and non-toxicity, EPS-160 shows great potential applications.

The effect of the combination of vanillin and chitosan (VC) coating on the microbiota composition and shelf-life of turbot (Scophthalmus maximus) filets during a 15-day storage period at 4 ± 1°day was investigated in this study. The control and coated fish samples were analyzed periodically for sensory and chemical attributes [total volatile basic nitrogen (TVB-N), thiobarbituric acid reactive substances (TBARS), and pH] and the presence of dominant spoilage microbiota. The results suggested that the sensory and the chemical quality of turbot filets effectively improved after treatment with vanillin (final concentration 2 mg/ml) combined with 1% chitosan, and the shelf-life was prolonged for 6 to 7 days compared with the control group. Furthermore, high-throughput sequencing showed that Proteobacteria (52.2%) and Firmicutes (29.8%) were the dominant bacteria at the phylum level in fresh turbot filets, while Pseudomonadaceae (40.2%) and Lactobacillaceae (39.4%) were the dominant bacteria at the family level in deteriorated turbot filets. However, after VC treatment, the relative abundance of Pseudomonadaceae and Lactobacillaceae decreased significantly due to the growth inhibition of potential bacteria, specifically spoilage bacteria, along with the rich bacterial diversity at the end of storage. Therefore, our data indicated that VC treatment might be effective in decreasing bacteria-induced quality deterioration and in extending the shelf-life of refrigerated turbot filets.

Some bacteria can modulate their spoilage potential by responding to environmental signaling molecules via the quorum sensing (QS) system. However, the ability of Pseudomonas fluorescens, the specific spoilage organism (SSO) of turbot, to response to environmental signaling molecules remains unclear. This study investigated the effects of six synthetic N-acyl homoserine lactones (AHLs) on typical behaviors mediated by QS in P. fluorescens, such as biofilm formation and extracellular protease activity. Total volatile basic nitrogen (TVB-N) was used as a spoilage indicator to evaluate quality changes in AHL-treated turbot filets during storage. The results confirm the enhancing effect of environmental AHLs on QS-dependent factors of P. fluorescens and quality deterioration of turbot filets, with C4-HSL and C14-HSL being the most effective. Moreover, the content decrease of exogenous AHLs was also validated by gas chromatography–mass spectrometry analysis. Further, changes in rhlR transcription levels in P. fluorescens suggest that this bacterium can sense environmental AHLs. Finally, molecular docking analysis demonstrates the potential interactions of RhlR protein with various exogenous AHLs. These findings strongly implicate environmental AHLs in turbot spoilage caused by P. fluorescens, suggesting preservation of turbot should not exclusively consider the elimination of SSO-secreted AHLs.

In an effort to balance the demands of plant growth promoting and biological control agents in a single product, the technology on the co-cultivation of two microbes, Trichoderma asperellum GDFS1009 and Bacillus amyloliquefaciens 1841 has been developed and demonstrated its effectiveness in synergistic interactions and its impact on the plant growth and biocontrol potential. In this study, optimization of T. asperellum and B. amyloliquefaciens growth in a single medium was carried out using response surface methodology (RSM). The optimal medium for enhanced growth was estimated as 2% yeast extract, 2% molasses and 2% corn gluten meal. T. asperellum evolved the complicated molecular mechanisms in the co-culture by the induction of BLR-1/BLR-2, VELVET, and NADPH oxidases genes. In performance with these genes, conserved signaling pathways, such as heterotrimeric G proteins and mitogen-activated protein kinases (MAPKs) had also involved in this molecular orchestration. The co-cultivation induced the expression of T. asperellum genes related to secondary metabolism, mycoparasitism, antioxidants and plant growth. On the other hand, the competition during co-cultivation induced the production of new compounds that are not detected in axenic cultures. In addition, the co-culture significantly enhanced the plant growth and protection against Fusarium graminearum. The present study demonstrated the potential of co-cultivation technology could be a used to grow the T. asperellum GDFS1009 and B. amyloliquefaciens 1841 synergistically to improve the production of mycoparasitism related enzymes, secondary metabolites, and plant growth promoting compounds to significantly enhance the plant growth and protection against plant pathogens.

Quorum sensing (QS), one of the most remarkable microbiological discoveries, is considered a global gene regulatory mechanism for various traits in bacteria, including virulence and spoilage. Hafnia alvei, an opportunistic pathogen and a dominant psychrophile, uses the lux-type QS system to regulate the production of virulence factors and biofilms, which are harmful to the food industry. Based on the QS interference approach, this study aimed to reveal the efficacy of L-carvone at sublethal concentrations on QS-regulated virulence factors and biofilm formation in H. alvei. QS inhibitory activity was demonstrated by the reduction in swinging motility (61.49%), swarming motility (74.94%), biofilm formation (52.41%) and acyl-homoserine lactone (AHL) production (0.5 μL/mL). Additionally, in silico analysis and RT-qPCR studies for AHL synthase HalI and QS transcriptional regulator HalR revealed a plausible molecular mechanism for QS inhibition by L-carvone. These findings suggest that L-carvone (a main component of spearmint essential oils) could be used as a novel quorum sensing inhibitor to control H. alvei in the food industry.

The gene family of thaumatin-like proteins (TLPs) plays a crucial role in the adaptation of organisms to environmental stresses. In recent years, fungal secreted proteins (SP) with inducing disease resistance activity in plants have emerged as important elicitors in the control of fungal diseases. Identifying SPs with inducing disease resistance activity and studying their mechanisms are crucial for controlling sheath blight. In the present study, 10 proteins containing the thaumatin-like domain were identified in strain AG4-JY of Rhizoctonia solani and eight of the 10 proteins had signal peptides. Analysis of the TLP genes of the 10 different anastomosis groups (AGs) showed that the evolutionary relationship of the TLP gene was consistent with that between different AGs of R. solani. Furthermore, it was found that RsTLP3, RsTLP9 and RsTLP10 were regarded as secreted proteins for their signaling peptides exhibited secretory activity. Prokaryotic expression and enzyme activity analysis revealed that the three secreted proteins possess glycoside hydrolase activity, suggesting they belong to the TLP family. Additionally, spraying the crude enzyme solution of the three TLP proteins could enhance maize resistance to sheath blight. Further analysis showed that genes associated with the salicylic acid and ethylene pathways were up-regulated following RsTLP3 application. The results indicated that RsTLP3 had a good application prospect in biological control.

Copper hydroxide is a broad-spectrum copper fungicide, which is often used to control crop fungal and bacterial diseases. In addition to controlling targeted pathogens, copper hydroxide may also affect other non-targeted microorganisms in the phyllosphere ecosystem. At four time points (before spraying, and 5, 10 and 15 days after fungicide application), the response of diseased and healthy tobacco phyllosphere microorganisms to copper hydroxide stress was studied by using Illumina high-throughput sequencing technology, and Biolog tools. The results showed that the microbiome communities of the healthy group were more affected than the disease group, and the fungal community was more sensitive than the bacterial community. The most common genera in the disease group were Alternaria, Boeremia, Cladosporium, Pantoea, Ralstonia, Pseudomonas, and Sphingomonas; while in the healthy group, these were Alternaria, Cladosporium, Symmetrospora, Ralstonia, and Pantoea. After spraying, the alpha diversity of the fungal community decreased at 5 days for both healthy and diseased groups, and then showed an increasing trend, with a significant increase at 15 days for the healthy group. The alpha diversity of bacterial community in healthy and diseased groups increased at 15 days, and the healthy group had a significant difference. The relative abundance of Alternaria and Cladosporium decreased while that of Boeremia, Stagonosporopsis, Symmetrospora, Epicoccum and Phoma increased in the fungal communities of healthy and diseased leaves. The relative abundance of Pantoea decreased first and then increased, while that of Ralstonia, Pseudomonas and Sphingomonas increased first and then decreased in the bacterial communities of healthy and diseased leaves. While copper hydroxide reduced the relative abundance of pathogenic fungi Alternaria and Cladosporium, it also resulted in the decrease of beneficial bacteria such as Actinomycetes and Pantoea, and the increase of potential pathogens such as Boeremia and Stagonosporopsis. After treatment with copper hydroxide, the metabolic capacity of the diseased group improved, while that of the healthy group was significantly suppressed, with a gradual recovery of metabolic activity as the application time extended. The results revealed changes in microbial community composition and metabolic function of healthy and diseased tobacco under copper hydroxide stress, providing a theoretical basis for future studies on microecological protection of phyllosphere.

Carbohydrate is the most common macronutrient consumed across all phases of the diet and acts as a potential regulator in modulating the gut microbiota in animals. However, the influences of dietary non-fibrous carbohydrate (NFC) to neutral detergent fiber (NDF) in different ratios on gut microbiota, metabolites, intestinal immunity, and growth performance have not been fully explored. A total of 135 healthy weaned rabbits (45.1 ± 0.7 d of age) with an average body weight of 1.08 ± 0.07 kg were randomly divided into five groups. Under the same other nutrient levels, rabbits were fed diets with NFC/NDF ratios of 0.7 (T1), 1.0 (T2), 1.3 (T3), 1.6 (T4), and 1.9 (T5). During the 28-day experiment, T3 rabbits showed the highest final body weight and the lowest feed-to-weight ratio than T5 rabbits (P < 0.05) but no significant difference with T1 or T2 rabbits. The expression of cecal pro-inflammatory factors IL-1β and TNF-α was increased in the T4 and T5 than in those of other groups (P < 0.05). Conversely, the tight junction proteins (ZO-1, Claudin-1, and Occludin) were decreased to varying degrees in the T4 and T5 groups. The pH value in the cecal digesta of T5 rabbits was lower than that of T1, T2, and T3 (P < 0.05), while the concentration of volatile fatty acids and propionate was higher than those of T1, T2, and T3 rabbits (P < 0.05). In terms of gut microbiota, at the phylum level, the relative burden of Firmicutes and Actinobacteria in T2 rabbits was the highest (P < 0.05), and the relative burden of Proteobacteria in T5 rabbits was higher than that of other groups (P < 0.05). At the genus level, the relative burden of Ruminococcus was higher in T2 and T3 rabbits than that of other groups, and T5 rabbits have the lowest relative burden of Ruminococcus. Combination analysis showed that cecal metabolites were positively associated with fermentation-related phenotypes and the burden of Firmicutes (P < 0.05). In conclusion, different dietary NFC/NDF ratios can affect the intestinal immune response and growth performance of rabbits, and there was a positive effect when dietary NFC/NDF = 1.0–1.3.

IntroductionThe diversity and dysregulation of intestinal microbiota is related to the pathology of epilepsy. Gut microbiota plays an important role in epilepsy, and regulating intestinal microbiota through exogenous intervention can alleviate symptoms. However, there are no studies about the effects of epilepsy-related diarrhea on gut microbiota.MethodsThe diversity and dysregulation of intestinal microbiota is related to the pathology of epilepsy. Gut microbiota plays an important role in epilepsy, and regulating intestinal microbiota through exogenous intervention can alleviate symptoms. However, there are no studies about the effects of epilepsy-related diarrhea on gut microbiota. To evaluate changes in gut microbiota structure and composition in patients with epilepsy and associated diarrhea, the structure and composition of the fecal microbiota among patients with epilepsy (EP, 13 cases), epilepsy with diarrhea (ED, 13 cases), and probiotic treatments (PT, 13 cases), and healthy controls (CK, seven cases) were investigated and validated by utilizing high-throughput 16S rRNA sequencing.ResultsThe results showed that the α-diversity indexes indicated that richness and phylogenetic diversity had no significant differences among groups. However, the variation of β-diversity indicated that the structure and composition of intestinal microbiota were significantly different among the CK, EP, ED, and PT groups (permutational multivariate analysis of variance, p-value = 0.001). Normalized stochasticity ratio and β-nearest taxon index indicated that stochastic mechanisms exerted increasing influence on community differences with epilepsy and associated diarrhea. ED microbiome alterations include increased Proteobacteria and decreased Actinobacteria and Firmicutes at the phylum level. Bifidobacterium was the core microbe in CK, EP, and PT, whereas it decreased significantly in ED. In contrast, Escherichia/Shigella was the core microbe in CK and ED, whereas it increased significantly in ED (Tukey’s multiple comparisons test, adjusted p-value

Tomato zonate spot virus (TZSV) is a phytopathogen of the genus Orthotospovirus (Bunyaviridae) that is widespread in many areas of Southwest China. TZSV is mainly transmitted by Frankliniella occidentalis, but its exact infection route remains unclear. To explore this issue, we detected the nucleocapsid protein of TZSV in the digestive systems of first-instar F. occidentalis nymphs fed with TZSV-infected pepper leaves. TZSV infection in the F. occidentalis digestive system begins within 4 h post-first access to diseased plants: The foregut is likely the primary site of infection, and primary salivary glands (PSGs) are the destination. There are three potential routes for TZSV transmission from the alimentary canal to the PSGs: (1) virus dissemination from the midgut to hemocoel followed by movement to the PSGs; (2) accumulation in midgut epithelial cells and arrival at PSGs via tubular salivary glands and efferent ducts; and (3) arrival at epitheliomuscular cells of the forepart of the midgut and movement along the ligament to the PSGs. We tested the transmission efficiency of F. occidentalis in second-instar nymphs and female and male adults. TZSV was transmitted in a persistent-propagative mode by both nymphs and adults, with adults appearing to show slightly higher transmission efficiency than nymphs. We confirmed the presence of all three routes for TZSV transmission in F. occidentalis and determined that like other Orthotospoviruses, TZSV is transmitted in a persistent-propagative manner. These results should facilitate the control of TZSV-related diseases and further our understanding of the transmission biology of Orthotospoviruses in general.

Clostridium botulinum is the causative pathogen of botulism. Laboratory detection of C. botulinum is essential for clinical therapy treatment of botulism due to the difficulty in diagnosis, especially in infant botulism. The extreme toxicity of botulinum neurotoxin (BoNT) requires a sensitive detection method. Due to the detection limit of real-time quantitative PCR (q-PCR), a more sensitive detection method, micro-drop digital PCR (ddPCR) was applied in C. botulinum main serotypes A and B. The following performance criteria were evaluated by ddPCR: analytical sensitivity; repeatability; and diagnostic specificity. The limit of detection (LOD) was 0.84 and 0.88 copies/μl for BoNT A and B genes, respectively, by ddPCR with high specificity, compared to 5.04×102 and 6.91×102 copies/μl by q-PCR. It was increased 10 times compared with q-PCR in spiked stool samples. This improvement in sensitivity was especially important in clinical samples as more positive samples were detected by digital PCR compared with q-PCR. Meanwhile, enrichment time for low bacteria content samples was shortened by four hours both in serotypes A and B C. botulinum by ddPCR compared with q-PCR, which are important for laboratory diagnosis and epidemiology work.

The potentially pathogenic species of the genus Vibrio pose a threat to both humans and animals, creating medical burdens and economic losses to the mariculture industry. Improvements in surveillance and diagnosis are needed to successfully manage vibriosis outbreaks. Matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) can provide rapid diagnosis and has been widely used in the identification of Vibrio spp. The main weakness of this technology is the limited number of strains and species of Vibrio in the existing commercial database. Here, we develop a new in-house database named PVBase containing 790 main spectra projections (MSP) of ten Vibrio species that come from various regions of China and include abundant clinical and environmental strains. PVBase was validated through a blind test of 65 Vibrio strains. The identification accuracy and scoring of Vibrio strains was greatly improved through the addition of PVBase. Identification accuracy increased from 73.4 to 100%. The number of strains with identification scores above 2.2 increased from 53.1% to 96.9% and 53.1% of strains had an identification score above 2.59. Moreover, perfect discrimination was obtained when using all of the MSPs created for the Vibrio species, even for very closely related species such as V. cholerae, V. albensis, and V. mimicus or V. alginolyticus, V. parahaemolyticus, and V. harveyi. In addition, we used phyloproteomic analysis to study whether there are differences in protein fingerprints of different regions or pathogenic strains. We found that MSP characteristics of Vibrio species were not related to their region or source. With the construction of PVBase, the identification efficiency of potentially pathogenic Vibrio species has been greatly improved, which is an important advance for epidemic prevention and control, and aquaculture disease detection.

Increasing evidences suggest that the gut microbiota have their contributions to the hypertension, but the metagenomic characteristics and potential regulating mechanisms in primary hypertension patients taking antihypertension drugs are not clear yet. We carried out a metagenomic analysis in 30 primary hypertension patients taking antihypertension medications and eight healthy adults without any medication. We found that bacterial strains from species, such as Bacteroides fragilis, Bacteroides vulgatus, Escherichia coli, Klebsiella pneumoniae, and Streptococcus vestibularis, were highly increased in patients; and these strains were reported to generate glycan, short-chain fatty acid (SCFA) and trimethylamine (TMA) or be opportunistic pathogens. Meanwhile, Dorea longicatena, Eubacterium hallii, Clostridium leptum, Faecalibacterium prausnitzii, and some other strains were greatly decreased in the patient group. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis found that ortholog groups and pathways related to glycan biosynthesis and multidrug resistance were significantly increased in the patient group, and some of the hub genes related to N-glycan biosynthesis were increased in the patient group, while those related to TMA precursor metabolism and amino acid metabolism both increased and decreased in the patient group. Metabolites tested by untargeted liquid chromatography–mass spectrometry (LC-MS) proved the decrease of acetic acid, choline, betaine, and several amino acids in patients’ fecal samples. Moreover, meta-analysis of recent studies found that almost all patients were taking at least one kind of drugs that were reported to regulate adenosine monophosphate-activated protein kinase (AMPK) pathway, so we further investigated if AMPK regulated the metagenomic changes by using angiotensin II-induced mouse hypertensive model on wild-type and macrophage-specific AMPK-knockout mice. We found that the changes in E. coli and Dorea and glycan biosynthesis-related orthologs and pathways were similar in our cohort and hypertensive wild-type mice but reversed after AMPK knockout. These results suggest that the gut microbiota-derived glycan, SCFA, TMA, and some other metabolites change in medication-taking primary hypertension patients and that medications might promote gut microbiota glycan biosynthesis through activating macrophage-AMPK.

The process of soybean fermentation has been practiced for more than 3,000 years. Although Dajiang and Sufu are two popular fermented soybean products consumed in North China, limited information is available regarding their microbial composition. Hence, the current study sought to investigate, and compare, the physicochemical indicators and microbial communities of traditional Dajiang and Sufu. Results showed that the titratable acidity (TA), and salinity, as well as the lactic acid, and malic acid contents were significantly higher in Sufu samples compared to Dajiang. Furthermore, Sufu samples contain abundant sucrose and fructose, while the acetic acid content was lower in Sufu compared to Dajiang samples. Moreover, the predominant bacterial phyla in Dajiang and Sufu samples were Firmicutes and Proteobacteria, while the major genera comprise Bacillus, Lactobacillus, Tetragenococcus, and Weissella. Moreover, Dajiang samples also contained abundant Pseudomonas, and Brevundimonas spp., while Halomonas, Staphylococcus, Lysinibacillus, Enterobacter, Streptococcus, Acinetobacter, and Halanaerobium spp. were abundant in Sufu samples. At the species level, Bacillus velezensis, Tetragenococcus halophilus, Lactobacillus rennini, Weissella cibaria, Weissella viridescens, Pseudomonas brenneri, and Lactobacillus acidipiscis represented the major species in Dajiang, while Halomonas sp., Staphylococcus equorum, and Halanaerobium praevalens were the predominant species in Sufu. Acetic acid and sucrose were found to be the primary major physicochemical factor influencing the bacterial communities in Dajiang and Sufu, respectively. Furthermore, Bacillus subtilis is strongly correlated with lactic acid levels, L. acidipiscis is positively correlated with acetic acid levels, while Staphylococcus sciuri and S. equorum are strongly, and positively, correlated with malic acid. Following analysis of carbohydrate and amino acid metabolism in all samples, cysteine and methionine metabolism, as well as fatty acid biosynthesis-related genes are upregulated in Dajiang compared to Sufu samples. However, such as the Staphylococcus, W. viridescens, and P. brenneri, as potentially foodborne pathogens, existed in Dajang and Sufu samples. Cumulatively, these results suggested that Dajiang and Sufu have unique bacterial communities that influence their specific characteristics. Hence, the current study provides insights into the microbial community composition in Dajiang and Sufu samples, which may facilitate the isolation of functional bacterial species suitable for Dajiang and Sufu production, thus improving their production efficiency.

Protein lysine acetylation (Kac) is an important post-translational modification mechanism in eukaryotes that is involved in cellular regulation. To investigate the role of Kac in virus-infected plants, we characterized the lysine acetylome of Nicotiana benthamiana plants with or without a Chinese wheat mosaic virus (CWMV) infection. We identified 4,803 acetylated lysine sites on 1,964 proteins. A comparison of the acetylation levels of the CWMV-infected group with those of the uninfected group revealed that 747 sites were upregulated on 422 proteins, including chloroplast localization proteins and histone H3, and 150 sites were downregulated on 102 proteins. Nineteen conserved motifs were extracted and 51 percent of the acetylated proteins located on chloroplast. Nineteen Kac sites were located on histone proteins, including 10 Kac sites on histone 3. Bioinformatics analysis results indicated that lysine acetylation occurs on a large number of proteins involved in biological processes, especially photosynthesis. Furthermore, we found that the acetylation level of chloroplast proteins, histone 3 and some metabolic pathway-related proteins were significantly higher in CWMV-infected plants than in uninfected plants. In summary, our results reveal the regulatory roles of Kac in response to CWMV infection.

We first performed a combination of metabolic engineering (deletion of ldh and poxB and overexpression of mmc) with evolutionary engineering (selection under oxygen stress, acid stress and osmotic stress) in Propionibacterium acidipropionici. The results indicated that the mutants had superior physiological activity, especially the mutant III obtained from P. acidipropionici-Δldh-ΔpoxB+mmc by evolutionary engineering, with 1.5–3.5 times higher growth rates, as well as a 37.1% increase of propionic acid (PA) titer and a 37.8% increase PA productivity compared to the wild type. Moreover, the integrative transcriptomics and proteomics analyses revealed that the differentially expressed genes (DEGs) and proteins (DEPs) in the mutant III were involved in energy metabolism, including the glycolysis pathway and tricarboxylic acid cycle (TCA cycle). These genes were up-regulated to supply increased amounts of energy and precursors for PA synthesis compared to the wild type. In addition, the down-regulation of fatty acid biosynthesis and fatty acid metabolism may indicate that the repressed metabolic flux was related to the production of PA. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was performed to verify the differential expression levels of 16 selected key genes. The results offer deep insights into the mechanism of high PA production, which provides the theoretical foundation for the construction of advanced microbial cell factories.

Seneca Valley virus (SVV) is the only member of the genus Senecavirus of the Picornaviridae family. SVV can selectively infect and lyse tumor cells with neuroendocrine features and is used as an oncolytic virus for treating small-cell lung cancers. However, the detailed mechanism underlying SVV-mediated destruction of tumor cells remains unclear. In this study, we found that SVV can increase the proportion of apoptotic 293T cells in a dose- and time-dependent manner. SVV-induced apoptosis was initiated via extrinsic and intrinsic pathways through activation of caspase-3, the activity of which could be attenuated by a pan-caspase inhibitor (Z-VAD-FMK). We confirmed that SVV 2C and 3Cpro play critical roles in SVV-induced apoptosis. The SVV 2C protein was located solely in the mitochondria and activated caspase-3 to induce apoptosis. SVV 3Cpro induced apoptosis through its protease activity, which was accompanied by release of cytochrome C into the cytoplasm, but did not directly cleave PARP1.