Identification of non-canonical antagonistic bacteria via interspecies contact-dependent killing.

BACKGROUND: Canonical biocontrol bacteria were considered to inhibit pathogenic bacteria mainly by secreting antibiotic metabolites or enzymes. Recent studies revealed that some biocontrol bacteria can inhibit pathogenic bacteria through contact-dependent killing (CDK) mediated by contact-dependent secretion systems. The CDK was independent of antibiotic metabolites and often ignored in normal biocontrol activity assay. RESULTS: In this study, we aimed to use a pathogen enrichment strategy to isolate non-canonical bacteria with CDK ability. Rhizosphere soil samples from Chinese cabbage showing soft rot symptom were collected and Pectobacterium carotovorum subsp. carotovorum (Pcc), the pathogen of cabbage soft rot, were added into these samples to enrich bacteria which attached on Pcc cells. By co-culture with Pcc, four bacteria strains (named as PcE1, PcE8, PcE12 and PcE13) showing antibacterial activity were isolated from Chinese cabbage rhizosphere. These four bacteria strains showed CDK abilities to different pathogenic bacteria of horticultural plants. Among them, PcE1 was identified as Chryseobacterium cucumeris. Genome sequencing showed that PcE1 genome encoded a type VI secretion system (T6SS) gene cluster. By heterologous expression, four predicted T6SS effectors of PcE1 showed antibacterial activity to Escherichia coli. CONCLUSION: Overall, this study isolated four bacteria strains with CDK activity to various horticultural plant pathogens, and revealed possible involvement of T6SS of Chryseobacterium cucumeris in antibacterial activity. These results provide valuable insight for potential application of CDK activity in biocontrol bacteria. © 2024 Society of Chemical Industry.

Adapting the inoculation methods of kiwifruit canker disease to identify efficient biocontrol bacteria from branch microbiome.

Pseudomonas syringae pv. actinidiae (Psa), the bacterium that causes kiwifruit bacterial canker, is a common field occurrence that is difficult to control globally. Currently, exploring the resources for efficient biocontrol bacteria is a hot spot in the field. The common strategy for isolating biocontrol bacteria is to directly isolate biocontrol bacteria that can secrete diffusible antibacterial substances, most of which are members of Bacillus, Pseudomonas and Streptomycetaceae, from disease samples or soil. Here, we report a new approach by adapting the typical isolation methods of kiwifruit canker disease to identify efficient biocontrol bacteria from the branch microbiome. Using this unique approach, we isolated a group of kiwifruit biocontrol agents (KBAs) from the branch microbiome of Psa-resistant varieties. Thirteen of these showed no antagonistic activity in vitro, which depends on the secretion of antibacterial compounds. However, they exhibited antibacterial activity via cell-to-cell contacts mimicked by co-culture on agar plates. Through biocontrol tests on plants, two isolates, KBA13 and KBA19, demonstrated their effectiveness by protecting kiwifruit branches from Psa infection. Using KBA19, identified as Pantoea endophytica, as a representative, we found that this bacterium uses the type VI secretion system (T6SS) as the main contact-dependent antibacterial weapon that acts via translocating toxic effector proteins into Psa cells to induce cell death, and that this capacity expressed by KBA19 is common to various Psa strains from different countries. Our findings highlight a new strategy to identify efficient biocontrol agents that use the T6SS to function in an antibacterial metabolite-independent manner to control wood diseases.

Loss of Flagella-Related Genes Enables a Nonflagellated, Fungal-Predating Bacterium To Strengthen the Synthesis of an Antifungal Weapon.

Loss of flagellar genes causes a nonmotile phenotype. The genus Lysobacter consists of numerous environmentally ubiquitous, nonflagellated bacteria, including Lysobacter enzymogenes, an antifungal bacterium that is beneficial to plants. L. enzymogenes still has many flagellar genes on its genome, although this bacterium does not engage in flagella-driven motility. Here, we report that loss of certain flagellar genes allows L. enzymogenes to strengthen its evolutionarily gained capacity in fungal killing. To clarify why this bacterium loses flagellar genes during the evolutionary process, we cloned several representative flagellar genes from Xanthomonas oryzae, a flagellated, phylogenetically related species of Lysobacter, and introduced them individually into L. enzymogenes to mimic genomic reacquisition of lost flagellar genes. Heterogeneous expression of the three X. oryzae flagellar structural genes (Xo-motA, Xo-motB, Xo-fliE) and one flagellar regulatory gene (Xo-fleQ) remarkably weakened the bacterial capacity to kill fungal pathogens by impairing the synthesis of an antifungal weapon, known as the heat-stable antifungal factor (HSAF). We further investigated the underlying mechanism by selecting Xo-FleQ as the representative because it is a master transcription factor responsible for flagellar gene expression. Xo-FleQ inhibited the transcription of operon genes responsible for HSAF synthesis via direct binding of Xo-FleQ to the promoter region, thereby decreasing HSAF biosynthesis by L. enzymogenes. These observations suggest a possible genome and function coevolution event, in which an antifungal bacterium deletes certain flagellar genes in order to enhance its ability to kill fungi. IMPORTANCE It is generally recognized that flagellar genes are commonly responsible for the flagella-driven bacterial motility. Thus, finding nonflagellated bacteria partially or fully lost flagellar genes is not a surprise. However, the present study provides new insights into this common idea. We found that loss of either certain flagellar structural or regulatory genes (such as motA, motB, fliE, and fleQ) allows a nonflagellated, antifungal bacterium (L. enzymogenes) to stimulate its fungal-killing capacity, outlining a genome-function coevolution event, where an antifungal bacterium "smartly" designed its genome to "delete" crucial flagellar genes to coordinate flagellar loss and fungal predation. This unusual finding might trigger bacteriologists to reconsider previously ignored functions of the lost flagellar genes in any nonflagellated, pathogenic, or beneficial bacteria.

Identification of atypical T4SS effector proteins mediating bacterial defense.

To remain competitive, proteobacteria use various contact-dependent weapon systems to defend against microbial competitors. The bacterial-killing type IV secretion system (T4SS) is one such powerful weapon. It commonly controls the killing/competition between species by secreting the lethal T4SS effector (T4E) proteins carrying conserved XVIPCD domains into competing cells. In this study, we sought knowledge to understand whether the bacterial-killing T4SS-producing bacteria encode T4E-like proteins and further explore their biological functions. To achieve this, we designed a T4E-guided approach to discover T4E-like proteins that are designated as atypical T4Es. Initially, this approach required scientists to perform simple BlastP search to identify T4E homologs that lack the XVIPCD domain in the genomes of T4SS-producing bacteria. These homologous genes were then screened in Escherichia coli to identify antibacterial candidates (atypical T4Es) and their neighboring detoxification proteins, followed by testing their gene cotranscription and validating their physical interactions. Using this approach, we did discover two atypical T4E proteins from the plant-beneficial Lysobacter enzymogenes and the phytopathogen Xanthomonas citri. We also provided substantial evidence to show that the atypical T4E protein Le1637-mediated bacterial defense in interspecies interactions between L. enzymogenes and its competitors. Therefore, the newly designed T4E-guided approach holds promise for detecting functional atypical T4E proteins in bacterial cells.

EDA2R mediates podocyte injury in high glucose milieu.

EDA2R is a member of the large family of tumor necrosis factor receptor (TNFR). Previous studies suggested that EDA2R expression might be increased in the kidneys of diabetic mice. However, its mRNA and protein expression in kidneys were not analyzed; moreover, its role in the development of diabetic kidney disease was not explored. Here we analyzed the mRNA and protein expressions of EDA2R in diabetic kidneys and examined its role in the podocyte injury in high glucose milieu. By analysis with real-time PCR, Western blotting, we found that both the mRNA and protein levels of EDA2R were increased in the kidneys of diabetic mice. Immunohistochemical studies revealed that EDA2R expression was enhanced in both glomerular and tubular cells of diabetic mice and humans. In vitro studies, high glucose increased EDA2R expression in cultured human podocytes. Overexpression of EDA2R in podocytes promoted podocyte apoptosis and decreased nephrin expression. Moreover, ED2AR increased ROS generation in podocytes, while inhibiting ROS generation attenuates EDA2R-mediated podocyte injury. In addition, EDA2R silencing partially suppressed high glucose-induced ROS generation, apoptosis, and nephrin decrease. Our study demonstrated that high glucose increases EDA2R expression in kidney cells and that EDA2R induces podocyte apoptosis and dedifferentiation in high glucose milieu partially through enhanced ROS generation.

Chronic intermittent hypoxia accelerates liver fibrosis in rats with combined hypoxia and nonalcoholic steatohepatitis via angiogenesis rather than endoplasmic reticulum stress.

In the present study, we aimed to investigate the role of endoplasmic reticulum stress (ERS) and its related inflammation and angiogenesis in liver fibrosis in a rat model of combined hypoxia and nonalcoholic steatohepatitis (NASH) and to confirm whether the intervention of hypoxia-inducible factor 1α (HIF1α) can improve fibrosis. Liver histological changes and biochemical indices, HIF1α, inflammatory factors, ERS-related parameters (GRP78, CHOP, caspase-3, and caspase-12), and angiogenesis indices (VEGFA, VEGFR2, and CD34) were evaluated. Compared with the control rats, the liver tissue of rats with hypoxia and NASH had obvious NASH characteristics and hepatic fibrosis was significantly aggravated, including bridging fibrosis in some rats. The mRNA expression levels of HIF1α, VEGFA, and VEGFR2 and total immunohistochemical staining scores of VEGFR2 and CD34 were significantly increased. In addition, HIF1α silencing significantly decreased HIF1α, biochemical indices (ALT, AST, and TG), inflammatory factors (TNFα, IL6, and IL1ß), and angiogenesis indices (CD34 and VEGFR2), consequently, improved the hepatic fibrosis score in the rat model of combined hypoxia and NASH. Taken together, chronic intermittent hypoxia accelerates liver fibrosis in rats with combined hypoxia and NASH via angiogenesis rather than ERS and HIF1α intervention can improve liver fibrosis, angiogenesis, inflammatory factors, and biochemical indices. Therefore, HIF1α is a key regulatory factor of liver fibrosis in rats with combined hypoxia and NASH.

Chronic intermittent hypoxia promotes the development of experimental non-alcoholic steatohepatitis by modulating Treg/Th17 differentiation.

The present study aims to characterize the effect of chronic intermittent hypoxia and HIF1α on the non-alcoholic steatohepatitis (NASH) process in mice, and to explore the role of the Treg/Th17 balance in the formation of NASH inflammation and fibrosis. To achieve this purpose, simple steatosis was induced in mice by high-fat diet administration. Subsequently, chronic intermittent hypoxia was simulated by intraperitoneally injecting sodium nitrite. The changes of inflammation, fibrosis, and Treg/Th17 balance in the liver were quantified under chronic intermittent hypoxia condition and after tail vein injection of HIF1α-siRNA. In addition, T cells were cultured in vitro, and HIF1α expression was either blocked or overexpressed under chronic intermittent hypoxia or normal conditions. Then, the changes of Treg/Th17 balance, inflammatory factors, and cell pathways were measured in each group. Our results demonstrated that chronic intermittent hypoxia accelerates the NASH process, while tail vein injection of HIF1α-siRNA improves liver histology and function. Chronic intermittent hypoxia alters the ratio of Th17 and Treg cells through HIF1α and mTOR signaling, and increases the expressions of NF-κB, IL-6, and IL-17, but decreases IL-10 expression. Inhibition of the mTOR-HIF1α-TLR4-IL-6 pathway increases the ratio of Treg/Th17. Thus, chronic intermittent hypoxia modulates the Treg/Th17 balance by inducing HIF1α, resulting in the activation of the mTOR-HIF1α-TLR4-IL-6 pathway, which accelerates the formation of NASH and fibrosis.