Yundan Wang

The genus Weissella is attracting an increasing amount of attention because of its multiple functions and probiotic potential. In particular, the species Weissella confusa is known to have great potential in industrial applications and exhibits numerous biological functions. However, the knowledge on this bacterium in insects is not investigated. Here, we isolated and identified W. confusa as the dominant lactic acid bacteria in the gut of the migratory locust. We named this strain W. confusa LM1, which is the first genome of an insect-derived W. confusa strain with one complete chromosome and one complete plasmid. Among all W. confusa strains, W. confusa LM1 had the largest genome. Its genome was the closest to that of W. confusa 1001271B_151109_G12, a strain from human feces. Our results provided accurate evolutionary relationships of known Weissella species and W. confusa strains. Based on genomic analysis, the pan-genome of W. confusa is in an open state. Most strains of W. confusa had the unique genes, indicating that these strains can adapt to different ecological niches and organisms. However, the variation of strain-specific genes did represent significant correlations with their hosts and ecological niches. These strains were predicted to have low potential to produce secondary metabolites. Furthermore, no antibiotic resistance genes were identified. At the same time, virulence factors associated with toxin production and secretion system were not found, indicating that W. confusa strains were not sufficient to perform virulence. Our study facilitated the discovery of the functions of W. confusa LM1 in locust biology and their potential application to locust management.

BACKGROUND: Improvements in the virulence of the fungal pathogen Metarhizium acridum can crucially promote its efficacy to control locusts and grasshoppers. The polysaccharide components of the cell wall remarkably contribute to fungal virulence.RESULTS: Here we found that M. acridum lacked the gene families of glycerate‐3‐kinase (GLYK) as the synthesis enzymes of saccharides. We then generated mutants by introducing the GLYK gene from the host‐generalist M. robertsii into the host‐specialist M. acridum. Consequently, compared with the wild‐type strain, the mutant strain (Ma::MrGLYK) increased the level of phospho‐6‐fructose in mycelia, the length and density of the mannan fibril layer on the cell wall. The mutant strains increased the mannan fibril in the cell wall and resistance to heat stress. Further transcriptome analysis showed that compared with the wild‐type strain, topical infection of Ma::MrGLYK strain induced higher expression of genes such as pattern‐recognition proteins, serine protease, and CYP450s in locusts, while reduced the expression of antimicrobial peptide and phenoloxidase activity. Moreover, topical infection and injection of Ma::MrGLYK significantly increased the mortality and shortened the lifespan of locusts compared with wild‐type M. acridum.CONCLUSION: Our study highlighted the application potential of the novel genetically modified fungal mutant of the host‐specialist M. acridum as a biocontrol agent against locust plagues. © 2020 Society of Chemical Industry

Metarhizium is a group of insect-pathogenic fungi that can produce insecticidal metabolites, such as destruxins. Interestingly, the acridid-specific fungus Metarhizium acridum (MAC) can kill locusts faster than the generalist fungus Metarhizium robertsii (MAA) even without destruxin. However, the underlying mechanisms of different pathogenesis between host-generalist and host-specialist fungi remain unknown. This study compared transcriptomes and metabolite profiles to analyze the difference in responsiveness of locusts to MAA and MAC infections. Results confirmed that the detoxification and tryptamine catabolic pathways were significantly enriched in locusts after MAC infection compared with MAA infection and that high levels of tryptamine could kill locusts. Furthermore, tryptamine was found to be capable of activating the aryl hydrocarbon receptor of locusts (LmAhR) to produce damaging effects by inducing reactive oxygen species production and immune suppression. Therefore, reducing LmAhR expression by RNAi or inhibitor (SR1) attenuates the lethal effects of tryptamine on locusts. In addition, MAA, not MAC, possessed the monoamine oxidase (Mao) genes in tryptamine catabolism. Hence, deleting MrMao-1 could increase the virulence of generalist MAA on locusts and other insects. Therefore, our study provides a rather feasible way to design novel mycoinsecticides by deleting a gene instead of introducing any exogenous gene or domain. Mycoinsecticides are widely used in place of chemical pesticides to protect crops from pest damage. Metarhizium spp. fungi specifically live inside the body cavity of insects and can produce insecticidal metabolites, such as beauvericin, destruxins, and taxol. The variable virulence between host-generalist fungus Metarhizium robertsii (MAA) and host-specialist fungus Metarhizium acridum (MAC) to locusts was studied. We found that MAC is more virulent than MAA on locusts, and MAC-infected locusts display higher amounts of tryptamine than do MAA-infected locusts. Furthermore, MAC cannot produce destruxins, but can produce abundant tryptamine to kill locusts when accumulated owing to the absence of a gene for tryptamine catabolism in the MAC genome. Tryptamine activates the aryl hydrocarbon receptor of locusts (LmAhR) that produces damaging effects by regulating reactive oxygen species production and suppressing the immune system of locusts. Therefore, the deletion of MrMao-1 in the generalist fungus MAA significantly improves the virulence of the fungus to locusts and other insect species. The resulting new insights into the core metabolism of high virulence of host-specialist fungus can provide an improved basis for designing mycoinsecticide strains.

Recognizing proteins via the production of highly specific monoclonal antibodies (mAbs) is crucial to identifying proteins for proteomic research. However, traditional mAb generation is time-consuming with low efficiency. In this study, we assessed the high throughput method of producing mAbs by immunizing mice with multiple antigens in order to obtain hybridomas against these multiple antigens in one cell fusion. We selected eight proteins that play important roles in human physiological or pathological processes. These proteins were mixed and simultaneously administered to one mouse. We observed the immunizing period for 10 d, and determined the effect of liquid medium and semi-solid medium in hybridoma generation. As a result, all eight immunogens induced antibodies in the immunized mouse in one cell fusion, we obtained hybridomas specific to all eight proteins by enzyme-linked immuno sorbent assay (ELISA) screening, hybridomas against five out of eight showed specific positive in Western-blotting assays. This indicates that we generated mAbs specific to eight proteins in one cell fusion, greatly increasing the efficiency of mAb generation. Furthermore, we observed that hybridomas selected from the liquid medium and semi-solid medium showed different reactivity to antigens. Our study established high-throughput and time-saving methods for production of mAbs. These results provide alternative approaches for increasing the efficacy of mAb generation.

The stress of living conditions, similar to infections, alters animal immunity. High population density is empirically considered to induce prophylactic immunity to reduce the infection risk, which was challenged by a model of low connectivity between infectious and susceptible individuals in crowded animals. The migratory locust, which exhibits polyphenism through gregarious and solitary phases in response to population density and displays different resistance to fungal biopesticide (Metarhizium anisopliae), was used to observe the prophylactic immunity of crowded animals. We applied an RNA-sequencing assay to investigate differential expression in fat body samples of gregarious and solitary locusts before and after infection. Solitary locusts devoted at least twice the number of genes for combating M. anisopliae infection than gregarious locusts. The transcription of immune molecules such as pattern recognition proteins, protease inhibitors, and anti-oxidation proteins, was increased in prophylactic immunity of gregarious locusts. The differentially expressed transcripts reducing gregarious locust susceptibility to M. anisopliae were confirmed at the transcriptional and translational level. Further investigation revealed that locust GNBP3 was susceptible to proteolysis while GNBP1, induced by M. anisopliae infection, resisted proteolysis. Silencing of gnbp3 by RNAi significantly shortened the life span of gregarious locusts but not solitary locusts. By contrast, gnbp1 silencing did not affect the life span of both gregarious and solitary locusts after M. anisopliae infection. Thus, the GNBP3-dependent immune responses were involved in the phenotypic resistance of gregarious locusts to fungal infection, but were redundant in solitary locusts. Our results indicated that gregarious locusts prophylactically activated upstream modulators of immune cascades rather than downstream effectors, preferring to quarantine rather than eliminate pathogens to conserve energy meanwhile increasing the “distance” of infectious and target individuals. Our study has obvious implications for bio-pesticides management of crowded pests, and for understanding disease epidemics and adaptiveness of pathogens. The wide application of fungal biopesticides for insect management has led to concerns over the development of biopesticide resistance. The migratory locust, a globally notorious agricultural pest, has density-dependent phase changes between solitary and gregarious states. The gregarious locusts displayed longer life spans than solitary locusts after biopesticide Metarhizium anisopliae infection. We analyzed prophylactic immunity of the locusts in phase change adaptation by transcriptome analysis. Gregarious locusts optimized immunity by investing more in molecules of upstream immune cascades including pattern recognition proteins, anti-oxidation proteins, protease inhibitors and serine protease. High levels of pattern recognition proteins guided deposition of immune products onto pathogens reducing growth, proliferation and transmission. This prophylactic immunity of gregarious locusts emphasized on quarenteening M. anisopliae pathogens in early infection, which decreased individuals' infection risk in a population and avoids disease epidemics. Pest outbreaks mostly occur in high population densities, thereby, diminishing entomopathogen biopesticide efficiency. Our results provide an insight to an organism's “enhanced” immunity induced by population densities and inspires new paradigms to understand biopesticide tolerance and disease epidemics in the future.

Urease B is an important virulence factor that is required for Helicobacter pylori to colonise the gastric mucosa. Mouse monoclonal antibodies (mAbs) that inhibit urease B enzymatic activity will be useful as vaccines for the prevention and treatment of H. pylori infection. Here, we produced murine mAbs against urease B that neutralize the enzyme's activity. We mapped their epitopes by phage display libraries and investigated the immunogenicity of the selected mimotopes in vivo. The urease B gene was obtained (GenBank accession No. DQ141576) and the recombinant pGEX-4T-1/UreaseB protein was expressed in Escherichia coli as a 92-kDa recombinant fusion protein with glutathione-S-transferase (GST). Five mAbs U001-U005 were produced by a hybridoma-based technique with urease B-GST as an immunogen. Only U001 could inhibit urease B enzymatic activity. Immunoscreening via phage display libraries revealed two different mimotopes of urease B protein; EXXXHDM from ph.D.12-library and EXXXHSM from ph.D.C7C that matched the urease B proteins at 347-353 aa. The antiserum induced by selected phage clones clearly recognised the urease B protein and inhibited its enzymatic activity, which indicated that the phagotope-induced immune responses were antigen specific. The present work demonstrated that phage-displayed mimotopes were accessible to the mouse immune system and triggered a humoral response. The urease B mimotope could provide a novel and promising approach for the development of a vaccine for the diagnosis and treatment of H. pylori infection.

Construction of a monoclonal antibody (mAb) bank containing a vast variety of antibodies against human tissue proteins is important for proteomic research. A novel strategy of subtractive immunization using fractionated native proteins was developed for high throughput generation of mAb against human plasma proteins. By this novel approach, the bottleneck of antigen preparation can be overcome by combining repeated immunization of animals with subtracted fractions of plasma or tissue proteins and identification of target antigen by immunoprecipitation/mass spectrum strategies. Plasma freshly collected from healthy adults was pooled and three fractions were prepared by size exclusion chromatography. Mice were immunized with the fractionated plasma proteins, and 205 strains of hybridomas secreting mAb were obtained after two‐round subtractive immunizations and cell fusions. In the first round, 110 strains of hybridomas were established, in which 77 strains secreting mAb were identified against 10 human plasma high‐abundant proteins. In the second round, plasma fraction I was absorbed with mAb against IgM, IgG, ceruloplasmin and haptoglobin. The absorbed fraction I was used as immunogen for the second round immunization and cell fusion. Ninety‐five strains of hybridomas secreting mAb were obtained. Although the target antigens of mAb from 82 strains of hybridomas were identified as IgM, IgA, α₂‐macroglobulin and fibrinogen, about 85% antibodies obtained from this round were identified as new antibodies when compared with mAb obtained in the first round immunization with plasma fraction I. The results suggest that subtractive immunization with fractionated plasma proteins followed by identification of antigens with immunoprecipitation/mass spectrum may be an effective approach for rapid preparation of mAb against high‐and medium‐abundant plasma or tissue proteins.