Journal: BMC biology

Article Title: MdSVWC1, a new pattern recognition receptor triggers multiple defense mechanisms against invading bacteria in Musca domestica.

doi: 10.1186/s12915-024-02042-5

Figure Lengend Snippet: Fig. 1 Bioinformatics analysis of single-domain von Willebrand factor type C (SVWC) genes. A Chromosomal maps of the MdSVWCs in Musca domestica. B Multiple sequence alignments of the SVWC domain of MdSVWCs. Conservation cysteine residues are shadowed in yellow. C The copy number of the SVWC gene varies across different species’ genomes. D Phylogenetic relationships, expression profiles, and protein motif analyses of SVWC from M. domestica. The basal expression of the MdSVWC genes in both house fly larvae and adults, as determined by RNA-seq analysis, is indicated as fragments per kilobase million (FPKM). The presented heat map illustrates the variation in MdSVWC expression following mixed Escherichia coli and Staphylococcus aureus stimulation. The slash (\) characters indicate that no expression was detected. E Phylogenetic tree comprising 2869 SVWC protein sequences derived from 407 species across 36 orders. The maximum-likelihood method was employed to construct the phylogenetic tree with a bootstrap value of 1000 using iqtree. Please refer to the supporting information section for the respective gene IDs and protein sequences analyzed in this study (Additional file 1: SI-1 and SI-2)

Article Snippet: The basal expression levels of MdSVWC genes in larvae were assessed and their alterations during bacterial infection were monitored by scrutinizing RNA-seq data obtained from six house fly samples (GBAB01000000) [32], including larvae and adults with or without bacterial challenge with mixed E. coli ATCC 25922 and St. aureus CGMCC 1.89.

Techniques: Sequencing, Expressing, RNA Sequencing, Derivative Assay, Construct

Journal: BMC biology

Article Title: MdSVWC1, a new pattern recognition receptor triggers multiple defense mechanisms against invading bacteria in Musca domestica.

doi: 10.1186/s12915-024-02042-5

Figure Lengend Snippet: Fig. 3 rMdSVWC1 binds to the surfaces of microbes and hemocytes. A rMdSVWC1 lacks direct bactericidal activity in vitro. The rMdSVWC1 was incubated with E. coli, Pseudomonas aeruginosa, St. aureus, or Bacillus subtilis for 12 h. Bacterial growth was evaluated by measuring the absorbance at 600 nm (n = 6). TBS and ampicillin were used as negative and positive controls, respectively. The error bar represents the mean ± SEM of three biological replicates. B The microbial binding of rMdSVWC1 was assessed using a western blot. Various microbes were incubated with rMdSVWC1, then washed four times with PBS. The resulting bacterial precipitate was analyzed via western blot using anti-His antibody. C Carbohydrate-binding assay of rMdSVWC1 by ELISA. Seven sugars were used for ELISA analysis (n = 3), including LPS, DAP-PGN, Lys-PGN, D-galactose, D-mannan, and β-1,3-glucan, while sucrose served as the negative control for ligands. D rMdSVWC1 was observed to agglutinate various microbes when Ca2+ was present. BSA was used as a negative control instead of rMdSVWC1. The size of the bar is 50 μm

Article Snippet: The basal expression levels of MdSVWC genes in larvae were assessed and their alterations during bacterial infection were monitored by scrutinizing RNA-seq data obtained from six house fly samples (GBAB01000000) [32], including larvae and adults with or without bacterial challenge with mixed E. coli ATCC 25922 and St. aureus CGMCC 1.89.

Techniques: Activity Assay, In Vitro, Incubation, Binding Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Negative Control

Journal: BMC biology

Article Title: MdSVWC1, a new pattern recognition receptor triggers multiple defense mechanisms against invading bacteria in Musca domestica.

doi: 10.1186/s12915-024-02042-5

Figure Lengend Snippet: Fig. 4 The enhancement of hemocyte phagocytosis activity towards bacteria is achieved by the coating of bacteria with rMdSVWC1. A Hemocyte phagocytosis was observed under a fluorescence microscope, where FITC-labeled B. subtilis (green) had been treated with rMdSVWC1 or BSA before injection into larvae. The bar represents 10 μm. The right panel shows a closer view of a particular section of the image. B The rate at which hemocytes phagocytose bacteria coated with rMdSVWC1 was measured. The mean ± SEM of the results (n = 6, one-way ANOVA, Dunnett’s test) was calculated based on three independent repeats. Statistical significance was indicated by different letters (p < 0.05). C The cellular localization of MdSVWC1 following phagocytosis by hemocytes in the presence of E. coli. A fluorescent immunocytochemical assay was conducted to investigate the localization of MdSVWC1 and bacteria expressing red fluorescence in the hemocytes of larvae. The red-fluorescing E. coli were injected into the larvae, and hemocytes were collected from 50 larvae at 30 min post-injection, then incubated with anti-His antibody (green). Nuclei were counterstained with DAPI (blue). The scale bar is 5 μm

Article Snippet: The basal expression levels of MdSVWC genes in larvae were assessed and their alterations during bacterial infection were monitored by scrutinizing RNA-seq data obtained from six house fly samples (GBAB01000000) [32], including larvae and adults with or without bacterial challenge with mixed E. coli ATCC 25922 and St. aureus CGMCC 1.89.

Techniques: Activity Assay, Bacteria, Fluorescence, Microscopy, Labeling, Injection, Expressing, Incubation

Journal: BMC biology

Article Title: MdSVWC1, a new pattern recognition receptor triggers multiple defense mechanisms against invading bacteria in Musca domestica.

doi: 10.1186/s12915-024-02042-5

Figure Lengend Snippet: Fig. 5 MdSVWC1 regulates the transcription of antibacterial peptide (AMP) genes by binding with MdSPN16. A Visualization of RNA-seq results with a heat map displaying gene expression of AMPs and Toll target transcription factors in WT, MdSVWC1−/−, and MdSPN16−/− groups following a mixed infection with E. coli and St. aureus for 6 h. Three biological replicate samples were collected for each genotype. B The interaction between MdSVWC1 and MdSPN16 was examined via co-immunoprecipitation. HEK293T cells were utilized to generate the proteins. The symbols ( +) or ( −) indicate the presence or absence of recombinant proteins in the system. The symbol (IP: HA) indicates that the HA antibody was used as the bait protein antibody for the IP experiment. The symbol (Input) is used as a positive control in the IP experiment. The symbols (IB: Myc) or (IB: HA) indicate the western blot detection of the samples with Myc or HA antibody. If the bait MdSVWC1-HA protein was present in the system, the MdSPN16-Myc protein was pulled down. C Schematic diagram of the gRNA’s targeting of the MdSPN16 locus. The gRNA was specifically designed to target exon I of the open reading frame. The red highlighted area indicates the PAM site. D Mutant and WT alleles of exon I from the MdSPN16 gene were separated using a 6% DNA-PAGE with acrylamide:bis (29:1) and run in 1 × TBE buffer at 150 V for 2.5 h. The gel contained lane M, a DNA marker. Lane (+ / +) showed the WT fragment while lane (+ / −) contained both the mutant and WT fragments. Finally, lane (− / −) contained the mutant fragment exclusively. E DNA sequencing of the MdSPN16 mutant revealed a deletion of 58 base pairs. F Venn diagram depicting the AMPs that demonstrated notable alterations in expression in the MdSVWC1−/− and MdSPN16−/− flies as a result of E. coli and St. aureus stimulation. G Expression profiles of AMPs in WT, MdSVWC1−/−, and MdSPN16.−/− flies through qRT-PCR following a mixed infection with E. coli and St. aureus for 6 h. The AMP genes and their respective IDs are as follows: diptericin (LOC101896897), defensin (LOC105261620), attacin (LOC109612355), cecropin (LOC101889972). Graphs show mean ± SEM. All trials were performed at least thrice. The statistical significance was determined utilizing a two-way ANOVA and Sidak’s test (n = 6) (*p < 0.05, **p < 0.01)

Article Snippet: The basal expression levels of MdSVWC genes in larvae were assessed and their alterations during bacterial infection were monitored by scrutinizing RNA-seq data obtained from six house fly samples (GBAB01000000) [32], including larvae and adults with or without bacterial challenge with mixed E. coli ATCC 25922 and St. aureus CGMCC 1.89.

Techniques: Binding Assay, RNA Sequencing, Gene Expression, Infection, Immunoprecipitation, Recombinant, Positive Control, Western Blot, Mutagenesis, Marker, DNA Sequencing, Expressing, Quantitative RT-PCR

Journal: Frontiers in cellular and infection microbiology

Article Title: How to Count Our Microbes? The Effect of Different Quantitative Microbiome Profiling Approaches.

doi: 10.3389/fcimb.2020.00403

Figure Lengend Snippet: FIGURE 1 | Microbiome profile comparisons. Genus-level fecal microbial composition of both replicates of all 16 healthy study subjects (n = 32 samples) based upon (A) relative microbiome profiling (RMP), (B) quantitative microbiome profiling (QMP, cells per gram feces), (C) QMP after PMAxx-treatment of fecal samples (QMP-PMA, cells per gram feces), and (D) QMP using qPCR for quantification of bacterial load (QMP-qPCR, cells per gram feces).

Article Snippet: We used the Gut Microbiome Whole cell Mix (ATCC R© MSA2006TM) containing an even mixture of whole bacterial cells (12 different species) in order to assess whether cell-based or molecular-based quantification was more accurate.

Techniques:

Journal: Frontiers in cellular and infection microbiology

Article Title: How to Count Our Microbes? The Effect of Different Quantitative Microbiome Profiling Approaches.

doi: 10.3389/fcimb.2020.00403

Figure Lengend Snippet: FIGURE 2 | Within method dissimilarity of sample replicates and between methods dissimilarity of samples. Fecal microbial community structure variation based upon Bray–Curtis (BC) dissimilarity between samples and sample replicates. (A) Principal coordinates analysis of the study cohort based upon BC dissimilarity. Each segment connects the two replicates of the same sample as profiled by QMP (blue), QMP-PMA (green), and QMP-qPCR (red), (B) Box-plot of BC distance between sample replicates for all quantitative profiling methods (within-method variability) and BC distance in microbial community structure from the same sample profiled with different quantitative methods (between-method variability). The significance was checked pairwise using the Wilcoxon test and then adjusted for multiple comparisons using the FDR correction. The significance coding is indicated as ***p < 0.005, **p < 0.01, *p < 0.05 and N.S. for p ≥0.05. For clarity only significance of the comparisons between within QMP-method dissimilarity and all other within- and between-method dissimilarities are indicated (all FDR-corrected p-values are presented in Table S5).

Article Snippet: We used the Gut Microbiome Whole cell Mix (ATCC R© MSA2006TM) containing an even mixture of whole bacterial cells (12 different species) in order to assess whether cell-based or molecular-based quantification was more accurate.

Techniques: