Cyclic di-GMP Regulates the Type III Secretion System and Virulence in Bordetella bronchiseptica

Abstract

The second messenger cyclic di-GMP (c-di-GMP) is a ubiquitous molecule in bacteria that regulates diverse phenotypes. Among them, motility and biofilm formation are the most studied. Furthermore, c-di-GMP has been suggested to regulate virulence factors, making it important for pathogenesis. Previously, we reported that c-di-GMP regulates biofilm formation and swimming motility in Bordetella bronchiseptica. Here, we present a multi-omics approach for the study of B. bronchiseptica strains expressing different cytoplasmic c-di-GMP levels, including transcriptome sequencing (RNA-seq) and shotgun proteomics with label-free quantification. We detected 64 proteins significantly up- or downregulated in either low or high c-di-GMP levels and 358 genes differentially expressed between strains with high c-di-GMP levels and the wild-type strain. Among them, we found genes for stress-related proteins, genes for nitrogen metabolism enzymes, phage-related genes, and virulence factor genes. Interestingly, we observed that a virulence factor like the type III secretion system (TTSS) was regulated by c-di-GMP. B. bronchiseptica with high c-di-GMP levels showed significantly lower levels of TTSS components like Bsp22, BopN, and Bcr4. These findings were confirmed by independent methods, such as quantitative reverse transcription-PCR (q-RT-PCR) and Western blotting. Higher intracellular levels of c-di-GMP correlated with an impaired capacity to induce cytotoxicity in a eukaryotic cell in vitro and with attenuated virulence in a murine model. This work presents data that support the role that the second messenger c-di-GMP plays in the pathogenesis of Bordetella.

Keywords: Bordetella; RNA-seq; c-di-GMP; immune response; label-free proteomics; type III secretion system.

FIG 1

BB2664 (PdeA) exhibits phosphodiesterase activity. (A) AlphaFold prediction structure for BB2664 (PdeA) and BdcA. (B) PDE activity in crude cell extracts of Bb-ppdeA and Bb-pEmpty, using the substrate bis-pNPP. The graph shows absorbance (410 nm). The results are based on two biologically independent replicates. **, P < 0.01. (C) Biofilm formation by Bb-ppdeA. Biofilm was allowed to form in U-bottom wells for 24 h and stained with crystal violet (CV) solution. Quantification was conducted by dissolving CV in acetic acid solution and measured absorbance (595 nm). The results are based on three biologically independent replicates. *, P < 0.01.

FIG 2

Quantitative analysis of proteins identified in Bb-pEmpty and Bb-pbdcA (A) or Bb-ppdeA (B) using a shotgun approach. Each plot represents a protein identified in 3 replicates of all conditions, plotted according to its P value [log₂(P)] and fold change [log₂(fold change)]. Blue dots represent proteins that meet neither the fold change nor the statistical criteria. Red and green dots correspond to upregulated and downregulated proteins, respectively, satisfying all statistical filters (see Table S1 for protein identity). The horizontal line represents a P value of 0.05. The vertical lines show 2- and 0.5-fold changes in protein expression.

FIG 3

(A) RT-qPCR. mRNA amounts were determined by quantitative real-time PCR. Fold changes were calculated by the ΔΔC_T method using recA levels as a control. Mean fold changes of triplicate cultures were compared using Student’s paired t test (two-tailed distribution), and a P value of <0.05 was considered significant. *, P < 0.05. (B) Clusters of Orthologous Groups of genes found in the RNA-seq approach. mRNA from Bb-pEmpty or Bb-pbdcA were purified, retrotranscribed to cDNA, and sequenced. Sequences were mapped to the B. bronchiseptica RB50 genome and classified according to the ortholog list. Numbers of genes that were upregulated (green bars) or downregulated (red bars) were clustered accordingly to orthologous groups.

FIG 4

High levels of c-di-GMP regulates the TTSS. (A) Diagram showing the TTSS locus, indicating significant (P < 0.05) fold changes. Blue arrows indicate genes for proteins involved in the secretion process. (B) Western blot with anti-Bsp22 antibodies performed on Bb-pEmpty, Bb-pbdcA, Bb-ppdeA, and BbbvgA⁻ (negative control) supernatant samples. Samples were normalized to cell optical density. Two biologically independent assays were performed. (C) Cytotoxicity assays on J774A.1 macrophages cells. Bb-Empty or Bb-pbdcA was added at the indicated MOI on J774A.1 macrophage monolayers and then incubated for 4 h in 5% CO₂ at 37°C. Cytotoxicity assays were conducted using a Pierce LDH cytotoxicity assay kit, and results were expressed relative to the maximum LDH release control provided by the manufacturer. *, P < 0.05 (unpaired one-tailed Student's t test).

FIG 5

B. bronchiseptica with high c-di-GMP levels presented attenuated virulence in the mouse model. Mice were inoculated intranasally with 1 × 10⁶ CFU of either Bb-pEmpty or Bb-pbdcA at 1, 4, and 7 days postinfection, and bacteria were recovered and quantified by serial dilution and colony counting. Bacterial burden was determined in lungs (A), tracheas (B), and nasal wash (C). CFU counts in nasal-associated lymphoid tissue (NALT) samples were determined at day 4 postinfection (D). Each symbol represents data from one mouse. Light gray dots correspond to CFU of mice infected with Bb-pEmpty found dead at day 7 and excluded from statistical analysis. Results are means and standard errors of the means (SEM). *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. P values were determined by two-tailed unpaired t test.

FIG 6

Bb-pbdcA recruited fewer infiltrating phagocytes to the site of infection. Immune cells were measured by flow cytometry at 1, 4, and 7 days postinfection. Neutrophils were identified as Gr-1^high CD11b⁺. Monocytes were identified as Gr-1^mid CD11b⁺. (A) Proportion of neutrophils in lung homogenates. (B) Proportion of monocytes in lung homogenates. (C) Proportion of neutrophils in blood. Group comparisons were analyzed by one-way ANOVA followed by Tukey’s multiple-comparison test. Each symbol represents data from one mouse. Results are means and SEM. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

FIG 7

Bb-pbdcA induced a milder proinflammatory immune response. Murine cytokines were detected in supernatants of lung homogenates at days 1, 4 and 7 postinfection. (A) IFN-γ, (B) TNF-α, (C) IL-1β, (D) IL-6, (E) IL-5, and (F) IL-10 were measured by Meso Scale Discovery’s V-plex Plus proinflammatory panel 1 mouse multiplex kit. Group comparisons were analyzed by one-way ANOVA followed by Tukey’s multiple-comparison test. Each symbol represents data from one mouse. Results are means and SEM. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.