The Contact-Dependent Growth Inhibition Pathways of Burkholderia pseudomallei 1026b and Escherichia coli EC93

Bacteria engage in social behavior by communicating through a variety of mechanisms. One method of communication is contact-dependent growth inhibition (CDI), a phenomenon in which one bacterium binds and delivers a toxin to a closely related target cell, using the proteins CdiB and CdiA. This toxin blocks cell growth unless the target cell contains an immunity protein, CdiI. The CDI pathway is the process by which toxins are delivered and activated, using distinct target-cell proteins such as outer membrane receptors and inner membrane transporters. The first chapter of this thesis focuses on the CDI system from Burkholderia pseudomallei 1026b. We identify three genes whose products appear to be necessary for growth inhibition, and describe a potential CDI pathway for this system. The second chapter discusses the mechanism by which Escherichia coli EC93 binds target cells. Mutations in BamA, the CdiAEC93 receptor, are described. These mutations confer resistance to CDI and block cell-cell binding. Both chapters demonstrate the variety and species specificity of CDI growth inhibition pathways

De novo design of modular protein oligomers to investigate cell signaling

Thesis (Ph.D.)--University of Washington, 2022Clustering of receptors is a critical step in activation of signaling, but this phenomenon is difficult to investigate with native soluble ligands that bind only 1-2 copies of a receptor. I designed a series of modular protein scaffolds with 4-, 6-, and 8-fold symmetry, offering up to 8 sites for display of a receptor-binding domain. Scaffolds were extended by adding repeat units to vary the spacing of the bound receptors. To target receptors, a de novo miniprotein binder to the fibroblast growth factor receptor (FGFR) was attached to scaffolds through genetic fusion. Treatment of cells with the designed scaffolds resulted in colocalization and decreased membrane diffusion of receptors, increased Erk phosphorylation, and intracellular calcium release indicating activation of FGF signaling. These designed protein scaffolds can be applied as a universal tool to a variety of systems to dissect the role of clustering in cell surface receptor-mediated signaling pathways

Genetic analysis of the CDI pathway from Burkholderia pseudomallei 1026b.

Contact-dependent growth inhibition (CDI) is a mode of inter-bacterial competition mediated by the CdiB/CdiA family of two-partner secretion systems. CdiA binds to receptors on susceptible target bacteria, then delivers a toxin domain derived from its C-terminus. Studies with Escherichia coli suggest the existence of multiple CDI growth-inhibition pathways, whereby different systems exploit distinct target-cell proteins to deliver and activate toxins. Here, we explore the CDI pathway in Burkholderia using the CDIIIBp1026b system encoded on chromosome II of Burkholderia pseudomallei 1026b as a model. We took a genetic approach and selected Burkholderia thailandensis E264 mutants that are resistant to growth inhibition by CDIIIBp1026b. We identified mutations in three genes, BTH_I0359, BTH_II0599, and BTH_I0986, each of which confers resistance to CDIIIBp1026b. BTH_I0359 encodes a small peptide of unknown function, whereas BTH_II0599 encodes a predicted inner membrane transport protein of the major facilitator superfamily. The inner membrane localization of BTH_II0599 suggests that it may facilitate translocation of CdiA-CTIIBp1026b toxin from the periplasm into the cytoplasm of target cells. BTH_I0986 encodes a putative transglycosylase involved in lipopolysaccharide (LPS) synthesis. ∆BTH_I0986 mutants have altered LPS structure and do not interact with CDI⁺ inhibitor cells to the same extent as BTH_I0986⁺ cells, suggesting that LPS could function as a receptor for CdiAIIBp1026b. Although ∆BTH_I0359, ∆BTH_II0599, and ∆BTH_I0986 mutations confer resistance to CDIIIBp1026b, they provide no protection against the CDIE264 system deployed by B. thailandensis E264. Together, these findings demonstrate that CDI growth-inhibition pathways are distinct and can differ significantly even between closely related species

Genetic analysis of the CDI pathway from Burkholderia pseudomallei 1026b.

Contact-dependent growth inhibition (CDI) is a mode of inter-bacterial competition mediated by the CdiB/CdiA family of two-partner secretion systems. CdiA binds to receptors on susceptible target bacteria, then delivers a toxin domain derived from its C-terminus. Studies with Escherichia coli suggest the existence of multiple CDI growth-inhibition pathways, whereby different systems exploit distinct target-cell proteins to deliver and activate toxins. Here, we explore the CDI pathway in Burkholderia using the CDIIIBp1026b system encoded on chromosome II of Burkholderia pseudomallei 1026b as a model. We took a genetic approach and selected Burkholderia thailandensis E264 mutants that are resistant to growth inhibition by CDIIIBp1026b. We identified mutations in three genes, BTH_I0359, BTH_II0599, and BTH_I0986, each of which confers resistance to CDIIIBp1026b. BTH_I0359 encodes a small peptide of unknown function, whereas BTH_II0599 encodes a predicted inner membrane transport protein of the major facilitator superfamily. The inner membrane localization of BTH_II0599 suggests that it may facilitate translocation of CdiA-CTIIBp1026b toxin from the periplasm into the cytoplasm of target cells. BTH_I0986 encodes a putative transglycosylase involved in lipopolysaccharide (LPS) synthesis. ∆BTH_I0986 mutants have altered LPS structure and do not interact with CDI⁺ inhibitor cells to the same extent as BTH_I0986⁺ cells, suggesting that LPS could function as a receptor for CdiAIIBp1026b. Although ∆BTH_I0359, ∆BTH_II0599, and ∆BTH_I0986 mutations confer resistance to CDIIIBp1026b, they provide no protection against the CDIE264 system deployed by B. thailandensis E264. Together, these findings demonstrate that CDI growth-inhibition pathways are distinct and can differ significantly even between closely related species

Complementation of CDI^R mutations.

<p>The indicated <i>B</i>. <i>thailandensis</i> strains were co-cultured with Bt81 inhibitors (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120265#pone.0120265.t001" target="_blank">Table 1</a>) that express the CDI_II^Bp1026b system for 24 h on solid medium, and the competitive index was calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120265#sec002" target="_blank">Materials and Methods</a>. The strain labeled <i>cdiI</i>^1026b expresses the cognate CdiI_II^Bp1026b immunity protein. Plasmid-borne copies of BTH_I0359, BTH_I0986 and BTH_II0599 genes were expressed from an L-arabinose inducible promoter. Data represent the mean ± SEM for three independent experiments. Sample values that were statistically different from one another (p < 0.05) are shown by bars with an asterisk (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120265#pone.0120265.g001" target="_blank">Fig. 1</a>).</p

Cell-cell binding.

<p>CDI⁺ (Bt81) and CDI^– (wild-type <i>B</i>. <i>thailandensis</i>) cells were labeled with GFP and mixed with the indicated DsRed-labeled target cells, then analyzed by flow cytometry to detect and quantify cell-cell aggregates. Binding was normalized to 1.0 for the interaction between Bt81 and wild-type <i>B</i>. <i>thailandensis</i> cells. Sample values that were statistically different from one another are shown by bars; ** = p < 0.01, and *** = p < 0.001 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120265#pone.0120265.g001" target="_blank">Fig. 1</a>). We then tested the three CDI^R target strains and found that ΔBTH_I0986 targets interacted poorly with inhibitor cells, similar to the level observed with CDI^– mock inhibitors (Fig. 6). In contrast, the ΔBTH_II0599 mutant showed wild-type binding levels, and ΔBTH_I0359 targets showed increased binding to inhibitor cells (Fig. 6). Together, these results suggest that mutations in BTH_I0986 confer CDI^R by altering the cell surface to prevent stable associations with CDI_II^Bp1026b inhibitor cells.</p

The CDI^R phenotype is specific for CDIII^Bp1026b.

<p>The indicated <i>B</i>. <i>thailandensis</i> strains were co-cultured with wild-type (<i>cdiAIB</i>⁺) <i>B</i>. <i>thailandensis</i> E264 cells for 24 h on solid medium, and the competitive index was calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120265#sec002" target="_blank">Materials and Methods</a> The strain labeled <i>cdiI</i>^E264 expresses the cognate CdiI^E264 immunity protein. Data represent the mean ± SEM for three independent experiments. Sample values that were statistically different from one another (p < 0.01) are shown by a bar with a double asterisk (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120265#pone.0120265.g001" target="_blank">Fig. 1</a>).</p

Toxicity of CdiA-CTII^Bp1026b expressed inside <i>B</i>. <i>thailandensis</i> cells.

<p>Plasmids pSCBAD and pSCBAD::<i>cdiA-CT</i>_II^Bp1026b were introduced into the indicated <i>B</i>. <i>thailandensis</i> strains by conjugation as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120265#sec002" target="_blank">Materials and Methods</a>. The mating mixtures were split into equal portions and plated onto LB agar with Polymyxin B and Trimethoprim supplemented with either D-glucose (left panels) or L-arabinose (right panels). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120265#sec002" target="_blank">Materials and Methods</a>.</p

Lipopolysaccharide (LPS) analysis.

<p>LPS was isolated from the indicated <i>B</i>. <i>thailandensis</i> strains and analyzed by SDS-PAGE using fluorescent detection. The LPS standard is from <i>Escherichia coli</i> serotype 055:B5.</p