A Structural Basis for Host Cytoskeletal Disruption and Virulence by Yersinia Protein Kinase A

Yersinia spp. cause gastroenteritis and the plague, representing historically devastating pathogens that are currently an important biodefense and antibiotic resistance concern. Although several antibiotic therapies exist, the emergence of strains that have garnered multiple drug resistances in combination with the weaponization of Yersinia, make understanding the biology of this pathogen a high priority. Yersinia, along with other pathogenic bacteria such as Salmonella, utilize a macromolecular complex, called a type III secretion apparatus, to deliver virulence proteins directly into cells. These factors commandeer several signaling pathways, often targeting the Rho family of small GTPases which regulate actin cytoskeletal dynamics. A critical virulence determinant in Yersinia species is the Yersinia protein kinase A, or YpkA, a multi-domain protein that disrupts the eukaryotic actin cytoskeleton. YpkA contains a Ser/Thr kinase domain whose activity modulates pathogenicity and a domain that binds to both Rac1 and RhoA of the Rho family of small GTPases. The crystal structure of a YpkA-Rac1 complex reveals that YpkA possesses a novel Rac1-binding domain that mimics the interactions of host guanine nucleotide dissociation inhibitors (GDIs) of the Rho GTPases. YpkA inhibits the exchange of nucleotide in Rac1 and RhoA, and mutations that disrupt the YpkA-GTPase interface abolish this activity in vitro and significantly impair in vivo YpkA-induced cytoskeletal disruption. A Yersinia pseudotuberculosis mutant lacking the GDI activity of YpkA was significantly attenuated for virulence in a mouse infection assay as compared to wild type bacteria. We conclude that virulence in Yersinia depends strongly upon a novel mimicry of host GDI proteins by YpkA. Finally, the YpkA kinase domain has homology to known eukaryotic Ser/Thr kinases and thus could be targeted for small molecule inhibitor design. An efficient approach integrating a machine learning method, homology modeling, and multiple conformational high throughput docking was used for the discovery of YpkA inhibitors. The resultant small molecule compounds, which are the first reported inhibitors for YpkA, not only provide a useful means in probing the function and mechanism of YpkA in bacterial pathogenesis, but also are potential candidates for further development of novel anti-plague drugs

The lifestyle switch protein Bd0108 of Bdellovibrio bacteriovorus is an intrinsically disordered protein

Bdellovibrio bacteriovorus is a δ-proteobacterium that preys upon Salmonella spp., E. coli, and other Gram-negative bacteria. Bdellovibrio can grow axenically (host-independent, HI, rare and mutation-driven) or subsist via a predatory lifecycle (host-dependent, HD, the usual case). Upon contact with prey, B. bacteriovorus enters the host periplasm from where it slowly drains the host cytosol of nutrients for its own replication. At the core of this mechanism is a retractile pilus, whose architecture is regulated by the protein Bd0108 and its interaction with the neighboring gene product Bd0109. Deletion of bd0108 results in negligible pilus formation, whereas an internal deletion (the one that instigates host-independence) causes mis-regulation of pilus length. These mutations, along with a suite of naturally occurring bd0108 mutant strains, act to control the entry to HI growth. To further study the molecular mechanism of predatory regulation, we focused on the apparent lifecycle switch protein Bd0108. Here we characterize the solution structure and dynamics of Bd0108 using nuclear magnetic resonance (NMR) spectroscopy complemented with additional biophysical methods. We then explore the interaction between Bd0108 and Bd0109 in detail utilizing isothermal titration calorimetry (ITC) and NMR spectroscopy. Together our results demonstrate that Bd0108 is an intrinsically disordered protein (IDP) and that the interaction with Bd0109 is of low affinity. Furthermore, we observe that Bd0108 retains an IDP nature while binding Bd0109. From our data we conclude that Bdellovibrio bacteriovorus utilizes an intrinsically disordered protein to regulate its pilus and control predation signaling

The conserved mosaic prophage protein paratox inhibits the natural competence regulator ComR in Streptococcus

Abstract Horizontal gene transfer is an important means of bacterial evolution. This includes natural genetic transformation, where bacterial cells become “competent” and DNA is acquired from the extracellular environment. Natural competence in many species of Streptococcus, is regulated by quorum sensing via the ComRS receptor-signal pair. The ComR-XIP (mature ComS peptide) complex induces expression of the alternative sigma factor SigX, which targets RNA polymerase to CIN-box promoters to activate genes involved in DNA uptake and recombination. In addition, the widely distributed Streptococcus prophage gene paratox (prx) also contains a CIN-box, and here we demonstrate it to be transcriptionally activated by XIP. In vitro experiments demonstrate that Prx binds ComR directly and prevents the ComR-XIP complex from interacting with DNA. Mutations of prx in vivo caused increased expression of the late competence gene ssb when induced with XIP as compared to wild-type, and Prx orthologues are able to inhibit ComR activation by XIP in a reporter strain which lacks an endogenous prx. Additionally, an X-ray crystal structure of Prx reveals a unique fold that implies a novel molecular mechanism to inhibit ComR. Overall, our results suggest Prx functions to inhibit the acquisition of new DNA by Streptococcus

Targeting bacterial nickel transport with aspergillomarasmine A suppresses virulence-associated Ni-dependent enzymes

Abstract Microbial Ni2+ homeostasis underpins the virulence of several clinical pathogens. Ni2+ is an essential cofactor in urease and [NiFe]-hydrogenases involved in colonization and persistence. Many microbes produce metallophores to sequester metals necessary for their metabolism and starve competing neighboring organisms. The fungal metallophore aspergillomarasmine A (AMA) shows narrow specificity for Zn2+, Ni2+, and Co2+. Here, we show that this specificity allows AMA to block the uptake of Ni2+ and attenuate bacterial Ni-dependent enzymes, offering a potential strategy for reducing virulence. Bacterial exposure to AMA perturbs H2 metabolism, ureolysis, struvite crystallization, and biofilm formation and shows efficacy in a Galleria mellonella animal infection model. The inhibition of Ni-dependent enzymes was aided by Zn2+, which complexes with AMA and competes with the native nickelophore for the uptake of Ni2+. Biochemical analyses demonstrated high-affinity binding of AMA-metal complexes to NikA, the periplasmic substrate-binding protein of the Ni2+ uptake system. Structural examination of NikA in complex with Ni-AMA revealed that the coordination geometry of Ni-AMA mimics the native ligand, Ni-(l-His)2, providing a structural basis for binding AMA-metal complexes. Structure-activity relationship studies of AMA identified regions of the molecule that improve NikA affinity and offer potential routes for further developing this compound as an anti-virulence agent

Molecular mechanism of quorum sensing inhibition in Streptococcus by the phage protein paratox

Open access article. Creative Commons Attribution 4.0 International License (CC BY 4.0) appliesStreptococcus pyogenes, or Group A Streptococcus, is a Gram-positive bacterium that can be both a human commensal and a pathogen. Central to this dichotomy are temperate bacteriophages that incorporate into the bacterial genome as prophages. These genetic elements encode both the phage proteins and the toxins harmful to the human host. One such conserved phage protein, paratox (Prx), is always found encoded adjacent to the toxin genes, and this linkage is preserved during all stages of the phage life cycle. Within S. pyogenes, Prx functions to inhibit the quorum-sensing receptor-signal pair ComRS, the master regulator of natural competence, or the ability to uptake endogenous DNA. However, the mechanism by which Prx directly binds and inhibits the receptor ComR is unknown. To understand how Prx inhibits ComR at the molecular level, we pursued an X-ray crystal structure of Prx bound to ComR. The structural data supported by solution X-ray scattering data demonstrate that Prx induces a conformational change in ComR to directly access its DNA-binding domain. Furthermore, electromobility shift assays and competition binding assays reveal that Prx effectively uncouples the interdomain conformational change required for activation of ComR via the signaling molecule XIP. Although to our knowledge the molecular mechanism of quorum-sensing inhibition by Prx is unique, it is analogous to the mechanism employed by the phage protein Aqs1 in Pseudomonas aeruginosa. Together, this demonstrates an example of convergent evolution between Gram-positive and Gram-negative phages to inhibit quorum-sensing and highlights the versatility of small phage proteins

NMR titration data of Bd0108 and Bd0109.

<p>(A) ¹H-¹⁵N HSQC of 25 µM Bd0108 titrated with 200 µM Bd0109. Blue is Bd0108 with 0 µM Bd0109 and magenta is with 200 µM Bd0109. (B) Close-up of region ¹H-¹⁵N HSQC of 25 µM Bd0108 titrated with increasing amounts of Bd0109 at the ratio shown in each panel. Arrows indicate the dose dependent appearance of new peaks in the spectra. (C) Graph representation of peak data height for each residue in the presence of 0 µM Bd0109 (blue), 25 µM Bd0109 (red), 50 µM Bd0109 (orange), 100 µM Bd0109 (green) and 200 µM Bd0109 (purple). Peak height has been normalized to the peak height of the C-terminal residue Q101 for each spectrum. (D) Sequence alignment of Bd0108 from known strains mapping sequence conservation. Strain HID22 is the <i>bd0108Δ42 bp</i> deletion mutant that lacks residues 73–86. The region corresponding to residues 73–86 is outlined in grey in both panels C and D. The alignment was created using Clustal Omega (<a href="http://www.ebi.ac.uk/Tools/msa/clustalo/" target="_blank">http://www.ebi.ac.uk/Tools/msa/clustalo/</a>) and Espript 3.0 (<a href="http://espript.ibcp.fr/ESPript/ESPript/" target="_blank">http://espript.ibcp.fr/ESPript/ESPript/</a>).</p

Bd0108 ¹H-¹⁵N HSQC.

<p>Assigned ¹H-¹⁵N HSQC of Bd0108 residues 23–101 taken at pH 7.0 and 25°C. The full chemical shift list can be accessed via the BMRB with accession code 25327.</p

Bd0108 adopts an extended conformation.

<p>Circular Dichroism spectra of isotopically labeled Bd0108 recorded at pH 6.5 (red), pH 7.0 (grey), and pH 7.5 (blue). CD spectrum recorded at pH 7.5 of Bd0108 purified from <i>E. coli</i> grown in LB media (green).</p

Isothermal titration calorimetry of Bd0108 and Bd0109.

<p>The top two panels show heats from control titrations of Bd0108 into experimental buffer and experimental buffer into Bd0109. The middle panel shows heats of titration of 1.2 mM Bd0108 into 20 µM Bd0109, and the bottom panel calculated enthalpies after data correction in origin software. The resulting stoichiometry (N-value) is 3.1, K_d of 45 µM, ΔH of −2075 kcal/mole, ΔS of 12.92 cal K⁻¹ mol⁻¹.</p

Purification of Bd0108.

<p>(A) Gel filtration profile of purified Bd0108. (Upper panel) Bd0108 elution from an SD75 column after affinity chromatography and cleavage of His-tag. Overlaid on the chromatogram is an SDS-PAGE gel showing purity of the corresponding fractions. (Lower Panel) Elution profile of the late elution peak re-run on an SD75 column. (B) Dynamic Light scattering of the Bd0108 late elution peak at approximately 2 mg/mL. (C) SDS-PAGE gel of purified Bd0108 with and without reducing agent (β-mercaptoethanol) and/or boiling indicated by a black circle.</p