Virulent Burkholderia species mimic host actin polymerases to drive actin-based motility.

Burkholderia pseudomallei and B. mallei are bacterial pathogens that cause melioidosis and glanders, whereas their close relative B. thailandensis is non-pathogenic. All use the trimeric autotransporter BimA to facilitate actin-based motility, host cell fusion, and dissemination. Here, we show that BimA orthologs mimic different host actin-polymerizing proteins. B. thailandensis BimA activates the host Arp2/3 complex. In contrast, B. pseudomallei and B. mallei BimA mimic host Ena/VASP actin polymerases in their ability to nucleate, elongate, and bundle filaments by associating with barbed ends, as well as in their use of WH2 motifs and oligomerization for activity. Mechanistic differences among BimA orthologs resulted in distinct actin filament organization and motility parameters, which affected the efficiency of cell fusion during infection. Our results identify bacterial Ena/VASP mimics and reveal that pathogens imitate the full spectrum of host actin-polymerizing pathways, suggesting that mimicry of different polymerization mechanisms influences key parameters of infection.

A Potent and Effective Suicidal Listeria Vaccine Platform.

Live-attenuated Listeria monocytogenes has shown encouraging potential as an immunotherapy platform in preclinical and clinical settings. However, additional safety measures will enable application across malignant and infectious diseases. Here, we describe a new vaccine platform, termed Lm-RIID (L. monocytogenes recombinase-induced intracellular death), that induces the deletion of genes required for bacterial viability yet maintains potent T cell responses to encoded antigens. Lm-RIID grows normally in broth but commits suicide inside host cells by inducing Cre recombinase and deleting essential genes flanked by loxP sites, resulting in a self-limiting infection even in immunocompromised mice. Lm-RIID vaccination of mice induces potent CD8+ T cells and protects against virulent challenges, similar to live L. monocytogenes vaccines. When combined with α-PD-1, Lm-RIID is as effective as live-attenuated L. monocytogenes in a therapeutic tumor model. This impressive efficacy, together with the increased clearance rate, makes Lm-RIID ideal for prophylactic immunization against diseases that require T cells for protection.

Helicobacter pylori NikR protein exhibits distinct conformations when bound to different promoters.

Helicobacter pylori NikR (HpNikR) is a ribbon-helix-helix (RHH) DNA-binding protein that binds to several different promoter regions. The binding site sequences are not absolutely conserved. The ability of HpNikR to discriminate specific DNA sites resides partly in its nine-amino acid N-terminal arm. Previously, indirect evidence indicated that the arm exists in different conformations when HpNikR is bound to the nixA and ureA promoters. Here, we directly examined HpNikR conformation when it was bound to nixA and ureA DNA fragments by tethering (S)-1{[bis(carboxymethyl)amino]methyl}-2-{4-[(2-bromoacetyl)amino]phenylethyl}(carboxymethyl)amino]acetic acid, iron(III) to different positions in the N-terminal arm and RHH DNA binding domain. Different cleavage patterns at each promoter directly demonstrated that both the RHH domain and the arm adopt different conformations on the nixA and ureA promoters. Additionally, the two RHH domain dimers of the HpNikR tetramer are in distinct conformations at ureA. Site-directed mutagenesis identified an interchain salt bridge (Lys(48)-Glu(47')) in the RHH domain remote from the DNA binding interface that is required for high affinity binding to ureA but not nixA. Finally, DNA affinity measurements of wild-type HpNikR and a salt bridge mutant (K48A) to hybrid nixA-ureA promoters demonstrated that inverted repeat half-sites, spacers, and flanking DNA are all required for sequence-specific DNA binding by HpNikR. Notably, the spacer region made the largest contribution to DNA affinity. HpNikR exhibits a substantially expanded regulon compared with other NikR proteins. The results presented here provide a molecular basis for understanding regulatory network expansion by NikR as well as other prokaryotic regulatory proteins.

Geobacter uraniireducens NikR displays a DNA binding mode distinct from other members of the NikR family.

NikR is a nickel-responsive ribbon-helix-helix transcription factor present in many bacteria and archaea. The DNA binding properties of Escherichia coli and Helicobacter pylori NikR (factors EcNikR and HpNikR, respectively) have revealed variable features of DNA recognition. EcNikR represses a single operon by binding to a perfect inverted repeat sequence, whereas HpNikR binds to promoters from multiple genes that contain poorly conserved inverted repeats. These differences are due in large part to variations in the amino acid sequences of the DNA-contacting beta-sheets, as well as residues preceding the beta-sheets of these two proteins. We present here evidence of another variation in DNA recognition by the NikR protein from Geobacter uraniireducens (GuNikR). GuNikR has an Arg-Gly-Ser beta-sheet that binds specifically to an inverted repeat sequence distinct from those recognized by Ec- or HpNikR. The N-terminal residues that precede the GuNikR beta-sheet residues are required for high-affinity DNA binding. Mutation of individual arm residues dramatically reduced the affinity of GuNikR for specific DNA. Interestingly, GuNikR tetramers are capable of binding cooperatively to the promoter regions of two different genes, nik(MN)1 and nik(MN)2. Cooperativity was not observed for the closely related G. bemidjiensis NikR, which recognizes the same operator sequence. The cooperative mode of DNA binding displayed by GuNikR could affect the sensitivity of transporter gene expression to changes in intracellular nickel levels.

An intact urease assembly pathway is required to compete with NikR for nickel ions in Helicobacter pylori.

We examined the effects of urease and hydrogenase assembly gene deletions on NikR activation in H. pylori strains 26695 and G27. The loss of any component of urease assembly increased NikR activity under Ni2+-limiting conditions, as measured by reduced transcript levels and 63Ni accumulation. Additionally, SlyD functioned in urease assembly in strain 26695.

Identification of Ni-(L-His)2 as a substrate for NikABCDE-dependent nickel uptake in Escherichia coli.

Nickel is an important cofactor for several microbial enzymes. The ATP-dependent NikABCDE transporter is one of several types of uptake pathways known to be important for nickel acquisition in microbes. The Escherichia coli NikA periplasmic binding protein is structurally homologous to the di- and oligopeptide binding proteins, DppA and OppA. This structural similarity raises interesting questions regarding the evolutionary relationships between the recognition of nickel ions and short peptides. We find that in defined minimal growth medium NikABCDE transports nickel ions in the presence of exogenously added L-histidine (L-His), but not D-histidine. Both nickel uptake in cells and nickel binding to purified NikA showed an L-His concentration dependence consistent with recognition of a Ni-(L-His)2 complex. This discovery reveals parallels to the transport of other metal complexes, notably iron, and suggests the structural diversity of nickel transporters may arise from the need to recognize extracellular nickel complexed with different organic ligands, whether they be exogenously or endogenously produced. Further, these results suggest that experiments examining the physiology and ecology of nickel-requiring microbes should account for the possibility that the growth medium may not support nickel uptake.

The N-terminal arm of the Helicobacter pylori Ni2+-dependent transcription factor NikR is required for specific DNA binding.

The Ni(2+)-dependent transcription factor NikR is widespread among microbes. The two experimentally characterized NikR orthologs, from Helicobacter pylori and Escherichia coli, display vastly different regulatory capabilities in response to increased intracellular Ni(2+). Here, we demonstrate that the nine-residue N-terminal arm present in H. pylori NikR plays a critical role in the expanded regulatory capabilities of this NikR family member. Specifically, the N-terminal arm is required to inhibit NikR binding to low affinity and nonspecific DNA sequences and is also linked to a cation requirement for NikR binding to the nixA promoter. Site-directed mutagenesis and arm-truncation variants of NikR indicate that two residues, Asp-7 and Asp-8, are linked to the cation requirement for binding. Pro-4 and Lys-6 are required for maximal DNA binding affinity of the full-length protein to both the nixA and ureA promoters. The N-terminal arm is highly variable among NikR family members, and these results suggest that it is an adaptable structural feature that can tune the regulatory capabilities of NikR to the nickel physiology of the microbe in which it is found.