NilD CRISPR RNA contributes to Xenorhabdus nematophila colonization of symbiotic host nematodes

The bacterium Xenorhabdus nematophila is a mutualist of entomopathogenic Steinernema carpocapsae nematodes and facilitates infection of insect hosts. X. nematophila colonizes the intestine of S. carpocapsae which carries it between insects. In the X. nematophila colonization-defective mutant nilD6::Tn5, the transposon is inserted in a region lacking obvious coding potential. We demonstrate that the transposon disrupts expression of a single CRISPR RNA, NilD RNA. A variant NilD RNA also is expressed by X. nematophila strains from S. anatoliense and S. websteri nematodes. Only nilD from the S. carpocapsae strain of X. nematophila rescued the colonization defect of the nilD6::Tn5 mutant, and this mutant was defective in colonizing all three nematode host species. NilD expression depends on the presence of the associated Cas6e but not Cas3, components of the Type I-E CRISPR-associated machinery. While cas6e deletion in the complemented strain abolished nematode colonization, its disruption in the wild-type parent did not. Likewise, nilD deletion in the parental strain did not impact colonization of the nematode, revealing that the requirement for NilD is evident only in certain genetic backgrounds. Our data demonstrate that NilD RNA is conditionally necessary for mutualistic host colonization and suggest that it functions to regulate endogenous gene expression

Role of the Membrane Localization Domain of the Pseudomonas aeruginosa Effector Protein ExoU in Cytotoxicity▿ †

ExoU is a potent effector protein that causes rapid host cell death upon injection by the type III secretion system of Pseudomonas aeruginosa. The N-terminal half of ExoU contains a patatin-like phospholipase A2 (PLA2) domain that requires the host cell cofactor superoxide dismutase 1 (SOD1) for activation, while the C-terminal 137 amino acids constitute a membrane localization domain (MLD). Previous studies had utilized insertion and deletion mutations to show that portions of the MLD are required for membrane localization and catalytic activity. Here we further characterize this domain by identifying six residues that are essential for ExoU activity. Substitutions at each of these positions resulted in abrogation of membrane targeting, decreased ExoU-mediated cytotoxicity, and reductions in PLA2 activity. Likewise, each of the six MLD residues was necessary for full virulence in cell culture and murine models of acute pneumonia. Purified recombinant ExoU proteins with substitutions at five of the six residues were not activated by SOD1, suggesting that these five residues are critical for activation by this cofactor. Interestingly, these same five ExoU proteins were partially activated by HeLa cell extracts, suggesting that a host cell cofactor other than SOD1 is capable of modulating the activity of ExoU. These findings add to our understanding of the role of the MLD in ExoU-mediated virulence

CRISPR required for host colonization

The bacterium Xenorhabdus nematophila is a mutualist of entomopathogenic Steinernema carpocapsae nematodes and facilitates infection of insect hosts. X. nematophila colonizes the intestine of S. carpocapsae which carries it between insects. In the X. nematophila colonization-defective mutant nilD6::Tn5, the transposon is inserted in a region lacking obvious coding potential. We demonstrate that the transposon disrupts expression of a single CRISPR RNA, NilD RNA. A variant NilD RNA also is expressed by X. nematophila strains from S. anatoliense and S. websteri nematodes. Only nilD from the S. carpocapsae strain of X. nematophila rescued the colonization defect of the nilD6::Tn5 mutant, and this mutant was defective in colonizing all three nematode host species. NilD expression depends on the presence of the associated Cas6e but not Cas3, components of the Type I-E CRISPR-associated machinery. While cas6e deletion in the complemented strain abolished nematode colonization, its disruption in the wild-type parent did not. Likewise, nilD deletion in the parental strain did not impact colonization of the nematode, revealing that the requirement for NilD is evident only in certain genetic backgrounds. Our data demonstrate that NilD RNA is conditionally necessary for mutualistic host colonization and suggest that it functions to regulate endogenous gene expression

Structure of the Type III Secretion Effector Protein ExoU in Complex with Its Chaperone SpcU

<div><p>Disease causing bacteria often manipulate host cells in a way that facilitates the infectious process. Many pathogenic gram-negative bacteria accomplish this by using type III secretion systems. In these complex secretion pathways, bacterial chaperones direct effector proteins to a needle-like secretion apparatus, which then delivers the effector protein into the host cell cytosol. The effector protein ExoU and its chaperone SpcU are components of the <em>Pseudomonas aeruginosa</em> type III secretion system. Secretion of ExoU has been associated with more severe infections in both humans and animal models. Here we describe the 1.92 Å X-ray structure of the ExoU–SpcU complex, a full-length type III effector in complex with its full-length cognate chaperone. Our crystallographic data allow a better understanding of the mechanism by which ExoU kills host cells and provides a foundation for future studies aimed at designing inhibitors of this potent toxin.</p> </div

Comparison of the ExoU–SpcU structures.

<p>(<b>A</b>) A stereo-view of the superposed 3TU3 and 4AKX (cyan) structures showing differences in the ExoU models. (<b>B</b>) Residues of the 395–402 region of the PLA₂ domain (carbon atoms are in red) and SpcU (carbon atoms are in yellow; asymmetric unit residues are underlined; symmetry-related residues are underlined italic) of 3TU3 are shown. Distances from the guanidinium group's nitrogen atoms of Arg 32 of ExoU to the N<i>ε</i>1 atom of Trp 87, the O<i>γ</i> atom of Ala 78 of SpcU from the asymmetric unit and the O<i>γ</i> atom of Ser 66 from a symmetry-related SpcU in 4AKX (carbon atoms are in cyan) are shown. The 1<i>σ</i> 2<i>F</i>_o – <i>F</i>_c electron density map of the 3TU3 structure is displayed. (<b>C</b>) The 3TU3's 1<i>σ</i> 2<i>F</i>_o – <i>F</i>_c electron density map with the C<i>α</i> traces of the superposed 3TU3 (red) and 4AKX (cyan) structures in the area of the active site “cap”. (<b>D</b>) The <i>Ω</i>-loop (residues 531–537) of domain 3 of ExoU of 3TU3 that are disordered in the 4AKX structure. The loop is positioned between SpcU-binding domain of one symmetry-related ExoU and domain 3 of another symmetry-related ExoU in 3TU3. (<b>E</b>) Water molecules stabilize the <i>Ω</i>-loop conformation.</p

Data collection, phasing and refinement statistics.

<p>Highest resolution shell is shown in parenthesis.</p>a<p><i>R</i>_merge = Σ_iΣ_j|<i>I</i>_ij−<<i>I</i>_j>|/Σ_iΣ_j/_ij, where i runs over multiple observations of the same intensity and j runs over all crystallographically unique intensities. If <i>R</i>_merge exceeds 1.0 <i>Scalepack</i> does not report its value because it is non-informative. Instead <i>I</i>/sigma criterion is used to define resolution cut-off.</p

Biophysical solution studies of the ExoU–SpcU interaction.

<p>(<b>A</b>) SEC-MALS elution profiles (solid lines) of the co-expressed/co-purified ExoU–SpcU complex, ExoU, SpcU, BSA and two different molar mixtures of ExoU and SpcU using the 10 mM Tris-HCl pH 8.3 with 200 mM NaCl conditions. Molecular mass distribution at 200 mM NaCl (dotted lines) and 500 mM NaCl (dashed lines) of the samples are shown. (<b>B</b>) The SPR experimental curve-fitting methodology for a simple 1∶1 binding model for immobilized SpcU with ExoU as the analyte. Pairs of colored traces at each concentration indicate duplicate experimental determinations; black traces show the corresponding binding model curves. A 1∶2 ExoU∶SpcU model was used to characterize binding of SpcU to immobilized ExoU (not shown; see text for the rates). (<b>C and D, upper panels</b>) Injection of SpcU (120 µM – dimer concentration) into ExoU (15 µM) and ExoU (50 µM) into SpcU (5 µM – monomer concentration) respectively, produced dose-dependent, exothermic responses. The integrated data (filled black squares, lower panels in <b>C</b> and <b>D</b>) could be fitted to a single set of sites (black fitting curve). N – binding stoichiometry, <i>K</i> – binding constant related to <i>K_d</i> by , Δ<i>S</i> – entropy change; and Δ<i>H</i> – enthalpy change.</p

Structure of the ExoU–SpcU complex.

<p>(<b>A</b>) Constructs used for co-crystallization. Disordered regions in the structure are shown as white boxes and numbered I through IX. “Tag” refers to the N-terminal 6×His purification tag and is not numbered. (<b>B</b>) The quaternary architecture of the ∼81×67×59 ų ExoU–SpcU complex. The two proteins bury a total surface area of 2890 Ų. (<b>C</b>) SpcU and individual ExoU domains colored as in (<b>A</b>). The MLD consists of two separate structural regions: domain 3 (light blue) and domain 4 (dark blue). Disordered regions are shown as dashed lines and numbered as in (<b>A</b>).</p

Details of the ExoU–SpcU interaction.

<p>Hydrogen bonded interactions are shown in panels A, C and E. (<b>A</b>) the SpcU-binding domain of ExoU (green); (<b>C</b>) the 395–402 region of the PLA₂ domain of ExoU (red); and (<b>E</b>) domain 4 of ExoU (blue). SpcU is yellow in all panels. Residues involved in non-bonded interactions are shown in B, D and F. (<b>B</b>) The chaperone-binding domain of ExoU (green); (<b>D</b>) the 395–402 region of ExoU (red); and (<b>F</b>) domain 4 of ExoU (blue). In all panels, depicted peptides of ExoU and SpcU that are longer than 3 residues are labeled with the N-terminal and C-terminal residues only. Residues of SpcU in (<b>B</b>, <b>D</b>, <b>F</b>) that do not make any contacts with ExoU are in grey. Residues of ExoU are labeled in three-letter code and SpcU in one-letter code.</p