Legionella pneumophila Type IV Effectors YlfA and YlfB Are SNARE-Like Proteins that Form Homo- and Heteromeric Complexes and Enhance the Efficiency of Vacuole Remodeling.

Legionella pneumophila is a Gram-negative bacterium that can colonize both freshwater protozoa and human alveolar macrophages, the latter infection resulting in Legionnaires' disease. The intracellular lifecycle of L. pneumophila requires extensive manipulation of its host cell, which is carried out by effector proteins that are translocated into the host cell through the Dot/Icm type IV secretion system. This study focuses on a pair of highly similar type IV substrates called YlfA/LegC7 and YlfB/LegC2 that were initially identified in a screen for proteins that cause growth inhibition in yeast. Analysis of truncation mutants revealed that the hydrophobic residues in the Ylf amino termini were required for localization of each protein to the membranes of host cells. Central and carboxy terminal coiled coil domains were found to mediate binding of YlfA and YlfB to themselves and to each other. In vivo, a ΔylfA ΔylfB double mutant strain of L. pneumophila was shown to be defective in establishing a vacuole that supports bacterial replication. This phenotype was subsequently correlated with a decrease in the association of endoplasmic reticulum (ER)-derived vesicles with vacuoles containing ΔylfA ΔylfB mutant bacteria. These data suggest that the Ylf proteins are membrane-associated effectors that enhance remodeling of the L. pneumophila -containing vacuole by promoting association and possibly fusion of ER-derived membrane vesicles with the bacterial compartment

Amino terminal hydrophobic residues mediate colocalization of YlfA and YlfB with eukaryotic membrane networks.

<p>(A) Predicted structural motifs of YlfA and YlfB. The amino terminal hydrophobic domains (HD), in grey, were identified by analysis of the YlfA and YlfB amino acid sequences using Kyte-Doolittle [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159698#pone.0159698.ref048" target="_blank">48</a>], TMBase25 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159698#pone.0159698.ref049" target="_blank">49</a>] and TMHMM2.0 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159698#pone.0159698.ref050" target="_blank">50</a>] algorithms. Regions predicted to form coiled coil domains (CC), in black, were defined using the COILS server. (B-E) Cellular localization of GFP-tagged Ylf proteins. CHO cells were transfected with plasmids encoding amino-terminal GFP-tagged proteins: full length YlfA (B), YlfA 122–425 (C), full length YlfB (D), and YlfB 121–405 (E). 18 h after transfection, cells were fixed and endogenous KDEL-containing proteins were stained with a mouse anti-KDEL antibody, followed by an AlexaFluor 594-conjugated anti-mouse secondary antibody. Grey scale images of confocal sections show the localization of GFP-tagged proteins and KDEL-containing proteins demarcating the early secretory pathway. Merged color images show GFP-Ylf fusion proteins (green) and anti-KDEL staining (red).</p

Co-production of YlfA and YlfB alters localization of YlfB.

<p>CHO cells were co-transfected for 18 h with plasmids encoding amino terminal RFP or GFP-tagged full length YlfA and YlfB. Cells were fixed in 2% PFA, mounted and imaged. Grey scale images show the localization of RFP and GFP-tagged proteins in confocal sections. Merged color images reveal colocalization of YlfA and YlfB.</p

YlfA and YlfB bind homo- and heterotypically <i>in vitro</i>.

<p>A) Lysates from <i>E</i>. <i>coli</i> co-transformed with plasmids encoding GST and M45-tagged Ylfs were incubated with GS4B resin. Bound proteins were eluted with reduced glutathione and eluates were analyzed by anti-M45 epitope immunoblot. To assess the relative efficiency of binding and elution of GST and GST-Ylf fusion proteins, PVDF membranes were subsequently stained with Ponceau S. B) Whole cell lysates of stationary phase <i>L</i>. <i>pneumophila</i> were separated by SDS-PAGE and immobilized on PVDF. <i>L</i>. <i>pneumophila</i> strains include wild type (lane 1), in-frame deletion Δ<i>ylfA</i> and Δ<i>ylfB</i> mutants (lanes 2, 3), a double in-frame deletion Δ<i>ylfA</i> Δ<i>ylfB</i> mutant (lane 4), and the Δ<i>ylfA</i> Δ<i>ylfB</i> mutant harboring plasmids encoding M45-YlfA or M45-YlfB (lanes 5, 6.) Membranes were incubated with recombinant purified GST, GST-YlfA 128–425, or GST-YlfB 131–405 suspended at 20 μg/ml in 2% NFDM 0.1% Tween PBS overnight at 4°C. Bound GST or GST fusion protein was detected by anti-GST immunoblot.</p

Amino terminal hydrophobic residues mediate association of YlfA and YlfB with eukaryotic lipid.

<p>HEK293T cells were transfected for 14 h with plasmids encoding full length (A and B) or truncation mutant Ylf proteins (A). A) Membrane-associated and soluble protein fractions were generated by centrifugation of whole cell lysate at 100,000 x g, followed by solubilization of membrane-associated proteins with 0.1% Triton X-100. The presence of Ylf protein in each subcellular fraction is detected by anti-YlfA and anti-YlfB immunoblot. To demonstrate effective fractionation of cell lysates, fractions were probed with antibodies against the transmembrane protein Calnexin and cytosolic alpha-Tubulin. B) Whole cell lysates were overlayed on a 4–26% continuous OptiPrep gradient and centrifuged at 100,000 x g for 3 h. The presence of Ylf protein in each odd fraction was detected by immunoblot using anti-YlfA or anti-YlfB antibodies. The sedimentation profile of membranes associated with the ER and early secretory pathway was revealed by anti-Calnexin immunoblot.</p

YlfA and YlfB are required for efficient formation of the <i>L</i>. <i>pneumophila</i>-containing vacuole.

<p>A) Bone marrow-derived macrophages derived from A/J mice were infected with wild type <i>L</i>. <i>pneumophila</i>, a Δ<i>ylfA</i> Δ<i>ylfB</i> mutant, and the Δ<i>ylfA</i> Δ<i>ylfB</i> mutant complemented by knock-in of both <i>ylf</i> ORFs onto the <i>L</i>. <i>pneumophila</i> chromosome. Duplicate samples were fixed at 2 and 10 h post-infection. The index of replicative vacuole formation was calculated by dividing the number of observed replicative vacuoles at 10 h by the total number of infected cells at 2 h. Due to variability in the overall efficiency of infection between individual experiments, the index for each strain is presented as a percent of wild type. Data represent the average of three independent experiments ± SD, n = 500 per time point, per experiment. Δ<i>ylfA</i> Δ<i>ylfB</i> mutant R.V. formation is significantly less efficient compared to wild type in an unpaired Student’s <i>t</i> test.*<i>p</i><0.0001. B) Whole cell lysates were prepared from stationary phase broth cultures of wild type <i>L</i>. <i>pneumophila</i>, Δ<i>ylfA</i>, Δ<i>ylfB</i>, Δ<i>ylfA</i> Δ<i>ylfB</i>, and Δ<i>ylfA</i> Δ<i>ylfB</i> in which one or both <i>ylf</i> open reading frames have been reintroduced into the genome. YlfA and YlfB protein levels in these strains were detected by anti-YlfA and anti-YlfB immunoblot. Relative loading efficiency was monitored by anti-DotA immunoblot.</p

Purified YlfA and YlfB form high molecular weight complexes that are resistant to boiling.

<p>6xHis-tagged YlfA 128–425 and YlfB 131–405 were applied to a Superdex 200 column. Peak fractions of each protein were combined with Laemmli buffer containing β-mercaptoethanol and a subset were heated for 10 min at 100°C. Protein complexes were detected by anti-YlfA and anti-YlfB immunoblot. Arrows indicate bands corresponding to monomeric, dimeric and trimeric species.</p

Molecular Characterization of Ancylostoma ceylanicum Kunitz-Type Serine Protease Inhibitor: Evidence for a Role in Hookworm-Associated Growth Delay

Hookworm infection is a major cause of iron deficiency anemia and malnutrition in developing countries. The Ancylostoma ceylanicum Kunitz-type inhibitor (AceKI) is a 7.9-kDa broad-spectrum inhibitor of trypsin, chymotrypsin, and pancreatic elastase that has previously been isolated from adult hookworms. Site-directed mutagenesis of the predicted P1 inhibitory reactive site amino acid confirmed the role of Met(26) in mediating inhibition of the three target serine proteases. By using reverse transcription-PCR, it was demonstrated that the level of AceKI gene expression increased following activation of third-stage larvae with serum and that the highest level of expression was reached in the adult stage of the parasite. Immunohistochemistry studies performed with polyclonal immunoglobulin G raised against recombinant AceKI showed that the inhibitor localized to the subcuticle of the adult hookworm, suggesting that it has a potential in vivo role in neutralizing intestinal proteases at the surface of the parasite. Immunization with recombinant AceKI was shown to confer partial protection against hookworm-associated growth delay without a measurable effect on anemia. Taken together, the data suggest that AceKI plays a role in the pathogenesis of hookworm-associated malnutrition and growth delay, perhaps through inhibition of nutrient absorption in infected hosts