Role of the Novel OprD Family of Porins in Nutrient Uptake in Pseudomonas aeruginosa

To circumvent the permeability barrier of its outer membrane, Pseudomonas aeruginosa has evolved a series of specific porins. These channels have binding sites for related classes of molecules that facilitate uptake under nutrient-limited conditions. Here, we report on the identification of a 19-member family of porins similar to the basic-amino-acid-specific porin OprD. The members of this family fell into one of two phylogenetically distinct clusters, one bearing high similarity to OprD and the other bearing most similarity to the putative phenylacetic acid uptake porin PhaK of Pseudomonas putida. Analysis of the genome context, operon arrangement, and regulation of the PhaK-like porin OpdK indicated that it might be involved in vanillate uptake. This result was confirmed by demonstrating that an opdK mutant had a deficiency in the ability to grow on vanillate as a carbon source. To extrapolate these data to other paralogues within this family, the substrate specificities of 6 of the 17 remaining OprD homologues were inferred using an approach similar to that used with opdK. The specificities determined were as follows: OpdP, glycine-glutamate; OpdC, histidine; OpdB, proline; OpdT, tyrosine; OpdH, cis-aconitate; and OpdO, pyroglutamate. Thus, members of the OprD subfamily took up amino acids and related molecules, and those characterized members most similar to PhaK were responsible for the uptake of a diverse array of organic acids. These results imply that there is a functional basis for the phylogenetic clustering of these proteins and provide a framework for studying OprD homologues in other organisms

Immunodominance in T cell responses elicited against different domains of detoxified pneumolysin PlyD1

Detoxified pneumolysin, PlyD1, is a protein vaccine candidate that induces protection against infections with Streptococcus pneumoniae in mouse models. Despite extensive knowledge on antibody responses against PlyD1, limited information is available about PlyD1 induced T cell recognition. Here we interrogated epitope breadth and functional characteristics of the T cell response to PlyD1 in two mouse strains. BALB/c (H-2d) and C57BL/6 (H-2b) mice were vaccinated with Al(OH)3-adjuvanted or non-adjuvanted PlyD1, or placebo, on day 0, 21 and 42 and were sacrificed at day 56 for collection of sera and spleens. Vaccination with adjuvanted and non-adjuvanted PlyD1 induced anti-pneumolysin IgG antibodies with neutralizing capacity in both mouse strains. Adjuvantation of PlyD1 enhanced the serological responses in both strains. In vitro restimulation of splenocytes with PlyD1 and 18-mer synthetic peptides derived from pneumolysin revealed specific proliferative and cytokine responses. For both mouse strains, one immunodominant and three subdominant natural epitopes were identified. Overlap between H-2d and H-2b restricted T cell epitopes was limited, yet similarities were found between epitopes processed in mice and predicted to be immunogenic in humans. H-2d restricted T cell epitopes were localized in pneumolysin domains 2 and 3, whereas H-2b epitopes were scattered over the protein. Cytokine responses show mostly a Th2 profile, with low levels of Th1 cytokines, in both mouse strains. In conclusion, PlyD1 evokes T cell responses in mice directed against multiple epitope regions, that is dependent on Major Histocompatibility Complex (MHC) background. These results are important to understand human PlyD1 T cell immunogenicity, to guide cell mediated immunity studies in the context of vaccine development

Ply sequence with localization of BALB/c and C57BL/6 T cell-immunogenic regions and predicted HLA-DR binding regions.

<p>Immunodominant epitopes of both BALB/c and C57BL/6 mice are indicated with a #, and subdominant epitopes with a *. ¶ shows the Epi-bars that are <i>in silico</i> predicted for HLA-DR-binding epitopes in humans. Mutated aa residues of PlyD1 are squared and positions with allelic variation as described by Jeffries <i>et al</i> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193650#pone.0193650.ref008" target="_blank">8</a>] are underlined. Colors, cyan, green, red and purple, indicate domains 1, 2, 3, and 4 of Ply, respectively.</p

3D modelling of T cell epitopes in Ply for BALB/c and C57BL/6 mice.

<p>Yellow spheres represent the C-ß atoms of the ID epitope and orange spheres represent the C-ß atoms of the subD epitopes. The dotted circle represents the overlap region between both mouse strains. Colors of the protein structure display the different domains of Ply: cyan indicates domain 1 (amino acids 1–21, 58–147,198–243,319–342), green indicates domain 2 (amino acids 22–57,343–359), red indicates domain 3 (amino acids 148–197,244–318), and purple indicates domain 4 (amino acids 360–469).</p