Journal: PLoS Pathogens
Article Title: SliC is a surface-displayed lipoprotein that is required for the anti-lysozyme strategy during Neisseria gonorrhoeae infectionNeisseria gonorrhoeae employs two protein inhibitors to evade killing by human lysozyme
Figure Lengend Snippet: Sequence alignment, phylogenetic analysis and conservation of SliC. (A) The amino acid sequence of SliC from N . gonorrhoeae FA1090 (WP 003688168) was aligned with MliC for E . coli (AJE56069), S . enterica (NP 460410), and P . aeruginosa (AAG04256) sequences downloaded from NCBI with the Clustal Omega online tool (Clustal 2.1; https://www.ebi.ac.uk/Tools/msa/clustalo/ ) using the default alignment parameters. The predicted lipoprotein signal peptide is shown in aquamarine. The conserved regions of the COG3895 domain in the MliC/PliC family, SxSGAxY and YxxxTKG are shown in red and green boxes, respectively. Preserved residues are marked in color within conserved regions and by asterisks (*) throughout the sequences. The residues S83 and K103, predicted to be involved in interaction with c-type lysozyme are designated by red and green arrows, respectively. (B) A phylogenetic comparison of SliC, MliC/PliC and ACP. The tree was constructed using the Jones-Taylor-Thornton model [ 58 ] in MEGA. Neighbor-Join and BioNJ algorithms were applied to a pairwise-distance matrix derived from the JTT model to obtain an initial tree for the heuristic search. The phylogenies were tested by 500 bootstrap iterations, and the tree with the highest log likelihood is presented. (C) A phylogenetic comparison of SliC alleles in 3787 isolates of N . gonorrhoeae deposited to the PubMLST was performed as described above. The dominant allele (29) present in over 97% gonococcal isolates and in FA1090 is denoted in a red box. (D) Analysis of single nucleotide polymorphisms of SliC (locus NEIS1425) in N . gonorrhoeae was performed by comparing DNA sequences between gonococcal isolates deposited to PubMLST ( https://pubmlst.org/neisseria/ , as of April 11, 2018). (E) A panel constituting 37 N . gonorrhoeae isolates that are distinct genetically, geographically, and temporally, including common laboratory strains; clinical isolates collected from public health clinics in Baltimore from 1991 to 1994 and Seattle from 2011 to 2016; and the 14 isolates constituting the 2016 WHO reference strains were grown concurrently on solid medium for 20 h in 5% CO 2 at 37° C. Cells were collected, lysed and processed for immunoblotting. (F) Whole-cell lysates of different Neisseria , including pathogenic N . meningitidis MC58, commensal N . lactamica NLI83/-01, and a human opportunistic pathogen N . weaveri 1032 were separated by SDS-PAGE and probed with antisera. In all experiments, samples were matched by equivalent OD 600 units, resolved in a 4 to 20% Tris-glycine gel, and transferred onto nitrocellulose. Immunoblots were performed using polyclonal rabbit antisera against SliC. Cell lysates of wild type FA1090 and isogenic Δ sliC were loaded as controls for the experiments.
Article Snippet: N . gonorrhoeae FA1090 (NC_002946) genomic DNA was isolated using the Wizard Genomic DNA Purification Kit (Promega).
Techniques: Sequencing, Construct, Derivative Assay, SDS Page, Western Blot