Phylogenomic and biochemical characterization of three Legionella pneumophila polypeptide deformylases

Abstract

Legionella pneumophila is a gram-negative facultative intracellular human pathogen that can cause fatal Legionnaires' disease. Polypeptide deformylase (PDF) is a novel broad-spectrum antibacterial target, and reports of inhibitors of PDF with potent activities against L. pneumophila have been published previously. Here, we report the identification of not one but three putative pdf genes, pdfA, pdfB, and pdfC, in the complete genome sequences of three strains of L. pneumophila. Phylogenetic analysis showed that L. pneumophila PdfA is most closely related to the commonly known gamma-proteobacterial PDFs encoded by the gene def. PdfB and PdfC are more divergent and do not cluster with any specific bacterial or eukaryotic PDF. All three putative pdf genes from L. pneumophila strain Philadelphia 1 have been cloned, and their encoded products have been overexpressed in Escherichia coli and purified. Enzymatic characterization shows that the purified PDFs with Ni2+ substituted are catalytically active and able to remove the N-formyl group from several synthetic polypeptides, although they appear to have different substrate specificities. Surprisingly, while PdfA and PdfB with Zn2+ substituted are much less active than the Ni2+ forms of each enzyme, PdfC with Zn2+ substituted was as active as the Ni2+ form for the fMA substrate and exhibited substrate specificity different from that of Ni2+ PdfC. Furthermore, the catalytic activities of these enzymes are potently inhibited by a known small-molecule PDF inhibitor, BB-3497, which also inhibits the extracellular growth of L. pneumophila. These results indicate that even though L. pneumophila has three PDFs, they can be effectively inhibited by PDF inhibitors which can, therefore, have potent anti-L. pneumophila activity.

FIG. 1.

Multiple sequence alignment of Legionella pneumophila PDFs with representative bacterial class I and II PDFs. The class I PDFs are from Escherichia coli (Esco), Pseudomonas aeruginosa (Psae), and Thermotoga maritima (Thma), while the class II PDFs are from Staphylococcus aureus (Stau) and Streptococcus pneumoniae (Stpn). These sequences are aligned with those of L. pneumophila PdfA (LpA), PdfB (LpB), and PdfC (LpC) from the L. pneumophila strains Paris (Paris), Lens (Lens), and Philadelphia 1 (Phil). The amino acid sequences of PdfA (LpA_PPL) are identical across all three strains. LpB_PhGSK is the amino acid sequence based on the DNA resequencing in this study of the pdfB gene from L. pneumophila strain Philadelphia 1, which differed from that of the previously published genome sequence (7). PdfA and PdfC were identical to previously determined sequences from L. pneumophila strain Philadelphia 1. The principal catalytic motifs of PDF, boxes 1, 2, and 3, are indicated. Progressively darker shading indicates conservation of amino acid residues in 60%, 80%, and 100% of the sequences. The program CLUSTALW (34) was used to construct the initial alignment, which was subsequently refined manually.

FIG. 2.

Genomic contexts of the three polypeptide deformylase genes (pdfA, pdfB, and pdfC) in three different strains of Legionella pneumophila. The arrows indicate the directions of transcriptional polarity. pdf genes are stippled, and proposed cotranscribed ORFs are solid. Flanking genes that are unlikely to be cotranscribed are crosshatched. Under each ORF is the recorded gene name (GeneID in GenBank) as annotated for the genomes of L. pneumophila Philadelphia 1 (Lpg), L. pneumophila Paris (Lpp), and L. pneumophila Lens (Lpl). Other gene names are peptide deformylase (def), methionyl-tRNA formyltransferase (fmt), and proline/betaine transporter 6 (proP6). In L. pneumophila Lens, N- and C-terminal portions of PdfB are annotated as two separate pseudogenes (Lpl0650a and Lpl0650b), which in fact is most likely a frameshift error in the DNA sequencing of a poly(A) 9-mer.

FIG. 3.

Phylogenetic tree of PDFs from representative species. The major organism groups are Legionella pneumophila (red; bold italic), proteobacteria (red; italic), low-G+C gram-positive bacteria (dark blue; italic), high-G+C gram-positive bacteria (light blue; italic), all other bacteria (black; italic), and eukaryotic mitochondrial PDF (green; italic). The major subgroups of class I and II PDFs are indicated, as are Legionella pneumophila PdfA, PdfB, and PdfC from three strains (Lens, Paris, and Philadelphia [strain Philadelphia 1]). The phylogenetic tree was reconstructed using the neighbor-joining method and pairwise protein divergence as implemented by the programs NEIGHBOR and PROTDIST of the PHYLIP package (10) (see Materials and Methods). Those nodes which occurred in 60% or more of 1,000 bootstrap replicates are indicated (). The scale bar represents 0.1 expected amino acid residue substitution per site.