Phylogenomic and Biochemical Characterization of Three Legionella pneumophila Polypeptide Deformylases

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 γ-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 Ni(2+) 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 Zn(2+) substituted are much less active than the Ni(2+) forms of each enzyme, PdfC with Zn(2+) substituted was as active as the Ni(2+) form for the fMA substrate and exhibited substrate specificity different from that of Ni(2+) 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

Rare predicted loss-of-function variants of type I IFN immunity genes are associated with life-threatening COVID-19

BackgroundWe previously reported that impaired type I IFN activity, due to inborn errors of TLR3- and TLR7-dependent type I interferon (IFN) immunity or to autoantibodies against type I IFN, account for 15-20% of cases of life-threatening COVID-19 in unvaccinated patients. Therefore, the determinants of life-threatening COVID-19 remain to be identified in similar to 80% of cases.MethodsWe report here a genome-wide rare variant burden association analysis in 3269 unvaccinated patients with life-threatening COVID-19, and 1373 unvaccinated SARS-CoV-2-infected individuals without pneumonia. Among the 928 patients tested for autoantibodies against type I IFN, a quarter (234) were positive and were excluded.ResultsNo gene reached genome-wide significance. Under a recessive model, the most significant gene with at-risk variants was TLR7, with an OR of 27.68 (95%CI 1.5-528.7, P=1.1x10(-4)) for biochemically loss-of-function (bLOF) variants. We replicated the enrichment in rare predicted LOF (pLOF) variants at 13 influenza susceptibility loci involved in TLR3-dependent type I IFN immunity (OR=3.70[95%CI 1.3-8.2], P=2.1x10(-4)). This enrichment was further strengthened by (1) adding the recently reported TYK2 and TLR7 COVID-19 loci, particularly under a recessive model (OR=19.65[95%CI 2.1-2635.4], P=3.4x10(-3)), and (2) considering as pLOF branchpoint variants with potentially strong impacts on splicing among the 15 loci (OR=4.40[9%CI 2.3-8.4], P=7.7x10(-8)). Finally, the patients with pLOF/bLOF variants at these 15 loci were significantly younger (mean age [SD]=43.3 [20.3] years) than the other patients (56.0 [17.3] years; P=1.68x10(-5)).ConclusionsRare variants of TLR3- and TLR7-dependent type I IFN immunity genes can underlie life-threatening COVID-19, particularly with recessive inheritance, in patients under 60 years old