Legionella pneumophila Secretes a Mitochondrial Carrier Protein during Infection

Abstract

The Mitochondrial Carrier Family (MCF) is a signature group of integral membrane proteins that transport metabolites across the mitochondrial inner membrane in eukaryotes. MCF proteins are characterized by six transmembrane segments that assemble to form a highly-selective channel for metabolite transport. We discovered a novel MCF member, termed Legionella nucleotide carrier Protein (LncP), encoded in the genome of Legionella pneumophila, the causative agent of Legionnaire's disease. LncP was secreted via the bacterial Dot/Icm type IV secretion system into macrophages and assembled in the mitochondrial inner membrane. In a yeast cellular system, LncP induced a dominant-negative phenotype that was rescued by deleting an endogenous ATP carrier. Substrate transport studies on purified LncP reconstituted in liposomes revealed that it catalyzes unidirectional transport and exchange of ATP transport across membranes, thereby supporting a role for LncP as an ATP transporter. A hidden Markov model revealed further MCF proteins in the intracellular pathogens, Legionella longbeachae and Neorickettsia sennetsu, thereby challenging the notion that MCF proteins exist exclusively in eukaryotic organisms.

MavN is a Legionella pneumophila vacuole-associated protein required for efficient iron acquisition during intracellular growth

“…The hydrophobic nature of MavN argues that additional bacterial or host factors may chaperone its translocation and integration into the LCV membrane. A bioinformatic analysis of predicted hydrophobic regions in IDTSs identified 71 putative transmembrane proteins, including a Legionella Icm/Dot substrate shown to integrate into the inner mitochondrial membrane (71 , and His 452 to alanine abrogates the function of MavN without altering steady-state levels or localization of the protein, indicating that these residues are required for MavN activity. His-rich motifs have been identified in numerous metal ion transporters and are present in the cytoplasmic loops of the functionally characterized metal transporters Zrt1 (Saccharomyces cerevisiae) and Irt1 (Arabidopsis thaliana) (72).…”

Legionella pneumophila Pathogenesis in the Galleria mellonella Infection Model

“…A fragment of the SidC homologue from L. pneumophila 130b containing the phosphatidylinositol-4 phosphate (PI4P) binding domain (amino acids 41 to 918 [SidC ]) was cloned into the XbaI and BamHI sites of the p4HA plasmid (13) to yield the IPTG-inducible 4HA-SidC 41-918 expression plasmid pICC562 using the forward primer 5=-CG TATTCTAGATAACACCTGCCAAACAGCAGTTGAG-3= and the reverse primer 5=-GGCTAGGATCCCTATTTCTTTATAACTCCCGTGTA C-3= and standard molecular biology techniques.…”

The Dot/Icm Effector SdhA Is Necessary for Virulence of Legionella pneumophila in Galleria mellonella and A/J Mice

“…The p4HA-SdhA (pICC1340) complementation plasmid was created using standard molecular biology techniques. Briefly, sdhA was amplified by PCR from L. pneumophila 130b chromosomal DNA using the primers GNS414 5=-CAG TCC CGG GAT ATT TCA GAA AAG ATC AAG CTT TTA GAA T-3= and GNS415 5=-CTA TCC CGG GTT ATG CTG ATG GCG CTA ATT GG-3=, digested using XmaI, and ligated into the cleaved pICC562 vector (pMMB207C-HAx4 [24]). The correct orientation and sequence identity of the sdhA insert were confirmed by DNA sequencing, and the p4HA-SdhA plasmid was transformed into L. pneumophila by electroporation.…”