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Legionella pneumophila secretes a mitochondrial carrier protein during infection.
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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.
Author Summary
Mitochondrial carrier proteins evolved during endosymbiosis to transport substrates across the mitochondrial inner membrane. As such the proteins are associated exclusively with eukaryotic organisms. Despite this, we identified putative mitochondrial carrier proteins in the genomes of different intracellular bacterial pathogens, including Legionella pneumophila, the causative agent of Legionnaire's disease. We named the mitochondrial carrier protein from L. pneumophila LncP and determined that the protein is translocated into host cells during infection by the bacterial Dot/Icm type IV secretion system. From there, LncP accesses the classical mitochondrial import pathway and is incorporated into the mitochondrial inner membrane as an integral membrane protein. Remarkably, LncP crosses five biological membranes to reach its final location. Biochemically, LncP is a unidirectional nucleotide transporter similar to Aac1 in yeast. Although not essential for intracellular replication, the high carriage rate of lncP among isolates of L. pneumophila suggests that the ability of the pathogen to manipulate mitochondrial ATP transport assists survival of the bacteria in an intracellular environment.
Introduction
Legionella pneumophila is an intracellular pathogen and the major causative agent of Legionnaire's disease, an acute form of pneumonia. The ability of the bacteria to replicate in environmental protozoa such as amoebae has equipped the bacteria with the capacity to replicate in human alveolar macrophages, leading to lung inflammation and disease [1], [2]. Within macrophages and amoebae, the bacteria replicate within a membrane bound vacuole, block phagolysosome fusion and intercept vesicles trafficking in the secretory pathway [3], [4]. Mitochondria are also transiently recruited to the L. pneumophila intracellular compartment [5]. The membrane of the mature Legionella-containing vacuole (LCV) shares many characteristics with membrane of the rough endoplasmic reticulum, reviewed in [6], [7] but interactions with the endocytic pathway are also evident [8]. Therefore formation of the intracellular replicative niche of L. pneumophila results from extensive remodelling of the intracellular vacuole and multiple interactions with vesicle trafficking pathways within the host cell [8], [9].
The formation of the LCV relies on a functional bacterial Dot/Icm Type IVB secretion system, which delivers at least 275 effectors into the host cell cytosol [10]–[13]. The effectors target multiple host cell functions including GTPase activity, ubiquitination, phosphoinositide metabolism, eukaryotic protein translation, autophagy and apoptosis, reviewed in [6], [14]–[17]. Many groups of effectors have overlapping and somewhat redundant activities making the use of reverse bacterial genetics to identify gene function difficult. Instead, many investigators have applied cell biology and protein biochemistry techniques to understand the biochemical activity of Dot/Icm effectors and their possible role during LCV formation and L. pneumophila intracellular replication [18]–[21].
Genomics has revealed that a substantial number of Dot/Icm effectors share simi