Tamara J. O'Connor

The Legionella pneumophila replication vacuole: making a cosy niche inside host cells

The pathogenesis of Legionella pneumophila is derived from its growth within lung macrophages after aerosols are inhaled from contaminated water sources. Interest in this bacterium stems from its ability to manipulate host cell vesicular-trafficking pathways and establish a membrane-bound replication vacuole, making it a model for intravacuolar pathogens. Establishment of the replication compartment requires a specialized translocation system that transports a large cadre of protein substrates across the vacuolar membrane. These substrates regulate vesicle traffic and survival pathways in the host cell. This Review focuses on the strategies that L. pneumophila uses to establish intracellular growth and evaluates why this microorganism has accumulated an unprecedented number of translocated substrates that are targeted at host cells.

The Legionella Effector RavZ Inhibits Host Autophagy Through Irreversible Atg8 Deconjugation

Axing Autophagy When intracellular pathogens like Legionella pneumophila take up residence in mammalian host cells, they must combat the efforts of the host cell to attack them. Autophagy is a process by which cells digest their own constituents, often involved in response to starvation or pathogen attack. Choy et al. (p. 1072, published online 25 October) now describe how L. pneumophila can inhibit the autophagy pathway in eukaryotic cells, and provide a detailed description of the biochemical mechanism. A Legionella effector protein, RavZ, acts as a very potent enzyme that specifically deconjugates a key autophagy protein, Atg8, from autophagosomal membranes, thus blocking autophagy. An intracellular pathogen disrupts autophagy by targeting an essential host protein on the early autophagosome. Eukaryotic cells can use the autophagy pathway to defend against microbes that gain access to the cytosol or reside in pathogen-modified vacuoles. It remains unclear if pathogens have evolved specific mechanisms to manipulate autophagy. Here, we found that the intracellular pathogen Legionella pneumophila could interfere with autophagy by using the bacterial effector protein RavZ to directly uncouple Atg8 proteins attached to phosphatidylethanolamine on autophagosome membranes. RavZ hydrolyzed the amide bond between the carboxyl-terminal glycine residue and an adjacent aromatic residue in Atg8 proteins, producing an Atg8 protein that could not be reconjugated by Atg7 and Atg3. Thus, intracellular pathogens can inhibit autophagy by irreversibly inactivating Atg8 proteins during infection.

The E Block motif is associated with Legionella pneumophila translocated substrates

Legionella pneumophila promotes intracellular growth by moving bacterial proteins across membranes via the Icm/Dot system. A strategy was devised to identify large numbers of Icm/Dot translocated proteins, and the resulting pool was used to identify common motifs that operate as recognition signals. The 3′ end of the sidC gene, which encodes a known translocated substrate, was replaced with DNA encoding 200 codons from the 3′ end of 442 potential substrate‐encoding genes. The resulting hybrid proteins were then tested in a high throughput assay, in which translocated SidC antigen was detected by indirect immunofluorescence. Among translocated substrates, regions of 6–8 residues called E Blocks were identified that were rich in glutamates. Analysis of SidM/DrrA revealed that loss of three Glu residues, arrayed in a triangle on an α‐helical surface, totally eliminated translocation of a reporter protein. Based on this result, a second strategy was employed to identify Icm/Dot substrates having carboxyl terminal glutamates. From the fusion assay and the bioinformatic queries, carboxyl terminal sequences from 49 previously unidentified proteins were shown to promote translocation into target cells. These studies indicate that by analysing subsets of translocated substrates, patterns can be found that allow predictions of important motifs recognized by Icm/Dot.

Minimization of the Legionella pneumophila genome reveals chromosomal regions involved in host range expansion

Legionella pneumophila is a bacterial pathogen of amoebae and humans. Intracellular growth requires a type IVB secretion system that translocates at least 200 different proteins into host cells. To distinguish between proteins necessary for growth in culture and those specifically required for intracellular replication, a screen was performed to identify genes necessary for optimal growth in nutrient-rich medium. Mapping of these genes revealed that the L. pneumophila chromosome has a modular architecture consisting of several large genomic islands that are dispensable for growth in bacteriological culture. Strains lacking six of these regions, and thus 18.5% of the genome, were viable but required secondary point mutations for optimal growth. The simultaneous deletion of five of these genomic loci had no adverse effect on growth of the bacterium in nutrient-rich media. Remarkably, this minimal genome strain, which lacked 31% of the known substrates of the type IVB system, caused only marginal defects in intracellular growth within mouse macrophages. In contrast, deletion of single regions reduced growth within amoebae. The importance of individual islands, however, differed among amoebal species. The host-specific requirements of these genomic islands support a model in which the acquisition of foreign DNA has broadened the L. pneumophila host range.

Beyond paralogs: The multiple layers of redundancy in bacterial pathogenesis

Redundancy has been referred to as a state of no longer being needed or useful. Microbiologists often theorize that the only case of true redundancy in a haploid organism would be a recent gene duplication event, prior to divergence through selective pressure. However, a growing number of examples exist where an organism encodes two genes that appear to perform the same function. For example, many pathogens translocate multiple effector proteins into hosts. While disruption of individual effector genes does not result in a discernable phenotype, deleting genes in combination impairs pathogenesis: this has been described as redundancy. In many cases, this apparent redundancy could be due to limitations of laboratory models of pathogenesis that do not fully recapitulate the disease process. Alternatively, it is possible that the selective advantage achieved by this perceived redundancy is too subtle to be measured in the laboratory. Moreover, there are numerous possibilities for different types of redundancy. The most common and recognized form of redundancy is functional redundancy whereby two proteins have similar biochemical activities and substrate specificities allowing each one to compensate in the absence of the other. However, redundancy can also exist between seemingly unrelated proteins that manipulate the same or complementary host cell pathways. In this article, we outline 5 types of redundancy in pathogenesis: molecular, target, pathway, cellular process, and system redundancy that incorporate the biochemical activities, the host target specificities and the impact of effector function on the pathways and cellular process they modulate. For each type of redundancy, we provide examples from Legionella pathogenesis as this organism employs over 300 secreted virulence proteins and loss of individual proteins rarely impacts intracellular growth. We also discuss selective pressures that drive the maintenance of redundant mechanisms, the current methods used to resolve redundancy and features that distinguish between redundant and non-redundant virulence mechanisms.

Iron Limitation Triggers Early Egress by the Intracellular Bacterial Pathogen Legionella pneumophila

ABSTRACT Legionella pneumophila is an intracellular bacterial pathogen that replicates in alveolar macrophages, causing a severe form of pneumonia. Intracellular growth of the bacterium depends on its ability to sequester iron from the host cell. In the L. pneumophila strain 130b, one mechanism used to acquire this essential nutrient is the siderophore legiobactin. Iron-bound legiobactin is imported by the transport protein LbtU. Here, we describe the role of LbtP, a paralog of LbtU, in iron acquisition in the L. pneumophila strain Philadelphia-1. Similar to LbtU, LbtP is a siderophore transport protein and is required for robust growth under iron-limiting conditions. Despite their similar functions, however, LbtU and LbtP do not contribute equally to iron acquisition. The Philadelphia-1 strain lacking LbtP is more sensitive to iron deprivation in vitro. Moreover, LbtP is important for L. pneumophila growth within macrophages while LbtU is dispensable. These results demonstrate that LbtP plays a dominant role over LbtU in iron acquisition. In contrast, loss of both LbtP and LbtU does not impair L. pneumophila growth in the amoebal host Acanthamoeba castellanii, demonstrating a host-specific requirement for the activities of these two transporters in iron acquisition. The growth defect of the ΔlbtP mutant in macrophages is not due to alterations in growth kinetics. Instead, the absence of LbtP limits L. pneumophila replication and causes bacteria to prematurely exit the host cell. These results demonstrate the existence of a preprogrammed exit strategy in response to iron limitation that allows L. pneumophila to abandon the host cell when nutrients are exhausted.