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

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.

Lessons Learned from the Development and Roll-Out of the rVSVΔG-ZEBOV-GP Zaire ebolavirus Vaccine to Inform Marburg Virus and Sudan ebolavirus Vaccines.

This review describes key aspects of the development of the rVSVΔG-ZEBOV-GP Ebola vaccine and key activities which are continuing to further expand our knowledge of the product. Extensive partnerships and innovative approaches were used to address the various challenges encountered during this process. The rVSVΔG-ZEBOV-GP Ebola vaccine was initially approved by the European Medicines Agency and prequalified by the World Health Organization in November 2019. It was approved by the United States Food and Drug Administration in December 2019 and approved in five African countries within 90 days of prequalification. The development resulted in the first stockpile of a registered Ebola vaccine that is available to support outbreak response. This also provides insights into how the example of rVSVΔG-ZEBOV-GP can inform the development of vaccines for Sudan ebolavirus, Marburg virus, and other emerging epidemic diseases in terms of the types of approaches and data needed to support product registration, availability, and the use of a filovirus vaccine.

Members of a Legionella pneumophila family of proteins with ExoU (phospholipase A) active sites are translocated to target cells.

Legionella pneumophila replicates within alveolar macrophages, causing a severe pneumonia termed Legionnaires' disease. The bacterium resides within a vacuole that escapes immediate transport to the host lysosome. Instead, the vacuole interacts with the early secretory pathway to establish an environment suitable for rapid multiplication. A type IV secretion system is central to the pathogenicity of the bacterium, and many protein substrates that are translocated by this system to the host cell have been identified. One of these, VipD, was found to interrupt the late secretory pathway when overproduced in Saccharomyces cerevisiae. We independently identified VipD in a previous study and have further characterized this protein as well as its three paralogs. The vipD gene belongs to a family of L. pneumophila open reading frames that are predicted to contain a phospholipase A domain with sequence similarity to the type III-secreted toxin ExoU from Pseudomonas aeruginosa. Similarly to other known translocated proteins of L. pneumophila, VipD is strongly induced in early stationary phase, a time when the bacterium is most virulent. Detergent extraction studies of infected macrophages confirm that VipD is translocated into host cells via the type IV secretion system. A second assay for translocation revealed that two paralogs of VipD, VpdA and VpdB, also have translocation signals recognized by the type IV system. A strain lacking VipD and its three paralogs grew at wild-type rates in murine macrophages, although secondary mutations that cause growth defects in strains lacking VipD accumulate. The quadruple mutant displayed a growth advantage in the amoebal host Dictyostelium discoideum, indicating that the protein family may modulate intracellular growth in a complex fashion. VipD is mildly toxic when overproduced in eukaryotic cells, and the toxicity is partially dependent on the putative phospholipase active site. VipD and its paralogs therefore define a family of translocated proteins that may assist in the establishment of a vacuole suitable for bacterial replication through functioning as a phospholipase.

IcmF and DotU are required for optimal effector translocation and trafficking of the Legionella pneumophila vacuole.

The gram-negative bacterium Legionella pneumophila causes a severe form of pneumonia called Legionnaires' disease, characterized by bacterial replication within alveolar macrophages. Prior to intracellular replication, the vacuole harboring the bacterium must first escape trafficking to the host lysosome, a process that is dependent on the Dot/Icm type IV secretion system. To identify genes required for intracellular growth, bacterial mutants were isolated that were delayed in escape from the macrophage but which retain a minimally functional Dot/Icm machinery. The mutations were found in eight distinct genes, including three genes known to be required for optimal intracellular growth. Two of these genes, icmF and dotU, are located at one end of a cluster of genes that encode the type IV secretion system, yet both icmF and dotU lack orthologs in other type IV translocons. DotU protein is degraded in the early postexponential phase in wild-type L. pneumophila and at all growth phases in an icmF mutant. IcmF contains an extracytoplasmic domain(s) based on accessibility to a membrane-impermeant amine-reactive reagent. In the absence of either gene, L. pneumophila targets inappropriately to LAMP-1-positive compartments during macrophage infection, is defective in the formation of replicative vacuoles, and is impaired in the translocation of the effector protein SidC. Therefore, although IcmF and DotU do not appear to be part of the core type IV secretion system, these proteins are necessary for an efficiently functioning secretion apparatus.