Legionella pneumophila strain 130b evades macrophage cell death independent of the effector SidF in the absence of flagellin

International audienceThe human pathogen Legionella pneumophila must evade host cell death signaling to enable replication in lung macrophages and to cause disease. After bacterial growth, however, L. pneumophila is thought to induce apoptosis during egress from macrophages. The bacterial effector protein, SidF, has been shown to control host cell survival and death by inhibiting pro-apoptotic BNIP3 and BCL-RAMBO signaling. Using live-cell imaging to follow the L. pneumophila-macrophage interaction, we now demonstrate that L. pneumophila evades host cell apoptosis independent of SidF. In the absence of SidF, L. pneumophila was able to replicate, cause loss of mitochondria membrane potential, kill macrophages, and establish infections in lungs of mice. Consistent with this, deletion of BNIP3 and BCL-RAMBO did not affect intracellular L. pneumophila replication, macrophage death rates, and in vivo bacterial virulence. Abrogating mitochondrial cell death by genetic deletion of the effectors of intrinsic apoptosis, BAX, and BAK, or the regulator of mitochondrial permeability transition pore formation, cyclophilin-D, did not affect bacterial growth or the initial killing of macrophages. Loss of BAX and BAK only marginally limited the ability of L. pneumophila to efficiently kill all macrophages over extended periods. L. pneumophila induced killing of macrophages was delayed in the absence of capsase-11 mediated pyroptosis. Together, our data demonstrate that L. pneumophila evades host cell death responses independently of SidF during replication and can induce pyroptosis to kill macrophages in a timely manner

Legionella pneumophila strain 130b possesses a unique combination of type IV secretion systems and novel Dot/Icm secretion system effector proteins

Legionella pneumophila is a ubiquitous inhabitant of environmental water reservoirs. The bacteria infect a wide variety of protozoa and, after accidental inhalation, human alveolar macrophages, which can lead to severe pneumonia. The capability to thrive in phagocytic hosts is dependent on the Dot/Icm type IV secretion system (T4SS), which translocates multiple effector proteins into the host cell. In this study, we determined the draft genome sequence of L. pneumophila strain 130b (Wadsworth). We found that the 130b genome encodes a unique set of T4SSs, namely, the Dot/Icm T4SS, a Trb-1-like T4SS, and two Lvh T4SS gene clusters. Sequence analysis substantiated that a core set of 107 Dot/Icm T4SS effectors was conserved among the sequenced L. pneumophila strains Philadelphia-1, Lens, Paris, Corby, Alcoy, and 130b. We also identified new effector candidates and validated the translocation of 10 novel Dot/Icm T4SS effectors that are not present in L. pneumophila strain Philadelphia-1. We examined the prevalence of the new effector genes among 87 environmental and clinical L. pneumophila isolates. Five of the new effectors were identified in 34 to 62% of the isolates, while less than 15% of the strains tested positive for the other five genes. Collectively, our data show that the core set of conserved Dot/Icm T4SS effector proteins is supplemented by a variable repertoire of accessory effectors that may partly account for differences in the virulences and prevalences of particular L. pneumophila strains. Copyright © 2010, American Society for Microbiology. All Rights Reserved

Identification and characterisation of novel Legionella pneumophila virulence genes

© 2015 Dr. Adam John VogrinLegionella pneumophila, the major causative agent of the severely pneumonic Legionnaires’ disease, is an intracellular bacterial pathogen that is able to exploit aquatic protozoa as well as human alveolar macrophages as hosts for replication. After internalisation, the Legionella containing vacuole (LCV) avoids fusion with the endosomal pathway and intercepts endoplasmic reticulum (ER)-derived early secretory vesicles prior to their transport to the Golgi. This aids in the formation of a vacuole that supports bacterial replication and is driven by effector proteins translocated by the Dot/Icm type IV secretion system. Apart from the Dot/Icm system, few genes have been identified that play a major role in intracellular replication. In this study, we constructed and screened a library of 10,006 L. pneumophila transposon mutants for attenuated intracellular replication within the amoebae Acanthamoeba castellanii. 34 mutants were identified, 21 of which carried insertions into the Dot/Icm Type IV secretion system. In addition to these, insertions into seven novel genes were discovered, including lpw27511 and sdeC. lpw27511 encodes a transcriptional regulator of the LuxR family. Its regulatory targets were identified in a DNA microarray, however, the majority of identified targets were weakly affected and there was no obvious explanation for the replication defect observed for the transposon mutant. Further analysis of an in-frame lpw27511 deletion mutant showed that the defined mutant did not show the intracellular replication defect seen for the transposon mutant. Unfortunately subsequent investigations could not find an explanation for the replication defect originally observed for the transposon mutant. sdeC encodes a Dot/Icm translocated effector and we investigated the contribution of this protein to L. pneumophila pathogenesis further. sdeC was required for full replication of L. pneumophila in amoebae, macrophages and in the lungs of A strain mice. HEK293T cells transfected with GFP-tagged SdeC showed that SdeC co-localised with the ER. 4HA-tagged SdeC translocated by L. pneumophila during infection localised with the Legionella containing vacuole and the ER in macrophages and amoebae. Using multiple approaches no binding partners of SdeC were identified. Several imaging techniques were used to observe LCV biogenesis of the sdeC deletion mutant and wild type L. pneumophila, and, apart from a minor replication defect no differences between the two strains were observed. However given the localisation of SdeC, we suggest that SdeC promotes the interactions between the LCV and ER, thereby aiding intracellular replication of L. pneumophila. During live cell imaging experiments multiple LCVs present in the same host cell were observed to fuse together. This was a novel observation of LCV development not previously reported in Legionella literature. Overall these studies have once again highlighted the importance of the Dot/Icm system for L. pneumophila intracellular replication and provided further insight into the molecular basis of this replication

Vegfd modulates both angiogenesis and lymphangiogenesis during zebrafish embryonic development

ABSTRACT Vascular endothelial growth factors (VEGFs) control angiogenesis and lymphangiogenesis during development and in pathological conditions. In the zebrafish trunk, Vegfa controls the formation of intersegmental arteries by primary angiogenesis and Vegfc is essential for secondary angiogenesis, giving rise to veins and lymphatics. Vegfd has been largely thought of as dispensable for vascular development in vertebrates. Here, we generated a zebrafish vegfd mutant by genome editing. vegfd mutants display significant defects in facial lymphangiogenesis independent of vegfc function. Strikingly, we find that vegfc and vegfd cooperatively control lymphangiogenesis throughout the embryo, including during the formation of the trunk lymphatic vasculature. Interestingly, we find that vegfd and vegfc also redundantly drive artery hyperbranching phenotypes observed upon depletion of Flt1 or Dll4. Epistasis and biochemical binding assays suggest that, during primary angiogenesis, Vegfd influences these phenotypes through Kdr (Vegfr2) rather than Flt4 (Vegfr3). These data demonstrate that, rather than being dispensable during development, Vegfd plays context-specific indispensable and also compensatory roles during both blood vessel angiogenesis and lymphangiogenesis

Evolutionary Differences in the Vegf/Vegfr Code Reveal Organotypic Roles for the Endothelial Cell Receptor Kdr in Developmental Lymphangiogenesis

Lymphatic vascular development establishes embryonic and adult tissue fluid balance and is integral in disease. In diverse vertebrate organs, lymphatic vessels display organotypic function and develop in an organ-specific manner. In all settings, developmental lymphangiogenesis is considered driven by vascular endothelial growth factor (VEGF) receptor-3 (VEGFR3), whereas a role for VEGFR2 remains to be fully explored. Here, we define the zebrafish Vegf/Vegfr code in receptor binding studies. We find that while Vegfd directs craniofacial lymphangiogenesis, it binds Kdr (a VEGFR2 homolog) but surprisingly, unlike in mammals, does not bind Flt4 (VEGFR3). Epistatic analyses and characterization of a kdr mutant confirm receptor-binding analyses, demonstrating that Kdr is indispensible for rostral craniofacial lymphangiogenesis, but not caudal trunk lymphangiogenesis, in which Flt4 is central. We further demonstrate an unexpected yet essential role for Kdr in inducing lymphatic endothelial cell fate. This work reveals evolutionary divergence in the Vegf/Vegfr code that uncovers spatially restricted mechanisms of developmental lymphangiogenesis. Lymphatic vessels display organotypic function and develop in an organ-specific manner. Vogrin et al. find that the zebrafish Kdr receptor is indispensible for craniofacial, but not trunk, lymphangiogenesis whereas Flt4 is essential for the latter. Thus, vascular endothelial growth factor (VEGF) receptor signaling pathways are differentially employed in different tissues to drive developmental lymphangiogenesis