scholarly journals The iron-regulated vacuolar Legionella pneumophila MavN protein is a transition-metal transporter

2019 ◽  
Vol 116 (36) ◽  
pp. 17775-17785 ◽  
Author(s):  
Eric T. Christenson ◽  
Dervla T. Isaac ◽  
Karin Yoshida ◽  
Erion Lipo ◽  
Jin-Sik Kim ◽  
...  
Keyword(s):  
Transition Metal ◽  
Legionella Pneumophila ◽  
Metal Ion ◽  
Critical Role ◽  
Effector Proteins ◽  
Bacterial Survival ◽  
Loss Of Function ◽  
Intracellular Growth ◽  
Iron Starvation ◽  
Metal Transporter

Legionella pneumophila causes a potentially fatal form of pneumonia by replicating within macrophages in the Legionella-containing vacuole (LCV). Bacterial survival and proliferation within the LCV rely on hundreds of secreted effector proteins comprising high functional redundancy. The vacuolar membrane-localized MavN, hypothesized to support iron transport, is unique among effectors because loss-of-function mutations result in severe intracellular growth defects. We show here an iron starvation response by L. pneumophila after infection of macrophages that was prematurely induced in the absence of MavN, consistent with MavN granting access to limiting cellular iron stores. MavN cysteine accessibilities to a membrane-impermeant label were determined during macrophage infections, revealing a topological pattern supporting multipass membrane transporter models. Mutations to several highly conserved residues that can take part in metal recognition and transport resulted in defective intracellular growth. Purified MavN and mutant derivatives were directly tested for transporter activity after heterologous purification and liposome reconstitution. Proteoliposomes harboring MavN exhibited robust transport of Fe2+, with the severity of defect of most mutants closely mimicking the magnitude of defects during intracellular growth. Surprisingly, MavN was equivalently proficient at transporting Fe2+, Mn2+, Co2+, or Zn2+. Consequently, flooding infected cells with either Mn2+ or Zn2+ allowed collaboration with iron to enhance intracellular growth of L. pneumophila ΔmavN strains, indicating a clear role for MavN in transporting each of these ions. These findings reveal that MavN is a transition-metal-ion transporter that plays a critical role in response to iron limitation during Legionella infection.

scholarly journals MavN is aLegionella pneumophilavacuole-associated protein required for efficient iron acquisition during intracellular growth

2015 ◽  
Vol 112 (37) ◽  
pp. E5208-E5217 ◽  
Author(s):  
Dervla T. Isaac ◽  
Rita K. Laguna ◽  
Nicole Valtz ◽  
Ralph R. Isberg
Keyword(s):  
Legionella Pneumophila ◽  
Transmembrane Protein ◽  
Iron Acquisition ◽  
Broth Culture ◽  
Growth Defect ◽  
Secretion Systems ◽  
Intracellular Growth ◽  
Iron Starvation ◽  
Macrophage Infection ◽  
Intracellular Iron

Iron is essential for the growth and virulence of most intravacuolar pathogens. The mechanisms by which microbes bypass host iron restriction to gain access to this metal across the host vacuolar membrane are poorly characterized. In this work, we identify a unique intracellular iron acquisition strategy used byLegionella pneumophila.The bacterial Icm/Dot (intracellular multiplication/defect in organelle trafficking) type IV secretion system targets the bacterial-derived MavN (more regions allowing vacuolar colocalization N) protein to the surface of theLegionella-containing vacuole where this putative transmembrane protein facilitates intravacuolar iron acquisition. TheΔmavNmutant exhibits a transcriptional iron-starvation signature before its growth is arrested during the very early stages of macrophage infection. This intracellular growth defect is rescued only by the addition of excess exogenous iron to the culture medium and not a variety of other metals. Consistent with MavN being a translocated substrate that plays an exclusive role during intracellular growth, the mutant shows no defect for growth in broth culture, even under severe iron-limiting conditions. Putative iron-binding residues within the MavN protein were identified, and point mutations in these residues resulted in defects specific for intracellular growth that are indistinguishable from the ΔmavNmutant. This model of a bacterial protein inserting into host membranes to mediate iron transport provides a paradigm for how intravacuolar pathogens can use virulence-associated secretion systems to manipulate and acquire host iron.

scholarly journals MultipleLegionella pneumophilaeffector virulence phenotypes revealed through high-throughput analysis of targeted mutant libraries

2017 ◽  
Vol 114 (48) ◽  
pp. E10446-E10454 ◽  
Author(s):  
Stephanie R. Shames ◽  
Luying Liu ◽  
James C. Havey ◽  
Whitman B. Schofield ◽  
Andrew L. Goodman ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Severe Pneumonia ◽  
Effector Proteins ◽  
Host Cells ◽  
Limiting Factor ◽  
Effector Protein ◽  
Host Immune System ◽  
Loss Of Function ◽  
High Throughput Analysis ◽  
Single Strain

Legionella pneumophilais the causative agent of a severe pneumonia called Legionnaires’ disease. A single strain ofL. pneumophilaencodes a repertoire of over 300 different effector proteins that are delivered into host cells by the Dot/Icm type IV secretion system during infection. The large number ofL. pneumophilaeffectors has been a limiting factor in assessing the importance of individual effectors for virulence. Here, a transposon insertion sequencing technology called INSeq was used to analyze replication of a pool of effector mutants in parallel both in a mouse model of infection and in cultured host cells. Loss-of-function mutations in genes encoding effector proteins resulted in host-specific or broad virulence phenotypes. Screen results were validated for several effector mutants displaying different virulence phenotypes using genetic complementation studies and infection assays. Specifically, loss-of-function mutations in the gene encoding LegC4 resulted in enhancedL. pneumophilain the lungs of infected mice but not within cultured host cells, which indicates LegC4 augments bacterial clearance by the host immune system. The effector proteins RavY and Lpg2505 were important for efficient replication within both mammalian and protozoan hosts. Further analysis of Lpg2505 revealed that this protein functions as a metaeffector that counteracts host cytotoxicity displayed by the effector protein SidI. Thus, this study identified a large cohort of effectors that contribute toL. pneumophilavirulence positively or negatively and has demonstrated regulation of effector protein activities by cognate metaeffectors as being critical for host pathogenesis.

scholarly journals Restriction of Legionella pneumophila Replication in Macrophages Requires Concerted Action of the Transcriptional Regulators Irf1 and Irf8 and Nod-Like Receptors Naip5 and Nlrc4

2009 ◽  
Vol 77 (11) ◽  
pp. 4794-4805 ◽  
Author(s):  
Anne Fortier ◽  
Karine Doiron ◽  
Maya Saleh ◽  
Sergio Grinstein ◽  
Philippe Gros
Keyword(s):  
Legionella Pneumophila ◽  
Transcriptional Activation ◽  
Genetic Interaction ◽  
Critical Role ◽  
Early Response ◽  
Loss Of Function ◽  
Macrophage Response ◽  
Effector Genes ◽  
Mouse Macrophages ◽  
Partner Gene

ABSTRACT The unique permissiveness of A/J mouse macrophages for replication of Legionella pneumophila is caused by a deficiency in the Nod-like receptor (NLR) protein and intracellular sensor for L. pneumophila flagellin (Naip5). The signaling pathways and proteins activated by Naip5 sensing in macrophages were investigated. Transcript profiling of macrophages from susceptible A/J mice and from resistant A/J mice harboring a transgenic wild-type copy of Naip5 at 4 h following L. pneumophila infection suggested that two members of the Irf transcriptional regulator family, Irf1 and Irf8, are regulated in response to Naip5 sensing of L. pneumophila. We show that macrophages having defective alleles of either Irf1 (Irf1 − / −) or its heterodimerization partner gene Irf8 (Irf8R294C ) become permissive for L. pneumophila replication, indicating that both the Irf1 and Irf8 proteins are essential for macrophage defense against L. pneumophila. Moreover, macrophages doubly heterozygous (Naip5AJ/WT Irf8R294C/WT or Nlrc4 −/+ Irf8R294C/WT ) for combined loss-of-function mutations in Irf8 and in either Naip5 or Nlrc4 are highly susceptible to L. pneumophila, indicating that there is a strong genetic interaction between Irf8 and the NLR protein family in the macrophage response to L. pneumophila. Legionella-containing phagosomes (LCPs) formed in permissive Irf1 − / − or Irf8R294C macrophages behave like LCPs formed in Naip5-insufficient and Nlrc4-deficient macrophages which fail to acidify. These results suggest that, in addition to Naip5 and Nlrc4, Irf1 and Irf8 play a critical role in the early response of macrophages to infection with L. pneumophila, including antagonizing the ability of L. pneumophila to block phagosome acidification. They also suggest that flagellin sensing by the NLR proteins Naip5 and Nlrc4 may be coupled to Irf1-Irf8-mediated transcriptional activation of key effector genes essential for macrophage resistance to L. pneumophila infection.

scholarly journals Evolution of the Arsenal ofLegionella pneumophilaEffectors To Modulate Protist Hosts

mBio ◽  
2018 ◽  
Vol 9 (5) ◽  
Author(s):  
Ashley Best ◽  
Yousef Abu Kwaik
Keyword(s):  
Legionella Pneumophila ◽  
Effector Proteins ◽  
Host Cells ◽  
Human Pathogen ◽  
Intracellular Growth ◽  
Content Type ◽  
Human Macrophages ◽  
Cellular Processes ◽  
Host Interaction

ABSTRACTWithin the human host,Legionella pneumophilareplicates within alveolar macrophages, leading to pneumonia. However,L. pneumophilais an aquatic generalist pathogen that replicates within a wide variety of protist hosts, including amoebozoa, percolozoa, and ciliophora. The intracellular lifestyles ofL. pneumophilawithin the two evolutionarily distant hosts macrophages and protists are remarkably similar. Coevolution with numerous protist hosts has shaped plasticity of the genome ofL. pneumophila, which harbors numerous proteins encoded by genes acquired from primitive eukaryotic hosts through interkingdom horizontal gene transfer. The Dot/Icm type IVb translocation system translocates ∼6,000 effectors amongLegionellaspecies and >320 effector proteins inL. pneumophilainto host cells to modulate a plethora of cellular processes to create proliferative niches. Since many of the effectors have likely evolved to modulate cellular processes of primitive eukaryotic hosts, it is not surprising that most of the effectors do not contribute to intracellular growth within human macrophages. Some of the effectors may modulate highly conserved eukaryotic processes, while others may target protist-specific processes that are absent in mammals. The lack of studies to determine the role of the effectors in adaptation ofL. pneumophilato various protists has hampered the progress to determine the function of most of these effectors, which are routinely studied in mouse or human macrophages. Since many protists restrictL. pneumophila, utilization of such hosts can also be instrumental in deciphering the mechanisms of failure ofL. pneumophilato overcome restriction of certain protist hosts. Here, we review the interaction ofL. pneumophilawith its permissive and restrictive protist environmental hosts and outline the accomplishments as well as gaps in our knowledge ofL. pneumophila-protist host interaction andL. pneumophila’s evolution to become a human pathogen.

scholarly journals Interaction of the Ankyrin H Core Effector of Legionella with the Host LARP7 Component of the 7SK snRNP Complex

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Juanita Von Dwingelo ◽  
Ivy Yeuk Wah Chung ◽  
Christopher T. Price ◽  
Lei Li ◽  
Snake Jones ◽  
...  
Keyword(s):  
Host Cell ◽  
Legionella Pneumophila ◽  
Effector Proteins ◽  
Host Protein ◽  
Host Cells ◽  
Transcriptional Elongation ◽  
Intracellular Growth ◽  
Pol Ii ◽  
Content Type ◽  
Small Nuclear Ribonucleoprotein

ABSTRACT Species of the Legionella genus encode at least 18,000 effector proteins that are translocated through the Dot/Icm type IVB translocation system into macrophages and protist hosts to enable intracellular growth. Eight effectors, including ankyrin H (AnkH), are common to all Legionella species. The AnkH effector is also present in Coxiella and Rickettsiella. To date, no pathogenic effectors have ever been described that directly interfere with host cell transcription. We determined that the host nuclear protein La-related protein 7 (LARP7), which is a component of the 7SK small nuclear ribonucleoprotein (snRNP) complex, interacts with AnkH in the host cell nucleus. The AnkH-LARP7 interaction partially impedes interactions of the 7SK snRNP components with LARP7, interfering with transcriptional elongation by polymerase (Pol) II. Consistent with that, our data show AnkH-dependent global reprogramming of transcription of macrophages infected by Legionella pneumophila. The crystal structure of AnkH shows that it contains four N-terminal ankyrin repeats, followed by a cysteine protease-like domain and an α-helical C-terminal domain. A substitution within the β-hairpin loop of the third ankyrin repeat results in diminishment of LARP7-AnkH interactions and phenocopies the ankH null mutant defect in intracellular growth. LARP7 knockdown partially suppresses intracellular proliferation of wild-type (WT) bacteria and increases the severity of the defect of the ΔankH mutant, indicating a role for LARP7 in permissiveness of host cells to intracellular bacterial infection. We conclude that the AnkH-LARP7 interaction impedes interaction of LARP7 with 7SK snRNP, which would block transcriptional elongation by Pol II, leading to host global transcriptional reprogramming and permissiveness to L. pneumophila. IMPORTANCE For intracellular pathogens to thrive in host cells, an environment that supports survival and replication needs to be established. L. pneumophila accomplishes this through the activity of the ∼330 effector proteins that are injected into host cells during infection. Effector functions range from hijacking host trafficking pathways to altering host cell machinery, resulting in altered cell biology and innate immunity. One such pathway is the host protein synthesis pathway. Five L. pneumophila effectors have been identified that alter host cell translation, and 2 effectors have been identified that indirectly affect host cell transcription. No pathogenic effectors have been described that directly interfere with host cell transcription. Here we show a direct interaction of the AnkH effector with a host cell transcription complex involved in transcriptional elongation. We identify a novel process by which AnkH interferes with host transcriptional elongation through interference with formation of a functional complex and show that this interference is required for pathogen proliferation.

scholarly journals Iron depletion limits intracellular bacterial growth in macrophages

Blood ◽  
2008 ◽  
Vol 112 (3) ◽  
pp. 866-874 ◽  
Author(s):  
Prasad N. Paradkar ◽  
Ivana De Domenico ◽  
Nina Durchfort ◽  
Irene Zohn ◽  
Jerry Kaplan ◽  
...  
Keyword(s):  
Bacterial Growth ◽  
Legionella Pneumophila ◽  
Bacterial Infections ◽  
Iron Chelator ◽  
Chlamydia Psittaci ◽  
Loss Of Function ◽  
Intracellular Growth ◽  
Intracellular Iron ◽  
Cellular Iron

AbstractMany intracellular pathogens infect macrophages and these pathogens require iron for growth. Here we demonstrate in vitro that the intracellular growth of Chlamydia psittaci, trachomatis, and Legionella pneumophila is regulated by the levels of intracellular iron. Macrophages that express cell surface ferroportin, the only known cellular iron exporter, limit the intracellular growth of these bacteria. Hepcidin is an antimicrobial peptide secreted by the liver in response to inflammation. Hepcidin binds to ferroportin mediating its internalization and degradation. Addition of hepcidin to infected macrophages enhanced the intracellular growth of these pathogens. Macrophages from flatiron mice, a strain heterozygous for a loss-of-function ferroportin mutation, showed enhanced intracellular bacterial growth independent of the presence of exogenous hepcidin. Macrophages, from wild-type or flatiron mice, incubated with the oral iron chelator deferriprone or desferasirox showed reduced intracellular bacterial growth suggesting that these chelators might be therapeutic in chronic intracellular bacterial infections.

scholarly journals Legionella pneumophila DotU and IcmF Are Required for Stability of the Dot/Icm Complex

2004 ◽  
Vol 72 (10) ◽  
pp. 5983-5992 ◽  
Author(s):  
Jessica A. Sexton ◽  
Jennifer L. Miller ◽  
Aki Yoneda ◽  
Thomas E. Kehl-Fie ◽  
Joseph P. Vogel
Keyword(s):  
Cell Surface ◽  
Legionella Pneumophila ◽  
Bacterial Species ◽  
Wide Distribution ◽  
Host Cells ◽  
Growth Defect ◽  
Intracellular Growth ◽  
Homologous Proteins ◽  
Type Iv ◽  
Cell Surface Structure

ABSTRACT Legionella pneumophila utilizes a type IV secretion system (T4SS) encoded by 26 dot/icm genes to replicate inside host cells and cause disease. In contrast to all other L. pneumophila dot/icm genes, dotU and icmF have homologs in a wide variety of gram-negative bacteria, none of which possess a T4SS. Instead, dotU and icmF orthologs are linked to a locus encoding a conserved cluster of proteins designated IcmF-associated homologous proteins, which has been proposed to constitute a novel cell surface structure. We show here that dotU is partially required for L. pneumophila intracellular growth, similar to the known requirement for icmF. In addition, we show that dotU and icmF are necessary for optimal plasmid transfer and sodium sensitivity, two additional phenotypes associated with a functional Dot/Icm complex. We found that these effects are due to the destabilization of the T4SS at the transition into the stationary phase, the point at which L. pneumophila becomes virulent. Specifically, three Dot proteins (DotH, DotG, and DotF) exhibit decreased stability in a ΔdotU ΔicmF strain. Furthermore, overexpression of just one of these proteins, DotH, is sufficient to suppress the intracellular growth defect of the ΔdotU ΔicmF mutant. This suggests a model where the DotU and IcmF proteins serve to prevent DotH degradation and therefore function to stabilize the L. pneumophila T4SS. Due to their wide distribution among bacterial species and their genetic linkage to known or predicted cell surface structures, we propose that this function in complex stabilization may be broadly conserved.

Transition-metal ion-mediated morphological transformation of pyridine-based peptide nanostructures

2021 ◽  
Vol 45 (1) ◽  
pp. 153-161
Author(s):  
Narendra Singh ◽  
Ramesh Singh ◽  
Swati Sharma ◽  
Khushboo Kesharwani ◽  
Khashti Ballabh Joshi ◽  
...  
Keyword(s):  
Transition Metal ◽  
Metal Ion ◽  
Chemical Structure ◽  
Morphological Transformation ◽  
Transition Metal Ion

Pyridine-mediated constitutionally isomeric artificial metallopeptides possess remarkable advantages over the natural counterparts mainly due to their tailor-made chemical structure.

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