scholarly journals The Legionella pneumophila metaeffector Lpg2505 (SusF) regulates SidI-mediated translation inhibition and GDP-dependent glycosyltransferase activity

2019 ◽  
Author(s):  
Ashley M. Joseph ◽  
Adrienne E. Pohl ◽  
Theodore J. Ball ◽  
Troy G. Abram ◽  
David K. Johnson ◽  
...  

AbstractLegionella pneumophila, the etiological agent of Legionnaires Disease, employs an arsenal of hundreds of Dot/Icm-translocated effector proteins to facilitate replication within eukaryotic phagocytes. Several effectors, called metaeffectors, function regulate the activity of other Dot/Icm-translocated effectors during infection. The metaeffector Lpg2505 is essential for L. pneumophila intracellular replication only when its cognate effector, SidI, is present. SidI is a cytotoxic effector that interacts with the host translation factor eEF1A and potently inhibits eukaryotic protein translation by an unknown mechanism. Here, we evaluated the impact of Lpg2505 on SidI-mediated phenotypes and investigated the mechanism of SidI function. We determined that Lpg2505 binds with nanomolar affinity to SidI and suppresses SidI-mediated inhibition of protein translation. SidI binding to eEF1A and SusF is not mutually exclusive and these proteins bind distinct regions of SidI. We also discovered that SidI possesses GDP-dependent glycosyltransferase activity and that this activity is regulated by Lpg2505. We have therefore renamed Lpg2505, SusF (suppressor of SidI function). This work reveals novel enzymatic activity for SidI and provides insight into how intracellular replication of L. pneumophila is regulated by a metaeffector.

2020 ◽  
Vol 88 (5) ◽  
Author(s):  
Ashley M. Joseph ◽  
Adrienne E. Pohl ◽  
Theodore J. Ball ◽  
Troy G. Abram ◽  
David K. Johnson ◽  
...  

ABSTRACT Legionella pneumophila, the etiological agent of Legionnaires’ disease, employs an arsenal of hundreds of Dot/Icm-translocated effector proteins to facilitate replication within eukaryotic phagocytes. Several effectors, called metaeffectors, function to regulate the activity of other Dot/Icm-translocated effectors during infection. The metaeffector Lpg2505 is essential for L. pneumophila intracellular replication only when its cognate effector, SidI, is present. SidI is a cytotoxic effector that interacts with the host translation factor eEF1A and potently inhibits eukaryotic protein translation by an unknown mechanism. Here, we evaluated the impact of Lpg2505 on SidI-mediated phenotypes and investigated the mechanism of SidI function. We determined that Lpg2505 binds with nanomolar affinity to SidI and suppresses SidI-mediated inhibition of protein translation. SidI binding to eEF1A and Lpg2505 is not mutually exclusive, and the proteins bind distinct regions of SidI. We also discovered that SidI possesses GDP-dependent glycosyl hydrolase activity and that this activity is regulated by Lpg2505. We have therefore renamed Lpg2505 MesI (metaeffector of SidI). This work reveals novel enzymatic activity for SidI and provides insight into how intracellular replication of L. pneumophila is regulated by a metaeffector.


2021 ◽  
Author(s):  
Luying Liu ◽  
Craig R. Roy

Legionella pneumophila is the causative agent of Legionnaires’ Disease and is capable replicating inside phagocytic cells such as mammalian macrophages. The Dot/Icm type IV secretion system is a L. pneumophila virulence factor that is essential for successful intracellular replication. During infection, L. pneumophila builds a replication permissive vacuole by recruiting multiple host molecules and hijacking host cellular signaling pathways, a process mediated by the coordinated functions of multiple Dot/Icm effector proteins. RavY is a predicted Dot/Icm effector protein found to be important for optimal L. pneumophila replication inside host cells. Here, we demonstrate that RavY is a Dot/Icm-translocated effector protein that is dispensable for axenic replication of L. pneumophila , but critical for optimal intracellular replication of the bacteria. RavY is not required for avoidance of endosomal maturation, nor does RavY contribute to the recruitment of host molecules found on replication-permissive vacuoles, such as ubiquitin, RAB1a, and RTN4. Vacuoles containing L. pneumophila ravY mutants promote intracellular survival but limit replication. The replication defect of the L. pneumophila ravY mutant was complemented when the mutant was in the same vacuole as wild type L. pneumophila . Thus, RavY is an effector that is essential for promoting intracellular replication of L. pneumophila once the specialized vacuole has been established.


2017 ◽  
Vol 62 (2) ◽  
Author(s):  
Romain Brunel ◽  
Ghislaine Descours ◽  
Isabelle Durieux ◽  
Patricia Doublet ◽  
Sophie Jarraud ◽  
...  

ABSTRACTtrans-Translation is a ribosome-rescue system that is ubiquitous in bacteria. Small molecules defining a new family of oxadiazole compounds that inhibittrans-translation have been found to have broad-spectrum antibiotic activity. We sought to determine the activity of KKL-35, a potent member of the oxadiazole family, against the human pathogenLegionella pneumophilaand other related species that can also cause Legionnaires' disease (LD). Consistent with the essential nature oftrans-translation inL. pneumophila, KKL-35 inhibited the growth of all tested strains at submicromolar concentrations. KKL-35 was also active against other LD-causingLegionellaspecies. KKL-35 remained equally active againstL. pneumophilamutants that have evolved resistance to macrolides. KKL-35 inhibited the multiplication ofL. pneumophilain human macrophages at several stages of infection. No resistant mutants could be obtained, even during extended and chronic exposure. Surprisingly, KKL-35 was not synergistic with other ribosome-targeting antibiotics and did not induce the filamentation phenotype observed in cells defective fortrans-translation. Importantly, KKL-35 remained active againstL. pneumophilamutants expressing an alternate ribosome-rescue system and lacking transfer-messenger RNA, the essential component oftrans-translation. These results indicate that the antibiotic activity of KKL-35 is not related to the specific inhibition oftrans-translation and its mode of action remains to be identified. In conclusion, KKL-35 is an effective antibacterial agent against the intracellular pathogenL. pneumophilawith no detectable resistance development. However, further studies are needed to better understand its mechanism of action and to assess further the potential of oxadiazoles in treatment.


eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Michael J Sheedlo ◽  
Clarissa L Durie ◽  
Jeong Min Chung ◽  
Louise Chang ◽  
Jacquelyn Roberts ◽  
...  

Legionella pneumophila is an opportunistic pathogen that causes the potentially fatal pneumonia known as Legionnaires' Disease. The pathology associated with infection depends on bacterial delivery of effector proteins into the host via the membrane spanning Dot/Icm type IV secretion system (T4SS). We have determined sub-3.0 Å resolution maps of the Dot/Icm T4SS core complex by single particle cryo-EM. The high-resolution structural analysis has allowed us to identify proteins encoded outside the Dot/Icm genetic locus that contribute to the core T4SS structure. We can also now define two distinct areas of symmetry mismatch, one that connects the C18 periplasmic ring (PR) and the C13 outer membrane cap (OMC) and one that connects the C13 OMC with a 16-fold symmetric dome. Unexpectedly the connection between the PR and OMC is DotH, with five copies sandwiched between the OMC and PR to accommodate the symmetry mismatch. Finally, we observe multiple conformations in the reconstructions that indicate flexibility within the structure.


2021 ◽  
Author(s):  
Tshegofatso Ngwaga ◽  
Deepika Chauhan ◽  
Abigail G Salberg ◽  
Stephanie R Shames

Legionella pneumophila causes Legionnaires' Disease via replication within host macrophages using an arsenal of hundreds of translocated virulence factors termed effector proteins. Effectors are critical for intracellular replication but can also enhance pathogen clearance in mammalian hosts via effector-triggered immunity. The effector LegC4 confers a fitness disadvantage on L. pneumophila within mouse models of Legionnaires' Disease and uniquely potentiates the antimicrobial activity of macrophages activated with either tumor necrosis factor (TNF) or interferon (IFN)-γ. Here, we investigated the mechanism of LegC4 function. We found that LegC4 binds proteasome activator (PA)28α, a subunit of the PA28αβ (11S) proteasome regulator, and that the LegC4 restriction phenotype is abolished within PA28αβ-deficient macrophages. PA28αβ facilitates ubiquitin-independent proteasomal degradation of oxidant-damaged proteins. Impaired proteasome activity results in compensatory upregulation of lysosomal degradation pathways to relieve oxidative proteotoxic stress. We found that LegC4 impairs the resolution of oxidative proteotoxic stress and enhances phagolysosomal fusion with the Legionella-containing vacuole. PA28αβ has been traditionally associated with antigen presentation and adaptive immunity; however, our data support a model whereby suppression of PA28αβ by LegC4 impairs resolution of oxidative proteotoxic stress, which culminates in the lysosomal killing of L. pneumophila within activated macrophages. This work provides a solid foundation on which to evaluate induced proteasome regulators as mediators of cell-autonomous immunity.


2019 ◽  
Author(s):  
Katherine H. Richardson ◽  
Lubov S. Grigoryeva ◽  
Paula Corsini ◽  
Richard C. White ◽  
Rosie Shaw ◽  
...  

AbstractChitinases are important enzymes that contribute to the generation of carbon and nitrogen from chitin, a long chain polymer of N-acetylglucosamine that is abundant in insects, fungi, invertebrates and fish. Although mammals do not produce chitin, chitinases have been identified in bacteria that are key virulence factors in severe respiratory, gastrointestinal and urinary diseases. However, it is unclear how these enzymes are able to carry out this dual function.Legionella pneumophilais the causative agent of Legionnaires’ disease, an often-fatal pneumonia and its chitinase ChiA is essential for the survival ofL. pneumophilain the lung. Here we report the first atomic resolution insight into the pathogenic mechanism of a bacterial chitinase. We derive an experimental model of intact ChiA and show how its N-terminal region targets ChiA to the bacterial surface after its secretion. We provide the first evidence thatL. pneumophilacan bind mucins on its surface but this is not dependent onchiA. This demonstrates that additional peripheral mucin binding proteins are also expressed inL. pneumophila. Finally, we show that the ChiA C-terminal chitinase domain has novel metal-dependent peptidase activity against mammalian mucins. These findings suggest that ChiA facilitates bacterial penetration of the alveolar mucosa and ChiA may be a promising target for vaccine development.


2014 ◽  
Vol 70 (a1) ◽  
pp. C583-C583
Author(s):  
Kathy Wong ◽  
Yinglu Zhang ◽  
Guennadi Kozlov ◽  
Kalle Gehring

Legionella pneumophila is a gram-negative bacterium that causes Legionnaires' disease. It uses a Dot/Icm type IV secretion system to inject effector proteins into the host cell to manipulate host processes. Currently, about 300 Icm/Dot dependent effectors of L.pneumophila have been identified. Lpg1496 is an effector protein, which contains a conserved domain from the SidE family. To date, the middle domain and the conserved SidE domain have been crystallized and the structure solved at a resolution of 1.15Å and 2.3Å, respectively. A structural homology search using the middle domain suggested a similarity to phosphoribosylaminoimidazolesuccinocarboxamide (SAICAR) synthase, an ATPase involved in purine nucleotide synthesis. We performed 1H–15N HSQC NMR titrations to show that this domain binds ATP, ADP and AMP, with the highest binding affinity for ADP. A structural homology search using the SidE domain showed a similarity to cyclic nucleotide phosphodiesterases. To further elucidate the function of lpg1496, other fragments have been cloned, expressed, and subjected to crystallization trials. Currently, we have successfully crystallized the N-terminal domain, with crystals diffracting to <2.0Å. Obtaining the crystal structure of lpg1496 and revealing its function will not only lead to a better understanding of the virulence of L. pneumophila, but also contribute to the development of novel therapeutic treatments of Legionnaires' disease.


2019 ◽  
Vol 201 (14) ◽  
Author(s):  
Tshegofatso Ngwaga ◽  
Alex J. Hydock ◽  
Sandhya Ganesan ◽  
Stephanie R. Shames

ABSTRACTLegionella pneumophilais ubiquitous in freshwater environments, where it replicates within unicellular protozoa. However,L. pneumophilais also an accidental human pathogen that can cause Legionnaires’ disease in immunocompromised individuals by uncontrolled replication within alveolar macrophages. To replicate within eukaryotic phagocytes,L. pneumophilautilizes a Dot/Icm type IV secretion system to translocate a large arsenal of over 300 effector proteins directly into host cells. In mammals, translocated effectors contribute to innate immune restriction ofL. pneumophila. We found previously that the effector LegC4 is important forL. pneumophilareplication within a natural host protist but is deleterious to replication in a mouse model of Legionnaires’ disease. In the present study, we used cultured mouse primary macrophages to investigate how LegC4 attenuatesL. pneumophilareplication. We found that LegC4 enhanced restriction ofL. pneumophilareplication within macrophages activated with tumor necrosis factor (TNF) or interferon gamma (IFN-γ). In addition, expression oflegC4was sufficient to restrictLegionella longbeachaereplication within TNF- or IFN-γ-activated macrophages. Thus, this study demonstrates that LegC4 contributes toL. pneumophilaclearance from healthy hosts by potentiating cytokine-mediated host defense mechanisms.IMPORTANCELegionellaspp. are natural pathogens of protozoa and accidental pathogens of humans. Innate immunity in healthy individuals effectively controlsLegionellainfection due in part to rapid and robust production of proinflammatory cytokines resulting from detection of Dot/Icm-translocated substrates, including effectors. Here, we demonstrate that the effector LegC4 enhances proinflammatory host restriction ofLegionellaby macrophages. These data suggest that LegC4 may augment proinflammatory signaling or antimicrobial activity of macrophages, a function that has not previously been observed for another bacterial effector. Further insight into LegC4 function will likely reveal novel mechanisms to enhance immunity against pathogens.


1998 ◽  
Vol 66 (9) ◽  
pp. 4450-4460 ◽  
Author(s):  
Lawrence A. Wiater ◽  
Kenneth Dunn ◽  
Frederick R. Maxfield ◽  
Howard A. Shuman

ABSTRACT During infection, the Legionnaires’ disease bacterium,Legionella pneumophila, survives and multiplies within a specialized phagosome that is near neutral pH and does not fuse with host lysosomes. In order to understand the molecular basis of this organism’s ability to control its intracellular fate, we have isolated and characterized a group of transposon-generated mutants which were unable to kill macrophages and were subsequently found to be defective in intracellular multiplication. These mutations define a set of 20 genes (19 icm [for intracellular multiplication] genes and dotA [for defect in organelle trafficking]). In this report, we describe a quantitative assay for phagosome-lysosome fusion (PLF) and its use to measure the levels of PLF in cells that have been infected with either wild-type L. pneumophila or one of several mutants defective in different icm genes ordotA. By using quantitative confocal fluorescence microscopy, PLF could be scored on a per-bacterium basis by determining the extent to which fluorescein-labeled L. pneumophilacolocalized with host lysosomes prelabeled with rhodamine-dextran. Remarkably, mutations in the six genes that were studied resulted in maximal levels of PLF as quickly as 30 min following infection. These results indicate that several, and possibly all, of the icmand dotA gene products act at an early step during phagosome establishment to determine whether L. pneumophila-containing phagosomes will fuse with lysosomes. Although not ruled out, subsequent activity of these gene products may not be necessary for successful intracellular replication.


Biomolecules ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 1802
Author(s):  
Kevin Voth ◽  
Shivani Pasricha ◽  
Ivy Yeuk Wah Chung ◽  
Rachelia R. Wibawa ◽  
Engku Nuraishah Huda E. Zainudin ◽  
...  

Legionella pneumophila is a Gram-negative intracellular pathogen that causes Legionnaires' disease in elderly or immunocompromised individuals. This bacterium relies on the Dot/Icm (Defective in organelle trafficking/Intracellular multiplication) Type IV Secretion System (T4SS) and a large (>330) set of effector proteins to colonize the host cell. The structural variability of these effectors allows them to disrupt many host processes. Herein, we report the crystal structure of MavL to 2.65 Å resolution. MavL adopts an ADP-ribosyltransferase (ART) fold and contains the distinctive ligand-binding cleft of ART proteins. Indeed, MavL binds ADP-ribose with Kd of 13 µM. Structural overlay of MavL with poly-(ADP-ribose) glycohydrolases (PARGs) revealed a pair of aspartate residues in MavL that align with the catalytic glutamates in PARGs. MavL also aligns with ADP-ribose “reader” proteins (proteins that recognize ADP-ribose). Since no glycohydrolase activity was observed when incubated in the presence of ADP-ribosylated PARP1, MavL may play a role as a signaling protein that binds ADP-ribose. An interaction between MavL and the mammalian ubiquitin-conjugating enzyme UBE2Q1 was revealed by yeast two-hybrid and co-immunoprecipitation experiments. This work provides structural and molecular insights to guide biochemical studies aimed at elucidating the function of MavL. Our findings support the notion that ubiquitination and ADP-ribosylation are global modifications exploited by L. pneumophila.


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