scholarly journals Structure and functional analysis of theLegionellachitinase ChiA reveals a novel mechanism of metal-dependent mucin degradation

2019 ◽  
Author(s):  
Katherine H. Richardson ◽  
Lubov S. Grigoryeva ◽  
Paula Corsini ◽  
Richard C. White ◽  
Rosie Shaw ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Vaccine Development ◽  
Dual Function ◽  
Peptidase Activity ◽  
Chain Polymer ◽  
Bacterial Surface ◽  
Carbon And Nitrogen ◽  
Promising Target ◽  
Legionnaires Disease ◽  
Insight Into

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.

scholarly journals Surface-Associated Heat Shock Proteins ofLegionella pneumophilaandHelicobacter pylori:Roles in Pathogenesis and Immunity

1999 ◽  
Vol 7 (1-2) ◽  
pp. 58-63 ◽  
Author(s):  
P. S. Hoffman ◽  
R. A. Garduno
Keyword(s):  
Heat Shock ◽  
Heat Shock Proteins ◽  
Legionella Pneumophila ◽  
Cell Model ◽  
Bacterial Surface ◽  
Acid Shock ◽  
Mucosal Infection ◽  
Microbial Infections ◽  
Legionnaires Disease ◽  
Shock Proteins

Bacterial heat shock proteins (Hsps) are abundantly produced during the course of most microbial infections and are often targeted by the mammalian immune response. While Hsps have been well characterized for their roles in protein folding and secretion activities, little attention has been given to their participation in pathogenesis. In the case ofLegionella pneumophila, an aquatic intracellular parasite of protozoa and cause of Legionnaires' disease, Hsp60 is uniquely located in the periplasm and on the bacterial surface. Surface-associated Hsp60 promotes attachment and invasion in a HeLa cell model and may alter an early step associated with the fusion of phagosomes with lysosomes. Avirulent strains ofL. pneumophilacontaining defined mutations in severaldot/icmgenes are defective in localizing Hsp60 onto their surface and are reduced approximately 1000-fold in their invasiveness towards HeLa cells. For the ulcer-causing bacteriumHelicobacter pylori, surfaceassociated Hsp60 and Hsp70 mediate attachment to gastric epithelial cells. The increased expression of these Hsps following acid shock correlates with both increased association with and inflammation of the gastric mucosa. A role for Hsps in colonization, mucosal infection and in promoting inflammation is discussed. Infect. Dis. Obstet. Gynecol. 7:58–63, 1999.

scholarly journals Population analysis of Legionella pneumophila reveals a basis for resistance to complement-mediated killing

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Bryan A. Wee ◽  
Joana Alves ◽  
Diane S. J. Lindsay ◽  
Ann-Brit Klatt ◽  
Fiona A. Sargison ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Population Analysis ◽  
Single Gene ◽  
Human Neutrophils ◽  
Classical Pathway ◽  
Genetic Traits ◽  
Bacterial Surface ◽  
Genomic Study ◽  
Legionnaires Disease ◽  
Severe Respiratory Infection

AbstractLegionella pneumophila is the most common cause of the severe respiratory infection known as Legionnaires’ disease. However, the microorganism is typically a symbiont of free-living amoeba, and our understanding of the bacterial factors that determine human pathogenicity is limited. Here we carried out a population genomic study of 902 L. pneumophila isolates from human clinical and environmental samples to examine their genetic diversity, global distribution and the basis for human pathogenicity. We find that the capacity for human disease is representative of the breadth of species diversity although some clones are more commonly associated with clinical infections. We identified a single gene (lag-1) to be most strongly associated with clinical isolates. lag-1, which encodes an O-acetyltransferase for lipopolysaccharide modification, has been distributed horizontally across all major phylogenetic clades of L. pneumophila by frequent recent recombination events. The gene confers resistance to complement-mediated killing in human serum by inhibiting deposition of classical pathway molecules on the bacterial surface. Furthermore, acquisition of lag-1 inhibits complement-dependent phagocytosis by human neutrophils, and promoted survival in a mouse model of pulmonary legionellosis. Thus, our results reveal L. pneumophila genetic traits linked to disease and provide a molecular basis for resistance to complement-mediated killing.

scholarly journals NAD(H)-mediated tetramerization controls the activity of Legionella pneumophila phospholipase PlaB

2020 ◽  
Author(s):  
Maurice Diwo ◽  
Wiebke Michel ◽  
Philipp Aurass ◽  
Katja Kuhle-Keindorf ◽  
Jan Pippel ◽  
...  
Keyword(s):  
Virulence Factor ◽  
Legionella Pneumophila ◽  
Enzyme Activation ◽  
Enzyme Inactivation ◽  
Activation Mechanism ◽  
Tissue Destruction ◽  
Bacterial Surface ◽  
Lung Colonization ◽  
Legionnaires Disease ◽  
Tetrameric Form

AbstractThe virulence factor and phospholipase PlaB promotes lung colonization, tissue destruction, and intracellular replication of Legionella pneumophila, the causative agent of Legionnaires’ disease. It is exposed at the bacterial surface and shows an extraordinary activation mechanism by tetramer deoligomerization. To unravel the molecular basis for enzyme activation and localization, we determined the crystal structure of PlaB in its tetrameric form. We found that the tetramer is a dimer of identical dimers, and a monomer consists of an N-terminal phospholipase α/β-hydrolase domain augmented by two non-canonical two-stranded β-sheets, β6/β7 and β9/β10. The C- terminal domain reveals a novel fold displaying a bilobed β-sandwich with a hook structure that is required for dimer formation and complementation of the phospholipase domain in the neighboring monomer. Unexpectedly, we observed eight NAD(H) molecules at the dimer/dimer interface, suggesting that these molecules stabilize the tetramer and hence lead to enzyme inactivation. Indeed, addition of NAD(H) increased the fraction of the tetrameric form and concomitantly reduced activity. β9/β10 mutants revealed a decrease in the tetrameric fraction, altered activity profiles, and mislocalization. Protein variants lacking the hook or strands β6/β7 were unaffected in terms of localization but lost their activity, and lid mutants changed substrate specificity. Together, these data reveal structural elements and an unprecedented NAD(H)- mediated tetramerization mechanism required for spatial and enzymatic control of a phospholipase virulence factor. The regulatory process identified is ideally suited to fine tune PlaB in a way that protects L. pneumophila from self-inflicted lysis while ensuring its activity at the pathogen–host interface.

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 ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Protein Translation ◽  
Effector Proteins ◽  
Intracellular Replication ◽  
Unknown Mechanism ◽  
Translation Inhibition ◽  
Legionnaires Disease ◽  
The Impact ◽  
Insight Into ◽  
Cytotoxic Effector

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.

scholarly journals Insights into the ubiquitin transfer cascade catalyzed by the Legionella effector SidC

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
David Jon Wasilko ◽  
Qingqiu Huang ◽  
Yuxin Mao
Keyword(s):  
Legionella Pneumophila ◽  
Ternary Complex ◽  
Common Mechanism ◽  
Binary Complex ◽  
Intracellular Membrane ◽  
E3 Ligases ◽  
Molecular Determinants ◽  
Membrane Bound ◽  
Legionnaires Disease ◽  
Insight Into

The causative agent of Legionnaires’ disease, Legionella pneumophila, delivers more than 330 virulent effectors to its host to establish an intracellular membrane-bound organelle called the Legionella containing vacuole. Among the army of Legionella effectors, SidC and its paralog SdcA have been identified as novel bacterial ubiquitin (Ub) E3 ligases. To gain insight into the molecular mechanism of SidC/SdcA as Ub ligases, we determined the crystal structures of a binary complex of the N-terminal catalytic SNL domain of SdcA with its cognate E2 UbcH5C and a ternary complex consisting of the SNL domain of SidC with the Ub-linked E2 UbcH7. These two structures reveal the molecular determinants governing the Ub transfer cascade catalyzed by SidC. Together, our data support a common mechanism in the Ub transfer cascade in which the donor Ub is immobilized with its C-terminal tail locked in an extended conformation, priming the donor Ub for catalysis.

scholarly journals Rab1-AMPylation by Legionella DrrA is allosterically activated by Rab1

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jiqing Du ◽  
Marie-Kristin von Wrisberg ◽  
Burak Gulen ◽  
Matthias Stahl ◽  
Christian Pett ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Adenosine Monophosphate ◽  
Vesicular Trafficking ◽  
Bacterial Protein ◽  
Post Translational Modification ◽  
Allosteric Control ◽  
Rab Protein ◽  
Biochemical Characterisation ◽  
A Site ◽  
Insight Into

AbstractLegionella pneumophila infects eukaryotic cells by forming a replicative organelle – the Legionella containing vacuole. During this process, the bacterial protein DrrA/SidM is secreted and manipulates the activity and post-translational modification (PTM) states of the vesicular trafficking regulator Rab1. As a result, Rab1 is modified with an adenosine monophosphate (AMP), and this process is referred to as AMPylation. Here, we use a chemical approach to stabilise low-affinity Rab:DrrA complexes in a site-specific manner to gain insight into the molecular basis of the interaction between the Rab protein and the AMPylation domain of DrrA. The crystal structure of the Rab:DrrA complex reveals a previously unknown non-conventional Rab-binding site (NC-RBS). Biochemical characterisation demonstrates allosteric stimulation of the AMPylation activity of DrrA via Rab binding to the NC-RBS. We speculate that allosteric control of DrrA could in principle prevent random and potentially cytotoxic AMPylation in the host, thereby perhaps ensuring efficient infection by Legionella.

scholarly journals Efficacy of SCH27899 in an Animal Model of Legionnaires' Disease Using Immunocompromised A/J Mice

2000 ◽  
Vol 44 (5) ◽  
pp. 1333-1336 ◽  
Author(s):  
Joan K. Brieland ◽  
David Loebenberg ◽  
Fred Menzel ◽  
Roberta S. Hare
Keyword(s):  
Animal Model ◽  
Murine Model ◽  
Legionella Pneumophila ◽  
Immunocompromised Host ◽  
Lung Infection ◽  
Cortisone Acetate ◽  
Contrast Administration ◽  
Once Daily ◽  
Legionnaires Disease ◽  
Lung Infections

ABSTRACT The efficacy of SCH27899, a new everninomicin antibiotic, against replicative Legionella pneumophila lung infections in an immunocompromised host was evaluated using a murine model of Legionnaires' disease. A/J mice were immunocompromised with cortisone acetate and inoculated intratracheally with L. pneumophilaserogroup 1 (105 CFU per mouse). At 24 h postinoculation, mice were administered either SCH27899 (6 to 60 mg/kg [MPK] intravenously) or a placebo once daily for 5 days, and mortality and intrapulmonary growth of L. pneumophila were assessed. In the absence of SCH27899, there was 100% mortality inL. pneumophila-infected mice, with exponential intrapulmonary growth of the bacteria. In contrast, administration of SCH27899 at a dose of ≥30 MPK resulted in ≥90% survival of infected mice, which was associated with inhibition of intrapulmonary growth ofL. pneumophila. In subsequent studies, the efficacy of SCH27899 was compared to ofloxacin (OFX) and azithromycin (AZI). Administration of SCH27899, OFX, or AZI at a dose of ≥30 MPK once daily for 5 days resulted in ≥85% survival of infected mice and inhibition of intrapulmonary growth of the bacteria. However, L. pneumophila CFU were recovered in lung homogenates following cessation of therapy with all three antibiotics. These studies demonstrate that SCH27899 effectively prevents fatal replicativeL. pneumophila lung infection in immunocompromised A/J mice by inhibition of intrapulmonary growth of the bacteria. However, in this murine model of pulmonary legionellosis, SCH27899, like OFX and AZI, was bacteriostatic.

Subtyping ofLegionella pneumophilaisolates by arbitrarily primed polymerase chain reaction

1995 ◽  
Vol 41 (9) ◽  
pp. 846-848 ◽  
Author(s):  
E. Ledesma ◽  
J. Llorca ◽  
M. A. Dasí ◽  
M. L. Camaró ◽  
E. Carbonell ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Legionella Pneumophila ◽  
Water Sources ◽  
Chain Reaction ◽  
Single Room ◽  
Legionnaires Disease ◽  
Polymerase Chain ◽  
Resort Hotel ◽  
Different Water Sources

Arbitrarily primed polymerase chain reaction (AP-PCR) was used to differentiate strains of Legionella pneumophila isolated from different water sources in a resort hotel in Benidorm, Alicante, Spain, where an outbreak of Legionnaires' disease occurred among a group of tourists between 65 and 80 years of age. All isolates were L. pneumophila serogroup 1, subtype Pontiac (Knoxville 1). Five different patterns (P1 to P5) were obtained by AP-PCR. The number of bands per pattern varied between 4 and 11. Patterns P1 and P2 represented 60 and 20% of L. pneumophila isolates, respectively. Since different subpopulations of L. pneumophila coexisted (up to three different AP-PCR patterns were identified in a single room), it was not possible to link an individual L. pneumophila strain to the occurrence of this outbreak.Key words: Legionella pneumophila, AP-PCR, subtyping, outbreak.

scholarly journals Human Lung Tissue Explants Reveal Novel Interactions during Legionella pneumophila Infections

2013 ◽  
Vol 82 (1) ◽  
pp. 275-285 ◽  
Author(s):  
Jens Jäger ◽  
Sebastian Marwitz ◽  
Jana Tiefenau ◽  
Janine Rasch ◽  
Olga Shevchuk ◽  
...  
Keyword(s):  
Lung Tissue ◽  
Legionella Pneumophila ◽  
Human Lung ◽  
Transcriptional Response ◽  
Human Infection ◽  
Bacterial Invasion ◽  
Human Lung Tissue ◽  
Content Type ◽  
Tissue Explants ◽  
Legionnaires Disease

ABSTRACTHistological and clinical investigations describe late stages of Legionnaires' disease but cannot characterize early events of human infection. Cellular or rodent infection models lack the complexity of tissue or have nonhuman backgrounds. Therefore, we developed and applied a novel model forLegionella pneumophilainfection comprising living human lung tissue. We stimulated lung explants withL. pneumophilastrains and outer membrane vesicles (OMVs) to analyze tissue damage, bacterial replication, and localization as well as the transcriptional response of infected tissue. Interestingly, we found that extracellular adhesion ofL. pneumophilato the entire alveolar lining precedes bacterial invasion and replication in recruited macrophages. In contrast, OMVs predominantly bound to alveolar macrophages. Specific damage to septa and epithelia increased over 48 h and was stronger in wild-type-infected and OMV-treated samples than in samples infected with the replication-deficient, type IVB secretion-deficient DotA−strain. Transcriptome analysis of lung tissue explants revealed a differential regulation of 2,499 genes after infection. The transcriptional response included the upregulation of uteroglobin and the downregulation of the macrophage receptor with collagenous structure (MARCO). Immunohistochemistry confirmed the downregulation of MARCO at sites of pathogen-induced tissue destruction. Neither host factor has ever been described in the context ofL. pneumophilainfections. This work demonstrates that the tissue explant model reproduces realistic features of Legionnaires' disease and reveals new functions for bacterial OMVs during infection. Our model allows us to characterize early steps of human infection which otherwise are not feasible for investigations.

scholarly journals Genome-wide identification of chitinase genes in Thalassiosira pseudonana and analysis of their expression under abiotic stresses

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Haomiao Cheng ◽  
Zhanru Shao ◽  
Chang Lu ◽  
Delin Duan
Keyword(s):  
Abiotic Stresses ◽  
Chitinase Activity ◽  
Thalassiosira Pseudonana ◽  
Centric Diatom ◽  
Carbon And Nitrogen ◽  
Genome Wide ◽  
Duplication Events ◽  
The Many ◽  
Chitinase Genes ◽  
Insight Into

Abstract Background The nitrogen-containing polysaccharide chitin is the second most abundant biopolymer on earth and is found in the cell walls of diatoms, where it serves as a scaffold for biosilica deposition. Diatom chitin is an important source of carbon and nitrogen in the marine environment, but surprisingly little is known about basic chitinase metabolism in diatoms. Results Here, we identify and fully characterize 24 chitinase genes from the model centric diatom Thalassiosira pseudonana. We demonstrate that their expression is broadly upregulated under abiotic stresses, despite the fact that chitinase activity itself remains unchanged, and we discuss several explanations for this result. We also examine the potential transcriptional complexity of the intron-rich T. pseudonana chitinase genes and provide evidence for two separate tandem duplication events during their evolution. Conclusions Given the many applications of chitin and chitin derivatives in suture production, wound healing, drug delivery, and other processes, new insight into diatom chitin metabolism has both theoretical and practical value.

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