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Protein sociology of ProA, Mip and other secreted virulence factors at the Legionella pneumophila surface.

ABSTRACT: The pathogenicity of L. pneumophila, the causative agent of Legionnaires' disease, depends on an arsenal of interacting proteins. Here we describe how surface-associated and secreted virulence factors of this pathogen interact with each other or target extra- and intracellular host proteins resulting in host cell manipulation and tissue colonization. Since progress of computational methods like AlphaFold, molecular dynamics simulation, and docking allows to predict, analyze and evaluate experimental proteomic and interactomic data, we describe how the combination of these approaches generated new insights into the multifaceted "protein sociology" of the zinc metalloprotease ProA and the peptidyl-prolyl cis/trans isomerase Mip (macrophage infectivity potentiator). Both virulence factors of L. pneumophila interact with numerous proteins including bacterial flagellin (FlaA) and host collagen, and play important roles in virulence regulation, host tissue degradation and immune evasion. The recent progress in protein-ligand analyses of virulence factors suggests that machine learning will also have a beneficial impact in early stages of drug discovery.

SUBMITTER: Scheithauer L

PROVIDER: S-EPMC10017501 | biostudies-literature | 2023

REPOSITORIES: biostudies-literature

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Protein sociology of ProA, Mip and other secreted virulence factors at the <i>Legionella pneumophila</i> surface.

Scheithauer Lina L Karagöz Mustafa Safa MS Mayer Benjamin E BE Steinert Michael M

Frontiers in cellular and infection microbiology 20230302

The pathogenicity of <i>L. pneumophila</i>, the causative agent of Legionnaires' disease, depends on an arsenal of interacting proteins. Here we describe how surface-associated and secreted virulence factors of this pathogen interact with each other or target extra- and intracellular host proteins resulting in host cell manipulation and tissue colonization. Since progress of computational methods like AlphaFold, molecular dynamics simulation, and docking allows to predict, analyze and evaluate e ...[more]

PMID: 36936764

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Project description:Macrophage infectivity potentiator (Mip) and Mip-like proteins are virulence factors in a wide range of pathogens including Legionella pneumophila. These proteins belong to the FK506 binding protein (FKBP) family of peptidyl-prolyl-cis/trans-isomerases (PPIases). In L. pneumophila, the PPIase activity of Mip is required for invasion of macrophages, transmigration through an in vitro lung-epithelial barrier, and full virulence in the guinea pig infection model. Additionally, Mip is a moonlighting protein that binds to collagen IV in the extracellular matrix. Here, we describe the development and synthesis of cycloheximide derivatives with adamantyl moieties as novel FKBP ligands, and analyze their effect on the viability of L. pneumophila and other bacteria. All compounds efficiently inhibited PPIase activity of the prototypic human FKBP12 as well as Mip with IC50-values as low as 180 nM and 1.7 μM, respectively. Five of these derivatives inhibited the growth of L. pneumophila at concentrations of 30-40 μM, but exhibited no effect on other tested bacterial species indicating a specific spectrum of antibacterial activity. The derivatives carrying a 3,5-dimethyladamantan-1-[yl]acetamide substitution (MT_30.32), and a 3-ethyladamantan-1-[yl]acetamide substitution (MT_30.51) had the strongest effects in PPIase- and liquid growth assays. MT_30.32 and MT_30.51 were also inhibitory in macrophage infection studies without being cytotoxic. Accordingly, by applying a combinatorial approach, we were able to generate novel, hybrid inhibitors consisting of cycloheximide and adamantane, two known FKBP inhibitors that interact with different parts of the PPIase domain, respectively. Interestingly, despite the proven Mip-inhibitory activity, the viability of a Mip-deficient strain was affected to the same degree as its wild type. Hence, we also propose that cycloheximide derivatives with adamantyl moieties are potent PPIase inhibitors with multiple targets in L. pneumophila.

Project description:Several novel Legionella pneumophila virulence genes were previously discovered by use of signature-tagged mutagenesis (P. H. Edelstein, M. A. Edelstein, F. Higa, and S. Falkow, Proc. Natl. Acad. Sci. 96:8190-8195, 1999). One of these mutants appeared to be defective in multiplication in guinea pig lungs and spleens, yet it multiplies normally in guinea pig alveolar macrophages. Here we report further characterization of the mutated gene and its protein and the virulence role of the gene. The complete sequence of the gene, now called lvgA, is 627 bp long, and its protein product is approximately 27 kDa in size. lvgA was present in all 50 strains of L. pneumophila tested. No significant nucleic acid or protein homology was found in the GenBank database for the gene, nor were any distinctive motifs discovered in a search of other databases. The expression of both DotA and IcmX in the lvgA mutant was normal. Subcellular fractionation studies localized LvgA to the outer membrane fraction, and protease digestion studies suggested that at least some of the protein is surface expressed. No change in bacterial lipopolysaccharide composition or reactivity to serogroup-specific antisera was detected in the mutant. Growth competition studies with alveolar macrophages showed that the mutant was outcompeted by its parent 3-fold in 24 h and 24-fold in 48 h, in contrast to what was observed with the null phenotype in parallel testing with alveolar macrophages or with the A549 alveolar epithelial cell line. This macrophage defect of the mutant bacterium was due to slower growth, as the mutant invaded alveolar macrophages normally. Electron microscopy showed that the mutant bacterium resided in a ribosome-studded phagosome in alveolar macrophages, with no distinction from its parent. The lvgA mutant was outcompeted by its parent about sixfold in guinea pig lungs and spleens; prolonged observation of infected animals showed no late-onset virulence of the mutant. Transcomplementation of the mutant restored the parental phenotype in guinea pigs. The lvgA mutant was twofold more susceptible to killing by human beta-defensin 2 but not to killing by other cationic peptides, serum complement, or polymorphonuclear neutrophils. lvgA is a novel virulence gene that is responsible for pleiotropic functions involving both extracellular and intracellular bacterial resistance mechanisms.

Project description:BackgroundLegionella pneumophila is a Gram-negative intracellular bacillus and is the causative agent of a severe form of pneumonia called Legionnaires' disease which accounts for 2-9% of cases of community acquired pneumonia. It produces an extremely large protein belonging to the RTX (Repeats in ToXin) family, called RtxA, and we previously reported that RtxA is transported by a dedicated type 1 secretion system (T1SS) to the cell surface. RTX proteins have been shown to participate in the virulence or biofilm formation of various bacteria, the most studied models being the pore forming hemolysin A (HlyA) of Escherichia coli and the biofilm associated protein LapA of P. fluorescens. LapA localization depends on the enzymatic release by LapD/LapG complex activity. This study aimed to elucidate the dual localization (cell surface associated or released state) of L. pneumophila RTX protein (RtxA) and whether this released versus sequestered state of RtxA plays a role in L. pneumophila virulence.ResultsThe hereby work reveals that, in vitro, LapG periplasmic protease cleaves RtxA N-terminus in the middle of a di-alanine motif (position 108-109). Consistently, a strain lacking LapG protease maintains RtxA on the cell surface, whereas a strain lacking the c-di-GMP receptor LapD does not exhibit cell surface RtxA because of its continuous cleavage and release, as in the LapA-D-G model of Pseudomonas fluorescens. Interestingly, our data point out a key role of RtxA in enhancing the infection process of amoeba cells, regardless of its location (embedded or released); therefore, this may be the result of a secondary role of this surface protein.ConclusionsThis is the first experimental identification of the cleavage site within the RTX protein family. The primary role of RtxA in Legionella is still questionable as in many other bacterial species, hence it sounds reasonable to propose a major function in biofilm formation, promoting cell aggregation when RtxA is embedded in the outer membrane and facilitating biofilm dispersion in case of RtxA release. The role of RtxA in enhancing the infection process may be a result of its action on host cells (i.e., PDI interaction or pore-formation), and independently of its status (embedded or released).

Dataset Information

Protein sociology of ProA, Mip and other secreted virulence factors at the Legionella pneumophila surface.

Publications

Protein sociology of ProA, Mip and other secreted virulence factors at the <i>Legionella pneumophila</i> surface.

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