[4.3.1]Bicyclic FKBP Ligands Inhibit Legionella Pneumophila Infection by LpMip-Dependent and LpMip-Independent Mechanisms.

Legionella pneumophila is the causative agent of Legionnaires' disease, a serious form of pneumonia. Its macrophage infectivity potentiator (Mip), a member of a highly conserved family of FK506-binding proteins (FKBPs), plays a major role in the proliferation of the gram-negative bacterium in host organisms. In this work, we test our library of >1000 FKBP-focused ligands for inhibition of LpMip. The [4.3.1]-bicyclic sulfonamide turned out as a highly preferred scaffold and provided the most potent LpMip inhibitors known so far. Selected compounds were non-toxic to human cells, displayed antibacterial activity and block bacterial proliferation in cellular infection-assays as well as infectivity in human lung tissue explants. The results confirm [4.3.1]-bicyclic sulfonamides as anti-legionellal agents, although their anti-infective properties cannot be explained by inhibition of LpMip alone.

Legionella pneumophila PPIase Mip Interacts with the Bacterial Proteins SspB, Lpc2061, and FlaA and Promotes Flagellation.

The peptidyl-prolyl-cis/trans-isomerase (PPIase) macrophage infectivity potentiator (Mip) contributes to the pathogenicity and fitness of L. pneumophila, the causative agent of Legionnaires' disease. Here, we identified the stringent starvation protein SspB, hypothetical protein Lpc2061, and flagellin FlaA as bacterial interaction partners of Mip. The macrolide FK506, which inhibits the PPIase activity of Mip, interfered with the binding of Lpc2061. Moreover, we demonstrated that the N-terminal dimerization region and amino acid Y185 in the C-terminal PPIase domain of Mip are required for the binding of Lpc2061 and FlaA. The modeling of the interaction partners and global docking with Mip suggested nonoverlapping binding interfaces, and a molecular dynamic simulation predicted an increased stability for the tripartite interaction of Lpc2061, Mip, and FlaA. On the functional level, we demonstrated that Mip promotes L. pneumophila flagellation, which is positively influenced by the binding of Lpc2061 and reduced by FK506. Also, L. pneumophila mutants expressing the Y185A or the monomeric Mip variant, which bind less Lpc2061, were nonmotile, were less flagellated, and yielded less FlaA when quantified. To our knowledge, this is the first report in which a PPIase and its bacterial interaction partners were demonstrated to influence flagellation.

Cellular adaptation of Clostridioides difficile to high salinity encompasses a compatible solute-responsive change in cell morphology.

Infections by the pathogenic gut bacterium Clostridioides difficile cause severe diarrhoeas up to a toxic megacolon and are currently among the major causes of lethal bacterial infections. Successful bacterial propagation in the gut is strongly associated with the adaptation to changing nutrition-caused environmental conditions; e.g. environmental salt stresses. Concentrations of 350 mM NaCl, the prevailing salinity in the colon, led to significantly reduced growth of C. difficile. Metabolomics of salt-stressed bacteria revealed a major reduction of the central energy generation pathways, including the Stickland-fermentation reactions. No obvious synthesis of compatible solutes was observed up to 24 h of growth. The ensuing limited tolerance to high salinity and absence of compatible solute synthesis might result from an evolutionary adaptation to the exclusive life of C. difficile in the mammalian gut. Addition of the compatible solutes carnitine, glycine-betaine, γ-butyrobetaine, crotonobetaine, homobetaine, proline-betaine and dimethylsulfoniopropionate restored growth (choline and proline failed) under conditions of high salinity. A bioinformatically identified OpuF-type ABC-transporter imported most of the used compatible solutes. A long-term adaptation after 48 h included a shift of the Stickland fermentation-based energy metabolism from the utilization to the accumulation of l-proline and resulted in restored growth. Surprisingly, salt stress resulted in the formation of coccoid C. difficile cells instead of the typical rod-shaped cells, a process reverted by the addition of several compatible solutes. Hence, compatible solute import via OpuF is the major immediate adaptation strategy of C. difficile to high salinity-incurred cellular stress.

Zinc metalloprotease ProA of Legionella pneumophila increases alveolar septal thickness in human lung tissue explants by collagen IV degradation.

ProA is a secreted zinc metalloprotease of Legionella pneumophila causing lung damage in animal models of Legionnaires' disease. Here we demonstrate that ProA promotes infection of human lung tissue explants (HLTEs) and dissect the contribution to cell type specific replication and extracellular virulence mechanisms. For the first time, we reveal that co-incubation of HLTEs with purified ProA causes a significant increase of the alveolar septal thickness. This destruction of connective tissue fibres was further substantiated by collagen IV degradation assays. The moderate attenuation of a proA-negative mutant in A549 epithelial cells and THP-1 macrophages suggests that effects of ProA in tissue mainly result from extracellular activity. Correspondingly, ProA contributes to dissemination and serum resistance of the pathogen, which further expands the versatile substrate spectrum of this thermolysin-like protease. The crystal structure of ProA at 1.48 Å resolution showed high congruence to pseudolysin of Pseudomonas aeruginosa, but revealed deviations in flexible loops, the substrate binding pocket S1 ' and the repertoire of cofactors, by which ProA can be distinguished from respective homologues. In sum, this work specified virulence features of ProA at different organisational levels by zooming in from histopathological effects in human lung tissue to atomic details of the protease substrate determination.

FKBPs in bacterial infections.

BACKGROUND: FK506-binding proteins (FKBPs) contain a domain with peptidyl-prolyl-cis/trans-isomerase (PPIase) activity and bind the immunosuppressive drugs FK506 and rapamycin. FKBPs belong to the immunophilin family and are found in eukaryotes and bacteria. SCOPE OF REVIEW: In this review we describe two major groups of bacterial virulence-associated FKBPs, the trigger factor and Mip-like PPIases. Moreover, we discuss the contribution of host FKBPs in bacterial infection processes. MAJOR CONCLUSIONS: Since PPIases are regarded as alternative antiinfective drug targets we highlight current research strategies utilizing pipecolinic acid and cycloheximide derivatives as well as substrate based inhibitors. GENERAL SIGNIFICANCE: The current research strategies suggest a beneficial synergism of drug development and basic research. This article is part of a Special Issue entitled Proline-directed Foldases: Cell Signaling Catalysts and Drug Targets.

Legionella-protozoa-nematode interactions in aquatic biofilms and influence of Mip on Caenorhabditis elegans colonization.

Legionella pneumophila, the causative agent of Legionnaires disease, is naturally found in aquatic habitats. The intracellular life cycle within protozoa pre-adapted the "accidental" human pathogen to also infect human professional phagocytes like alveolar macrophages. Previous studies employing the model organism Caenorhabditis elegans suggest that also nematodes might serve as a natural host for L. pneumophila. Here, we report for the first time from a natural co-habitation of L. pneumophila and environmental nematode species within biofilms of a warm water spring. In addition, we identified the protozoan species Oxytricha bifaria, Stylonychia mytilus, Ciliophrya sp. which have never been described as potential interaction partners of L. pneumophila before. Modeling and dissection of the Legionella-protozoa-nematode interaction revealed that C. elegans ruptures Legionella-infected amoebal cells and by this means incorporate the pathogen. Further infection studies revealed that the macrophage infectivity potentiator (Mip) protein of L. pneumophila, which is known to bind collagen IV during human lung infection, promotes the colonization of the intestinal tract of L4 larvae of C. elegans and negatively influences the life span of the worms. The Mip-negative L. pneumophila mutant exhibited a 32-fold reduced colonization rate of the nematodes after 48h when compared to the wild-type strain. Taken together, these studies suggest that nematodes may serve as natural hosts for L. pneumophila, promote their persistence and dissemination in the environment, and co-evolutionarily pre-adapt the pathogen for interactions with extracellular constituents of human lung tissue.

Peptidylprolyl cis-trans isomerases of Legionella pneumophila: virulence, moonlighting and novel therapeutic targets.

Legionella pneumophila, typically a parasite of free-living protozoa, can also replicate in human alveolar macrophages and lung epithelial cells causing Legionnaires' disease in humans, a severe atypical pneumonia. The pathogen encodes six peptidylprolyl cis-trans isomerases (PPIases), which generally accelerate folding of prolyl peptide bonds, and influence protein folding. PPIases can be divided into three classes, cyclophilins, parvulins and FK506-binding proteins (FKBPs). They contribute to a multitude of cellular functions including bacterial virulence. In the present review, we provide an overview of L. pneumophila PPIases, discussing their known and anticipated functions as well as moonlighting phenomena. By taking the example of the macrophage infectivity potentiator (Mip) of L. pneumophila, we highlight the potential of PPIases as promising drug targets.

Polyketide synthase (PKS) reduces fusion of Legionella pneumophila-containing vacuoles with lysosomes and contributes to bacterial competitiveness during infection.

L. pneumophila-containing vacuoles (LCVs) exclude endocytic and lysosomal markers in human macrophages and protozoa. We screened a L. pneumophila mini-Tn10 transposon library for mutants, which fail to inhibit the fusion of LCVs with lysosomes by loading of the lysosomal compartment with colloidal iron dextran, mechanical lysis of infected host cells, and magnetic isolation of LCVs that have fused with lysosomes. In silico analysis of the mutated genes, D. discoideum plaque assays and infection assays in protozoa and U937 macrophage-like cells identified well established as well as novel putative L. pneumophila virulence factors. Promising candidates were further analyzed for their co-localization with lysosomes in host cells using fluorescence microscopy. This approach corroborated that the O-methyltransferase, PilY1, TPR-containing protein and polyketide synthase (PKS) of L. pneumophila interfere with lysosomal degradation. Competitive infections in protozoa and macrophages revealed that the identified PKS contributes to the biological fitness of pneumophila strains and may explain their prevalence in the epidemiology of Legionnaires' disease.

QseC controls biofilm formation of non-typeable Haemophilus influenzae in addition to an AI-2-dependent mechanism.

Non-typeable Haemophilus influenzae (NTHi) is a common pathogen associated with diseases such as acute otitis media or exacerbations in patients with chronic obstructive pulmonary disease. The biofilm-forming capability substantially contributes to the persistence of NTHi. However, the regulation of biofilm formation is not completely understood. Quorum sensing regulated by autoinducer-2 produced by luxS is until now the only described regulatory mechanism. In this study, we show that the two-component signalling system QseB/C is involved in the biofilm formation of NTHi in vitro. An isogenic NTHi mutant of qseC (Hi3655KR2) showed a significant decrease in biofilm formation under static and semi-static conditions as assessed by crystal violet staining. In addition, under constant flow conditions, Hi3655KR2 formed less biofilm after 48 h. The biofilm defects were irrespective of autoinducer-2 levels. Hence, here we suggest for the first time a regulatory circuit in NTHi, which controls biofilm formation by mechanisms other than or in addition to luxS-dependent factors.

Collagen IV-derived peptide binds hydrophobic cavity of Legionella pneumophila Mip and interferes with bacterial epithelial transmigration.

The Legionella virulence factor Mip (macrophage infectivity potentiator) contributes to bacterial dissemination within infected lung tissue. The Mip protein, which belongs to the enzyme family of FK506-binding proteins (FKBP), binds specifically to collagen IV. We identified a surface-exposed Mip-binding sequence in the NC1 domain of human collagen IV α1. The corresponding collagen IV-derived peptide (P290) co-precipitated with Mip and competitively inhibited the Mip-collagen IV binding. Transmigration of Legionella pneumophila across a barrier of NCI-H292 lung epithelial cells and extracellular matrix was efficiently inhibited by P290. This significantly reduced transmigration was comparable to the inefficient transmigration of PPIase-negative Mip mutant or rapamycin-treated L. pneumophila. Based on NMR data and docking studies a model for the mode of interaction of P290 and Mip was developed. The amino acids of the hydrophobic cavity of Mip, D142 and to a lesser extent Y185 were identified to be part of the interaction surface. In the complex structure of Mip(77-213) and P290, both amino acid residues form hydrogen bonds to P290. Utilizing modelling, molecular dynamics (MD) simulations and structural data of human PPIase FKBP12, the most related human orthologue of Mip, we were able to propose optimized P290 variants with increased binding specificity and selectivity for the putative bacterial drug target Mip.

Redundant and unique roles of coronin proteins in Dictyostelium.

Dictyostelium discoideum harbors a short (CRN12) and a long coronin (CRN7) composed of one and two beta-propellers, respectively. They are primarily present in the cell cortex and cells lacking CRN12 (corA⁻) or CRN7 (corB⁻) have defects in actin driven processes. We compared the characteristics of a mutant cell line (corA⁻/corB⁻) lacking CRN12 and CRN7 with the single mutants focusing on cytokinesis, phagocytosis, chemotaxis and development. Cytokinesis, uptake of small particles, and developmental defects were not enhanced in the corA⁻/corB⁻ strain as compared to the single mutants, whereas motility and phagocytosis of yeast particles were more severely impaired. It appears that although both proteins affect the same processes they do not act in a redundant manner. Rather, they often act antagonistically, which is in accordance with their proposed roles in the actin cytoskeleton where CRN12 acts in actin disassembly whereas CRN7 stabilizes actin filaments and protects them from disassembly.

Zinc Metalloprotease ProA from Legionella pneumophila Inhibits the Pro-Inflammatory Host Response by Degradation of Bacterial Flagellin.

The environmental bacterium Legionella pneumophila is an intracellular pathogen of various protozoan hosts and able to cause Legionnaires' disease, a severe pneumonia in humans. By encoding a wide selection of virulence factors, the infectious agent possesses several strategies to manipulate its host cells and evade immune detection. In the present study, we demonstrate that the L. pneumophila zinc metalloprotease ProA functions as a modulator of flagellin-mediated TLR5 stimulation and subsequent activation of the pro-inflammatory NF-κB pathway. We found ProA to be capable of directly degrading immunogenic FlaA monomers but not the polymeric form of bacterial flagella. These results indicate a role of the protease in antagonizing immune stimulation, which was further substantiated in HEK-BlueTM hTLR5 Detection assays. Addition of purified proteins, bacterial suspensions of L. pneumophila mutant strains as well as supernatants of human lung tissue explant infection to this reporter cell line demonstrated that ProA specifically decreases the TLR5 response via FlaA degradation. Conclusively, the zinc metalloprotease ProA serves as a powerful regulator of exogenous flagellin and presumably creates an important advantage for L. pneumophila proliferation in mammalian hosts by promoting immune evasion.

A Coronin7 homolog with functions in actin-driven processes.

Dictyostelium discoideum Coronin7 (DdCRN7) together with human Coronin7 (CRN7) and Pod-1 of Drosophila melanogaster and Caenorhabditis elegans belong to the coronin family of WD-repeat domain-containing proteins. Coronin7 proteins are characterized by two WD-repeat domains that presumably fold into two beta-propeller structures. DdCRN7 shares highest homology with human CRN7, a protein with roles in membrane trafficking. DdCRN7 is present in the cytosol and accumulates in cell surface projections during movement and phago- and pinocytosis. Cells lacking CRN7 have altered chemotaxis and phagocytosis. Furthermore, loss of CRN7 affects the infection process by the pathogen Legionella pneumophila and allows a more efficient internalization of bacteria. To provide a mechanism for CNR7 action, we studied actin-related aspects. We could show that CRN7 binds directly to F-actin and protects actin filaments from depolymerization. CRN7 also associated with F-actin in vivo. It was present in the Triton X-100-insoluble cytoskeleton, colocalized with F-actin, and its distribution was sensitive to drugs affecting the actin cytoskeleton. We propose that the CRN7 role in chemotaxis and phagocytosis is through its effect on the actin cytoskeleton.

Vitronectin binds to the head region of Moraxella catarrhalis ubiquitous surface protein A2 and confers complement-inhibitory activity.

The serum resistance of the common respiratory pathogen Moraxella catarrhalis is mainly dependent on ubiquitous surface proteins (Usp) A1 and A2 that interact with complement factor 3 (C3) and complement inhibitor C4b binding protein (C4BP) preventing the alternative and classical pathways of the complement system respectively. UspA2 also has the capacity to attract vitronectin that in turn binds C9 and hereby inhibits membrane attack complex (MAC) formation. We found UspA2 as a major vitronectin binding protein and hence the UspA2/vitronectin interaction was studied in detail. The affinity constant (K(D)) for vitronectin binding to UspA2 was 2.3 x 10(-8) M, and the N-terminal region encompassing residues UspA2 30-170 bound vitronectin with a K(D) of 7.9 x 10(-8) M. Electron microscopy verified that the active binding domain (UspA2(30-177)) was located at the head region of UspA2. Experiments with recombinantly expressed vitronectin also revealed that UspA2(30-177) bound to the C-terminal region of vitronectin residues 312-396. Finally, when human serum was pre-incubated with UspA2, bacteria showed significantly less serum resistance. Our study directly reveals the binding mode between the N-terminal domain of UspA2 and the C-terminal part of vitronectin and thus sheds light upon the mechanism of M. catarrhalis-dependent serum resistance.

Loss of Dictyostelium ATG9 results in a pleiotropic phenotype affecting growth, development, phagocytosis and clearance and replication of Legionella pneumophila.

Infection of Dictyostelium discoideum with Legionella pneumophila resulted in a large number of differentially regulated genes among them three core autophagy genes, ATG8, ATG9 and ATG16. Macroautophagy contributes to many physiological and pathological processes and might also constitute an important mechanism in cell-autonomous immunity. For further studies we selected the highly conserved ATG9. In colocalization studies with GFP-tagged ATG9 and different organelle marker proteins we neither observed colocalization with mitochondria, the ER nor lysosomes. However, there was partial colocalization with the Golgi apparatus and many ATG9-GFP-containing vesicles localized along microtubules and accumulated around the microtubule organizing centre. ATG9-deficient cells had pleiotropic defects. In addition to growth defects they displayed severe developmental defects, consistent with the known role of autophagy in Dictyostelium development. Unexpectedly, the ATG9 mutant also had a strong phagocytosis defect that was particularly apparent when infecting the cells with L. pneumophila. However, those Legionellae that entered the host could multiply better in mutant than in wild-type cells, because of a less efficient clearance in the early and a more efficient replication in the late phase of infection. We conclude that ATG9 and hence macroautophagy has a protective role during pathogen infection.

Necrotizing myositis and septic shock caused by Haemophilus influenzae type f in a previously healthy man diagnosed with an IgG3 and a mannose-binding lectin deficiency.

An increased incidence of infections by Haemophilus influenzae type f (Hif) has recently been suggested, but such infections have mainly been regarded as opportunistic. We present here a dramatic case of Hif necrotizing myositis and septic shock. A subsequently diagnosed IgG3 and mannose-binding lectin deficiency possibly contributed to the severe outcome.

Development of a Larval Zebrafish Infection Model for Clostridioides difficile.

Clostridioides difficile infection (CDI) is considered to be one of the most common healthcare-associated gastrointestinal infections in the United States. The innate immune response against C. difficile has been described, but the exact roles of neutrophils and macrophages in CDI are less understood. In the current study, Danio rerio (zebrafish) larvae are used to establish a C. difficile infection model for imaging the behavior and cooperation of these innate immune cells in vivo. To monitor C. difficile, a labeling protocol using a fluorescent dye has been established. A localized infection is achieved by microinjecting labeled C. difficile, which actively grows in the zebrafish intestinal tract and mimics the intestinal epithelial damage in CDI. However, this direct infection protocol is invasive and causes microscopic wounds, which can affect experimental results. Hence, a more noninvasive microgavage protocol is described here. The method involves delivery of C. difficile cells directly into the intestine of zebrafish larvae by intubation through the open mouth. This infection method closely mimics the natural infection route of C. difficile.

Pleiotropic Clostridioides difficile Cyclophilin PpiB Controls Cysteine-Tolerance, Toxin Production, the Central Metabolism and Multiple Stress Responses.

The Gram-positive pathogen Clostridioides difficile is the main bacterial agent of nosocomial antibiotic associated diarrhea. Bacterial peptidyl-prolyl-cis/trans-isomerases (PPIases) are well established modulators of virulence that influence the outcome of human pathologies during infections. Here, we present the first interactomic network of the sole cyclophilin-type PPIase of C. difficile (CdPpiB) and show that it has diverse interaction partners including major enzymes of the amino acid-dependent energy (LdhA, EtfAB, Had, Acd) and the glucose-derived (Fba, GapA, Pfo, Pyk, Pyc) central metabolism. Proteins of the general (UspA), oxidative (Rbr1,2,3, Dsr), alkaline (YloU, YphY) and cold shock (CspB) response were found bound to CdPpiB. The transcriptional (Lrp), translational (InfC, RFF) and folding (GroS, DnaK) control proteins were also found attached. For a crucial enzyme of cysteine metabolism, O-acetylserine sulfhydrylase (CysK), the global transcription regulator Lrp and the flagellar subunit FliC, these interactions were independently confirmed using a bacterial two hybrid system. The active site residues F50, F109, and F110 of CdPpiB were shown to be important for the interaction with the residue P87 of Lrp. CysK activity after heat denaturation was restored by interaction with CdPpiB. In accordance, tolerance toward cell wall stress caused by the exposure to amoxicillin was reduced. In the absence of CdPpiB, C. difficile was more susceptible toward L-cysteine. At the same time, the cysteine-mediated suppression of toxin production ceased resulting in higher cytotoxicity. In summary, the cyclophilin-type PPIase of C. difficile (CdPpiB) coordinates major cellular processes via its interaction with major regulators of transcription, translation, protein folding, stress response and the central metabolism.

PrsA2 (CD630_35000) of Clostridioides difficile Is an Active Parvulin-Type PPIase and a Virulence Modulator.

Clostridioides difficile is the main cause for nosocomial antibiotic associated diarrhea and has become a major burden for the health care systems of industrial countries. Its main virulence factors, the small GTPase glycosylating toxins TcdA and TcdB, are extensively studied. In contrast, the contribution of other factors to development and progression of C. difficile infection (CDI) are only insufficiently understood. Many bacterial peptidyl-prolyl-cis/trans-isomerases (PPIases) have been described in the context of virulence. Among them are the parvulin-type PrsA-like PPIases of Gram-positive bacteria. On this basis, we identified CD630_35000 as the PrsA2 homolog in C. difficile and conducted its enzymatic and phenotypic characterization in order to assess its involvement during C. difficile infection. For this purpose, wild type CdPrsA2 and mutant variants carrying amino acid exchanges mainly in the PPIase domain were recombinantly produced. Recombinant CdPrsA2 showed PPIase activity toward the substrate peptide Ala-Xaa-Pro-Phe with a preference for positively charged amino acids preceding the proline residue. Mutation of conserved residues in its active site pocket impaired the enzymatic activity. A PrsA2 deficient mutant was generated in the C. difficile 630Δerm background using the ClosTron technology. Inactivation of prsA2 resulted in a reduced germination rate in response to taurocholic acid, and in a slight increase in resistance to the secondary bile acids LCA and DCA. Interestingly, in the absence of PrsA2 colonization of mice by C. difficile 630 was significantly reduced. We concluded that CdPrsA2 is an active PPIase that acts as a virulence modulator by influencing crucial processes like sporulation, germination and bile acid resistance resulting in attenuated mice colonization.