The intracellular pathogen Legionella pneumophila replicates in a membrane-bound compartment known as the Legionella-containing vacuole (LCV) where it abundantly releases its chaperonin HtpB, suggesting that HtpB may have virulence-related functions. To assess these functions, I attempted to construct an L. pneumophila ?htpBmutant but was unable to do so, likely because htpB is essential. In the absence of genetic deletion, functional tests were used to study the released HtpB. A small portion of the HtpB in L. pneumophila-infected cells was found in the cytoplasm of the infected cells, as judged by the CyaA reporter assay. To identify potential functions of the HtpB present in the eukaryotic cytoplasm, htpB was ectopically expressed in Saccharomyces cerevisiae. HtpB induced pseudohyphal growth (PHG) in yeast, suggesting it interacts with eukaryotic targets. A yeast two-hybrid screen showed that HtpB interacted with SAMDC, an essential yeast enzyme encoded by SPE2 that is required for polyamine biosynthesis. Overexpression of SPE2 induced PHG in S. cerevisiae, suggesting that HtpB induces PHG by activating polyamine synthesis, and that L. pneumophila may require exogenous polyamines for growth. A pharmacological inhibitor of SAMDC reduced L. pneumophila replication in host cells, whereas exogenous polyamines enhanced intracellular growth. Bioinformatics revealed that most known enzymes required for polyamine biosynthesis in bacteria are absent in L. pneumophila, suggesting that L. pneumophila depends on exogenous polyamines transported from host cells. L. pneumophila possesses only one putative operon,potABCD, which encodes a polyamine transporter. Using GFP as a reporter of potABCD promoter (PpotA), we found that PpotA activity was turned on during exponential phase of growth in vitro. To test the potential function of this transporter in pathogenesis, potD was deleted.Although deletion of potD did not affect L. pneumophila growth in vitro, it reduced L. pneumophila attachment to phagocytic cells, intracellular growth, and the ability of the LCV to recruit vesicles. Collectively, these findings have contributed to a better understanding of the biology of L. pneumophila by suggesting that HtpB and PotD might collaborate to ensure a supply of polyamines required for the optimal intracellular growth of L. pneumophila.

Würzburg, Univ., Diss., 2009. / Zsfassung in dt. Sprache.

Opportunistic pathogens (e.g., Legionella pneumophila, Mycobacterium avium complex, Acanthamoeba polyphaga, Pseudomonas aeruginosa) residing in human-made water systems, particularly premise plumbing, are now the primary source of water-borne disease in developed countries. The prevention and control of opportunistic pathogens is a new challenge in premise plumbing due to the limited knowledge concerning the factors driving their occurrence and regrowth mechanisms, and also the complexity of premise plumbing conditions. The goal of this study is to identify key factors governing occurrence of opportunistic pathogens in drinking water distribution systems, particularly premise plumbing, via field investigations and lab-scale experiments. A molecular survey of three opportunistic pathogens (L. pneumophila, M. avium, P. aeruginosa), related groups (Legionella and mycobacteria) and two amoeba hosts (Acanthamoeba spp. and Hartmanella vermiformis) was performed in two real-word chloraminated drinking water distribution systems using quantitative polymerase chain reaction (q-PCR). A high occurrence of Legionella (" 69.0%) and mycobacteria (100%), lower occurrence of L. pneumophila (" 20%) and M. avium (" 33.3%), and rare detection of Pseudomonas aeruginosa (" 13.3%) was observed in both systems. Hartmanella vermiformis was more prevalent than Acanthamoeba. Three-minute flushing resulted in reduced gene copies of Legionella, mycobacteria, H. vermiformis and 16S rRNA genes (P<0.05) and distinct microbial community structure in postflushing water, implying strong regrowth potential of opportunistic pathogens in premise pluming. In order to examine the influence of pipe material, disinfectant type, and water age on occurrence and persistence of the target microorganisms, triplicate simulated distribution systems (SDSs) comparing iron, cement and PVC pipe materials were fed either chlorinated or chloraminated tap water, and were sampled at water ages ranging from 1d to 5.7d. q-PCR quantification of target microorganisms in both biofilm and bulk water revealed that Legionella, mycobacteria, P. aeruginosa and both amoebas naturally colonized the six SDSs, but L. pneumophila and M. avium were not detected. Disinfectant type and dose have the strongest influence on the microbiota. Disinfectant decay was noted with water age, particularly in chloraminated SDSs (due to nitrification), generally resulting in increased microbial detection frequencies and densities with water age. Influence of pipe material became apparent at water ages corresponding to low disinfectant residual. Natural colonization of Legionella spp., Mycobacterium spp., Acanthamoeba spp., H. vermiformis and M. avium was also observed in biofilms from five annular reactors, which were used to investigate effects of prior granular activated carbon (GAC) biofiltration and disinfectant type (chlorine, chloramine) on opportunistic pathogens under premise plumbing conditions. GAC pre-treatment effectively reduced total organic carbon (TOC). In most cases, total bacteria and opportunistic pathogens were higher in undisinfected annular reactors, but the levels were not proportional to the level of GAC pre-treatment/TOC. Chlorine was more effective for controlling mycobacteria and Acanthamoeba, whereas chloramine was more effective for controlling Legionella. Both chlorine and chloramine effectively reduced M. avium and H. vermiformis numbers. Pyrosequencing of 16S rRNA genes in biofilms revealed a significant effect of GAC pre-treatment and disinfectant type on the microbial community structure. Overall, the study provides insights to critical factors triggering proliferation of opportunistic pathogens in drinking water systems. Knowledge gained from this study can assist in formulating practical guidance for drinking water utilities and water consumers in terms of opportunistic pathogen prevention and control. / Ph. D.

No description available.

Legionella pneumophila and other opportunistic pathogens of drinking water pose important problems at the interface of biology, environmental engineering, public health, and governance. In this thesis, I explore the molecular mechanisms permitting survival of L. pneumophila in built water systems is the nature of its physiology under different conditions and different life-phases. In the first chapter, I discuss how various physiological states of L. pneumophila affect the propensity for survival and virulence in relation to drinking water environments. This literature review should provide a perspective important for designing controlled laboratory experiments rooted in a robust understanding of how phenotype dictates experimental results. In the second chapter, I describe sequence and phylogenetic analyses performed to investigate the presence of a type 1 secretion system and virulence factor throughout the Legionella genus. While this system was previously believed to be conserved to L. pneumophila, our analysis indicates that this system is well-distributed throughout the Legionella genus, blurring the lines between "pathogenic" and "non-pathogenic" species. In the third chapter, I report the secretome of endemic Flint, Michigan L. pneumophila in corrosive water, simulating the environmental impact of the Flint Water Crisis on local L. pneumophila populations. Our results from this study have expanded the secretome of L. pneumophila, provided insight on mechanisms it may employ to resist stress in water, and created several novel lines of inquiry at the merging frontier of biochemistry and environmental engineering. / Master of Science in Life Sciences

Premise plumbing, the pipes and fixtures at the building level, present a unique challenge for maintaining drinking water quality. Of particular concern are opportunistic pathogens, including Legionella pneumophila which can regrow in premise plumbing and cause disease in immunocompromised populations. The goal of this work was to explore engineering methods for control of L. pneumophila and total regrowth. The first line of study involved a series of experiments with simulated glass water heaters (SGWHs) to investigate interactions between specific water chemistry factors and L. pneumophila regrowth, and the second used laboratory grade purified water to investigate the limits of a nutrient control approach for biological stability. Several water chemistry factors including assimilable organic carbon (AOC) content, granular activated carbon (GAC) biofiltration, plumbing materials, copper concentrations and temperature were investigated using SGWHs. AOC is the carbon available for bacteria growth in drinking water. Results indicated that AOC reduction may be a promising method for controlling L. pneumophila and total bacteria regrowth, but there may be a point at which AOC reduction is no longer effective. Prior GAC biofiltration removed organic carbon and was effective in controlling total bacterial regrowth in SGWHs, but actually encouraged L. pneumophila regrowth. A wide variety of materials typically encountered in premise plumbing was investigated and only had limited effect on proliferation of L. pneumophila and total bacteria. The effects were dynamic, even with long-term studies. Copper pipes held promise for control of L. pneumophila, as did copper concentration across a range of pHs. Aqueous copper concentration released from pipes was dependent on temperature, however, and thus this control method may not be applicable in all hot water lines. The peak temperatures for L. pneumophila proliferation fell between 41 and 45 °C, temperatures which could be encountered in a hot water distribution system when the water heater is set to 48 °C, as is often recommended with scalding and energy concerns. A constant temperature of 53 °C seemed to provide control of L. pneumophila, but recolonization is possible even at these high temperatures. Work with laboratory grade water indicated that extreme control of nutrients was not enough to completely control regrowth in premise plumbing. With stagnation in the cleanest conditions, a 2-log increase of a diverse group of bacteria was observed within 10 days. As drinking water can never achieve such nutrient removal, this study presents the limits of nutrient removal as a strategy for regrowth control. This work explored both the potential and the limitations of several mechanisms for controlling regrowth in premise plumbing. Understanding how these water chemistry factors affect L. pneumophila and total bacterial regrowth is critical to identifying the most effective engineering controls. / Master of Science

In dieser Arbeit wurde in Legionella pneumophila Corby ein bislang nicht in L. pneumophila beschriebener Bereich im Genom kloniert und sequenziert, der für ein putatives Konjugations- Typ IV Sekretionssystem kodiert. Alle für ein Typ IV Sekretionssystem notwendigen Gene sind vorhanden. Zum einen kodieren diese ein „mating pair formation“ System, also Proteine für die Pilusgenese und energieabhängigen Transport von Substraten aus der Bakterienzelle. Konjugationsexperimente zeigen, dass es sich bei den „DNA transfer and replication“ Genen trb/tra System um einen funktionierenden Mechanismus zur Mobilisierung von DNA handelt. / This study explains a System for gene transfer in Legionella pneumophila, which is abscent in the recently published Legionella pneumophila genomes. All genes needed for energy dependent transfer of genetic material are included in this gene cluster. Mating experiments show that those genes are able to mobilize DNA in E.coli strains.

In diesem Projekt wurde die Wechselwirkung des PPIase-Enzyms MIP mit Kollagen IV unter- sucht. MIP ist maßgeblich für die Infektiösität von Legionella pneumophila verantwortlich, einem Bakterium, welches im Menschen schwere Lungenentzündungen auslösen kann. Das Enzym zeigt eine hohe Affinität gegenüber einem kurzen Peptidsequenzabschnitt in Kolla- gen IV (genannt „P290”), welches unter anderem im Epithel der Lunge zu finden ist. Die Interaktionsoberfläche der Moleküle wurde durch den Einsatz eines paramagnetischen Spin-Labels in NMR-Experimenten charakterisiert. Mit Hilfe von Docking und Moleküldy- namiksimulationen konnte aus diesen Daten ein Modell des MIP-Kollagen-Komplexes be- rechnet werden. Es wurde gezeigt, dass MIP als Dimer in der Lage ist, nach Kollagen IV zu „greifen” und sich dann an das Molekül heranzuziehen. Wahrscheinlich dient dieser Mechanismus der Adhä- sion von L. pneumophila an die Wirtszelle. Neben der zuvor postulierten Destabilisierung von Kollagen IV durch MIP, welche hier nicht beobachtet wurde, könnte die Adhäsion ein wichtiger Faktor für die Virulenz von L. pneumophila sein. Weiterhin wurde die inhibitorische Wirkung des isolierten Peptids P290 auf die biologische PPIase-Aktivität von MIP untersucht. Durch NMR-Messungen und anschließenden Mole- küldynamiksimulationen konnte gezeigt werden, dass P290 sich stabil in die Bindungsta- sche von MIP einlagert und durch den Sequenzabschnitt -CYS130-PRO131---TRP134- das Enzym blockiert. Die übrigen Aminosäuren in P290 dienen der Stabilisierung des Kom- plexes und sorgen für die Selektivität von P290, welches, im Unterschied zu bekannten Wirkstoffen, das humane Homolog zu MIP nicht inhibiert. Die Vorhersagen der Simulatio- nen konnten durch ein Peptid Microarray und Messungen der enzymatischen Aktivität von MIP in PPIase-Assays bestätigt werden. Die Ergebnisse wurden zur Optimierung von P290 eingesetzt, indem die Peptidsequenz durch den Austausch zweier Aminosäuren verändert und das Molekül zu einem Ring geschlossen wurde. Die entstandene Struktur besitzt deut- lich verbesserte Bindungseigenschaften und könnte künftig als Basis für eine neue Klasse von Wirkstoffen gegen L. pneumophila dienen. In diesem Projekt wurde eine Methode zur Aufklärung der Molekülstruktur neuartiger Wirkstoffe gegen Malaria im Komplex mit ihrem paramagnetischen Zielmolekül etabliert und weiterentwickelt. Die Vorgehensweise leitet intermolekulare Distanzinformationen aus der sog. paramagnetischen Relaxation ab, einem Effekt, der den Einsatz klassischer Me- thoden zur Molekülstrukturaufklärung mittels NMR verhindert. Es werden drei Parameter durch NMR-Spektroskopie bestimmt: 1. die longitudinale Relaxationszeit der Wasserstoff- atome in Wirkstoffmolekül, 2. die effektive Korrelationszeit des Komplexes und 3. der Spin- Zustand des Eisenions im Zielmolekül. Mit Hilfe dieser Messmethode konnte die Komplexstruktur mehrerer bekannter Medika- mente gegen Malaria aufgeklärt werden. Weiterhin wurden zwei neue Klassen von Wirkstof- fen untersucht, die C,C-gekoppelten Naphthylisoquinolin-Alkaloide und die N,C-gekoppelte Naphthylisoquinolin-Alkaloide. In Übereinstimmung mit theoretischen Vorhersagen aus der Literatur lagern sich die Wirkstoffe stets um einen Winkel geneigt und in Richtung des Randes des Zielmoleküls verschoben an. Diese Konfiguration maximiert die attraktiven &#960;- &#960;-Wechselwirkungen zwischen den Molekülen. Aufgrund der gewonnenen Ergebnisse aus NMR, UV-Spektroskopie und Massenspektrome- trie konnte die Existenz eines bisher nicht bekannten Tetramer-Komplexes nachgewiesen werden, welcher eine wichtige Zwischenstufe in der Biokristallisation von Hämozoin durch die Malariaparasiten darstellen könnte, und Ansatzpunkte für den weiterhin nicht vollstän- dig bekannten Wirkmechanismus der meisten Antimalaria-Wirkstoffe liefert. Für die Naphthylisoquinolin-Alkaloide zeigte sich weiterhin, dass Wasser eine essenzielle Rolle in der Komplexbildung spielt. In Moleküldynamiksimulationen der N,C-gekoppelten Naphthylisoquinolin-Alkaloide konnte die Entstehung einer Wasserstoffbrücke zwischen Wirkstoff und Zielmolekül gezeigt werden, welche einen zusätzlichen Weg der Komplex- stabilisierung neben den bereits bekannten &#960;-&#960;-Wechselwirkungen aufzeigt. Die N,C-NIQs konnten erstmals auch bei einem pH-Wert von 5,6 beobachtet werden, einer chemischen Umgebung wie sie auch in-vivo in der Verdauungsvakuole des Malariaparasiten herrscht. / Summary Even in the 21st century, infectious diseases remain the predominant cause of death world- wide. According to reports of the World Health Organisation, 2 million people die of Malaria every year, most of which are children under the age of five years. Respiratory infections claim an additional 3.9 million lives. Other infections are held responsible for a total of more than 10 million deaths. Global climate change leads to the occurrence of tropical in- fections well beyond their former endemic regions. Additional challenges arise due to the growing number of resistant organisms, rendering most known treatments ineffective. To achieve sustained success in the fight against infectious diseases, a detailed understan- ding on the mode of action of newly developed substances on a molecular level is essential. In this thesis, magnetic resonance spectroscopy is used as a tool for molecular structure determination. My results may offer incentives for the development of new agents against infectious diseases and their continuous optimization. 4.1 The MIP-collagen IV complex The scope of this project was to investigate the interaction between the PPIase enzyme MIP and the NC1 (non-collagenous 1) domain of collagen IV. The MIP (macrophage infectivity potentiator) protein is the major virulence factor of Legionella pneumophila, a bacterium causing severe lung infections in humans. MIP exhibits high affinity towards a short peptide sequence in collagen IV (“P290”). Amongst others, this type of collagen is found in the epithelial cells of the lung. In this work, the interface of interaction between P290 and MIP was mapped using a pa- ramagnetic spin label in combination with nuclear magnetic resonance spectroscopy expe- riments. Labeled P290 strongly enhances the relaxation rates of individual amino acids in MIP, which are in the immediate vicinity (within 1 nm) of the spin label. The enhancedrelaxation rates were detected through T2-sensitive HSQC experiments. Subsequently, re- sults were incorporated in docking and molecular dynamic (MD) simulations to compute a model of the MIP-collagen IV complex. Results show the MIP dimer “grabbing” collagen IV with both enzymatic domains and pul- ling the molecules closer together. We suggest that this molecular adhesion mechanism may play a key role in the invasion of host tissue by L. pneumophila. A possible destabilization of collagen IV through the enzymatic activity of MIP, as suggested previously by other groups, was not observed. Additionally, our co-operation partners were able to demonstrate that P290, as an indi- vidual peptide, inhibits the biological PPIase activity of MIP, while leaving human homo- logue enzymes untouched. My findings from NMR measurements and subsequent MD si- mulations showed that P290 occupies the MIP binding pocket via the amino acid sequence -CYS130-PRO131---TRP134-. This sequence element is stabilized via the attachment of the terminal residues of P290 to the surface of MIP, thereby enabling P290 to distinguish between MIP and human enzymes. Based on these results, we constructed optimized versions of P290 by ring closure and repla- cement of two amino acids. Our co-operation partners showed that the resulting structures exhibit improved binding properties on a peptide microarray and may provide the basis for a new class of inhibitors targeting Legionella pneumophila. 4.2 Structure elucidation of paramagnetic complexes for- med by novel antimalarial agents We used paramagnetic NMR spectroscopy to characterize the formation of complexes of several antimalarial compounds with their presumed target “heme”. A paramagnetic Fe(III) ion is located at the center of heme, which influences the longitudinal relaxation rates of nearby proton spins. This effect interferes with common strategies for NMR structure elucidation, but in this study was taken advantage of in a newly developed method to map intermolecular distances with high precision using NMR inversion recovery experiments at 9.4 T, 14.1 T, 17.6 T, and 18.8 T. This method was utilized to solve the molecular structure of known drugs against Mala- ria as well as two new classes of antimalarial agents (the C,C-coupled naphthylisoquinoline alkaloids and the N,C-coupled naphthylisoquinoline alkaloids) in complex with their target molecule: heme. In accordance with theoretical predictions from the literature, we sho- wed that the drug molecules align in a configuration maximizing attractive &#960;-&#960; stacking interactions between the molecules. In combination with findings from NMR, UV spectroscopy and mass spectrometry, we de- monstrated the formation of a previously unknown tetrameric complex. This complex may represent an important step in the mode of action of antimalarial drugs. Additionally, results from NMR measurements and molecular dynamics simulations provided insight into the important role of H2O for complex stabilization. We were able to demonstrate the formation of a so far undescribed hydrogen bond between drug and target. Furthermore, it was possible to investigate the N,C-coupled naphthylisoquinoline alkaloids at pH 5.6, which exactly matches the chemical environment in the food vacuole of the ma- larial parasite in-vivo. All these findings may contribute to a deeper understanding of the mode of action of new antimalarial agents.

Die vorliegende Arbeit thematisiert das computergestützte strukturbasierte Design auf dem Gebiet der HIV-1-Integrase und des Macrophage Infectivity Potentiator (MIP) von Legionella pneumophila. Die durchgeführten Studien geben wertvolle Aufschlüsse über den Wirk-mechanismus einer bekannten Integrase-Inhibitorenklasse and zeigt darüber hinaus einen neuartigen Ansatz zur Integrase-Inhibition auf. Im Falle des MIP-Enzyms konnten zwei niedermolekulare Inhibitoren ermittelt werden. Die Integrase-Studien ergaben wertvolle Informationen im Hinblick auf das Design neuer Inhibitoren. Docking-Experimente konnten die Hypothese weiter untermauern, nach der die Klasse der Diketosäure-Inhibitoren nicht als freie Liganden, sondern als Metallion-Komplexe an das aktive Zentrum der Integrase binden. Die Ergebnisse dieser Studie helfen dabei, das Verständnis über den Wirkmechanismus dieser wichtigen Klasse von Integrase-Inhibitoren weiter zu vertiefen. Um der Entwicklung von Integrase-Inhibitoren einen neuen Impuls zu geben, wurde eine neue Strategie zur Inhibition dargelegt: Anstatt an das aktive Zentrum soll eine neue Inhibitor-Klasse an das Dimerisierungs-Interface eines Integrase-Monomers binden, die katalytisch notwendige Dimerisierung verhindern und somit die enzymatische Aktivität stören. Das Hauptproblem hierbei bestand in den fehlenden Strukturdaten des freien Monomers. Hierzu wurden Molekulardynamik-Simulationen durchgeführt, um nähere strukturelle Informationen zu erhalten. Momentaufnahmen unterschiedlicher Konformationen dienten als Input-Strukturen für eine Docking-Studie mit dem peptidischen Inhibitor YFLLKL, um dessen Bindemodus aufzuklären. Hierbei zeigte sich, dass dieser Ligand an eine Interface-Konformation bindet, die durch eine Y-förmige Bindestelle charakterisiert ist. Im nächsten Schritt sollte diese Protein-Konformation mit kleinen, nicht-peptidischen Molekülen adressiert werden. Die erste Strategie bestand darin, ein Pharmakophor-Modell zu erstellen, das zur Suche nach Molekülen mit einer guten Komplementarität zur Y-förmigen Bindetasche geeignet ist. Das folgende virtuelle Screening ergab zehn Verbindungen, die eine gute Komplementarität und günstige hydrophobe Wechselwirkungen aufwiesen. Leider zeigte keine der Verbindungen eine reproduzierbare Aktivität im Integrase-Assay. Hierbei verbleiben jedoch gewisse Zweifel, da in dem Assay die Zugabe von BSA vorgeschrieben war, das möglicherweise die hydrophoben Inhibitor-Kandidaten gebunden hat. Die erwähnte erste Strategie wurde überdacht: In einem zweiten Ansatz galt die Hauptaufmerksamkeit der Absättigung von wasserstoffbrückenbildenden Resten. Diese waren zuvor von den eher hydrophoben Verbindungen nicht optimal abgesättigt worden. Zwei Pharmakophor-Modelle wurden erstellt und in einem virtuellen Screening eingesetzt: Docking-Studien der Hits zeigten jedoch, dass nach wie vor viele wasserstoffbrückenbildende Reste des Proteins nicht vom Liganden abgesättigt wurden. Nach abschließender eingehender Betrachtung der Bindemoden der verbliebenen Moleküle aus dem virtuellen Screening konnten nur acht für weitere Testungen ausgewählt werden (Ergebnisse der experimentellen Testung durch Kooperationspartner stehen noch aus). Diese geringe „Ausbeute“ an geeigneten Verbindungen für das Integrase-Dimerisierungsinterface zeigt, wie schwer dieses Target zu adressieren ist: Das Interface weist eine schnell wechselnde Abfolge von basischen, sauren und hydrophoben Resten auf. Im Gegensatz zu anderen Protein-Protein-Interfaces zeigt das Integrase-Interface keine „aufgeräumte“ Bindetasche mit klar voneinander getrennten hydrophoben und hydrophilen Bereichen. Für das zweite Enzym, MIP, konnten mit Hilfe des strukturbasierten Designs zwei niedermolekulare Inhibitoren gefunden werden. Beide Verbindungen führten zu einer deutlichen Abnahme der katalytischen Aktivität. Soweit bekannt, sind bisher keinerlei niedermolekulare MIP-Inhibitoren veröffentlicht. Der Vergleich von MIP mit der humanen PPIase FKBP12 zeigte eine größtenteils ähnliche Tasche, die jedoch einen entscheidenden Unterschied aufweist, nämlich in der Orientierung des Restes Tyr109. Die detaillierte Betrachtung der Strukturdaten beider Enzyme konnte schließlich eine Erklärung liefern, warum ein ketoacyl-substituiertes Pipecolinderivat nicht an MIP bindet, ein sulfonsubstituiertes Pipecolinderivat hingegen das Enzym inhibiert. Die Erkenntnisse über das Inhibitoren-Design für Legionella-MIP können auch auf andere Organismen (z.B. Trypanosomen) übertragen werden, bei denen ebenfalls (homologes) MIP ein Pathogenitätsfaktor ist. / In this thesis, computational structure-based design approaches were employed to target the HIV-1 integrase and the macrophage infectivity potentiator (MIP) of Legionella pneumophila. The thesis yields valuable information about the mechanism of action of a known class of integrase inhibitors and a novel approach towards enzyme inhibition, which still is mainly unaddressed in current integrase research. For the MIP enzyme, two small-molecule MIP inhibitors were discovered. The computational studies of HIV-1 integrase have provided valuable information for IN inhibitor design. Docking experiments supported the hypothesis that the well-known diketo acid inhibitors enter the IN active site not as free ligands, but rather as metal complexes. These results help to reveal the mechanism of action of this important class of IN inhibitors.To give an impulse for the development of a novel class of inhibitors, a new strategy towards IN inhibition was introduced: An alternative binding site, the dimerization interface of an IN catalytic core domain monomer, was explored for inhibitor design. The lack of structural data of the free monomer was overcome by extensive MD studies. Snapshots derived from the MD simulation were used as protein input structures in a docking study with the inhibitory peptide YFLLKL to reveal its potential binding mode. The docking procedure showed that the peptidic ligand binds to a dimerization interface conformation which shows a Y-shaped binding site.. The next step was to address this protein conformation with small, non-peptidic molecules. The first strategy towards finding small-molecule interface binders was to create a pharmacophore model with hydrophobic features and shape constraints, aiming to find molecules with a good complementarity to the Y-shaped dimerization interface. Virtual screening yielded a total of 10 compounds, which all displayed good shape complementarity and favorable hydrophobic interactions. Unfortunately, none of the compounds showed a reproducible inhibitory activity in biological assays. Some doubts remain about the validity of the assay results: The use of BSA was critical, since it is not unlikely that BSA “intercepted” the hydrophobic candidate compounds. The first strategy towards finding small-molecule dimerization inhibitors was reconsidered: In the second approach, the satisfaction of hydrogen bonding residues at the dimerization interface, was of major interest. Two pharmacophore models were employed, which retrieved several hundred hit molecules. However, docking of these molecules showed that still many hydrogen bonding groups of the protein remained unaddressed by the ligands. Eventually, after visual inspection, only eight molecules were selected as candidate compounds for further testing (results pending). This small “yield” underlines the difficulties in finding interface binders: The IN dimerization interface is a peculiar target with frequently alternating basic, acidic, and hydrophobic residues. It is not a well-ordered binding site with continuous hydrophobic areas and distinct hydrogen bond donors / acceptors. Other protein-protein interfaces show such well-ordered binding sites. Accordingly, the peculiarity of the IN dimerization interface, in addition to the delicate task of disrupting protein-protein interactions at all, makes the development of IN dimerization inhibitors very challenging. For MIP, the studies revealed two experimentally validated MIP inhibitors, which significantly reduce MIP enzymatic activity. To our knowledge, no small-molecule MIP inhibitor has been reported in the literature so far. A detailed analysis of the available structural data of MIP and a comparison to the human PPIase counterpart, FKBP12, pointed out a conformational diversity among the MIP structures and a crucial difference between the two PPIases, which could be traced to mainly one residue (Tyr109). The detailed comparison of FKBP12 and MIP complex structures made it possible to give an explanation, why a ketoacyl-substituted pipecoline derivative most probably does not bind to MIP, but a sulfone-substituted pipecoline derivative does bind to MIP. Knowledge of Legionella MIP inhibitors could be transferred also to other organisms (e.g. trypanosoms), where homologous MIP proteins are also pathological factors.

<p>The bacterial pathogen <i>Legionella pneumophila</i> modulates host immunity using effectors translocated by its Dot/Icm transporter to facilitate its intracellular replication. A number of these effectors employ diverse mechanisms to interfere with protein ubiquitination, a post-translational modification essential for immunity. Here, we have found that <i>L. pneumophila</i> induces monoubiquitination of the E2 enzyme UBE2N by its Dot/Icm substrate MavC(Lpg2147). Ubiquitination of UBE2N by MavC abolishes its activity in the formation of K63-linked polyubiquitin chains, which dampens NF-kB signaling in the initial phase of bacterial infection. The inhibition of UBE2N activity by MavC creates a conundrum because this E2 enzyme is important in multiple signaling pathways, including some that are important for intracellular <i>L. pneumophila</i> replication. Here we also show that the activity of UBE2N is restored by MvcA(Lpg2148), an ortholog of MavC. MvcA functions to deubiquitinate UBE2N-Ub using the same catalytic triad required for its deamidase activity. Structural analysis of the MvcA-UBE2N-Ub complex reveals a crucial role of the insertion domain in MvcA in substrate recognition. Our findings reveal that two remarkably similar proteins catalyze the forward and reverse reactions to impose temporal regulation of the activity of UBE2N during <i>L. pneumophila</i> infection.</p>