December 11, 2017 – 17.15 h | Seminar room N260/3.13
Abstract: Acinetobacter baumannii is an important nosocomial pathogen that accounts for a significant percentage of infections in intensive care units worldwide. Furthermore, A. baumannii strains have emerged that are resistant to all available antimicrobials. These facts highlight the need for new therapeutic strategies to combat this growing public health threat. Given the critical role for transition metals at the pathogen-host interface, interrogating the role for these metals in A. baumannii physiology and pathogenesis could elucidate novel therapeutic strategies. Toward this end, my laboratory is interrogating the impact of host metal binding proteins in defense against A. baumannii pneumonia, and the bacterial factors that compete with this powerful host defense. In particular, we are interested in the neutrophil protein calprotectin (Cp) that inhibits microbial growth through the chelation of nutrient manganese (Mn) and zinc (Zn). We have found that CP accompanies neutrophil recruitment to the lung and accumulates at foci of infection in a murine model of A. baumannii pneumonia. CP contributes to host survival and control of bacterial replication in the lung and limits dissemination to secondary sites. Furthermore, we discovered that A. baumannii coordinates transcription of an NRAMP family Mn transporter and a urea carboxylase to resist the antimicrobial activities of metal chelation. We have also found that this system combats CP in vivo. These findings reveal that A. baumannii has evolved mechanisms to subvert host-mediated metal sequestration and they uncover a connection between metal starvation and metabolic stress.
When: December 11, 2017 – 17.15 h
Where: Seminar room N260/3.13
Related Publication: Kinsella RL, Lopez J, Palmer LD, Salinas ND, Skaar EP, Tolia NH, Feldman MF. Defining the interaction of the protease CpaA with its type II secretion-chaperone CpaB and its contribution to virulence in Acinetobacter species. The Journal of biological chemistry. 2017 Oct 5. PMID: 28982978 [PubMed]
The source of genomic innovation in the human pathogen Acinetobacter baumannii
In the proposed project we aim at delineating the factors that have transformed A. baumannii from an environmental bacterium into a potent human pathogen. We will begin with setting up the bioinformatics infrastructure facilitating an automated establishment of function-aware phylogenetic profiles for proteins. In parallel we will use comparative genomics between successful epidemic clonal lineages and non-epidemic lineages complemented with data from non-pathogenic close relatives of A. baumannii to investigate the general evolution of gene repertoires in this genus. This will obtain for each gene in the pan-genome of Acinetobacter information about its phylogenetic distribution in the genus, of when it has been introduced into the genus and of its propensity for gene loss. Integrating the results with functional annotations of Acinetobacter genes and their involvement in functional networks will form the foundation of linking phenotypic characteristics of the various Acinetobacter species and strains to their genotype. We expect that this novel catalogue will form an unprecedented basis for delineating the genetic network of A. baumannii virulence. We will then proceed with integrating the pan-genome of Acinetobacter into a proteinfunction-aware phylogenomic network connecting this genus to an exhaustive collection of bacteria, archaea and eukaryotes. In this network we will identify edges connecting proteins that most likely share the same biochemical activity. This will allow, in particular, the tracing of virulence related factors, e.g. efflux pumps, motility functions, and functional protein networks, such as the A. baylyi natural transformation system or metabolic pathways across species. We will then investigate the evolutionary origins of A. baumannii virulence factors and the routes these factors have taken to enter the A. baumannii genome. More precisely, we will address the question how A. baumannii mines environmental genetic diversity for adaptive genes. We will concentrate on investigating the role of natural competence as the most versatile source of genetic information for bacterial evolution. To this end, we will establish phylogenetic profiles of the known natural competence systems to investigate their evolutionary history and to assess their prevalence in contemporary bacteria. In a complementary approach we will investigate the footprint of natural competence in the genomes of competent bacteria. We will assess prevalence, distribution and origin of laterally acquired genes in bacteria competent under a broad variety of conditions, relative to that in bacteria with inducible competence and non-competent bacteria. Both results in combination will contribute to the understanding to what extent A. baumannii makes use of its facultative natural competence for acquiring novel genes. These analyses will form the basis for an initial model of genetic innovation flux into and within the system A. baumannii.
Prof. Dr. Ingo Ebersberger
Bardya Djahanschiri (PhD student)
Research Interest: A comparative approach to trace the genetic basis of A. baumannii
Sachli Zafari (PhD student)
Research Interest: The role of natural competence in bacterial evolution
Ruben Iruegas (Master student)
Research Interest: The role of natural competence in bacterial evolution
Role of AdeRS and EsvA in the regulation of multidrug resistance in A. baumannii
Antibiotic resistance in bacteria is fast becoming one of the biggest threats to modern medicine; in the hospital setting modern medicine has introduced a subgroup of patients that are severely immunocompromised and subsequently very susceptible to infection. This has led to an ever increasing use of antimicrobials, with a corresponding selective pressure, followed by the emergence of so called “super bugs” that are resistant to most, if not all, available antimicrobials. The era of untreatable infections is now upon us. It is in this background that Acinetobacter baumannii has emerged as a serious hospital-acquired pathogen.
A. baumannii has gained notoriety for its multi-drug (MDR), pan-drug, and in some cases extreme-drug resistance, with carbapenem- and colistin-resistance causing the greatest concern. Now we are faced with an organism that is sometimes untreatable. This MDR phenotype is strongly associated with particular clonal lineages, and is mediated through acquired mechanisms such as Class-D oxacillinases, and mutations in genes encoding the antibiotic targets. In addition there are intrinsic mechanisms such as drug efflux combined with reduced permeability. Of the several different classes of efflux pumps, in Gram-negative microorganisms the RND (resistance nodulation and cell division) pumps pose the greatest threat. These tri-partite pumps span the inner and outer membranes and typically have a broad substrate specificity and their over-expression can lead to an MDR phenotype; resistance to fluoroquinolones, beta-lactams, tetracyclines, aminoglycosides, macrolides and trimethoprim as well as antiseptics and disinfectants like triclosan and benzalkonium chloride.
Currently there are three RND pumps characterized in A. baumannii: AdeABC, AdeFGH and AdeIJK. Expression of these pumps are regulated by a two-component regulatory system (AdeRS), a LysR-type transcriptional regulator (AdeL) or a TetR type regulator (AdeN), respectively. Analysis of A. baumannii genomes reveals there are several uncharacterised RND efflux pump systems.
Our goal is to investigate these RND efflux pumps, looking at their regulation and expression in clinical isolates, and to determine their relative contribution to the MDR phenotype.
Dr. Paul G. Higgins
Prof. Dr. Harald Seifert
Stefanie Gerson (PhD student)
Research Interest: Investigating the mechanisms of tigecycline and colistin resistance in A. baumannii
Coordinated network of multidrug resistance transporters in Acinetobacter baumannii
Multidrug resistant bacteria survive and adapt to hostile environments like hospitals where the use of antibiotics strongly selects for organisms, which have acquired multiple defense mechanisms against these toxic compounds. The rise of Gram-negative pathogens like Acinetobacter baumannii causing serious infections in immune-compromised patients is a major concern in hospitals. The first line of defense in the adaptation strategy of Gram-negative bacteria is the expression of antibiotic efflux genes, resulting in the equipment of the inner and outer membrane with transporters and channels, which catalyze the concerted efflux of toxic compounds. The combined effort of efflux pumps from members of four major transporter families designated Resistance Nodulation and Cell Division (RND) Superfamily, Multi Antimicrobial Extrusion family (MATE), the Major Facilitator Superfamily (MFS) and the Small Multidrug Resistance (SMR) family leads to the observed resistance against multiple drugs including various classes of antibiotics, detergents and dyes. The A. baumannii AYE genome contains from all four families 4, 3, 4 and 5 member genes, respectively, which are expected to act in concert to produce the observed resistance phenotype. We plan to address the combined role and interdependency of different sets of drug transporters in vivo and in vitro and in addition embark on the structural elucidation of transporters, which can be considered main players in the multiple drug resistance phenotype.
Prof. Dr. Klaas Martinus Pos
Wuen Ee (Wendy) Foong (PhD student)
Research Interest: Structure and function of multidrug efflux in A. baumannii
Surface-associated motility of Acinetobacter baumannii
Colonization of surface environments is of the outmost importance in the bacterial world, notably also during infection. In many cases, surface-associated motility facilitates initial stages of biofilm formation, a well-known capability of the nosocomial pathogen Acinetobacter baumannii which can contribute to persistence in the hospital environment, increased antibiotic resistance or protection from immune responses. Bacterial communities also express particular traits while moving to colonize new habitats. For instance, swarmer cells have been shown to exhibit reduced susceptibility to antibiotics. Motility is thus considered as part of the intrinsic resistance endowment of bacterial pathogens. Moreover, motility can significantly contribute to virulence of bacteria. Of further interest, we could recently demonstrate that many isolates of A. baumannii become naturally competent while they move along surfaces. This transformability of A. baumannii might lead to acquisition of novel genetic traits including virulence and antibiotic resistance determinants. We recently established that a flagella-independent, surface-associated form of motility is a common trait of clinical isolates of A. baumannii. This widespread ability could play a central role in the establishment of infection, in persistence and acquisition of resistance. We aim at elucidating the molecular basis of this motility by characterizing a collection of motility-deficient mutants. Notably, we intend to study in detail the role of the polyamine 1,3-diaminopropane (DAP) which we found to play an essential role in motility. We reason about additional important functions of DAP given that it is the predominant polyamine found in members of the genus Acinetobacter. Taken together, we expect that elucidating surface-associated motility of A. baumannii will contribute to our understanding of the pathogen-host interaction, to persistence in the hospital environment and to antibiotic resistance development.
PD Dr. Gottfried Wilharm
Ulrike Blaschke (PhD student)
Research Interest: Surface-associated motility of A. baumannii
Magdalena Heindorf (PhD student)
Research Interest: Contribution of sulfite reductase to antibiotic resistance and virulence in A. baumannii
Analysis of Acinetobacter trimeric adhesin (Ata) in multi-drug resistant clinical ilsolates and its contribution to the pathogenic potential of A. baumannii
Pathogenic bacteria specifically adhere to cell and tissue surfaces in their hosts to initiate infection. After invasion, several virulence mechanisms support persistent infection and bacterial dissemination. Adhesion to host cells and proteins of the extracellular matrix is mediated by the interaction of bacterial adhesins with corresponding host cell ligands.
Trimeric autotransporter adhesins (TAAs) are important pathogenicity factors of Gram-negative bacteria. Recently, we identified TAAs in Acinetobacter spp. by in silico screening methods. The TAA of Acinetobacter baumannii, Acinetobacter trimeric autoransporter (Ata), is modularly constructed consisting of head, stalk and membrane anchor domains with a duplication of the head domain and neck-stalk regions of different lengths. Ata is expressed on the surface of A. baumannii, mediates adherence to matrix proteins and is necessary for pathogenicity in several in vitro and in vivo infection models.
The explanation for the successful spread of multidrug-resistant A. baumannii in hospitals is assumed, primarily, to be associated with the inability to adequately treat infections caused by these multidrug-resistant bacteria and only to a lesser degree due to an increase in virulence. However, there is clearly a lack of knowledge whether and how multidrug-resistance affects pathogenicity of A. baumannii.
Our studies (i) will help to understand to what extend Ata contributes to the pathogenicity of multidrug-resistant A. baumannii and (ii) might lead to new, innovative therapeutic concepts for these difficult to treat multi-resistant pathogens (e.g., by blocking the bacterial adhesion to the host with “antiligands”).
Prof. Dr. med Volkhard A. J. Kempf
Dr. med. Stephan Göttig
Marko Weidensdorfer (PhD student)
Research Interest: Functional characterization of the trimeric autotransporter adhesin Ata in A. baumannii upon infection of the human host
Sara Riedel-Christ (technician)
Research Interest: Investigating antibiotic resistance and pathogenicity of clinical Acinetobacter isolates
Metabolic adaptation of Acinetobacter baumannii – role of phospholipids in nutrition and infection
Membranous environments are colonized by various strains of A. baumannii, which raises the question on how A. baumannii manages to adapt its metabolism to the skin environment. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are the most abundant phospholipids found in human cell membranes and make them good candidates as carbon and energy source. Initial degradation of PC or PE is facilitated by a set of different phospholipases whose substrate specificity is determined by the acyl side chains and the hydrophilic group. A. baumannii ATCC 19606 encodes six phospholipases belonging to the phospholipase superfamilies PLA, PLC and PLD. Two of them are known from the literature as virulence factors. To elucidate the function of all phospholipases they will be produced in E. coli, purified and their enzymatic properties will be determined. Transcriptional and immunological analyses will identify conditions under which the encoding genes are expressed. Phospholipase mutants will be generated using the established markerless mutagenesis strategy and mutant studies will unravel the role of the phospholipases in phospholipid degradation, survival of in cell culture, adhesion to eukaryotic cells and complement resistance as well as their role in cytotoxicity on prokaryotic and eukaryotic cells. Furthermore, we aim to identify and characterize phospholipase exporters by genetic and biochemical techniques. Our own preparatory work demonstrated that A. baumannii takes up the PC cleavage product choline and oxidizes it to glycine betaine. The genome of A. baumannii 19606 predicts a two-step oxidation pathway, a transcriptional regulator and two choline transporters (BetT1 and BetT2). BetT1 in A. baylyi was found to facilitate osmolarity-independent choline transport, most likely by an uniport mechanism. Analogously BetT1 in A. baumannii might play a role in metabolic adaptation to choline rich environments. To analyze the role of the choline transporters and choline oxidation in metabolic adaptation and pathogenicity of A. baumannii the encoding genes will be deleted. Mutant studies will be performed to elucidate the biochemistry of choline transport and to determine the role in virulence in cell cultures.
Another focus will be to identify the substrate of the transcriptional regulator BetI and study its mode of interaction with the DNA. To unravel the molecular transport mechanism of H+-coupled choline symport and proton-independent choline uniport in the BCCT family we will purify both choline transporters and attempt to solve their atomic structures by X-ray crystallography on 3D crystals in a high-throughput process. Furthermore, we will investigate their interactions with specific lipids by 2D crystallization and cryo-electron microscopy. To elucidate the transport mechanism we will perform transport experiments in vivo and in vitro in cells proteoliposomes, respectively, as well as binding studies by ITC and TRP-fluorescence.
Prof. Dr. Beate Averhoff
Prof. Dr. Christine Ziegler
Jennifer Breisch (PhD student)
Research Interest: Role of BCC transporter in virulence of Acinetobacter baumannii
Katharina Pfefferle (Master student)
Research Interest: Contribution of phospholipases to virulence of Acinetobacter baumannii