Genetic Approaches for Identifying and Characterizing Effectors in Bacterial Pathogens.

Microbial pathogens have coevolved with their hosts, often for millions of years, and in the process have developed a variety of virulence mechanisms to ensure their survival, typically at the host's expense. At the center of this host-pathogen warfare are proteins called effectors that are delivered by bacteria into their host where they alter the intracellular environment to promote bacterial proliferation. Many effectors are believed to have been acquired by the bacteria from their host during evolution, explaining why researchers are keen to understand their function, as this information may provide insight into both microbial virulence strategies and biological processes that happen within our own cells.

The Legionella pneumophila effector DenR hijacks the host NRas proto-oncoprotein to downregulate MAPK signaling.

Small GTPases of the Ras subfamily are best known for their role as proto-oncoproteins, while their function during microbial infection has remained elusive. Here, we show that Legionella pneumophila hijacks the small GTPase NRas to the Legionella-containing vacuole (LCV) surface. A CRISPR interference screen identifies a single L.

A randomized multiplex CRISPRi-Seq approach for the identification of critical combinations of genes.

Identifying virulence-critical genes from pathogens is often limited by functional redundancy. To rapidly interrogate the contributions of combinations of genes to a biological outcome, we have developed a ltiplex, andomized RISPR nterference equencing (MuRCiS) approach. At its center is a new method for the randomized self-assembly of CRISPR arrays from synthetic oligonucleotide pairs.

A randomized multiplex CRISPRi-Seq approach for the identification of critical combinations of genes.

Identifying virulence-critical genes from pathogens is often limited by functional redundancy. To rapidly interrogate the contributions of combinations of genes to a biological outcome, we have developed a multiplex, randomized CRISPR interference sequencing (MuRCiS) approach. At its center is a new method for the randomized self-assembly of CRISPR arrays from synthetic oligonucleotide pairs.

VpdC is a ubiquitin-activated phospholipase effector that regulates vacuole expansion during infection.

Intravacuolar pathogens need to gradually expand their surrounding vacuole to accommodate the growing number of bacterial offspring during intracellular replication. Here we found that controls vacuole expansion by fine-tuning the generation of lysophospholipids within the vacuolar membrane. Upon allosteric activation by binding to host ubiquitin, the type IVB (Dot/Icm) effector VpdC converts phospholipids into lysophospholipids which, at moderate concentrations, are known to promote membrane fusion but block it at elevated levels by generating excessive positive membrane curvature.

A Comprehensive Phenotypic Screening Strategy to Identify Modulators of Cargo Translocation by the Bacterial Type IVB Secretion System.

Bacterial type IV secretion systems (T4SSs) are macromolecular machines that translocate effector proteins across multiple membranes into infected host cells. Loss of function mutations in genes encoding protein components of the T4SS render bacteria avirulent, highlighting the attractiveness of T4SSs as drug targets. Here, we designed an automated high-throughput screening approach for the identification of compounds that interfere with the delivery of a reporter-effector fusion protein from Legionella pneumophila into RAW264.

A multiplex CRISPR interference tool for virulence gene interrogation in Legionella pneumophila.

Catalytically inactive dCas9 imposes transcriptional gene repression by sterically precluding RNA polymerase activity at a given gene to which it was directed by CRISPR (cr)RNAs. This gene silencing technology, known as CRISPR interference (CRISPRi), has been employed in various bacterial species to interrogate genes, mostly individually or in pairs. Here, we developed a multiplex CRISPRi platform in the pathogen Legionella pneumophila capable of silencing up to ten genes simultaneously.

Structural insight into the membrane targeting domain of the Legionella deAMPylase SidD.

AMPylation, the post-translational modification with adenosine monophosphate (AMP), is catalyzed by effector proteins from a variety of pathogens. Legionella pneumophila is thus far the only known pathogen that, in addition to encoding an AMPylase (SidM/DrrA), also encodes a deAMPylase, called SidD, that reverses SidM-mediated AMPylation of the vesicle transport GTPase Rab1. DeAMPylation is catalyzed by the N-terminal phosphatase-like domain of SidD.

The kinase LegK7 exploits the Hippo pathway scaffold protein MOB1A for allostery and substrate phosphorylation.

During infection, the bacterial pathogen manipulates a variety of host cell signaling pathways, including the Hippo pathway which controls cell proliferation and differentiation in eukaryotes. Our previous studies revealed that encodes the effector kinase LegK7 which phosphorylates MOB1A, a highly conserved scaffold protein of the Hippo pathway. Here, we show that MOB1A, in addition to being a substrate of LegK7, also functions as an allosteric activator of its kinase activity.

The Legionella Effector Kinase LegK7 Hijacks the Host Hippo Pathway to Promote Infection.

The intracellular pathogen Legionella pneumophila encodes translocated effector proteins that modify host cell processes to support bacterial survival and growth. Here, we show that the L. pneumophila effector protein LegK7 hijacks the conserved Hippo signaling pathway by molecularly mimicking host Hippo kinase (MST1 in mammals), which is the key regulator of pathway activation.

Legionella effector AnkX interacts with host nuclear protein PLEKHN1.

Background: The intracellular bacterial pathogen Legionella pneumophila proliferates in human alveolar macrophages, resulting in a severe pneumonia termed Legionnaires' disease. Throughout the course of infection, L. pneumophila remains enclosed in a specialized membrane compartment that evades fusion with lysosomes.

RavN is a member of a previously unrecognized group of Legionella pneumophila E3 ubiquitin ligases.

The eukaryotic ubiquitylation machinery catalyzes the covalent attachment of the small protein modifier ubiquitin to cellular target proteins in order to alter their fate. Microbial pathogens exploit this post-translational modification process by encoding molecular mimics of E3 ubiquitin ligases, eukaryotic enzymes that catalyze the final step in the ubiquitylation cascade. Here, we show that the Legionella pneumophila effector protein RavN belongs to a growing class of bacterial proteins that mimic host cell E3 ligases to exploit the ubiquitylation pathway.

Molecular mechanism for the subversion of the retromer coat by the effector RidL.

Microbial pathogens employ sophisticated virulence strategies to cause infections in humans. The intracellular pathogen encodes RidL to hijack the host scaffold protein VPS29, a component of retromer and retriever complexes critical for endosomal cargo recycling. Here, we determined the crystal structure of RidL in complex with the human VPS29-VPS35 retromer subcomplex.

Exploitation of the host cell ubiquitin machinery by microbial effector proteins.

Pathogenic bacteria are in a constant battle for survival with their host. In order to gain a competitive edge, they employ a variety of sophisticated strategies that allow them to modify conserved host cell processes in ways that favor bacterial survival and growth. Ubiquitylation, the covalent attachment of the small modifier ubiquitin to target proteins, is such a pathway.

Discovering Protein-Protein Interactions Using Nucleic Acid Programmable Protein Arrays.

We have developed a protocol enabling the study of protein-protein interactions (PPIs) at the proteome level using in vitro-synthesized proteins. Assay preparation requires molecular cloning of the query gene into a vector that supports in vitro transcription/translation (IVTT) and appends a HaloTag to the query protein of interest. In parallel, protein microarrays are prepared by printing plasmids encoding glutathione S-transferase (GST)-tagged target proteins onto a carrier matrix/glass slide coated with antibody directed against GST.

Cardiac reoperations in octogenarians: Do they really benefit?

Aim: According to the demographic development of our society, the numbers of octogenarians referred to cardiac surgery are continuously growing. Although the benefit of first-time cardiac procedures for these patients is well documented, the fate of octogenarians after redo-procedures, with special regard to long-term survival, functional status and quality of life, is poorly investigated.

Methods: We retrospectively identified 84 consecutive patients aged ≥80 years, who underwent a cardiac reoperation at the department for Cardiothoracic Surgery in the Heart & Vessel Center Bad Bevensen between January 2007 and 2013.

Infection biology: Small RNA with a large impact.

A simultaneous comparison of the RNA molecules expressed by Salmonella bacteria and human cells during infection reveals how a bacterial small RNA alters the transcript profiles of both the bacteria and the host cells.

Host Cell-catalyzed S-Palmitoylation Mediates Golgi Targeting of the Legionella Ubiquitin Ligase GobX.

The facultative intracellular pathogen Legionella pneumophila, the causative agent of Legionnaires disease, infects and replicates within human alveolar macrophages. L. pneumophila delivers almost 300 effector proteins into the besieged host cell that alter signaling cascades and create conditions that favor intracellular bacterial survival.

Host-pathogen interaction profiling using self-assembling human protein arrays.

Host-pathogen protein interactions are fundamental to every microbial infection, yet their identification has remained challenging due to the lack of simple detection tools that avoid abundance biases while providing an open format for experimental modifications. Here, we applied the Nucleic Acid-Programmable Protein Array and a HaloTag-Halo ligand detection system to determine the interaction network of Legionella pneumophila effectors (SidM and LidA) with 10 000 unique human proteins. We identified known targets of these L.

Structural basis for the recruitment and activation of the Legionella phospholipase VipD by the host GTPase Rab5.

A challenge for microbial pathogens is to assure that their translocated effector proteins target only the correct host cell compartment during infection. The Legionella pneumophila effector vacuolar protein sorting inhibitor protein D (VipD) localizes to early endosomal membranes and alters their lipid and protein composition, thereby protecting the pathogen from endosomal fusion. This process requires the phospholipase A1 (PLA1) activity of VipD that is triggered specifically on VipD binding to the host cell GTPase Rab5, a key regulator of endosomes.

Distortion of a hybrid stent graft following a frozen elephant trunk procedure.

We describe a case of an acute type A dissection, where technical problems during the frozen elephant trunk technique led to a distortion of the hybrid stent graft with severe stenosis of the thoracic aortic endoprosthesis. Interventional aortoplasty was performed to re-establish flow. This new technique bears some risk of technical failure and therefore should be applied only after careful considerations.

A novel probe for phosphatidylinositol 4-phosphate reveals multiple pools beyond the Golgi.

Polyphosphoinositides are an important class of lipid that recruit specific effector proteins to organelle membranes. One member, phosphatidylinositol 4-phosphate (PtdIns4P) has been localized to Golgi membranes based on the distribution of lipid binding modules from PtdIns4P effector proteins. However, these probes may be biased by additional interactions with other Golgi-specific determinants.

VipD is a Rab5-activated phospholipase A1 that protects Legionella pneumophila from endosomal fusion.

A crucial step in the elimination of invading microbes by macrophages is phagosomal maturation through heterotypic endosomal fusion. This process is controlled by the guanine nucleotide binding protein Rab5, which assembles protein microdomains that include the tethering protein early endosomal antigen (EEA) 1 and the phosphatidylinositol (PI) 3-kinase hVps34, which generates PI(3)P, a phospholipid required for membrane association of EEA1 and other fusion factors. During infection of macrophages, the pathogen Legionella pneumophila bypasses the microbicidal endosomal compartment by an unknown mechanism.