Andréa Dessen

The V Antigen of Pseudomonas aeruginosa Is Required for Assembly of the Functional PopB/PopD Translocation Pore in Host Cell Membranes

ABSTRACT Pseudomonas aeruginosa efficiently intoxicates eukaryotic cells through the activity of the type III secretion-translocation system (TTSS). Gene deletions within the translocation operon pcrGVH-popBD abolish pore-forming activity of P. aeruginosa strains with macrophages and TTSS-dependent hemolysis. Here we investigated the requirements for PcrV, PopB, and PopD in pore formation by analyzing specific mutants using red blood cells (RBCs) and fibroblasts expressing green fluorescent protein fused to actin. Simultaneous secretion of three proteins, PopB, PopD, and PcrV, was required to achieve wild-type hemolysis and effector translocation. Deletion of pcrV in a cytotoxic strain did not affect secretion of PopB and PopD but abolished hemolytic activity and translocation of effectors into fibroblasts. Notably, the PcrV-deficient mutant was not capable of inserting PopD into host cell membranes, whereas PopB and PopD, but not PcrV, were readily found within membranes of wild-type-infected RBCs. Immunoprecipitation experiments performed by using a liposome model of pore assembly revealed a direct interaction between PopD and PopB but not between PopD and PcrV. Consequently, PcrV is necessary for the functional assembly of the PopB/D translocon complex but does not interact directly with pore-forming Pop proteins.

Oligomerization of type III secretion proteins PopB and PopD precedes pore formation in Pseudomonas

Pseudomonas aeruginosa is the agent of opportunistic infections in immunocompromised individuals and chronic respiratory illnesses in cystic fibrosis patients. Pseudomonas aeruginosa utilizes a type III secretion system for injection of toxins into the host cell cytoplasm through a channel on the target membrane (the ‘translocon’). Here, we have functionally and structurally characterized PopB and PopD, membrane proteins implicated in the formation of the P.aeruginosa translocon. PopB and PopD form soluble complexes with their common chaperone, PcrH, either as stable heterodimers or as metastable heterooligomers. Only oligomeric forms are able to bind to and disrupt cholesterol‐rich membranes, which occurs within a pH range of 5–7 in the case of PopB/PcrH, and only at acidic pH for PcrH‐free PopD. Electron microscopy reveals that upon membrane association PopB and PopD form 80 Å wide rings which encircle 40 Å wide cavities. Thus, formation of metastable oligomers precedes membrane association and ring generation in the formation of the Pseudomonas translocon, a mechanism which may be similar for other pathogens that employ type III secretion systems.

The SciZ protein anchors the enteroaggregative Escherichia coli Type VI secretion system to the cell wall

Type VI secretion systems (T6SS) are multi‐component machines encoded within the genomes of most Gram‐negative bacteria that associate with plant, animal and/or human cells, and therefore are considered as potential virulence factors. We recently launched a study on the Sci‐1 T6SS of enteroaggregative Escherichia coli (EAEC). The Sci‐1 T6SS is composed of all or a subset of the 21 gene products encoded within the cluster, 13 of which are shared by all T6SS identified so far. In the present work, we focussed our attention on the SciZ protein. We first showed that SciZ is required for the release of the Hcp protein in the culture supernatant and for efficient biofilm formation, demonstrating that SciZ is necessary for EAEC T6SS function. Indeed, SciZ forms a complex with SciP, SciS and SciN, three core components of the transport apparatus. Fractionation and topology studies showed that SciZ is a polytopic inner membrane protein with three trans‐membrane segments. Computer analyses identified a motif shared by peptidoglycan binding proteins of the OmpA family in the SciZ periplasmic domain. Using in vivo and in vitro binding assays, we showed that this motif anchors the SciZ protein to the cell wall and is required for T6SS function.

A Bacterial Protein Targets the BAHD1 Chromatin Complex to Stimulate Type III Interferon Response

A virulence factor secreted by Listeria monocytogenes induces expression of interferon-stimulated genes Intracellular pathogens such as Listeria monocytogenes subvert cellular functions through the interaction of bacterial effectors with host components. Here we found that a secreted listerial virulence factor, LntA, could target the chromatin repressor BAHD1 in the host cell nucleus to activate interferon (IFN)–stimulated genes (ISGs). IFN-λ expression was induced in response to infection of epithelial cells with bacteria lacking LntA; however, the BAHD1-chromatin associated complex repressed downstream ISGs. In contrast, in cells infected with lntA-expressing bacteria, LntA prevented BAHD1 recruitment to ISGs and stimulated their expression. Murine listeriosis decreased in BAHD1+/– mice or when lntA was constitutively expressed. Thus, the LntA-BAHD1 interplay may modulate IFN-λ−mediated immune response to control bacterial colonization of the host.

Membrane targeting and pore formation by the type III secretion system translocon

The type III secretion system (T3SS) is a complex macromolecular machinery employed by a number of Gram‐negative species to initiate infection. Toxins secreted through the system are synthesized in the bacterial cytoplasm and utilize the T3SS to pass through both bacterial membranes and the periplasm, thus being introduced directly into the eukaryotic cytoplasm. A key element of the T3SS of all bacterial pathogens is the translocon, which comprises a pore that is inserted into the membrane of the target cell, allowing toxin injection. Three macromolecular partners associate to form the translocon: two are hydrophobic and one is hydrophilic, and the latter also associates with the T3SS needle. In this review, we discuss recent advances on the biochemical and structural characterization of the proteins involved in translocon formation, as well as their participation in the modification of intracellular signalling pathways upon infection. Models of translocon assembly and regulation are also discussed.

Biogenesis, Regulation, and Targeting of the Type III Secretion System

The type III secretion system (T3SS) is employed by a number of Gram-negative bacterial pathogens to inject toxins into eukaryotic cells. The biogenesis of this complex machinery requires the regulated interaction between over 20 cytosolic, periplasmic, and membrane-imbedded proteins, many of which undergo processes such as polymerization, partner recognition, and partial unfolding. Elements of this intricate macromolecular system have been characterized through electron microscopy, crystallography, and NMR techniques, allowing for an initial understanding of the spatiotemporal regulation of T3SS-related events. Here, we report recent advances in the structural characterization of T3SS proteins from a number of bacteria, and provide an overview of recently identified small molecule T3SS inhibitors that could potentially be explored for novel antibacterial development.

Sortase-Mediated Pilus Fiber Biogenesis in Streptococcus Pneumoniae.

Streptococcus pneumoniae is a piliated pathogen whose ability to circumvent vaccination and antibiotic treatment strategies is a cause of mortality worldwide. Pili play important roles in pneumococcal infection, but little is known about their biogenesis mechanism or the relationship between components of the pilus-forming machinery, which includes the fiber pilin (RrgB), two minor pilins (RrgA, RrgC), and three sortases (SrtC-1, SrtC-2, SrtC-3). Here we show that SrtC-1 is the main pilus-polymerizing transpeptidase, and electron microscopy analyses of RrgB fibers reconstituted in vitro reveal that they structurally mimic the pneumococcal pilus backbone. Crystal structures of both SrtC-1 and SrtC-3 reveal active sites whose access is controlled by flexible lids, unlike in non-pilus sortases, and suggest that substrate specificity is dictated by surface recognition coupled to lid opening. The distinct structural features of pilus-forming sortases suggest a common pilus biogenesis mechanism that could be exploited for the development of broad-spectrum antibacterials.

Structural and kinetic analysis of Escherichia coli GDP-mannose 4,6 dehydratase provides insights into the enzyme’s catalytic mechanism and regulation by GDP-fucose

BACKGROUND GDP-mannose 4,6 dehydratase (GMD) catalyzes the conversion of GDP-(D)-mannose to GDP-4-keto, 6-deoxy-(D)-mannose. This is the first and regulatory step in the de novo biosynthesis of GDP-(L)-fucose. Fucose forms part of a number of glycoconjugates, including the ABO blood groups and the selectin ligand sialyl Lewis X. Defects in GDP-fucose metabolism have been linked to leukocyte adhesion deficiency type II (LADII). RESULTS The structure of the GDP-mannose 4,6 dehydratase apo enzyme has been determined and refined using data to 2.3 A resolution. GMD is a homodimeric protein with each monomer composed of two domains. The larger N-terminal domain binds the NADP(H) cofactor in a classical Rossmann fold and the C-terminal domain harbors the sugar-nucleotide binding site. We have determined the GMD dissociation constants for NADP, NADPH and GDP-mannose. Each GMD monomer binds one cofactor and one substrate molecule, suggesting that both subunits are catalytically competent. GDP-fucose acts as a competitive inhibitor, suggesting that it binds to the same site as GDP-mannose, providing a mechanism for the feedback inhibition of fucose biosynthesis. CONCLUSIONS The X-ray structure of GMD reveals that it is a member of the short-chain dehydrogenase/reductase (SDR) family of proteins. We have modeled the binding of NADP and GDP-mannose to the enzyme and mutated four of the active-site residues to determine their function. The combined modeling and mutagenesis data suggests that at position 133 threonine substitutes serine as part of the serine-tyrosine-lysine catalytic triad common to the SDR family and Glu 135 functions as an active-site base.

Bridging cell wall biosynthesis and bacterial morphogenesis

The bacterial cell wall is a complex three-dimensional structure that protects the cell from environmental stress and ensures its shape. The biosynthesis of its main component, the peptidoglycan, involves the coordination of activities of proteins present in the cytoplasm, the membrane, and the periplasm, some of which also interact with the bacterial cytoskeleton. The sheer complexity of the cell wall elongation process, which is the main focus of this review, has created a significant challenge for the study of the macromolecular interactions that regulate peptidoglycan biosynthesis. The availability of new structural and biochemical data on a number of components of peptidoglycan assembly machineries, including a complex between MreB and RodZ as well as structures of penicillin-binding proteins (PBPs) from a number of pathogenic species, now provide novel insight into the underpinnings of an intricate molecular machinery.

Structure of the heterotrimeric complex that regulates type III secretion needle formation

Type III secretion systems (T3SS), found in several Gram-negative pathogens, are nanomachines involved in the transport of virulence effectors directly into the cytoplasm of target cells. T3SS are essentially composed of basal membrane-embedded ring-like structures and a hollow needle formed by a single polymerized protein. Within the bacterial cytoplasm, the T3SS needle protein requires two distinct chaperones for stabilization before its secretion, without which the entire T3SS is nonfunctional. The 2.0-Å x-ray crystal structure of the PscE-PscF55–85-PscG heterotrimeric complex from Pseudomonas aeruginosa reveals that the C terminus of the needle protein PscF is engulfed within the hydrophobic groove of the tetratricopeptide-like molecule PscG, indicating that the macromolecular scaffold necessary to stabilize the T3SS needle is totally distinct from chaperoned complexes between pilus- or flagellum-forming molecules. Disruption of specific PscG–PscF interactions leads to impairment of bacterial cytotoxicity toward macrophages, indicating that this essential heterotrimer, which possesses homologs in a wide variety of pathogens, is a unique attractive target for the development of novel antibacterials.