Mario F. Feldman

Selective Sorting of Cargo Proteins into Bacterial Membrane Vesicles

In contrast to the well established multiple cellular roles of membrane vesicles in eukaryotic cell biology, outer membrane vesicles (OMV) produced via blebbing of prokaryotic membranes have frequently been regarded as cell debris or microscopy artifacts. Increasingly, however, bacterial membrane vesicles are thought to play a role in microbial virulence, although it remains to be determined whether OMV result from a directed process or from passive disintegration of the outer membrane. Here we establish that the human oral pathogen Porphyromonas gingivalis has a mechanism to selectively sort proteins into OMV, resulting in the preferential packaging of virulence factors into OMV and the exclusion of abundant outer membrane proteins from the protein cargo. Furthermore, we show a critical role for lipopolysaccharide in directing this sorting mechanism. The existence of a process to package specific virulence factors into OMV may significantly alter our current understanding of host-pathogen interactions.

Two distinct but interchangeable mechanisms for flipping of lipid-linked oligosaccharides

Translocation of lipid‐linked oligosaccharide (LLO) intermediates across membranes is an essential but poorly understood process in eukaryotic and bacterial glycosylation pathways. Membrane proteins defined as translocases or flippases are implicated to mediate the translocation reaction. The membrane protein Wzx has been proposed to mediate the translocation across the plasma membrane of lipopolysaccharide (LPS) O antigen subunits, which are assembled on an undecaprenyl pyrophosphate lipid carrier. Similarly, PglK (formerly WlaB) is a Campylobacter jejuni‐encoded ABC‐type transporter proposed to mediate the translocation of the undecaprenylpyrophosphate‐linked heptasaccharide intermediate involved in the recently identified bacterial N‐linked protein glycosylation pathway. A combination of genetic and carbohydrate structural analyses defined and characterized flippase activities in the C. jejuni N‐linked protein glycosylation and the Escherichia coli LPS O antigen biosynthesis. PglK displayed relaxed substrate specificity with respect to the oligosaccharide structure of the LLO intermediate and complemented a wzx deficiency in E. coli O‐antigen biosynthesis. Our experiments provide strong genetic evidence that LLO translocation across membranes can be catalyzed by two distinct proteins that do not share any sequence similarity.

Identification of a General O-linked Protein Glycosylation System in Acinetobacter baumannii and Its Role in Virulence and Biofilm Formation

Acinetobacter baumannii is an emerging cause of nosocomial infections. The isolation of strains resistant to multiple antibiotics is increasing at alarming rates. Although A. baumannii is considered as one of the more threatening “superbugs” for our healthcare system, little is known about the factors contributing to its pathogenesis. In this work we show that A. baumannii ATCC 17978 possesses an O-glycosylation system responsible for the glycosylation of multiple proteins. 2D-DIGE and mass spectrometry methods identified seven A. baumannii glycoproteins, of yet unknown function. The glycan structure was determined using a combination of MS and NMR techniques and consists of a branched pentasaccharide containing N-acetylgalactosamine, glucose, galactose, N-acetylglucosamine, and a derivative of glucuronic acid. A glycosylation deficient strain was generated by homologous recombination. This strain did not show any growth defects, but exhibited a severely diminished capacity to generate biofilms. Disruption of the glycosylation machinery also resulted in reduced virulence in two infection models, the amoebae Dictyostelium discoideum and the larvae of the insect Galleria mellonella, and reduced in vivo fitness in a mouse model of peritoneal sepsis. Despite A. baumannii genome plasticity, the O-glycosylation machinery appears to be present in all clinical isolates tested as well as in all of the genomes sequenced. This suggests the existence of a strong evolutionary pressure to retain this system. These results together indicate that O-glycosylation in A. baumannii is required for full virulence and therefore represents a novel target for the development of new antibiotics.

Identification and characterization of the Brucella abortus phosphoglucomutase gene : Role of lipopolysaccharide in virulence and intracellular multiplication

ABSTRACT Smooth lipopolysaccharide (LPS) of Brucella abortus has been reported to be an important virulence factor, although its precise role in pathogenesis is not yet clear. While the protective properties of LPS against complement are well accepted, there is still some controversy about the capacity of rough mutants to replicate intracellularly. The B. abortus phosphoglucomutase gene (pgm) was cloned, sequenced, and disrupted. The gene has a high index of identity to Agrobacterium tumefaciens pgm but is not part of the glycogen operon. A B. abortus null mutant lacks LPS O antigen but has an LPS core with an electrophoretic profile undistinguishable from that of the wild-type core, suggesting that glucose, galactose, or a derivative of these sugars may be part of the linkage between the core and the O antigen. This mutant is unable to survive in mice but replicates in HeLa cells, indicating that the complete LPS is not essential either for invasion or for intracellular multiplication. This behavior suggests that the LPS may play a role in extracellular survival in the animal, probably protecting the cell against complement-mediated lysis, but is not involved in intracellular survival.

The discovery of SycO highlights a new function for type III secretion effector chaperones

Bacterial injectisomes deliver effector proteins straight into the cytosol of eukaryotic cells (type III secretion, T3S). Many effectors are associated with a specific chaperone that remains inside the bacterium when the effector is delivered. The structure of such chaperones and the way they interact with their substrate is well characterized but their main function remains elusive. Here, we describe and characterize SycO, a new chaperone for the Yersinia effector kinase YopO. The chaperone‐binding domain (CBD) within YopO coincides with the membrane localization domain (MLD) targeting YopO to the host cell membrane. The CBD/MLD causes intrabacterial YopO insolubility and the binding of SycO prevents this insolubility but not folding and activity of the kinase. Similarly, SycE masks the MLD of YopE and SycT covers an aggregation‐prone domain of YopT, presumably corresponding to its MLD. Thus, SycO, SycE and most likely SycT mask, inside the bacterium, a domain needed for proper localization of their cognate effector in the host cell. We propose that covering an MLD might be an essential function of T3S effector chaperones.

Analogies and homologies in lipopolysaccharide and glycoprotein biosynthesis in bacteria

Bacteria generate and attach countless glycan structures to diverse macromolecules. Despite this diversity, the mechanisms of glycoconjugate biosynthesis are often surprisingly similar. The focus of this review is on the commonalities between lipopolysaccharide (LPS) and glycoprotein assembly pathways and their evolutionary relationship. Three steps that are essential for both pathways are completed by membrane proteins. These include the initiation of glycan assembly through the attachment of a first sugar residue onto the lipid carrier undecaprenyl pyrophosphate, the translocation across the plasma membrane and the final transfer onto proteins or lipid A-core. Two families of initiating enzymes have been described: the polyprenyl-P N-acetylhexosamine-1-P transferases and the polyprenyl-P hexosamine-1-P transferases, represented by Escherichia coli WecA and Salmonella enterica WbaP, respectively. Translocases are either Wzx-like flippases or adenosine triphosphate (ATP)-binding cassette transporters (ABC transporters). The latter can consist either of two polypeptides, Wzt and Wzm, or of a single polypeptide homolog to the Campylobacter jejuni PglK. Finally, there are two families of conjugating enzymes, the N-oligosaccharyltransferases (N-OTase), best represented by C. jejuni PglB, and the O-OTases, including Neisseria meningitidis PglL and the O antigen ligases involved in LPS biosynthesis. With the exception of the N-OTases, probably restricted to glycoprotein synthesis, members of all these transmembrane protein families can be involved in the synthesis of both glycoproteins and LPS. Because many translocation and conjugation enzymes display relaxed substrate specificity, these bacterial enzymes could be exploited in engineered living bacteria for customized glycoconjugate production, generating potential vaccines and therapeutics.

A novel mechanism for the biogenesis of outer membrane vesicles in Gram-negative bacteria.

Bacterial outer membrane vesicles (OMVs) have important biological roles in pathogenesis and intercellular interactions, but a general mechanism of OMV formation is lacking. Here we show that the VacJ/Yrb ABC (ATP-binding cassette) transport system, a proposed phospholipid transporter, is involved in OMV formation. Deletion or repression of VacJ/Yrb increases OMV production in two distantly related Gram-negative bacteria, Haemophilus influenzae and Vibrio cholerae. Lipidome analyses demonstrate that OMVs from VacJ/Yrb-defective mutants in H. influenzae are enriched in phospholipids and certain fatty acids. Furthermore, we demonstrate that OMV production and regulation of the VacJ/Yrb ABC transport system respond to iron starvation. Our results suggest a new general mechanism of OMV biogenesis based on phospholipid accumulation in the outer leaflet of the outer membrane. This mechanism is highly conserved among Gram-negative bacteria, provides a means for regulation, can account for OMV formation under all growth conditions, and might have important pathophysiological roles in vivo.

SycE allows secretion of YopE–DHFR hybrids by the Yersinia enterocolitica type III Ysc system

The Ysc type III secretion system allows Yersinia enterocolitica to translocate virulence proteins, called Yop effectors, into the cytosol of eukaryotic cells. Some of the Yop effectors possess an individual chaperone called a Syc protein. The first 15 amino acids of the YopE effector constitute a secretion signal that is sufficient to promote secretion of several reporter proteins. Residues 15–50 of YopE comprise the minimal binding domain for the SycE chaperone. In this study, we investigated the secretion by the Ysc system of several YopE–DHFR hybrid proteins with different folding properties, and evaluated the role of SycE, the cognate chaperone of YopE, in this context. We have analysed the secretion of hybrids containing 16 (YopE16), 52 (YopE52) and 80 (the complete region covered by the chaperone, YopE80) amino acids of YopE or full‐length YopE (YopEFL) with wild‐type DHFR and two mutants with altered folding properties. The hybrids containing DHFRΔ77, the mutant whose folding properties are the most drastically affected, could be secreted in all the conditions tested, even in the absence of the chaperone SycE. In contrast, DHFRwt could only be secreted fused to the first 52 amino acids of YopE, and its secretion was strictly dependent on SycE. The hybrids YopE80–DHFRwt and YopEFL–DHFRwt were not secreted. YopEFL–DHFRwt completely jammed the channel in an SycE‐dependent fashion. Our experiments indicate that, in order to be secreted, proteins must be unfolded or only partially folded, and that TSS chaperones could keep their substrates in a secretion‐competent conformation, probably by preventing their folding. In addition, they show that the secretion apparatus can reject folded proteins if they are not deeply engaged into the injectisome.

Extreme Substrate Promiscuity of the Neisseria Oligosaccharyl Transferase Involved in Protein O-Glycosylation

Neisseria meningitidis PglL belongs to a novel family of bacterial oligosaccharyltransferases (OTases) responsible for O-glycosylation of type IV pilins. Although members of this family are widespread among pathogenic bacteria, there is little known about their mechanism. Understanding the O-glycosylation process may uncover potential targets for therapeutic intervention, and can open new avenues for the exploitation of these pathways for biotechnological purposes. In this work, we demonstrate that PglL is able to transfer virtually any glycan from the undecaprenyl pyrophosphate (UndPP) carrier to pilin in engineered Escherichia coli and Salmonella cells. Surprisingly, PglL was also able to interfere with the peptidoglycan biosynthetic machinery and transfer peptidoglycan subunits to pilin. This represents a previously unknown post-translational modification in bacteria. Given the wide range of glycans transferred by PglL, we reasoned that substrate specificity of PglL lies in the lipid carrier. To test this hypothesis we developed an in vitro glycosylation system that employed purified PglL, pilin, and the lipid farnesyl pyrophosphate (FarPP) carrying a pentasaccharide that had been synthesized by successive chemical and enzymatic steps. Although FarPP has different stereochemistry and a significantly shorter aliphatic chain than the natural lipid substrate, the pentasaccharide was still transferred to pilin in our system. We propose that the primary roles of the lipid carrier during O-glycosylation are the translocation of the glycan into the periplasm, and the positioning of the pyrophosphate linker and glycan adjacent to PglL. The unique characteristics of PglL make this enzyme a promising tool for glycoengineering novel glycan-based vaccines and therapeutics.

Pour some sugar on it: the expanding world of bacterial protein O‐linked glycosylation

Protein glycosylation was once considered as an eccentricity of a few bacteria. However in the recent years multiple O‐glycosylation mechanisms have been identified in bacterial species from the most diverse genera, including various important human pathogens. This review focuses on summarizing the structural diversity, the various pathways and the physiological roles of this post‐translational protein modification. We propose a classification of O‐glycosylation based on the requirement of an oligosaccharyltransferase (OTase). OTase‐dependent glycosylation utilizes an oligosaccharide synthesized on a lipid carrier that is transferred to proteins en bloc by an OTase. Multiple proteins, including the pilins, are glycosylated using this mechanism. OTase‐independent glycosylation refers to the pathway in which glycosyltransferases sequentially add monosaccharides onto the target proteins. This pathway is employed for glycosylation of flagella and autotransporters. Both systems play key roles in pathogenesis. Exploiting glycosylation machineries it is now possible to generate glycoconjugates made of different proteins attached to polysaccharides derived from LPS or capsule biosynthesis. These recombinant glycoproteins can be exploited for vaccines and diagnostics of bacterial infections. Furthermore, O‐glycosylation systems are promising targets for antibiotic development. Technological advances in MS and NMR will facilitate the discovery of novel glycosylation systems. Likely, the O‐glycosylation pathways we currently know constitute just the tip of the iceberg of a still largely uncharacterized bacterial glycosylation world.