Bacteroides fragilis toxin expression enables lamina propria niche acquisition in the developing mouse gut.

Bacterial toxins are well-studied virulence factors; however, recent studies have revealed their importance in bacterial niche adaptation. Enterotoxigenic Bacteroides fragilis (ETBF) expresses B. fragilis toxin (BFT) that we hypothesized may contribute to both colonic epithelial injury and niche acquisition. We developed a vertical transmission model for ETBF in mice that showed that BFT enabled ETBF to access a lamina propria (LP) niche during colonic microbiome development that was inaccessible to non-toxigenic B. fragilis. LP entry by ETBF required BFT metalloprotease activity, and showed temporal restriction to the pre-weaning period, dependent on goblet-cell-associated passages. In situ single-cell analysis showed bft expression at the apical epithelial surface and within the LP. BFT expression increased goblet cell number and goblet-cell-associated passage formation. These findings define a paradigm by which bacterial toxin expression specifies developmental niche acquisition, suggesting that a selective advantage conferred by a toxin may impact long-term host health.

Lipidome of the Bacteroides Genus Containing New Peptidolipid and Sphingolipid Families Revealed by Multiple-Stage Mass Spectrometry.

The anaerobic bacteria of the Bacteroides fragilis group including Bacteroides thetaiotaomicron, B. fragilis, Bacteroides vulgatus, and Bacteroides ovatus in genus Bacteroides are among the most commonly found human gut microbiota. They are generally commensal but are also opportunistic pathogens. Both the inner and outer membranes of the Bacteroides cell envelope contain abundant lipids with diversified structures, and dissection of the lipid composition of the inner and outer membrane fractions is important for understanding the biogenesis of this multilaminate wall structure. Here, we describe mass spectrometry-based approaches to delineate in detail the lipidome of the membrane and the outer membrane vesicle of the bacteria cells. We identified 15 lipid class/subclasses (>100 molecular species), including sphingolipid families [dihydroceramide (DHC), glycylseryl (GS) DHC, DHC-phosphoinositolphosphoryl-DHC (DHC-PIP-DHC), ethanolamine phosphorylceramide, inositol phosphorylceramide (IPC), serine phosphorylceramide, ceramide-1-phosphate, and glycosyl ceramide], phospholipids [phosphatidylethanolamine, phosphatidylinositol (PI), and phosphatidylserine], peptide lipids (GS-, S-, and G-lipids) and cholesterol sulfate, of which several have not been reported previously, or have similar structures to those found in Porphyromonas gingivalis, the periodontopathic bacterium in oral microbiota. The new DHC-PIPs-DHC lipid family is found only in B. vulgatus, which, however, lacks the PI lipid family. The galactosyl ceramide family is exclusively present in B. fragilis, which nevertheless lacks IPC and PI lipids. The lipidomes as revealed in this study demonstrate the lipid diversity among the various strains and the utility of multiple-stage mass spectrometry (MSn) with high-resolution mass spectrometry in the structural elucidation of complex lipids.

Lipidomics Analysis of Outer Membrane Vesicles and Elucidation of the Inositol Phosphoceramide Biosynthetic Pathway in Bacteroides thetaiotaomicron.

Approximately one-third of the human colonic microbiome is formed by bacteria from the genus Bacteroides. These bacteria produce a large amount of uniformly sized outer membrane vesicles (OMVs), which are equipped with hydrolytic enzymes that play a role in the degradation of diet- and host-derived glycans. In this work, we characterize the lipid composition of membranes and OMVs from Bacteroides thetaiotaomicron VPI-5482. Liquid chromatography-mass spectrometry (LC-MS) analysis indicated that OMVs carry sphingolipids, glycerophospholipids, and serine-dipeptide lipids. Sphingolipid species represent more than 50% of the total lipid content of OMVs. The most abundant sphingolipids in OMVs are ethanolamine phosphoceramide (EPC) and inositol phosphoceramide (IPC). Bioinformatics analysis allowed the identification of the BT1522-1526 operon putatively involved in IPC synthesis. Mutagenesis studies revealed that BT1522-1526 is essential for the synthesis of phosphatidylinositol (PI) and IPC, confirming the role of this operon in the biosynthesis of IPC. BT1522-1526 mutant strains lacking IPC produced OMVs that were indistinguishable from the wild-type strain, indicating that IPC sphingolipid species are not involved in OMV biogenesis. Given the known role of sphingolipids in immunomodulation, we suggest that OMVs may act as long-distance vehicles for the delivery of sphingolipids in the human gut. IMPORTANCE Sphingolipids are essential membrane lipid components found in eukaryotes that are also involved in cell signaling processes. Although rare in bacteria, sphingolipids are produced by members of the phylum Bacteroidetes, human gut commensals. Here, we determined that OMVs carry sphingolipids and other lipids of known signaling function. Our results demonstrate that the BT1522-1526 operon is required for IPC biosynthesis in B. thetaiotaomicron.

Good Gone Bad: One Toxin Away From Disease for Bacteroides fragilis.

The human gut is colonized by hundreds of trillions of microorganisms whose acquisition begins during early infancy. Species from the Bacteroides genus are ubiquitous commensals, comprising about thirty percent of the human gut microbiota. Bacteroides fragilis is one of the least abundant Bacteroides species, yet is the most common anaerobe isolated from extraintestinal infections in humans. A subset of B. fragilis strains carry a genetic element that encodes a metalloprotease enterotoxin named Bacteroides fragilis toxin, or BFT. Toxin-bearing strains, or Enterotoxigenic B. fragilis (ETBF) cause acute and chronic intestinal disease in children and adults. Despite this association with disease, around twenty percent of the human population appear to be asymptomatic carriers of ETBF. BFT damages the colonic epithelial barrier by inducing cleavage of the zonula adherens protein E-cadherin and initiating a cell signaling response characterized by inflammation and c-Myc-dependent pro-oncogenic hyperproliferation. As a consequence, mice harboring genetic mutations that predispose to colonic inflammation or tumor formation are uniquely susceptible to toxin-mediated injury. The recent observation of ETBF-bearing biofilms in colon biopsies from humans with colon cancer susceptibility loci strongly suggests that ETBF is a driver of colorectal cancer. This article will address ETBF biology from a host-pathobiont perspective, including clinical data, analysis of molecular mechanisms of disease, and the complex ecological context of the human gut.

Surface Exposure and Packing of Lipoproteins into Outer Membrane Vesicles Are Coupled Processes in Bacteroides.

Outer membrane vesicles (OMVs) are spherical structures derived from the outer membranes (OMs) of Gram-negative bacteria. Bacteroides spp. are prominent components of the human gut microbiota, and OMVs produced by these species are proposed to play key roles in gut homeostasis. OMV biogenesis in Bacteroides is a poorly understood process. Here, we revisited the protein composition of Bacteroides thetaiotaomicron OMVs by mass spectrometry. We confirmed that OMVs produced by this organism contain large quantities of glycosidases and proteases, with most of them being lipoproteins. We found that most of these OMV-enriched lipoproteins are encoded by polysaccharide utilization loci (PULs), such as the sus operon. We examined the subcellular locations of the components of the Sus system and found a split localization; the alpha-amylase SusG is highly enriched in OMVs, while the oligosaccharide importer SusC remains mostly in the OM. We found that all OMV-enriched lipoproteins possess a lipoprotein export sequence (LES), and we show that this signal mediates translocation of SusG from the periplasmic face of the OM toward the extracellular milieu. Mutations in the LES motif caused defects in surface exposure and recruitment of SusG into OMVs. These experiments link, for the first time, surface exposure to recruitment of proteins into OMVs. We also show that surface-exposed SusG in OMVs is active and rescues the growth of bacterial cells incapable of growing on starch as the only carbon source. Our results support the role of OMVs as "public goods" that can be utilized by other organisms with different metabolic capabilities.IMPORTANCE Species from the Bacteroides genus are predominant members of the human gut microbiota. OMVs in Bacteroides have been shown to be important for the homeostasis of complex host-commensal relationships, mainly involving immune tolerance and protection from disease. OMVs carry many enzymatic activities involved in the cleavage of complex polysaccharides and have been proposed as public goods that can provide growth to other bacterial species by release of polysaccharide breakdown products into the gut lumen. This work shows that the presence of a negatively charged rich amino acid motif (LES) is required for efficient packing of the surface-exposed alpha-amylase SusG into OMVs. Our findings strongly suggest that surface exposure is coupled to packing of Bacteroides lipoproteins into OMVs. This is the first step in the generation of tailor-made probiotic interventions that can exploit LES-related sequences to generate Bacteroides strains displaying proteins of interest in OMVs.

RomA, A Periplasmic Protein Involved in the Synthesis of the Lipopolysaccharide, Tunes Down the Inflammatory Response Triggered by Brucella.

Brucellaceae are stealthy pathogens with the ability to survive and replicate in the host in the context of a strong immune response. This capacity relies on several virulence factors that are able to modulate the immune system and in their structural components that have low proinflammatory activities. Lipopolysaccharide (LPS), the main component of the outer membrane, is a central virulence factor of Brucella, and it has been well established that it induces a low inflammatory response. We describe here the identification and characterization of a novel periplasmic protein (RomA) conserved in alpha-proteobacteria, which is involved in the homeostasis of the outer membrane. A mutant in this gene showed several phenotypes, such as membrane defects, altered LPS composition, reduced adhesion, and increased virulence and inflammation. We show that RomA is involved in the synthesis of LPS, probably coordinating part of the biosynthetic complex in the periplasm. Its absence alters the normal synthesis of this macromolecule and affects the homeostasis of the outer membrane, resulting in a strain with a hyperinflammatory phenotype. Our results suggest that the proper synthesis of LPS is central to maximize virulence and minimize inflammation.

Glycoengineered Outer Membrane Vesicles as a Platform for Vaccine Development.

As we enter into the postantibiotic era, vaccines to prevent bacterial infections previously treatable with antibiotics are urgently needed. Most successful antibacterial vaccines are glycoconjugates, composed of cell surface carbohydrates chemically attached to a carrier protein. Glycoconjugate vaccines provide a safe and consistent strategy against polysaccharide-encapsulated pathogens. The best examples are the conjugate vaccines against Haemophilus influenzae type b, Streptococcus pneumoniae, and Neisseria meningitidis, all based on capsular polysaccharides. Although these types of vaccines are effective, their current manufacturing process presents multiple drawbacks, such as biosafety risks and batch-to-batch variability. Furthermore, inclusion of additional serotypes is extremely slow, mainly due to the intricate chemical methods of conjugation. Thus, novel platforms for antibacterial vaccines are required. Gram-negative bacteria are able to produce outer membrane vesicles (OMVs). OMVs are mainly composed of lipopolysaccharide (LPS), outer membrane and periplasmic proteins, and phospholipids. Although their biogenesis is poorly understood, it is known that OMVs are formed by blebbing of the outer membrane. OMVs are attractive candidates for novel vaccine delivery platforms due to their immunogenic properties, self-adjuvanticity, and capacity for enhancement by recombinant engineering. We have shown that OMVs can be engineered to display surface glycans from different bacteria and that these glycoengineered OMVs (geOMVs) are effective in diverse animal models of infection. Here we provide a detailed method for the design and preparation of geOMV displaying the O-antigen from a prominent uropathogenic Escherichia coli (UPEC) serotype, O25b, as a proof of concept for the use of geOMVs as vaccine candidates.

New Features in the Lipid A Structure of Brucella suis and Brucella abortus Lipopolysaccharide.

Brucellaceae are Gram-negative bacteria that cause brucellosis, one of the most distributed worldwide zoonosis, transmitted to humans by contact with either infected animals or their products. The lipopolysaccharide exposed on the cell surface has been intensively studied and is considered a major virulence factor of Brucella. In the last years, structural studies allowed the determination of new structures in the core oligosaccharide and the O-antigen of this lipopolysaccharide. In this work, we have reinvestigated the lipid A structure isolated from B. suis and B. abortus lipopolysaccharides. A detailed study by MALDI-TOF mass spectrometry in the positive and negative ion modes of the lipid A moieties purified from both species was performed. Interestingly, a new feature was detected: the presence of a pyrophosphorylethanolamine residue substituting the backbone. LID-MS/MS analysis of some of the detected ions allowed assurance that the Lipid A structure composed by the diGlcN3N disaccharide, mainly hexa-acylated and penta-acylated, bearing one phosphate and one pyrophosphorylethanolamine residue. Graphical abstract ᅟ.

LPS Remodeling Triggers Formation of Outer Membrane Vesicles in Salmonella.

UNLABELLED: Outer membrane vesicles (OMV) are proposed to mediate multiple functions during pathogenesis and symbiosis. However, the mechanisms responsible for OMV formation remain poorly understood. It has been shown in eukaryotic membranes that lipids with an inverted-cone shape favor the formation of positive membrane curvatures. Based on these studies, we formulated the hypothesis that lipid A deacylation might impose shape modifications that result in the curvature of the outer membrane (OM) and subsequent OMV formation. We tested the effect of lipid A remodeling on OMV biogenesis employing Salmonella enterica serovar Typhimurium as a model organism. Expression of the lipid A deacylase PagL resulted in increased vesiculation, without inducing an envelope stress response. Mass spectrometry analysis revealed profound differences in the patterns of lipid A in OM and OMV, with accumulation of deacylated lipid A forms exclusively in OMV. OMV biogenesis by intracellular bacteria upon macrophage infection was drastically reduced in a pagL mutant strain. We propose a novel mechanism for OMV biogenesis requiring lipid A deacylation in the context of a multifactorial process that involves the orchestrated remodeling of the outer membrane. IMPORTANCE: The role of lipid remodeling in vesiculation is well documented in eukaryotes. Similarly, bacteria produce membrane-derived vesicles; however, the molecular mechanisms underlying their production are yet to be determined. In this work, we investigated the role of outer membrane remodeling in OMV biogenesis in S Typhimurium. We showed that the expression of the lipid A deacylase PagL results in overvesiculation with deacylated lipid A accumulation exclusively in OMV. An S Typhimurium ΔpagL strain showed a significant reduction in intracellular OMV secretion relative to the wild-type strain. Our results suggest a novel mechanism for OMV biogenesis that involves outer membrane remodeling through lipid A modification. Understanding how OMV are produced by bacteria is important to advance our understanding of the host-pathogen interactions.

Sugar and Spice Make Bacteria Not Nice: Protein Glycosylation and Its Influence in Pathogenesis.

Protein glycosylation is a post-translational modification that occurs across the whole tree of life. In the recent years, multiple N- and O-glycosylation mechanisms have been identified and characterized in diverse bacterial species, including human pathogens. This review focuses on bacterial protein glycosylation and its impact in pathogenesis. Bacteria carry N- and O-glycosylation systems that are mediated by an oligosaccharyltransferase (OTase). In OTase-dependent glycosylation mechanisms, an oligosaccharide is synthesized on a lipid carrier and subsequently transferred to proteins en bloc by an OTase. Multiple proteins are glycosylated using this mechanism. OTase-independent glycosylation refers to the pathway in which Protein N- and O-glycosyltransferases (PGTases) sequentially add monosaccharides onto the target proteins. This pathway is employed for glycosylation of flagella and autotransporters. By exploiting glycosylation machineries, it is now possible to generate tailor-made glycoconjugates by attaching polysaccharides derived from lipopolysaccharide or capsule biosynthesis onto a protein of choice. These glycoproteins can be used in developing vaccines and diagnostics of bacterial infections. Furthermore, both N- and O-glycosylation systems are promising targets for antibiotic development. Recently, the discovery of GTase toxins produced by bacterial pathogens and secreted into the host cells has greatly expanded. These proteins are a key factor in host-pathogen interactions and are required by certain pathogenic bacteria to establish a successful infection. The exact functions of bacterial glycoproteins in pathogenesis are just starting to emerge. Understanding these roles is key for new opportunities in the prevention of bacterial infections, which is crucial in times when antibiotic resistance continues to increase.

Glycoengineered Outer Membrane Vesicles: A Novel Platform for Bacterial Vaccines.

The World Health Organization has indicated that we are entering into a post-antibiotic era in which infections that were routinely and successfully treated with antibiotics can now be lethal due to the global dissemination of multidrug resistant strains. Conjugate vaccines are an effective way to create a long-lasting immune response against bacteria. However, these vaccines present many drawbacks such as slow development, high price, and batch-to-batch inconsistencies. Alternate approaches for vaccine development are urgently needed. Here we present a new vaccine consisting of glycoengineered outer membrane vesicles (geOMVs). This platform exploits the fact that the initial steps in the biosynthesis of most bacterial glycans are similar. Therefore, it is possible to easily engineer non-pathogenic Escherichia coli lab strains to produce geOMVs displaying the glycan of the pathogen of interest. In this work we demonstrate the versatility of this platform by showing the efficacy of geOMVs as vaccines against Streptococcus pneumoniae in mice, and against Campylobacter jejuni in chicken. This cost-effective platform could be employed to generate vaccines to prevent infections caused by a wide variety of microbial agents in human and animals.

Galactofuranose in Mycoplasma mycoides is important for membrane integrity and conceals adhesins but does not contribute to serum resistance.

Mycoplasma mycoides subsp. capri (Mmc) and subsp. mycoides (Mmm) are important ruminant pathogens worldwide causing diseases such as pleuropneumonia, mastitis and septicaemia. They express galactofuranose residues on their surface, but their role in pathogenesis has not yet been determined. The M. mycoides genomes contain up to several copies of the glf gene, which encodes an enzyme catalysing the last step in the synthesis of galactofuranose. We generated a deletion of the glf gene in a strain of Mmc using genome transplantation and tandem repeat endonuclease coupled cleavage (TREC) with yeast as an intermediary host for the genome editing. As expected, the resulting YCp1.1-Δglf strain did not produce the galactofuranose-containing glycans as shown by immunoblots and immuno-electronmicroscopy employing a galactofuranose specific monoclonal antibody. The mutant lacking galactofuranose exhibited a decreased growth rate and a significantly enhanced adhesion to small ruminant cells. The mutant was also 'leaking' as revealed by a ß-galactosidase-based assay employing a membrane impermeable substrate. These findings indicate that galactofuranose-containing polysaccharides conceal adhesins and are important for membrane integrity. Unexpectedly, the mutant strain showed increased serum resistance.

Identification of a type IV secretion substrate of Brucella abortus that participates in the early stages of intracellular survival.

Brucella abortus, the aetiological agent of bovine brucellosis, is an intracellular pathogen whose virulence is completely dependent on a type IV secretion system. This secretion system translocates effector proteins into the host cell to modulate the intracellular fate of the bacterium in order to establish a secure niche were it actively replicates. Although much has been done in understanding how this secretion system participates in the virulence process, few effector proteins have been identified to date. We describe here the identification of a type IV secretion substrate (SepA) that is only present in Brucella spp. and has no detectable homology to known proteins. This protein is secreted in a virB-dependent manner in a two-step process involving a periplasmic intermediate and secretion is necessary for its function. The deletion mutant showed a defect in the early stages of intracellular replication in professional and non-professional phagocytes although it invades the cells more efficiently than the wild-type parental strain. Our results indicate that, even though the mutant was more invasive, it had a defect in excluding the lysosomal marker Lamp-1 and was inactivated more efficiently during the early phases of the intracellular life cycle.

Identification of an atypical peptidyl-prolyl cis/trans isomerase from trypanosomatids.

The parvulin family of peptidyl-prolyl cis/trans isomerases (PPIases) catalyzes the cis/trans isomerization of the peptide bonds preceding Pro residues. Eukaryotic parvulin-type PPIases have been shown to be involved in cell proliferation and cell cycle progression. Here we present the biochemical and molecular characterization of a novel multi-domain parvulin-type PPIase from the human pathogenic Trypanosoma cruzi, annotated as TcPar45. Like most other parvulins, Par45 has an N-terminal extension, but, in contrast to human Pin1, it contains a forkhead-associated domain (FHA) instead of a WW domain at the N-terminal end. Par45 shows a strong preference for a substrate with the basic Arg residue preceding Pro (Suc-Ala-Arg-Pro-Phe-NH-Np: k(cat)/K(M)=97.1 /M/s), like that found for human Par14. In contrast to human Pin1, but similarly to Par14, Par45 does not accelerate the cis/trans interconversion of acidic substrates containing Glu-Pro bonds. It is preferentially located in the parasite nucleus. Single RNA interference (RNAi)-mediated knock-down showed that there was a growth inhibition in procyclic Trypanosoma brucei cells. These results identify Par45 as a phosphorylation-independent parvulin required for normal cell proliferation in a unicellular eukaryotic cell.