Glycoengineering with neuraminic acid analogs to label lipooligosaccharides and detect native sialyltransferase activity in gram-negative bacteria.

Lipooligosaccharides are the most abundant cell surface glycoconjugates on the outer membrane of Gram-negative bacteria. They play important roles in host-microbe interactions. Certain Gram-negative pathogenic bacteria cap their lipooligosaccharides with the sialic acid, N-acetylneuraminic acid (Neu5Ac), to mimic host glycans that among others protects these bacteria from recognition by the hosts immune system. This process of molecular mimicry is not fully understood and remains under investigated. To explore the functional role of sialic acid-capped lipooligosaccharides at the molecular level, it is important to have tools readily available for the detection and manipulation of both Neu5Ac on glycoconjugates and the involved sialyltransferases, preferably in live bacteria. We and others have shown that the native sialyltransferases of some Gram-negative bacteria can incorporate extracellular unnatural sialic acid nucleotides onto their lipooligosaccharides. We here report on the expanded use of native bacterial sialyltransferases to incorporate neuraminic acids analogs with a reporter group into the lipooligosaccharides of a variety of Gram-negative bacteria. We show that this approach offers a quick strategy to screen bacteria for the expression of functional sialyltransferases and the ability to use exogenous CMP-Neu5Ac to decorate their glycoconjugates. For selected bacteria we also show this strategy complements two other glycoengineering techniques, Metabolic Oligosaccharide Engineering and Selective Exo-Enzymatic Labeling, and that together they provide tools to modify, label, detect and visualize sialylation of bacterial lipooligosaccharides.

Genes involved in the adhesion and invasion of Arcobacter butzleri.

Arcobacter butzleri is a foodborne pathogen that mainly causes enteritis in humans, but the number of cases of bacteraemia has increased in recent years. However, there is still limited knowledge on the pathogenic mechanisms of this bacterium. To investigate how A. butzleri causes disease, single knockout mutants were constructed in the cadF, ABU_RS00335, ciaB, and flaAB genes, which might be involved in adhesion and invasion properties. These mutants and the isogenic wild-type (WT) were then tested for their ability to adhere and invade human Caco-2 and HT29-MTX cells. The adhesion and invasion of A. butzleri RM4018 strain was also visualized by a Leica CTR 6500 confocal microscope. The adhesion and invasion abilities of mutants lacking the invasion antigen CiaB or a functional flagellum were lower than those of the WTs. However, the extent of the decrease varied depending on the strain and/or cell line. Mutants lacking the fibronectin (FN)-binding protein CadF consistently exhibited reduced abilities, while the inactivation of the other studied FN-binding protein, ABU_RS00335, led to a reduction in only one of the two strains tested. Therefore, the ciaB and flaAB genes appear to be important for A. butzleri adhesion and invasion properties, while cadF appears to be indispensable.

Sweet impersonators: Molecular mimicry of host glycans by bacteria.

All bacteria display surface-exposed glycans that can play an important role in their interaction with the host and in select cases mimic the glycans found on host cells, an event called molecular or glycan mimicry. In this review, we highlight the key bacteria that display human glycan mimicry and provide an overview of the involved glycan structures. We also discuss the general trends and outstanding questions associated with human glycan mimicry by bacteria. Finally, we provide an overview of several techniques that have emerged from the discipline of chemical glycobiology, which can aid in the study of the composition, variability, interaction and functional role of these mimicking glycans.

Selective Exoenzymatic Labeling of Lipooligosaccharides of Neisseria gonorrhoeae with α2,6-Sialoside Analogues.

The interactions between bacteria and their host often rely on recognition processes that involve host or bacterial glycans. Glycoengineering techniques make it possible to modify and study the glycans on the host's eukaryotic cells, but only a few are available for the study of bacterial glycans. Here, we have adapted selective exoenzymatic labeling (SEEL), a chemical reporter strategy, to label the lipooligosaccharides of the bacterial pathogen Neisseria gonorrhoeae, using the recombinant glycosyltransferase ST6Gal1, and three synthetic CMP-sialic acid derivatives. We show that SEEL treatment does not affect cell viability and can introduce an α2,6-linked sialic acid with a reporter group on the lipooligosaccharides by Western blot, flow cytometry and fluorescent microscopy. This new bacterial glycoengineering technique allows for the precise modification, here with α2,6-sialoside derivatives, and direct detection of specific surface glycans on live bacteria, which will aid in further unravelling the precise biological functions of bacterial glycans.

Bacteroides fragilis fucosidases facilitate growth and invasion of Campylobacter jejuni in the presence of mucins.

The enteropathogenic bacterium, Campylobacter jejuni, was considered to be non-saccharolytic, but recently it emerged that l-fucose plays a central role in C. jejuni virulence. Half of C. jejuni clinical isolates possess an operon for l-fucose utilisation. In the intestinal tract, l-fucose is abundantly available in mucin O-linked glycan structures, but C. jejuni lacks a fucosidase enzyme essential to release the l-fucose. We set out to determine how C. jejuni can gain access to these intestinal l-fucosides. Growth of the fuc + C. jejuni strains, 129,108 and NCTC 11168, increased in the presence of l-fucose while fucose permease knockout strains did not benefit from additional l-fucose. With fucosidase assays and an activity-based probe, we confirmed that Bacteriodes fragilis, an abundant member of the intestinal microbiota, secretes active fucosidases. In the presence of mucins, C. jejuni was dependent on B. fragilis fucosidase activity for increased growth. Campylobacter jejuni invaded Caco-2 intestinal cells that express complex O-linked glycan structures that contain l-fucose. In infection experiments, C. jejuni was more invasive in the presence of B. fragilis and this increase is due to fucosidase activity. We conclude that C. jejuni fuc + strains are dependent on exogenous fucosidases for increased growth and invasion.

Metabolic Labeling of Legionaminic Acid in Flagellin Glycosylation of Campylobacter jejuni Identifies Maf4 as a Putative Legionaminyl Transferase.

Campylobacter jejuni is the major human food-borne pathogen. Its bipolar flagella are heavily O-glycosylated with microbial sialic acids and essential for its motility and pathogenicity. However, both the glycosylation of flagella and the exact contribution of legionaminic acid (Leg) to flagellar activity is poorly understood. Herein, we report the development of a metabolic labeling method for Leg glycosylation on bacterial flagella with probes based on azide-modified Leg precursors. The hereby azido-Leg labeled flagellin could be detected by Western blot analysis and imaged on intact bacteria. Using the probes on C. jejuni and its isogenic maf4 mutant we also further substantiated the identification of Maf4 as a putative Leg glycosyltransferase. Further evidence was provided by UPLC-MS detection of labeled CMP-Leg and an in silico model of Maf4. This method and the developed probes will facilitate the study of Leg glycosylation and the functional role of this modification in C. jejuni motility and invasiveness.

Generation of the membrane potential and its impact on the motility, ATP production and growth in Campylobacter jejuni.

The generation of a membrane potential (Δψ), the major constituent of the proton motive force (pmf), is crucial for ATP synthesis, transport of nutrients and flagellar rotation. Campylobacter jejuni harbors a branched electron transport chain, enabling respiration with different electron donors and acceptors. Here, we demonstrate that a relatively high Δψ is only generated in the presence of either formate as electron donor or oxygen as electron acceptor, in combination with an acceptor/donor respectively. We show the necessity of the pmf for motility and growth of C. jejuni. ATP generation is not only accomplished by oxidative phosphorylation via the pmf, but also by substrate-level phosphorylation via the enzyme AckA. In response to a low oxygen tension, C. jejuni increases the transcription and activity of the donor complexes formate dehydrogenase (FdhABC) and hydrogenase (HydABCD) as well as the transcription of the alternative respiratory acceptor complexes. Our findings suggest that in the gut of warm-blooded animals, C. jejuni depends on at least formate or hydrogen as donor (in the anaerobic lumen) or oxygen as acceptor (near the epithelial cells) to generate a pmf that sustains efficient motility and growth for colonization and pathogenesis.

Catabolite repression in Campylobacter jejuni correlates with intracellular succinate levels.

Bacteria have evolved different mechanisms to catabolize carbon sources from nutrient mixtures. They first consume their preferred carbon source, before others are used. Regulatory mechanisms adapt the metabolism accordingly to maximize growth and to outcompete other organisms. The human pathogen Campylobacter jejuni is an asaccharolytic Gram-negative bacterium that catabolizes amino acids and organic acids for growth. It prefers serine and aspartate as carbon sources, however it lacks all regulators known to be involved in regulating carbon source utilization in other organisms. In which manner C. jejuni adapts its metabolism towards the presence or absence of preferred carbon sources is unknown. In this study, we show with transcriptomic analysis and enzyme assays how C. jejuni adapts its metabolism in response to its preferred carbon sources. In the presence of serine as well as lactate and pyruvate C. jejuni inhibits the utilization of other carbon sources, by repressing the expression of a number of central metabolic enzymes. The regulatory proteins RacR, Cj1000 and CsrA play a role in the regulation of these metabolic enzymes. This metabolism dependent transcriptional repression correlates with an accumulation of intracellular succinate. Hence, we propose a demand-based catabolite repression mechanism in C. jejuni, depended on intracellular succinate levels.

Feedback control of Campylobacter jejuni flagellin levels through reciprocal binding of FliW to flagellin and the global regulator CsrA.

Bacterial flagella assembly is tightly regulated to ensure a timely and sequential production of the various flagellum constituents. In the pathogen Campylobacter jejuni the hierarchy in flagella biosynthesis is largely determined at the transcriptional level through the activity of the alternative sigma factors sigma54 and sigma28 . Here, we report that C. jejuni flagellin levels are also controlled at the post-transcriptional level via the thus far poorly-characterized flagellar assembly factor FliW. Analysis of flagellin synthesis in C. jejuni 81116 and a ΔfliW knock-out mutant showed reduced flagellin protein levels in the mutant strain while ectopic expression of FliW resulted in enhanced levels. Real-time RT-PCR revealed relatively minor changes in flaA and flaB mRNA levels for the recombinant and parent strain consistent with post-transcriptional regulation. Purified FliW was found to bind to FlaA and FlaB flagellin as well as to the global post-transcriptional regulator CsrA. Inactivation of CsrA resulted in increased levels of flagellin translation. An in vitro translation assay confirmed the regulatory role of CsrA in flagellin biosynthesis. We propose that competitive reciprocal binding of FliW to flagellins and the RNA binding protein CsrA serves as a feedback mechanism to control the number of cytosolic flagellin copies at the protein level.

The Campylobacter jejuni†RacRS system regulates fumarate utilization in a low oxygen environment.

The natural environment of the human pathogen Campylobacter jejuni is the gastrointestinal tract of warm-blooded animals. In the gut, the availability of oxygen is limited; therefore, less efficient electron acceptors such as nitrate or fumarate are used by C. jejuni. The molecular mechanisms that regulate the activity of the highly branched respiratory chain of C. jejuni are still a mystery mainly because C. jejuni lacks homologues of transcription factors known to regulate energy metabolism in other bacteria. Here we demonstrate that dependent on the available electron acceptors the two-component system RacRS controls the production of fumarate from aspartate, as well as its transport and reduction to succinate. Transcription profiling, DNAse protection and functional assays showed that phosphorylated RacR binds to and represses at least five promoter elements located in front of genes involved in the uptake and synthesis of fumarate. The RacRS system is active in the presence of nitrate and trimethyl-amine-N-oxide under oxygen-limited conditions when fumarate is less preferred as an alternative electron acceptor. In the inactive state, RacRS allows utilization of fumarate for respiration. The unique C. jejuni RacRS regulatory system illustrates the disparate evolution of Campylobacter and aids the survival of this pathogen.

Identification of a functional type VI secretion system in Campylobacter jejuni conferring capsule polysaccharide sensitive cytotoxicity.

The pathogen Campylobacter jejuni is the principal cause of bacterial food-borne infections. The mechanism(s) that contribute to bacterial survival and disease are still poorly understood. In other bacterial species, type VI secretion systems (T6SS) are increasingly recognized to contribute to bacterial pathogenesis by toxic effects on host cells or competing bacterial species. Here we report the presence of a functional Type VI secretion system in C. jejuni. Proteome and genetic analyses revealed that C. jejuni strain 108 contains a 17-kb T6SS gene cluster consisting of 13 T6SS-conserved genes, including the T6SS hallmark genes hcp and vgrG. The cluster lacks an ortholog of the ClpV ATPase considered important for T6SS function. The sequence and organization of the C. jejuni T6SS genes resemble those of the T6SS located on the HHGI1 pathogenicity island of Helicobacter hepaticus. The C. jejuni T6SS is integrated into the earlier acquired Campylobacter integrated element CJIE3 and is present in about 10% of C. jejuni isolates including several isolates derived from patients with the rare clinical feature of C. jejuni bacteremia. Targeted mutagenesis of C. jejuni T6SS genes revealed T6SS-dependent secretion of the Hcp needle protein into the culture supernatant. Infection assays provided evidence that the C. jejuni T6SS confers contact-dependent cytotoxicity towards red blood cells but not macrophages. This trait was observed only in a capsule-deficient bacterial phenotype. The unique C. jejuni T6SS phenotype of capsule-sensitive contact-mediated hemolysis represents a novel evolutionary pathway of T6SS in bacteria and expands the repertoire of virulence properties associated with T6SS.

Low-Level Tetracycline Resistance Gene tet(O)_3 in Campylobacter jejuni.

Campylobacter (C.) spp. are the most important foodborne, bacterial, and zoonotic pathogens worldwide. Resistance monitoring of foodborne bacterial pathogens is an important tool to control antimicrobial resistance as a part of the "One Health" approach. The detection and functionality of new resistance genes are of paramount importance in applying more effective screening methods based on whole genome sequencing (WGS). Most tetracycline-resistant C. spp. isolates harbor tet(O), a gene that encodes a ribosomal protection protein. Here we describe tet(O)_3, which has been identified in two food isolates of C. jejuni and is very similar to the tet(O) gene in Streptococcus pneumoniae, having a truncated promoter sequence. This gene confers resistance to tetracycline below 1 mg/L, which is the epidemiological cut-off value. We have analyzed the entire genome of these two isolates, together with a C. jejuni isolate found to have high-level resistance to tetracycline. In contrast to the highly resistant isolate, the promoter of tet(O)_3 is highly responsive to tetracycline, as observed by reverse transcription polymerase chain reaction (RT-PCR). In addition, the two isolates possess a CRISPR repeat, fluoroquinolone resistance due to the gyrA point mutation C257T, a ß-lactamase resistance gene blaOXA-184, a multidrug efflux pump CmeABC and its repressor CmeR, but no plasmid. Low-level antibiotic resistant C. jejuni might therefore have an advantage for surviving in non-host environments.

Campylobacter jejuni benefits from the bile salt deoxycholate under low-oxygen condition in a PldA dependent manner.

Enteric bacteria need to adapt to endure the antibacterial activities of bile salts in the gut. Phospholipase A (PldA) is a key enzyme in the maintenance of bacterial membrane homeostasis. Bacteria respond to stress by modulating their membrane composition. Campylobacter jejuni is the most common cause of human worldwide. However, the mechanism by which C. jejuni adapts and survives in the gut environment is not fully understood. In this study, we investigated the roles of PldA, bile salt sodium deoxycholate (DOC), and oxygen availability in C. jejuni biology, mimicking an in vivo situation. Growth curves were used to determine the adaptation of C. jejuni to bile salts. RNA-seq and functional assays were employed to investigate the PldA-dependent and DOC-induced changes in gene expression that influence bacterial physiology. Survival studies were performed to address oxidative stress defense in C. jejuni. Here, we discovered that PldA of C. jejuni is required for optimal growth in the presence of bile salt DOC. Under high oxygen conditions, DOC is toxic to C. jejuni, but under low oxygen conditions, as is present in the lumen of the gut, C. jejuni benefits from DOC. C. jejuni PldA seems to enable the use of iron needed for optimal growth in the presence of DOC but makes the bacterium more vulnerable to oxidative stress. In conclusion, DOC stimulates C. jejuni growth under low oxygen conditions and alters colony morphology in a PldA-dependent manner. C. jejuni benefits from DOC by upregulating iron metabolism in a PldA-dependent manner.

Biological functions of bacterial lysophospholipids.

Lysophospholipids (LPLs) are lipid-derived metabolic intermediates in the cell membrane. The biological functions of LPLs are distinct from their corresponding phospholipids. In eukaryotic cells LPLs are important bioactive signaling molecules that regulate many important biological processes, but in bacteria the function of LPLs is still not fully defined. Bacterial LPLs are usually present in cells in very small amounts, but can strongly increase under certain environmental conditions. In addition to their basic function as precursors in membrane lipid metabolism, the formation of distinct LPLs contributes to the proliferation of bacteria under harsh circumstances or may act as signaling molecules in bacterial pathogenesis. This review provides an overview of the current knowledge of the biological functions of bacterial LPLs including lysoPE, lysoPA, lysoPC, lysoPG, lysoPS and lysoPI in bacterial adaptation, survival, and host-microbe interactions.

Protective Effects of Alginate and Chitosan Oligosaccharides against Clostridioides difficile Bacteria and Toxin.

Clostridioides difficile infection is expected to become the most common healthcare-associated infection worldwide. C. difficile-induced pathogenicity is significantly attributed to its enterotoxin, TcdA, which primarily targets Rho-GTPases involved in regulating cytoskeletal and tight junction (TJ) dynamics, thus leading to cytoskeleton breakdown and ultimately increased intestinal permeability. This study investigated whether two non-digestible oligosaccharides (NDOs), alginate (AOS) and chitosan (COS) oligosaccharides, possess antipathogenic and barrier-protective properties against C. difficile bacteria and TcdA toxin, respectively. Both NDOs significantly reduced C. difficile growth, while cell cytotoxicity assays demonstrated that neither COS nor AOS significantly attenuated the TcdA-induced cell death 24 h post-exposure. The challenge of Caco-2 monolayers with increasing TcdA concentrations increased paracellular permeability, as measured by TEER and LY flux assays. In this experimental setup, COS completely abolished, and AOS mitigated, the deleterious effects of TcdA on the monolayer's integrity. These events were not accompanied by alterations in ZO-1 and occludin protein levels; however, immunofluorescence microscopy revealed that both AOS and COS prevented the TcdA-induced occludin mislocalization. Finally, both NDOs accelerated TJ reassembly upon a calcium-switch assay. Overall, this study established the antipathogenic and barrier-protective capacity of AOS and COS against C. difficile and its toxin, TcdA, while revealing their ability to promote TJ reassembly in Caco-2 cells.

Campylobacter jejuni permeabilizes the host cell membrane by short chain lysophosphatidylethanolamines.

Lysophospholipids (LPLs) are crucial for regulating epithelial integrity and homeostasis in eukaryotes, however the effects of LPLs produced by bacteria on host cells is largely unknown. The membrane of the human bacterial pathogen Campylobacter jejuni is rich in LPLs. Although C. jejuni possesses several virulence factors, it lacks traditional virulence factors like type III secretion systems, present in most enteropathogens. Here, we provide evidence that membrane lipids lysophosphatidylethanolamines (lysoPEs) of C. jejuni are able to lyse erythrocytes and are toxic for HeLa and Caco-2 cells. Lactate dehydrogenase (LDH) release assays and confocal microscopy revealed that lysoPE permeabilizes the cells. LysoPE toxicity was partially rescued by oxidative stress inhibitors, indicating that intracellular reactive oxygen species may contribute to the cell damage. Our results show that especially the short-chain lysoPEs (C:14) which is abundantly present in the C. jejuni membrane may be considered as a novel virulence factor.

Altered linkage of hydroxyacyl chains in lipid A of Campylobacter jejuni reduces TLR4 activation and antimicrobial resistance.

Modification of the lipid A moiety of bacterial lipopolysaccharide influences cell wall properties, endotoxic activity, and bacterial resistance to antimicrobial peptides. Known modifications are variation in the number or length of acyl chains and/or attached phosphoryl groups. Here we identified two genes (gnnA and gnnB) in the major foodborne pathogen Campylobacter jejuni that enable the synthesis of a GlcN3N precursor UDP 2-acetamido-3-amino-2,3-dideoxy-alpha-D-glucopyranose (UDP-GlcNAc3N) in the lipid A backbone. Mass spectrometry of purified lipooligosaccharide verified that the gene products facilitate the formation of a 2,3-diamino-2,3-dideoxy-D-glucose (GlcN3N) disaccharide lipid A backbone when compared with the beta-1'-6-linked D-glucosamine (GlcN) disaccharide observed in Escherichia coli lipid A. Functional assays showed that inactivation of the gnnA or gnnB gene enhanced the TLR4-MD2-mediated NF-kappaB activation. The mutants also displayed increased susceptibility to killing by the antimicrobial peptides polymyxin B, colistin and the chicken cathelicidin-1. The gnnA and gnnB genes are organized in one operon with hemH, encoding a ferrochelatase catalyzing the last step in heme biosynthesis. These results indicate that lipid A modification resulting in amide-linked acyl chains in the lipid A is an effective mechanism to evade activation of the innate host defense and killing by antimicrobial peptides.

Temperature-dependent FlgM/FliA complex formation regulates Campylobacter jejuni flagella length.

Regulation of the biosynthesis of the flagellar filament in bacteria containing multiple flagellin genes is not well understood. The major food-borne pathogen Campylobacter jejuni possesses on both poles a flagellum that consists of two different flagellin subunits, FlaA and FlaB. Here we identify the protein Cj1464 as a regulator of C. jejuni flagellin biosynthesis. The protein shares characteristics of the FlgM family of anti-sigma factor proteins: it represses transcription of sigma(28)-dependent genes, forms a complex with sigma factor FliA, and is secreted through the flagellar filament. However, unlike other FlgM proteins, the interaction of C. jejuni FlgM with FliA is regulated by temperature and the protein does not inhibit FliA activity during the formation of the hook-basal body complex (HBB). Instead, C. jejuni FlgM limits the length of the flagellar filament by suppressing the synthesis of both the sigma(28)- and the sigma(54)-dependent flagellins. The main function of the C. jejuni FlgM therefore is not to silence sigma(28)-dependent genes until the HBB is completed, but to prevent unlimited elongation of the flagellum, which otherwise leads to reduced bacterial motility.

Control of n-Butanol Induced Lipidome Adaptations in E. coli.

The versatile compound n-butanol is one of the most promising biofuels for use in existing internal combustion engines, contributing to a smooth transition towards a clean energy society. Furthermore, n-butanol is a valuable resource to produce more complex molecules such as bioplastics. Microbial production of n-butanol from waste materials is hampered by the biotoxicity of n-butanol as it interferes with the proper functioning of lipid membranes. In this study we perform a large-scale investigation of the complete lipid-related enzyme machinery and its response to exposure to a sublethal concentration of n-butanol. We profiled, in triplicate, the growth characteristics and phospholipidomes of 116 different genetic constructs of E. coli, both in the presence and absence of 0.5% n-butanol (v/v). This led to the identification of 230 lipid species and subsequently to the reconstruction of the network of metabolites, enzymes and lipid properties driving the homeostasis of the E. coli lipidome. We were able to identify key lipids and biochemical pathways leading to altered n-butanol tolerance. The data led to new conceptual insights into the bacterial lipid metabolism which are discussed.

Growth phase-dependent activation of the DccRS regulon of Campylobacter jejuni.

Two-component systems are widespread prokaryotic signal transduction devices which allow the regulation of cellular functions in response to changing environmental conditions. The two-component system DccRS (Cj1223c-Cj1222c) of Campylobacter jejuni is important for the colonization of chickens. Here, we dissect the DccRS system in more detail and provide evidence that the sensor DccS selectively phosphorylates the cognate effector, DccR. Microarray expression profiling, real-time reverse transcription-PCR (RT-PCR), electrophoretic mobility shift assay, and primer extension analyses revealed that the DccRS regulon of strain 81116 consists of five promoter elements, all containing the consensus direct repeat sequence WTTCAC-N6-TTCACW covering the putative -35 promoter regions. One of these promoters is located in front of an operon encoding a putative macrolide efflux pump while the others are in front of genes coding for putative periplasmic or membrane proteins. The DccRS-regulated genes in C. jejuni strain 81116 are needed to enhance early in vivo growth of C. jejuni in 7-day-old chickens. The DccRS system is activated in the late stationary bacterial growth phase, probably by released metabolic products. Whole-genome mRNA profiling and real-time RT-PCR analysis under these conditions demonstrated that the system has no influence on the transcription of genes outside the DccRS regulon.