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 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.

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.

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.

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.

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.

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.

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.

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.

Dataset of the phospholipidome and transcriptome of Campylobacter jejuni under different growth conditions.

The membrane phospholipid composition is not a stable bacterial characteristic but can change in response to altered environmental conditions. Here we provide the dataset of the phospholipidome and transcriptome of the microaerophilic human pathogen Campylobacter jejuni under different environmental conditions. These data have been used in Cao (2020), The unique phospholipidome of the enteric pathogen C. jejuni: Lysolipids are required for motility at low oxygen availability. Here the abundance of each phospholipid is shown during the growth of C. jejuni for 0-108 h under low and high oxygen conditions (0.3 vs 10% O2). The phospholipid data were obtained by applying high performance liquid chromatography tandem-mass spectrometry (LC-MS/MS). The transcriptomic data obtained by RNA-seq show the differential expressed genes between logarithmic and stationary grown bacteria. In addition, our data might serve as a reference information for further in-depth investigation to understand the relation between specific phospholipids and the activity of membrane associated proteins.

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.

The Unique Phospholipidome of the Enteric Pathogen Campylobacter jejuni: Lysophosholipids Are Required for Motility at Low Oxygen Availability.

In response to changes in their environment bacteria need to change both their protein and phospholipid repertoire to match environmental requirements, but the dynamics of bacterial phospholipid composition under different growth conditions is still largely unknown. In the present study, we investigated the phospholipidome of the bacterial pathogen Campylobacter jejuni. Transcription profiling on logarithmic and stationary phase grown cells of the microaerophilic human pathogen C. jejuni using RNA-seq revealed differential expression of putative phospholipid biosynthesis genes. By applying high-performance liquid chromatography tandem-mass spectrometry, we identified 203 phospholipid species representing the first determination of the phospholipidome of this pathogen. We identified nine different phospholipid classes carrying between one and three acyl chains. Phospholipidome analysis on bacteria of different ages (0-5 days) showed rapid changes in the ratio of phospholipids containing ethanolamine, or glycerol as phospholipid head group and in the number of cyclopropane bond containing fatty acids. Oxygen concentration influenced the percentage of lysophospholipids, and cyclo-propane bonds containing acyl chains. We show that large amounts of the phospholipids are lysophospholipids (30-45%), which mutant studies reveal are needed for normal C. jejuni motility at low oxygen conditions. C. jejuni possesses an unusual phospholipidome that is highly dynamic in response to environmental changes.

Regulation of Respiratory Pathways in Campylobacterota: A Review.

The Campylobacterota, previously known as Epsilonproteobacteria, are a large group of Gram-negative mainly, spiral-shaped motile bacteria. Some members like the Sulfurospirillum spp. are free-living, while others such as Helicobacter spp. can only persist in strict association with a host organism as commensal or as pathogen. Species of this phylum colonize diverse habitats ranging from deep-sea thermal vents to the human stomach wall. Despite their divergent environments, they share common energy conservation mechanisms. The Campylobacterota have a large and remarkable repertoire of electron transport chain enzymes, given their small genomes. Although members of recognized families of transcriptional regulators are found in these genomes, sofar no orthologs known to be important for energy or redox metabolism such as ArcA, FNR or NarP are encoded in the genomes of the Campylobacterota. In this review, we discuss the strategies that members of Campylobacterota utilize to conserve energy and the corresponding regulatory mechanisms that regulate the branched electron transport chains in these bacteria.

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.

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.

Importance of Campylobacter jejuni FliS and FliW in Flagella Biogenesis and Flagellin Secretion.

Flagella-driven motility enables bacteria to reach their favorable niche within the host. The human foodborne pathogen Campylobacter jejuni produces two heavily glycosylated structural flagellins (FlaA and FlaB) that form the flagellar filament. It also encodes the non-structural FlaC flagellin which is secreted through the flagellum and has been implicated in host cell invasion. The mechanisms that regulate C. jejuni flagellin biogenesis and guide the proteins to the export apparatus are different from those in most other enteropathogens and are not fully understood. This work demonstrates the importance of the putative flagellar protein FliS in C. jejuni flagella assembly. A constructed fliS knockout strain was non-motile, displayed reduced levels of FlaA/B and FlaC flagellin, and carried severely truncated flagella. Pull-down and Far Western blot assays showed direct interaction of FliS with all three C. jejuni flagellins (FlaA, FlaB, and FlaC). This is in contrast to, the sensor and regulator of intracellular flagellin levels, FliW, which bound to FlaA and FlaB but not to FlaC. The FliS protein but not FliW preferred binding to glycosylated C. jejuni flagellins rather than to their non-glycosylated recombinant counterparts. Mapping of the binding region of FliS and FliW using a set of flagellin fragments showed that the C-terminal subdomain of the flagellin was required for FliS binding, whereas the N-terminal subdomain was essential for FliW binding. The separate binding subdomains required for FliS and FliW, the different substrate specificity, and the differential preference for binding of glycosylated flagellins ensure optimal processing and assembly of the C. jejuni flagellins.

Function and Regulation of the C4-Dicarboxylate Transporters in Campylobacter jejuni.

C4-dicarboxylates are important molecules for the human pathogen C.jejuni, as they are used as carbon and electron acceptor molecules, as sugars cannot be utilized by this microaerophilic organism. Based on the genome analysis, C. jejuni may possess five different C4-dicarboxylate transporters: DctA, DcuA, DcuB, and two homologs of DcuC. Here, we investigated the regulation and function of various C4-dicarboxylate transporters in C. jejuni. Transcription of the dctA and dcuC homologs is constitutive, while dcuA and dcuB are both directly regulated by the two-component RacR/RacS system in response to limited oxygen availability and the presence of nitrate. The DctA transporter is the only C4-dicarboxylate transporter to allow C. jejuni to grow on C4-carbon sources such as aspartate, fumarate, and succinate at high oxygen levels (10% O2) and is indispensable for the uptake of succinate from the medium under these conditions. Both DcuA and DcuB can sequester aspartate from the medium under low-oxygen conditions (0.3% O2). However, under these conditions, DcuB is the only transporter to secrete succinate to the environment. Under low-oxygen conditions, nitrate prevents the secretion of succinate to the environment and was able to overrule the phenotype of the C4-transporter mutants, indicating that the activity of the aspartate-fumarate-succinate pathway in C. jejuni is strongly reduced by the addition of nitrate in the medium.

Chicken Immune Response after In Ovo Immunization with Chimeric TLR5 Activating Flagellin of Campylobacter jejuni.

Campylobacter jejuni is the main cause of bacterial food-borne diseases in developed countries. Chickens are the most important source of human infection. Vaccination of poultry is an attractive strategy to reduce the number of C. jejuni in the intestinal tract of chickens. We investigated the immunogenicity and protective efficacy of a recombinant C. jejuni flagellin-based subunit vaccine with intrinsic adjuvant activity. Toll-like receptor activation assays demonstrated the purity and TLR5 stimulating (adjuvant) activity of the vaccine. The antigen (20-40 µg) was administered in ovo to 18 day-old chicken embryos. Serum samples and intestinal content were assessed for antigen-specific systemic and mucosal humoral immune responses. In ovo vaccination resulted in the successful generation of IgY and IgM serum antibodies against the flagellin-based subunit vaccine as determined by ELISA and Western blotting. Vaccination did not induce significant amounts of flagellin-specific secretory IgA in the chicken intestine. Challenge of chickens with C. jejuni yielded similar intestinal colonization levels for vaccinated and control animals. Our results indicate that in ovo delivery of recombinant C. jejuni flagellin subunit vaccine is a feasible approach to yield a systemic humoral immune response in chickens but that a mucosal immune response may be needed to reduce C. jejuni colonization.

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 two-component system activates the glutamate synthesis by directly upregulating γ-glutamyltranspeptidase (GGT).

The highly conserved enzyme γ-glutamyltranspeptidase (GGT) plays an important role in metabolism of glutathione and glutamine. Yet, the regulation of ggt transcription in prokaryotes is poorly understood. In the human pathogen Campylobacter jejuni, GGT is important as it contributes to persistent colonization of the gut. Here we show that the GGT activity in C. jejuni is dependent on a functional RacRS (reduced ability to colonize) two-component system. Electrophoretic mobility shift and luciferase reporter assays indicate that the response regulator RacR binds to a promoter region ~80 bp upstream of the ggt transcriptional start site, which contains a recently identified RacR DNA binding consensus sequence. RacR needs to be phosphorylated to activate the transcription of the ggt gene, which is the case under low oxygen conditions in presence of alternative electron acceptors. A functional GGT and RacR are needed to allow C. jejuni to grow optimally on glutamine as sole carbon source under RacR inducing conditions. However, when additional carbon sources are present C. jejuni is capable of utilizing glutamine independently of GGT. RacR is the first prokaryotic transcription factor known to directly up-regulate both the cytoplasmic [glutamine-2-oxoglutarate aminotransferase (GOGAT)] as well as the periplasmic (GGT) production of glutamate.