Human Pathogen Campylobacter Jejuni Research Articles

Evolution and Role of Proteases in Campylobacter jejuni Lifestyle and Pathogenesis.

Infection with the main human food-borne pathogen Campylobacter jejuni causes campylobacteriosis that accounts for a substantial percentage of gastrointestinal infections. The disease usually manifests as diarrhea that lasts for up to two weeks. C. jejuni possesses an array of peptidases and proteases that are critical for its lifestyle and pathogenesis. These include serine proteases Cj1365c, Cj0511 and HtrA; AAA+ group proteases ClpP, Lon and FtsH; and zinc-dependent protease PqqE, proline aminopeptidase PepP, oligopeptidase PepF and peptidase C26. Here, we review the numerous critical roles of these peptide bond-dissolving enzymes in cellular processes of C. jejuni that include protein quality control; protein transport across the inner and outer membranes into the periplasm, cell surface or extracellular space; acquisition of amino acids and biofilm formation and dispersal. In addition, we highlight their role as virulence factors that inflict intestinal tissue damage by promoting cell invasion and mediating cleavage of crucial host cell factors such as epithelial cell junction proteins. Furthermore, we reconstruct the evolution of these proteases in 34 species of the Campylobacter genus. Finally, we discuss to what extent C. jejuni proteases have initiated the search for inhibitor compounds as prospective novel anti-bacterial therapies.

Characterisation of N-linked protein glycosylation in the bacterial pathogen Campylobacter hepaticus

Campylobacter hepaticus is an important pathogen which causes Spotty Liver Disease (SLD) in layer chickens. SLD results in an increase in mortality and a significant decrease in egg production and therefore is an important economic concern of the global poultry industry. The human pathogen Campylobacter jejuni encodes an N-linked glycosylation system that plays fundamental roles in host colonization and pathogenicity. While N-linked glycosylation has been extensively studied in C. jejuni and is now known to occur in a range of Campylobacter species, little is known about C. hepaticus glycosylation. In this study glycoproteomic analysis was used to confirm the functionality of the C. hepaticus N-glycosylation system. It was shown that C. hepaticus HV10T modifies > 35 proteins with an N-linked heptasaccharide glycan. C. hepaticus shares highly conserved glycoproteins with C. jejuni that are involved in host colonisation and also possesses unique glycoproteins which may contribute to its ability to survive in challenging host environments. C. hepaticus N-glycosylation may function as an important virulence factor, providing an opportunity to investigate and develop a better understanding the system’s role in poultry infection.

IL-23 Contributes to Campylobacter jejuni-Induced Intestinal Pathology via Promoting IL-17 and IFNγ Responses by Innate Lymphoid Cells.

Human pathogen Campylobacter jejuni is a significant risk factor for the development of long-term intestinal dysfunction although the cellular and molecular mechanisms remain scantily defined. IL-23 is an emerging therapeutic target for the treatment of inflammatory intestinal diseases, however its role in C. jejuni-driven intestinal pathology is not fully understood. IL-10 deficient mice represent a robust model to study the pathogenesis of C. jejuni infection because C. jejuni infection of mice lacking IL-10 results in symptoms and pathology that resemble human campylobacteriosis. To determine the role of IL-23 in C. jejuni-driven intestinal inflammation, we studied the disease pathogenesis in IL-23-/- mice with inhibited IL-10Rα signaling. These mice exhibited reduced intestinal pathology independent from bacterial clearance. Further, levels of IFNγ, IL-17, IL-22, TNF, and IL-6 were reduced and associated with reduced accumulation of neutrophils, monocytes and macrophages in the colon. Flow cytometry analysis revealed reduced production of IL-17 and IFNγ by group 1 and 3 innate lymphoid cells. Thus, our data suggest that IL-23 contributes to intestinal inflammation in C. jejuni infected mice by promoting IL-17 and IFNγ production by innate lymphoid cells.

Campylobacter jejuni Cas9 Modulates the Transcriptome in Caco-2 Intestinal Epithelial Cells.

The zoonotic human pathogen Campylobacter jejuni is known for its ability to induce DNA-damage and cell death pathology in humans. The molecular mechanism behind this phenomenon involves nuclear translocation by Cas9, a nuclease in C. jejuni (CjeCas9) that is the molecular marker of the Type II CRISPR-Cas system. However, it is unknown via which cellular pathways CjeCas9 drives human intestinal epithelial cells into cell death. Here, we show that CjeCas9 released by C. jejuni during the infection of Caco-2 human intestinal epithelial cells directly modulates Caco-2 transcriptomes during the first four hours of infection. Specifically, our results reveal that CjeCas9 activates DNA damage (p53, ATM (Ataxia Telangiectasia Mutated Protein)), pro-inflammatory (NF-κB (Nuclear factor-κB)) signaling and cell death pathways, driving Caco-2 cells infected by wild-type C. jejuni, but not when infected by a cas9 deletion mutant, towards programmed cell death. This work corroborates our previous finding that CjeCas9 is cytotoxic and highlights on a RNA level the basal cellular pathways that are modulated.

Random sorting of Campylobacter jejuni phase variants due to a narrow bottleneck during colonization of broiler chickens.

Phase variation (PV), involving stochastic switches in gene expression, is exploited by the human pathogen Campylobacter jejuni to adapt to different environmental and host niches. Phase-variable genes of C. jejuni modulate expression of multiple surface determinants, and hence may influence host colonization. Population bottlenecks can rapidly remove the diversity generated by PV, and strict single-cell bottlenecks can lead to propagation of PV states with highly divergent phenotypes. Using a combination of high-throughput fragment size analysis and comparison with in vivo and in silico bottleneck models, we have characterized a narrow population bottleneck during the experimental colonization of broiler chickens with C. jejuni strain 81-176. We identified high levels of variation in five PV genes in the inoculum, and subsequently, massively decreased population diversity following colonization. Each bird contained a dominant five-gene phasotype that was present in the inoculum indicative of random sorting through a narrow, non-selective bottleneck during colonization. These results are evidence of the potential for confounding effects of PV on in vivo studies of Campylobacter colonization factors and poultry vaccine studies. Our results are also an argument for population bottlenecks as mediators of stochastic variability in the propensity to survive through the food chain and cause clinical human disease.

Design, solid-phase synthesis and evaluation of enterobactin analogs for iron delivery into the human pathogen Campylobacter jejuni

The human enteropathogen Campylobacter jejuni, like many bacteria, employs siderophores such as enterobactin for cellular uptake of ferric iron. This transport process has been shown to be essential for virulence and presents an attractive opportunity for further study of the permissiveness of this pathway to small-molecule intervention and as inspiration for the development of synthetic carriers that may effectively transport cargo into Gram-negative bacteria. In this work, we have developed a facile and robust microscale assay to measure growth recovery of C. jejuni NCTC 11168 in liquid culture as a result of ferric iron uptake. In parallel, we have established the solid-phase synthesis of catecholamide compounds modeled on enterobactin fragments. Applying these methodological developments, we show that small synthetic iron chelators of minimal dimensions provide ferric iron to C. jejuni with equal or greater efficiency than enterobactin.

Excretion patterns of Campylobacter jejuni by dairy cows

ABSTRACTExcretion of the human bacterial pathogen Campylobacter jejuni by naturally infected dairy cows was investigated by monitoring 18 and 17 cows on two different commercial farms fortnightly for up to 12 months. C. jejuni was enumerated in the collected faeces by a most probable number technique and genotyped by enterobacterial repetitive intergenic consensus (ERIC) sequences. On both farms, C. jejuni excretion was highly variable among the studied cows, with excretion patterns ranging from chronic to sporadic. Chronic excretion of C. jejuni was associated with long-term predominance of a given genotype, co-excretion of a few genotypes, or succession over time of dominant genotypes. Sporadic excretion was with re-excretion of the same C. jejuni genotype when there was less than 1.5 months between C. jejuni-positive samples. Overall, the results showed the complexity of C. jejuni excretion pattern by dairy cows with an animal and farm specificity.

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.

Western Faculty Profile: Dr. Carole Creuzenet

Dr. Carole Creuzenet is an Associate Professor in the Department of Microbiology and Immunology at the University of Western Ontario. A biochemist by training prior to transitioning into Microbiology during her post-doctoral studies at the University of Guelph, Dr. Creuzenet combines these disciplines through studying the mechanisms of the biosynthesis of surface carbohydrates and their role in the virulence of the human pathogens Campylobacter jejuni and Helicobacter pylori. Kathleen Qu, an Academic Affairs Committee Coordinator, interviewed Dr. Creuzenet about her experiences in research and to provide advice for students who wish to pursue research.

Transcriptome of Arabidopsis thaliana plants treated with the human pathogen Campylobacter jejuni

Abstract Recognition of pathogen infection in plants and animals occurs through interaction of pathogen-associated molecular patterns (PAMPs) with pattern recognition receptors (PRR), such as, for example, the recognition of flagellin by Toll-like receptor (TLR) 5 in animals and FLS2 in plants. In this work we exposed wild type and fls2 Arabidopsis thaliana mutant to Campylobacter jejuni . Transcriptome profiling revealed differentially expressed genes in both genetic backgrounds, with ~ 30% overlap in the upregulated group category. The response of fls2 mutant was more robust as more genes changed their expression and larger number of GO term categories was enriched in the mutant. Comparison of the response to Arabidopsis to C. jejuni with response to Escherichia coli O157:H7 showed only partial overlap, with the response in Col-0 plants having more similarities in commonly upregulated genes and enriched GO term categories than the response in fls2 plants. Whereas in the Col-0 , GO terms “response to bacteria”, “leucine-rich repeats” and “plant-type cell wall” were commonly enriched between two animal pathogens tested. In the mutant, GO terms associated with abiotic stress response were enriched for both pathogens. Analysis of motifs of commonly regulated genes showed a large diversity of motifs found in up- and down-regulated genes, and correlation analysis showed more similarity in comparison of motifs between C. jejuni and E. coli O157:H7, rather than between Col-0 and fls2 groups of the same bacterial treatment. Our work thus shows that plants are able to respond to C. jejuni infection in FLS2-dependent and independent manner.

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.

HtrA-mediated E-cadherin cleavage is limited to DegP and DegQ homologs expressed by gram-negative pathogens.

BackgroundThe serine proteases HtrA/DegP secreted by the human gastrointestinal pathogens Helicobacter pylori (H. pylori) and Campylobacter jejuni (C. jejuni) cleave the mammalian cell adhesion protein E-cadherin to open intercellular adhesions. A wide range of bacteria also expresses the HtrA/DegP homologs DegQ and/or DegS, which significantly differ in structure and function.MethodsE-cadherin shedding was investigated in infection experiments with the Gram-negative pathogens H. pylori, enteropathogenic Escherichia coli (EPEC), Salmonella enterica subsp. Enterica (S. Typhimurium), Yersinia enterocolitica (Y. enterocolitica), and Proteus mirabilis (P. mirabilis), which express different combinations of HtrAs. Annotated wild-type htrA/degP, degQ and degS genes were cloned and proteolytically inactive mutants were generated by a serine—to—alanine exchange in the active center. All HtrA variants were overexpressed and purified to compare their proteolytic activities in casein zymography and in vitro E-cadherin cleavage experiments.ResultsInfection of epithelial cells resulted in a strong E-cadherin ectodomain shedding as reflected by the loss of full length E-cadherin in whole cell lysates and formation of the soluble 90 kDa extracellular domain of E-cadherin (NTF) in the supernatants of infected cells. Importantly, comparing the caseinolytic and E-cadherin cleavage activities of HtrA/DegP, DegQ and DegS proteins revealed that DegP and DegQ homologs from H. pylori, S. Typhimurium, Y. enterocolitica, EPEC and P. mirabilis, but not activated DegS, cleaved E-cadherin as a substrate in vitro.ConclusionsThese data indicate that E-cadherin cleavage is confined to HtrA/DegP and DegQ proteins representing an important prevalent step in bacterial pathogenesis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12964-016-0153-y) contains supplementary material, which is available to authorized users.

The CsrA-FliW network controls polar localization of the dual-function flagellin mRNA in Campylobacter jejuni.

The widespread CsrA/RsmA protein regulators repress translation by binding GGA motifs in bacterial mRNAs. CsrA activity is primarily controlled through sequestration by multiple small regulatory RNAs. Here we investigate CsrA activity control in the absence of antagonizing small RNAs by examining the CsrA regulon in the human pathogen Campylobacter jejuni. We use genome-wide co-immunoprecipitation combined with RNA sequencing to show that CsrA primarily binds flagellar mRNAs and identify the major flagellin mRNA (flaA) as the main CsrA target. The flaA mRNA is translationally repressed by CsrA, but it can also titrate CsrA activity. Together with the main C. jejuni CsrA antagonist, the FliW protein, flaA mRNA controls CsrA-mediated post-transcriptional regulation of other flagellar genes. RNA-FISH reveals that flaA mRNA is expressed and localized at the poles of elongating cells. Polar flaA mRNA localization is translation dependent and is post-transcriptionally regulated by the CsrA-FliW network. Overall, our results suggest a role for CsrA-FliW in spatiotemporal control of flagella assembly and localization of a dual-function mRNA.

The Campylobacter jejuni MarR-like transcriptional regulators RrpA and RrpB both influence bacterial responses to oxidative and aerobic stresses.

The ability of the human intestinal pathogen Campylobacter jejuni to respond to oxidative stress is central to bacterial survival both in vivo during infection and in the environment. Re-annotation of the C. jejuni NCTC11168 genome revealed the presence of two MarR-type transcriptional regulators Cj1546 and Cj1556, originally annotated as hypothetical proteins, which we have designated RrpA and RrpB (regulator of response to peroxide) respectively. Previously we demonstrated a role for RrpB in both oxidative and aerobic (O2) stress and that RrpB was a DNA binding protein with auto-regulatory activity, typical of MarR-type transcriptional regulators. In this study, we show that RrpA is also a DNA binding protein and that a rrpA mutant in strain 11168H exhibits increased sensitivity to hydrogen peroxide oxidative stress. Mutation of either rrpA or rrpB reduces catalase (KatA) expression. However, a rrpAB double mutant exhibits higher levels of resistance to hydrogen peroxide oxidative stress, with levels of KatA expression similar to the wild-type strain. Mutation of either rrpA or rrpB also results in a reduction in the level of katA expression, but this reduction was not observed in the rrpAB double mutant. Neither the rrpA nor rrpB mutant exhibits any significant difference in sensitivity to either cumene hydroperoxide or menadione oxidative stresses, but both mutants exhibit a reduced ability to survive aerobic (O2) stress, enhanced biofilm formation and reduced virulence in the Galleria mellonella infection model. The rrpAB double mutant exhibits wild-type levels of biofilm formation and wild-type levels of virulence in the G mellonella infection model. Together these data indicate a role for both RrpA and RrpB in the C. jejuni peroxide oxidative and aerobic (O2) stress responses, enhancing bacterial survival in vivo and in the environment.

Campylobacter jejuni biofilms contain extracellular DNA and are sensitive to DNase I treatment.

Biofilms make an important contribution to survival and transmission of bacterial pathogens in the food chain. The human pathogen Campylobacter jejuni is known to form biofilms in vitro in food chain-relevant conditions, but the exact roles and composition of the extracellular matrix are still not clear. Extracellular DNA has been found in many bacterial biofilms and can be a major component of the extracellular matrix. Here we show that extracellular DNA is also an important component of the C. jejuni biofilm when attached to stainless steel surfaces, in aerobic conditions and on conditioned surfaces. Degradation of extracellular DNA by exogenous addition of DNase I led to rapid biofilm removal, without loss of C. jejuni viability. Following treatment of a surface with DNase I, C. jejuni was unable to re-establish a biofilm population within 48 h. Similar results were obtained by digesting extracellular DNA with restriction enzymes, suggesting the need for high molecular weight DNA. Addition of C. jejuni genomic DNA containing an antibiotic resistance marker resulted in transfer of the antibiotic resistance marker to susceptible cells in the biofilm, presumably by natural transformation. Taken together, this suggest that eDNA is not only an important component of C. jejuni biofilms and subsequent food chain survival of C. jejuni, but may also contribute to the spread of antimicrobial resistance in C. jejuni. The degradation of extracellular DNA with enzymes such as DNase I is a rapid method to remove C. jejuni biofilms, and is likely to potentiate the activity of antimicrobial treatments and thus synergistically aid disinfection treatments.

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.

Characterization and localization of the Campylobacter jejuni transformation system proteins CtsE, CtsP, and CtsX.

The human pathogen Campylobacter jejuni is naturally competent for transformation with its own DNA. Genes required for efficient transformation in C. jejuni include those similar to components of type II secretion systems found in many Gram-negative bacteria (R. S. Wiesner, D. R. Hendrixson, and V. J. DiRita, J Bacteriol 185:5408-5418, 2003, http://dx.doi.org/10.1128/JB.185.18.5408-5418.2003). Two of these, ctsE and ctsP, encode proteins annotated as putative nucleotide binding nucleoside triphosphatases (NTPases) or nucleoside triphosphate (NTP) binding proteins. Here we demonstrate that the nucleotide binding motifs of both proteins are essential for their function in transformation of C. jejuni. Localization experiments demonstrated that CtsE is a soluble protein while CtsP is membrane associated in C. jejuni. A bacterial two-hybrid screen identified an interaction between CtsP and CtsX, an integral membrane protein also required for transformation. Topological analysis of CtsX by the use of LacZ and PhoA fusions demonstrated it to be a bitopic, integral membrane protein with a cytoplasmic amino terminus and a periplasmic carboxyl terminus. Notwithstanding its interaction with membrane-localized CtsX, CtsP inherently associates with the membrane, requiring neither CtsX nor several other Cts proteins for this association.

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.

Comprehensive proteomic profiling of outer membrane vesicles from Campylobacter jejuni.

This work demonstrates the importance of understanding vesicular proteomes from a human pathogen Campylobacter jejuni. From the secreted outer membrane vesicles (OMVs) of C. jejuni NCTC11168, we found a variety of virulence factors and essential proteins for bacterial survival. Bioinformatics analysis of these proteins predicted functional enrichment and localization. The most highly enriched were redox enzymes, which are considered to be essential for survival in oxygen-limiting environments and are predicted to be on the twin-arginine translocation (Tat) pathway suggesting a role for this pathway in the biogenesis of OMVs. This study additionally implicates a biological role for N-linked glycoproteins in OMVs. These approaches allow for a better understanding of the physiology of this important human pathogen.

Hydrogen-Bonded Networks Along and Bifurcation of the E-Pathway in Quinol:Fumarate Reductase

The E-pathway of transmembrane proton transfer has been demonstrated previously to be essential for catalysis by the diheme-containing quinol:fumarate reductase (QFR) of Wolinella succinogenes. Two constituents of this pathway, Glu-C180 and heme bD ring C (bD-C-) propionate, have been validated experimentally. Here, we identify further constituents of the E-pathway by analysis of molecular dynamics simulations. The redox state of heme groups has a crucial effect on the connectivity patterns of mobile internal water molecules that can transiently support proton transfer from the bD-C-propionate to Glu-C180. The short H-bonding paths formed in the reduced states can lead to high proton conduction rates and thus provide a plausible explanation for the required opening of the E-pathway in reduced QFR. We found evidence that the bD-C-propionate group is the previously postulated branching point connecting proton transfer to the E-pathway from the quinol-oxidation site via interactions with the heme bD ligand His-C44. An essential functional role of His-C44 is supported experimentally by site-directed mutagenesis resulting in its replacement with Glu. Although the H44E variant enzyme retains both heme groups, it is unable to catalyze quinol oxidation. All results obtained are relevant to the QFR enzymes from the human pathogens Campylobacter jejuni and Helicobacter pylori.