Salmonella Typhimurium is a major cause of foodborne illness worldwide, with pigs a major zoonotic reservoir. Two phylogenetically distinct variants, U288 and ST34, emerged in UK pigs around the same time but present different risk to food safety. Here we show using genomic epidemiology that ST34 accounts for over half of all S. Typhimurium infections in people while U288 less than 2%. That the U288 clade evolved in the recent past by acquiring AMR genes, indels in the virulence plasmid pU288-1, and accumulation of loss-of-function polymorphisms in coding sequences. U288 replicates more slowly and is more sensitive to desiccation than ST34 isolates and exhibited distinct pathogenicity in the murine model of colitis and in pigs. U288 infection was more disseminated in the lymph nodes while ST34 were recovered in greater numbers in the intestinal contents. These data are consistent with the evolution of S. Typhimurium U288 adaptation to pigs that may determine their reduced zoonotic potential.The emergence of new bacterial pathogens is a continuing challenge for agriculture and food safety.  Salmonella enterica in affecting livestock and potentially humans. Their results indicate that the molecular evolution of these isolates is associated with altered pathogenicity in pigs and reduced zoonotic potential of U288.Mark Kirkwood et al. take an epidemiological, genomic, phylogenetic, and experimental approach to assess the evolutionary origins and virulence of the U288 and ST34 isolates of  Typhimurium) ST313 associated with invasive non-typhoidal Salmonella (iNTS) disease in sub-Saharan Africa, and extensively drug resistant (XDR) S. Typhi.4Salmonella was estimated to have caused around 87 million human infections resulting in approximately 1.2 million deaths globally in the year 2010. Non-typhoidal Salmonella alone has the greatest impact on health with 4 million disability adjusted life years lost, the greatest burden on human health among foodborne diseases.5 Pigs are one of the major zoonotic reservoirs, with 10–20% of human salmonellosis in Europe attributable to them.7 An understanding of the evolutionary processes leading to the emergence of new infectious diseases has the potential to improve pathogen diagnostics and surveillance, and guide policy and interventions aimed at decreasing the burden of human and animal infection.Emergence of infectious diseases presents new challenges for the management of human and livestock health, with substantial human and economic costs through morbidity and mortality, and lost productivity in agriculture. The emergence of 335 human infectious diseases between 1945 and 2004 was dominated by zoonoses of bacterial aetiological agents.Salmonella consists of over 2500 different serovars that have diverse host ranges, pathogenicity and risk to human health. One of these serovars, S. Typhimurium (including monophasic variants), has consistently been a dominant serovar in pigs globally, and currently accounts for around two thirds of isolates in the UK.9 Despite the ostensibly stable prevalence of S. Typhimurium in pig populations over time, the epidemiological record indicates a dynamic process where distinct variants, identified by phage typing, increase and decrease in prevalence over time.8 Since the middle of the 20th century in Europe the dominant phage types were definitive type 9 (DT9), DT204, DT104 and most recently DT193 that is a monophasic S. Typhimurium  with sequence type 34 (ST34).11 At their peak incidence, each accounted for over half of all human isolates of S. Typhimurium. Phage typing has been useful for surveillance and outbreak detection, but only provides limited information about the relationship of the Salmonella isolates due to their polyphyletic nature and potential for rapid changes in phage type as a result of mutations and horizontal gene transfer.12 Nonetheless, sub-genomic and whole genome sequence analysis confirmed that the emergence of new phage types over time does represent the emergence of distinct clonal groups.14 The drivers of their emergence and the consequences for human and animal health are largely unknown.The genus S. Typhimurium isolates of U288 and DT193 have dominated UK pigs.8 U288 appeared in UK pig populations around 2003 followed around the year 2006 by monophasic S. Typhimurium  ST34 rapidly emerging in pig populations around the world.16 U288 and ST34 co-existed in the UK pig population and together accounted for around 80% of isolates.17 Despite, approximately half of all pork consumed in the UK being from UK pig herds,18 since its emergence U288 have rarely been isolated from human infections in the UK.17 In contrast, by the year 2013 in the UK over half of all S. Typhimurium infections in the UK were due to ST34, reflecting its capacity to be transmitted through the food chain and cause human infections.20 U288 is not a definitive type and the designation is not widely adopted outside of the UK. Consequently, the prevalence of U288 outside of the UK is unclear. However, we previously detected U288 in pigs in Ireland,21 a study reported that it was widespread in Danish Pig herds,22 and was present in Italy.23Since around the year 2003, 25 It is believed that contamination of pig carcasses with faeces and gut contents at slaughter, and the ability of Salmonella to spread from the gut to other organs, results in contamination of meat products that enter the food chain and pose a risk to humans if improperly handled or cooked. However, the relative risk from contamination of meat by gut contents during slaughter or from tissue colonised by Salmonella prior to slaughter is not known and could be affected by differences in pathogenesis depending on the genotype of Salmonella involved. Survival of Salmonella in food depends upon adaptive response to environmental stresses including osmotic stress from biocides and desiccation, antimicrobial activity of preservatives and fluctuating temperatures during storage or cooking. In order to cause disease, Salmonella may also need to replicate in food to achieve a population size able to overcome the colonisation resistance of the host.A baseline survey reported prevalence of 21.2% and 30.5% in mesenteric lymph nodes and caecal contents for UK slaughter pigs in studies from 2007 to 2013, respectively.S. Typhimurium are thought to have evolved from a broad host range ancestor resulting in distinct host range, outcome of infection and risk to food safety,27 similar to that observed for distinct serovars.28 An understanding of the molecular basis of risk to food safety of S. Typhimurium pathovariants is critical to improve assessment of risk and devise intervention strategies aimed at decreasing Salmonella presence in food. Furthermore, the identification of genomic signatures of zoonotic risk of Salmonella has the potential to further improve source attribution in outbreak investigations, as recently shown using machine learning approaches.30 We therefore investigated the population structure of S. Typhimurium U288 and the genomic evolution accompanying the clonal expansion of U288 by analysis of whole genome sequences. The objective was to identify representative isolates of the U288 epidemic clade and compare their interaction with the environment and the pig host to gain insight into the phenotypic consequences of their distinct evolutionary trajectories.Multiple pathovariants of S. Typhimurium U288 was first reported in pigs in the UK around the year 2000 and thereafter became the dominant phage-type isolated for much of the following decade.8 Monophasic S. Typhimurium ST34 emerged around seven years later in UK pigs, and these two variants have co-existed in pig populations since. Retrospective analysis of the frequency of U288 and monophasic S. Typhimurium ST34 isolated from animals in the UK by the Animal and Plant Health Agency (APHA) between 2006 and 2015, revealed distinct host ranges  were U288 and of these, 20 were present in a distinct clonal group, composed of 33 isolates in total , reported previously from Denmark in 2016.32 The phylogenetic structure of S. Typhimurium was consistent with that described previously,12 with a number of deeply rooted lineages, some of which exhibited evidence of clonal expansion at terminal branches  differed by fewer than 5 SNPs from the common ancestor of the U288 clade and the 13 related non-U288 strains. S. Typhimurium strain ATCC700720 (LT2) was originally isolated from a human clinical infection at Stoke Mandeville hospital, London in 1948, and subsequently has been used for studying the genetics of Salmonella worldwide.33 The three U288 isolates from human clinical infections in the minor U288 clade clustered together with isolates of other non-U288 phage types.To investigate the relationship of contemporaneous S. Typhimurium U288 clade isolates and monophasic S. Typhimurium ST34 clade isolates to human clinical infections in the UK between April 2014 and December 2015. Of 1826 S. Typhimurium isolated in this period 33 (1.8%) were from the U288 clade. In contrast, 894 isolates (49%) were from the monophasic S. Typhimurium ST34 clade.We next estimated the relative contribution of the S. Typhimurium whole genome sequences in the Enterobase database. We identified a hierarchical cluster (HC20-201) composed of 455 genomes, of which 345 reported the country of origin, that contained all strains reported as U288. This cluster was consistent with the main U288 phylogenetic cluster34  greater than 1 kb due to distinct prophage occupancy, recombination within shared prophage in one or other genome, the presence of a resistance region encoding multidrug resistance, and an integrative conjugative element (SGI-4) in strain S04698-09  and cat.S. Typhimurium ST34 strain S04698-09 genome contained three HDCS outside of prophage, with reference to S. Typhimurium SL1344. In contrast, S. Typhimurium U288 strain S01960-05 contained 19 HDCS outside of prophage , while eleven had predicted functions based on sequence similarity with proteins of known function .A notable difference in coding capacity affecting the core genome of the S01960-05 and S04698-09 strains resulted from hypothetically disrupted coding sequences (HDCS) due to the introduction of a premature nonsense codon from small insertions or deletions (indels) resulting in a frameshift or a single nucleotide polymorphisms (SNP) giving rise to a new nonsense codon. The monophasic Analysis of the pangenome revealed a similar-sized core and accessory genome, with 3962 and 4056 core gene families, out of pangenomes of 5501 and 5578 gene families, for U288 and ST34, respectively  genes, encoding resistance to streptomycin, sulphonamide and tetracycline antibiotics. AMR genes cmlA, sul3, dfrA12, aadA2 and blaTEM that confer resistance to chloramphenicol, sulphonamides, trimethoprim, aminoglycoside and β-lactam antibiotics respectively were present on pU288-1 in all but 16 U288 clade isolates. These AMR genes were absent from six of the most deeply rooted isolates in the S. Typhimurium U288 clade.The pattern of AMR gene presence was consistent with acquisition in two distinct evolutionary events. First, an IncQ1 plasmid (pU288-2) was acquired concurrent with initial clonal expansion of the clade, followed by subsequent acquisition of a transposon on the pSLT-like plasmid, pU288-1 Fig. . The IncS. Typhimurium U288 strain S01960-05 and within diverse S. Typhimurium strains in the context of their phylogenetic relationship indicated their sequential acquisition during the evolution of the U288 clade  were also present in the genome sequence of closely related isolates including LT2, and four  were present in isolates from two relatively distinctly related clades. Two additional HDCS in S. Typhimurium U288 strain S01960-05, were either sporadically present as HDCS throughout the S. Typhimurium collection (oatA and rocR), or only present as HDCS in strain S01960-05 and 14 closely related isolates. Six HDCS  were present in most U288 clade isolates, although a wild type allele of one of these (ygbE) was present in a subclade containing 35 isolates, and two other U288 isolates, suggesting that these may have subsequently reverted.Investigation of the distribution of the 19 HDCS identified in ade Fig. . Of the S. Typhimurium strain SL1344 outgroup was constructed from recombination-purged SNPs in the core genome. Root-to-tip accumulation of SNPs exhibited a molecular clock signal with a statistically significant fit to a linear regression model   Fig. . A time 01) Fig. . This anS. Typhimurium ST34 isolates. The mean doubling time for three U288 isolates was 0.6 h and 0.54 h in aerobic and anaerobic environments, respectively, compared to 0.52 h and 0.47 h for three ST34 isolates . The inability or diminished ability of strain 4/74 to utilise m-Tartaric, Tricarballylic acid and D-xylose was a major factor that distinguished this strain from U288 and ST34 strains. Three ST34 strains clustered together, but the U288 isolates exhibited considerably greater diversity in carbon source utilization. Utilization of myo-inositol as a sole carbon source was the most pronounced phenotype that distinguished the two clusters of U288 isolates. Of note, strain S05968-02 and 11020-1996 that were able to use myo-inositol were isolated earlier and were more deeply rooted than strains S01960-09 and S07292-07 that were unable to use this source of carbon.Since strains of each clade exhibited distinct replication rates, we compared respiration for three isolates of ST34 and four isolates of U288, utilizing a range of substrates as the sole carbon source for metabolism using the BIOLOG phenotyping microarray. All of the strains tested were able to use the majority of 95 carbon sources tested, but there was variation among approximately a quarter of substrates Fig. . The patWe observed a clade-specific variation in tolerance to desiccation by comparing ten U288 strains and three ST34 strains. Following desiccation for 24 hours, approximately 2% of the initial inoculum remained viable for all three ST34 strains Fig. . In compSalmonella enterica38  or porcine (IPEC-J2) epithelial cells in culture was observed Fig. E, F. AltIn an initial experiment using the streptomycin pre-treated C57bl/6 mouse model of colitis we found that mice infected with randomly selected strains of the same phylogroup (ST34 or U288) exhibited similar colonisation level  and ST34 (S04698-09) and two additional randomly selected strains of U288 (S07292-07 and 11020-1996) and ST34 (S00065-06 and S01569-10) were modified by insertion of unique sequence in the chromosome to facilitate identification by sequencing, and four pigs were inoculated orally with an equal mixture of all six isolates. Colonisation was investigated after 48 hours by sequencing cultured homogenates of faeces and tissue and enumeration of the sequence reads for the strain-specific tags. U288 and ST34 exhibited a distinct pattern of colonisation Fig. . ST34 isSalmonella CFU present in faeces was also lower at all time points in pigs inoculated with U288, compared with ST34  were from the U288 clade. In contrast, 894 isolates (54%) were from the monophasic S. Typhimurium ST34 clade. Whole genome sequence or sequence polymorphisms specific to the U288 clade have the potential to improve surveillance to identify the U288 pathovariant in pig herds.Estimation of relative risk of human infection based on phage-typing is potentially misleading, due to potential polyphyletic clusters of common phage-types. Our phylogenetic analysis indicated that the majority of U288 isolates are from a single clonal group, but that this clade also contained a number of isolates that were not identifiable by phage typing as U288, therefore, the contribution to human infection may be underestimated. Conversely, a proportion of the S. Typhimurium U288 isolates from livestock and human infections were present in a phylogenetic clade that evolved from a common ancestor that was closely related to strain LT2. LT2 was isolated at Stoke Mandeville hospital in 1948 and has been used in a large number of studies on the genetics and biochemistry of S. Typhimurium.39 Approximately 50 years elapsed since the isolation of LT2 before U288 was first detected by epidemiological surveillance in the UK pig population, around the year 2003.8 The zoonotic source of the infection caused by the LT2 strain and that of the common ancestor with U288, is not known.39 Evolution of successive descendants of the LT2-like hypothetical ancestor gave rise to multiple lineages, including one that gave rise to the U288 epidemic clade during the 1980s. Evolution of U288 was characterized by the step-wise acquisition of genes involved in resistance to multiple antibiotics, and the accumulation of genome sequence polymorphisms, some of which resulted in interruption of coding sequences. Evolution culminated with the acquisition of AMR genes on pU288-1 and disrupted of the tsr gene around 1995, the date of the common ancestor of the majority of the isolates in the U288 clade. The U288 clade may therefore have been evolving in the pig population before its first detection and rapid spread around the year 2003. The slow emergence of the U288 clade may have been associated with a gradual adaptation to a unique niche in the pig population, but it may also reflect a lag in time from emergence to detection by surveillance, as was proposed for other epidemic serotypes such as S. Enteritidis in poultry layer flocks around 1980, after the eradication of S. Gallinarum more than a decade previously.40The majority of 41 A U288 isolate was previously reported to encode antimicrobial resistance genes on a pSLT-like plasmid pU288-1  and cat) on an IncQ plasmid pU288-2.35 The pU288-2 plasmid was likely acquired first as it is present in isolates from throughout the U288 clade. Insertions carrying AMR genes in pU288-1 may have occurred later than the acquisition of pU288-2, since a group of seven U288 isolates that form a more deeply rooted, basal clade, lacked the pU288-1-associated AMR genes. The majority of the U288 isolates in our analysis were direct descendants of the hypothetical ancestor that acquired AMR genes on pU288-1, and few from that of the basal clade that lacked these genes, suggesting pU288-1 evolution was an important event in the success of the U288 epidemic clade. However, despite a number of examples of apparent loss of AMR genes associated with both pU288-1 or loss of the pU288-2 plasmid (loss of AMR genes and the IncQ replicon), there were just three isolates that had lost both concurrently, highlighting the importance of MDR.The acquisition of resistance to antimicrobials is a key factor in the emergence of bacterial pathogens over the past 50 years, and is often the evolutionary event immediately preceding the spread and clonal expansion of a new clone.S. Typhimurium U288 strain S01960-05 resulted in disrupted coding sequences affecting 26 genes, with reference to strain SL1344, a commonly used lab strain. Sixteen of these polymorphisms were predicted to have occurred before the hypothetical LT2-like ancestor of the U288 and related clades  were disrupted in all descendants of the hypothetical LT2-like ancestor, and one other (oadA) in all but one deeply rooted lineage. However, eight genes  were disrupted in either the hypothetical ancestor of all U288 clade isolates, or subsequently in a subset of descendant lineages, in a stepwise manner, similar to that reported previously for the S. Typhimurium ST313 pathovariant.43 Ancestral state reconstruction indicated that the first gene to be disrupted specifically in the U288 lineage was pncA, an event that coincided with the acquisition of pU288-2, encoding AMR genes. Disruption of pncA is therefore, characteristic of the U288 clade. PncA is a nicotinamidase, a component of one of the pyridine nucleotide cycle (PNC) pathways, involved in the recycling nicotinamide adenine dinucleotide (NAD). The PncA dependent PNC pathway is probably more active in scavenging of pyridine compounds present in the environment.44 NAD is central to metabolism in all living systems, participating in over 300 enzymatic oxidation-reduction reactions,44 and therefore, conditions where de novo synthesis of NAD is limited by the availability of tryptophan or aspartate, the inability to use exogenous pyridines may limit metabolism. Perhaps significantly, the pncA gene is also disrupted in S. Choleraesuis, a serotype that is highly host-adapted to pigs and replicates more slowly than S. Typhimurium in the pig intestinal mucosa.45 Chronologically, the next events were the disruption of the yqaA, ybaA, sadA and ygbE genes. SadA is a surface localised adhesin that contributes to cell-cell interactions and therefore multicellular behaviour,46 while the function of yqaA, ybaA and ygbE are unknown. Disruption of tsr, that encodes a methyl accepting chemotaxis protein involved in energy taxis and colonisation of Peyer’s patches in the murine model of infection, was acquired by a common ancestor of the majority of the U288 clade. This polymorphism occurred on an internal branch of the tree that coincided with the acquisition AMR genes inserted on pU288-1 and a clone that spread successfully through the pig population. Insight into the role of genes present as HDCS in U288 comes from a functional genome screen of genes required for colonisation of the pig colon using a transposon insertion library.47 Transposon insertions in yqaA, ybaA, ygbE had no affect on colon colonisation and insertions in pncA and tsr were not present in the transposon insertion library and so their role is not known. The potential role of sadA is unclear from the study since just one of eight insertion mutants was recovered in significantly lower numbers from the colon of pigs, while the remaining sadA mutants were recovered in similar proportions to those present in the inoculum. However, several additional genes disrupted in U288 but intact had ST34 were implicated in colonisation of the pig colon in the study. In particular, several insertion mutants in assT3 and assT5 were recovered in significantly lower numbers indicated by a fitness score of −2 and −5.76, and may therefore contribute to the reduced intestinal colonisation of U288 isolates.Sequence polymorphisms in Salmonella have been traced back to low moisture, ready-to-eat (RTE) foods,48 including dried pork products.49 Resistance to desiccation may be of particular significance because low oxygen tension is associated with increased resistance to a number of secondary stressors such as pH, salt, alcohol and heat.48 Monophasic S. Typhimurium ST34 also replicated at a significantly higher rate than U288 in culture, a characteristic that may result in a higher level of contamination in food. This is important because bacteria of the family Enterobacteriaceae are known to replicate in food stored at 7 °C, increasing up to 10,000-fold in sausage meat in two weeks.50 As naturally contaminated meat samples typically contained around 103 CFU/g51 and the infective dose of Salmonella enterica in humans is in the range of 105 to 109 CFU, considerable replication in food may be necessary for transmission along the food chain until the final consumer.U288 and ST34 exhibited important differences in the way they interacted with the non-host environment, that could affect the likelihood that it survives in food and is transmitted on to consumers. First, considerably more viable ST34 bacteria were recovered following desiccation for 24 h, compared to U288. Many foodborne disease outbreaks due to S. Typhimurium U288 and monophasic S. Typhimurium ST34 are associated with distinct risk to human health, despite circulating in the same pig population in the UK.8 Our data suggest that this may be due to the differences in tissue tropism and levels of each variant in the pig host that has the potential to impact the likelihood that Salmonella enters the food during the slaughter and butchering process. The greatest risk to Salmonella entering pork products is the contamination of the carcass at slaughter due to errors in the evisceration process and inadequate cleaning of polishing machines.52 The relative contribution of Salmonella present in the faeces and tissues to contamination of food is not known, but increased colonisation of Salmonella in the caecum due to longer time in lairage correlated with contamination of the carcass at slaughter.53 If the level of contamination of different organs of the pig is important for transmission of U288 and ST34 through the food chain, our data is consistent with a greater role for faecal contamination of the final food product. Mean viable counts of a U288 isolate were up to two orders of magnitude lower in the faeces, but 10-fold greater in mesenteric lymph nodes, compared to an ST34 isolate. The reason for the difference in colonisation of pigs is not known. However, ST34 lack the pSLT virulence plasmid, encoding the spv locus that is required for invasive disease in mice and severe gastroenteritis in cattle.56 Polymorphisms in some U288 strains also affect this locus. Acquisition of the IS26 element appears to have been accompanied by deletion of the spvR and spvA genes, also likely affecting expression of spvB and spvC as they are under transcriptional control by SpvR. Further analysis of the impact of the genotypic variation of pig colonisation of U288 and ST34 will reveal the key determinants.Salmonella pathovariant. In the case of S. Typhimurium U288, adaptation in pigs appears to have been accompanied by a decreased risk to food safety for the consumption of pork or cross-contaminated food products by the human population. However, the consequences to the health and productivity of pigs as a result of a more invasive disease are not known but may be an important consideration for the pork production industry.Taken together, our data contribute to a better understanding of the evolutionary history and phenotypes associated with the emergence of a new Salmonella Typhimurium U288 and ST34 isolates used in this study were isolated from human clinical infections during routine diagnostic testing by Public Health England (PHE), or from animals during routine surveillance or epidemiological investigation by Animal and Plant Health Agency (APHA)  broth or MacConkey containing 5% Agar, and supplemented with chloramphenicol (30 mg/l) or kanamycin (50 mg/l) as appropriate.Genomic DNA for short-read sequencing reported in this study was extracted using Wizard® Genomic DNA Purification (Promega) from a culture inoculated from a single colony and incubated for 18 hours at 37 °C. Low Input Transposase Enabled (LITE) Illumina libraries were constructed using a modified protocol based on the Illumina Nextera kit . A total of 1 ng of DNA was combined with 0.9 µl of Nextera reaction buffer and 0.1 µl Nextera enzyme in a reaction volume of 5 µl and incubated for 10 minutes at 55 °C. To this 5 µl mixture containing the DNA we added 2.5 µl of 2 µM custom barcoded P5 and P7 compatible primers, 5 µl 5x Kapa Robust 2 G reaction buffer, 0.5 µl 10 mM dNTPs, 0.1 µl Kapa Robust 2 G enzyme and 10.4 µl water were mixed and DNA amplified by incubating the sample at 72 °C for 3 minutes, followed by 14 PCR cycles consisting of 95 °C for 1 minute, 65 °C for 20 seconds and 72 °C for 3 minutes. 20 µl of amplified DNA was added to 20 µl of Kapa beads and incubated at room temperature for 5 minutes to precipitate DNA molecules >200 bp onto the beads. The beads were then pelleted on a magnetic particle concentrator (MPC), the supernatant removed, and two 70% ethanol washes performed. Beads were left to dry for 5 minutes at room temperature before being re-suspended in 20 µl of 10 mM Tris-HCl, pH8. This was then incubated at room temperature for 5 minutes to elute the DNA molecules. Beads were harvested with an MPC and the aqueous phase containing the size selected DNA molecules transferred to a new tube. The size distribution of each purified library was determined on a PerkinElmer GX by diluting 3 µl of the size-selected library in 18 µl 10 mM Tris-HCl, pH8. Equimolar pool purified libraries were then subjected to size selection on a Sage Science 1.5% BluePippin cassette recovering molecules between 400 and 600 bp. QC of the size selected pool was performed by running 1 µl aliquots on a Life Technologies Qubit high sensitivity assay and an Agilent DNA High Sense BioAnalyser chip and the concentration of viable library molecules measured using qPCR. 10 pM library pools were loaded on a HiSeq4000  based on an average of the qubit and qPCR concentrations using a mean molecule size of 425 bp.58 or S01960-05 (PRJEB34597)12 using SNIPPY (version 3.0) (https://github.com/tseemann/snippy). The size of the core genome was determined using snp-sites (version 2.3.3),59 outputting monomorphic as well as variant sites and only sites containing A,C,T or G. A multifasta alignment of variant sites was used to generate a maximum likelihood phylogenetic tree with RAxML using the GTRCAT model implemented with an extended majority-rule consensus tree criterion.60 The genome sequence of S. Heidelberg strain SL476 (NC_011083.1) was used as an outgroup in the analysis to identify the root and common ancestor of all S. Typhimurium strains. To identify S. Typhimurium genomes in the Enterobase database in the same hierarchical clustering level as the main U288 clade34 we determined the lowest genetic difference cluster level (HCC20-201) that contained all isolates that were reported as U288 (accessed December 2020). The relationship of core genome sequence types was visualised using grapetree implemented within Enterobase.61Paired-end raw sequence data for each isolate were mapped to the SL1344 reference genome (FQ312003)62 sequence variation in the core genome with recombination purged using Gubbins with five iterations.63 The resulting sequence alignments were used to construct a maximum likelihood phylogenetic tree using RAxML rooted on S. Typhimurium SL1344 genome as the outgroup. The Markov chain Monte Carlo was run for 1 million iterations and the convergence and mixing of chains were 113.3, 129.5, 145.8  calculated using the R package corda.64To infer the time of nodes on the phylogeny, we used the BactDating software package implemented in R,S. Typhimurium from a representative collection described14  and unique sequence tag inserted in the genome to distinguish and quantify pooled populations of strains in mixed inoculum experiments of pigs by sequencing. Briefly, primers where used to amplify the aphI gene from pKD4 and recombination into the genome was directed by the inclusion of 50 nucleotide sequence flanking the insertion site  environments and viable bacteria in colony-forming units (CFU) enumerated by serial dilution and culture on LB agar at 1, 3, 5 and 7 h post-inoculation. Doubling time was calculated in the exponential range of growth using the mean from three biological replicates. Determination of survival after desiccation of strains SL1344, S04698-09, S00065-06, S01569-10, S01960-05, H09152-0230, S02724-05, 10584-1997, 12005-1995, S05968-02, 11020-1996 and 3203-1997 was based on a method previously described.71 Briefly, bacteria were cultured in LB broth at 37 °C with shaking for 18 h, harvested by centrifugation, washed with phosphate-buffered saline pH7.4 (PBS) and re-suspended in PBS and adjusted to OD600nm of 1. 0.05 ml of cell suspension were added to polystyrene 96 well plate (Nunc) and desiccated at 22 °C, 36% relative humidity (RH). Desiccated plates were stored in a sealed vessel containing saturated potassium acetate solution, to maintain RH at 36% and incubated at 22 °C for 24 h. Cells were re-suspended in 0.2 ml PBS and viable counts were determined by culture of serial 10-fold dilutions on LB agar. The percentage survival was calculated from at least three biological replicates. To study biofilm formation of the same strains as for desiccation, liquid cultures were incubated statically in polystyrene 96-well plates at 22 °C for 24 hours, washed once with PBS pH7.4, and attached bacteria were stained with crystal violet and absorbance read at 340 nm. Data points represent the mean of at least three biological replicates.For determination of growth rate of randomly selected U288 strains , and ST34 strains , bacterial cultures in LB broth were diluted to approximately 1 × 1072 The area-under-the-curve was a metric for total respiration of the indicated carbon sources and plotted as a heatmap using R software.To assess utilisation of carbon sources, eight strains ST4/74, S04698-09, S00065-06, S01569-10, S05968-02, S01960-05, 11020-1996 and S07292-07 were cultured on LB agar overnight at 37 °C, inoculated into IF-0 medium containing tetrazolium dye, and added to a PM-1 plate (with 95 different carbon sources), according to the manufacturer’s instructions (IBiolC). Accumulation of purple indicator dye as a consequence of redox activity was measured every 15 minutes for 48 hours. Raw absorbance data were processed using R software and the opm package.2 overnight. Stationary phase cultures of S. Typhimurium strains SL1344, SL1344 ΔinvA::aph, S04698-09, S00065-06, S01569-10, S01960-05, H091520230, S02724-05, 10584-1997, 12005-1995, S05968-02, 11020-1996 and 3203-1997 were balanced to OD600nm ~1.0 in PBS and used to inoculate epithelial cells at a multiplicity of infection (MOI) of 20. Cells were incubated for 30 minutes before washing 5 times with PBS, re-suspending in DMEM + gentamicin (100 mg/l) and incubating for a further 30 minutes at 37 °C in 5% CO2 atmosphere to kill extracellular bacteria. The medium was then replaced with DMEM + gentamicin (10 mg/l) and the plates were incubated for 60 minutes at 37 °C in 5% CO2 atmosphere. Plates were then incubated again at 37 °C in 5% CO2 atmosphere for 90 minutes, before a final wash with PBS + 0.1% Triton-X100. Plates were left for 2 minutes and cells were disrupted by vigorous pipetting for one minute followed by serial dilution and culture on LB agar to determine viable counts.IPEC-J2 porcine epithelial cells and T84 human epithelial cells were cultured and routinely passaged in Dulbecco Modified Eagles Medium (DMEM), containing high glucose or low glucose for each cell line, respectively. 24-well tissue culture plates were seeded with 1 × 105 cells/ml of epithelial cell lines, and incubated at 37 °C 5% CO73 Groups of five female 6–9 week old specific pathogen free C57bl/6 mice were randomly chosen and housed together in individually ventilated cages with food and water ad libitum. Mice were administered 20 mg of streptomycin sulphate by oral gavage 24 h prior to inoculation of S. Typhimurium. Five S. Typhimurium strains S00065-06, S01569-10, S01960-05, 11020-1996 and S07292-07 were cultured for 18 hours in 50 ml LB broth with shaking and approximately 5 × 106 CFU were inoculated orally in 0.2 ml of PBS pH7.4 by gavage. A control group of five mice were administered only the PBS buffer. PBS pH7.4 was used to mimic physiological osmolarity and pH to maintain viability. On day 3 post-inoculation mice were killed by asphyxiation with slowly rising CO2 concentration and then the caecum was aseptically removed. An approximately 3 mm section of the caecum halfway down the organ from the ileal junction was removed and fixed in formalin for histopathology examination of 5 μm thin sections stained with hemotoxylin and eosin. Approximately 5 mg of caecum tissue was removed and placed in RNAlater (Thermo Fisher) and stored at −80 °C. The remaining caecum (approximately two thirds) was homogenised in sterile PBS pH7.4 and serial dilutions plated on LB agar containing 0.05 mg/ml kanamycin, incubated at 37 °C for 18 h and viable counts enumerated.Ethical approval for the experimental mouse infections was granted following review by the Animal Welfare and Ethical Review Body  under project licence PPL 70/8597. A streptomycin pretreated model of colitis was used.74RNA from caecum tissue was prepared using guanidinium thiocyanate-phenol-chloroform extraction with Tri Reagent (Merck). Tissue was homogenized in 1 ml of Tri reagent and disrupted using 1.4 mm zinc oxide beads in a bead beater and tissue debris removed by centrifugation. 0.2 ml of nuclease free water and 0.2 ml of chloroform were added to the supernatant, mixed and centrifuged for 15 minutes at 12,000 x g. 0.5 ml of isopropanol was added to the upper aqueous phase and centrifuged for 15 minutes at 12,000 x g. The resulting RNA pellet was washed twice with 70% ethanol, briefly dried and resuspended in 0.02 ml of RNase free water. The relative abundance of Nos2 and Cxl1 mRNA was determined by quantitative RT-PCR using primers specific to the test genes and the Gapdh house-keeping gene as the control  in a mixed inoculum assay. A mixed-strain inoculum was prepared by combining equal volumes of individual cultures of the six strains grown statically at 37°C for 16 hours in LB broth supplemented with 50 mg/l kanamycin, which were optical density (OD600nm) standardized to contain 8.9 log10 CFU/ml. The number of CFU in the inoculum was determined by plating 10-fold serial dilutions of the inoculum on MacConkey agar containing 50 μg/ml kanamycin. Aliquots of the inoculum were stored at −20 °C for DNA extraction. Five ml of the mixed-strain inoculum was mixed with 5 ml of antacid  to promote colonization and administered orally by syringe before the morning feed. Pigs were fed as normal following challenge. Rectal temperatures were recorded every 24 hours and faecal samples were collected at 24 and 48 hours post-infection. The endpoint of the experiment was humane euthanasia at 72 hours post-inoculation. A section of distal ileal mucosa, mesenteric lymph nodes (MLNs) draining the distal ileal loop, a section of spiral colon, colonic lymph nodes (CLNs) and a section of liver were collected. Lymph nodes were trimmed of excess fat and fascia, and the sections of distal ileum and spiral colon were washed gently in PBS to remove nonadherent bacteria. One gram of each tissue was homogenized in 9 ml of PBS in gentleMACS M tubes using the appropriate setting on the gentleMACS dissociator (Miltenyi Biotec). Homogenates were filtered through 40-μm-pore-size filters and an aliquot was used to determine viable counts. The remaining homogenate was spread onto 10 MacConkey agar plates (500 μl per plate) containing 50 μg/ml kanamycin and incubated overnight at 37 °C. The bacterial lawns recovered from each sample were collected by washing with PBS, and the pellets were stored at −20 °C for DNA extraction. Genomic DNA (gDNA) was extracted from the pellets using the NucleoSpin tissue kit (Macherey-Nagel), according to the manufacturer’s instructions. The quality and quantity of DNA were assessed initially by NanoDrop 3300 (Thermo Scientific), and samples with an A260/280 of < =1.8 were considered suitable for library preparation. These were confirmed further by using the DNA ScreenTape (Agilent Technologies) and the Qubit double-stranded DNA (dsDNA) BR assay kit (Life Technologies), respectively. One microgram of gDNA with a DNA integrity number (DIN) of < =6 was used for library preparation using the TruSeq PCR-free library preparation kit (Illumina) according to the manufacturer’s protocol. Whole genome sequencing on the HiSeq system (Illumina) followed by bioinformatics analysis were performed as previously described,76 with the exception that strains were quantified by mapping sequence data to the unique WITS tag sequence incorporated chromosomally into each strain. Sequence data was submitted to the NCBI SRA database  were standardized to contain 9.2 log10 CFU/ml, which was confirmed retrospectively by plating 10-fold serial dilutions on MacConkey agar. Groups of four Salmonella-free pigs were challenged orally with 5 ml of each strain as described above. Rectal temperatures were recorded every 12 hours and faecal samples were collected every 24 hours post-infection. Clinical scores were calculated for each animal using their temperatures, physiological signs and faecal consistency. At 72 hours post-infection, tissue samples were collected and processed for viable counts as described above. The bacterial load of each strain in each tissue of the infected pigs was determined. Data are presented as the mean ± SEM.From the strain phenotypes identified in the mixed-strain infection, one representative strain of each ST34 and U288 clade that exhibited the greatest colonisation was selected for in vivo phenotype validation, S04698-09 and 11020-1996, respectively. The strains were grown statically at 37 °C for 16 hours in LB broth supplemented with 50 mg/l kanamycin and the optical densities . The viable counts of bacteria in each case is presented as mean ± SEM, and differences between strains were analysed using a two-sided Mann-Whitney test of significance. Area under the curve analysis followed by a two-sided Mann-Whitney test was used to analyse the cumulative clinical scores of the infected pigs during single-strain infections. Supplementary InformationSupplementary Data 1Description of Additional Supplementary Files


P < 0.0001 respectively; median RFS, 27.1 months in high-TIL, 10.0 months in low-TIL, P < 0.0001 respectively). In the high-CD8+ TIL BTC group, the tumor expression of β-catenin and DKK1 had a significant negative impact on either OS or RFS. In the low-TIL BTC group, there were no differences according to ß-catenin and DKK1 expression. Cox regression multivariate analysis demonstrated that CD8+ TIL and β-catenin retained significant association with OS. Among patients with resected BTC, the β-catenin and DKK1 protein and high CD8+ TIL levels were associated with poor and good clinical outcomes, respectively.The role of β-catenin and Dickkopf-1 (DKK1) is dependent on the specific immunobiology of T cell inflammation in biliary tract cancer (BTC). We aimed to analyze the role of DKK1 or β-catenin as a prognostic factor in BTC, and determine the clinical associations of ß-catenin and DKK1 with CD8+ tumor-infiltrating lymphocytes (TIL). We used data from The Cancer Genome Atlas Research Network and the clinicopathological data of 145 patients with BTC who had undergone primary radical resection between 2006 and 2016. CD8+ TIL expression was a significant predictor of favorable overall survival (OS) and relapse-free survival (RFS) (median OS, 34.9 months in high-TIL, 16.7 months in low-TIL,  BTC is a fatal and rare adenocarcinoma that arises in the biliary tree, including the intrahepatic (ICC) and extrahepatic (ECC) bile duct and gallbladder (GBC)3. Surgical resection is the primary treatment modality for early-stage BTC, but the majority of patients still develop a recurrence4. Further, the mortality rate is high primarily because patients are diagnosed at a late stage5. Despite recent advancements in our understanding of the molecular biology of BTC, limited progress has been made in cytotoxic systemic therapy for this disease. Agents targeting IDH1, fibroblast growth factor receptor, and EGFR have demonstrated only limited efficacy7. In addition, immunotherapy has minimal usefulness in BTC, showing only modest benefits in advanced BTC patients with deficient mismatch repair and high microsatellite instability status8. Therefore, new therapeutic molecular studies are required to improve the survival of patients by overcoming the heterogeneity of BTC.Biliary tract cancer (BTC) accounts for approximately 3% of all gastrointestinal malignancies and is the most common hepatobiliary cancer after hepatocellular carcinoma9. The mutational status of this pathway’s components is highly relevant to the pathogenesis of BTC2. Particularly, activation of the Wnt pathway has been shown to be associated with chemoresistance and metastatic spread in ECC and ICC11. Different mutations found in ECC and ICC sequencing are known to act on Wnt/β-catenin to induce chromosomal instability and oncogenesis13. Some genetic variants in the Wnt/β-catenin pathway are associated with decreased apoptosis of GBC and influenced susceptibilities2.The Wnt/β-catenin signaling cascade controls cell proliferation, cell polarity, and cell fate during embryonic development and homeostasis in human tissues14. DKK1 plays a role in cancer proliferation, invasion, and growth via the modulation of Wnt signaling15. DKK1 is expressed in multiple tumor types, including BTC, but its impact and the clinical role of this pathway’s components are not completely understood16. Recent studies have suggested that high DKK1 expression in BTC is associated with immunosuppressive conditioning through myeloid-derived suppressor cells(MDSC) and tumor-associated macrophages17. It is also associated with high expression of matrix metalloproteinase 9 proteins, which play a role in tumor cell invasion, angiogenesis, and lymph node metastasis in BTC and are correlated with poor prognosis18.Dickkopf-related protein1 (DKK1), the most well-known Wnt antagonist of the canonical Wnt/β-catenin pathway, is a 35-kDa protein that contains a secreted signal peptide sequence. DKK1 can inhibit Wnt signaling by interacting with the Wnt receptors of the Frizzled family21. Several studies have demonstrated that BTC contains abundant tumor-infiltrating lymphocytes (TILs) and cancer-associated fibroblasts in the tumor microenvironment (TME)21. In addition, Wnt/β-catenin signaling and DKK1 expression are associated with immunosuppressive modulation and protumoral conditioning in BTC through several mechanisms19, including activation of innate immune cells such as tumor-associated macrophages or MDSCs23 or loss of effector functions by T cell de-differentiation24. However, the clinical implications for β-catenin according to tumor infiltrative immune cells and DKK1, as an antagonist of Wnt/β-catenin signaling, remains controversial. Therefore, this study aimed to investigate the role of DKK1 and β-catenin as prognostic factors in BTC with reference to CD8+ TILs.Given that BTC is constantly exposed to intestinal microbial products, it has both the immune tolerance capability to suppress inappropriate inflammatory responses and the immune capability to protect against harmful stimuli, such as infection and tumorhttps://tcga-data.nci.nih.gov/tcga/). All mRNA expression data were log2-transformed. The expressions of CD8A, DKK1, and CTNNB1 genes were analyzed in 45 samples.This retrospective study analyzed data from The Cancer Genome Atlas (TCGA) Research Network to identify the associations of Wnt/β-catenin gene expression in CD8 cells and clinical outcomes in BTC. mRNA expression data and clinical data were downloaded from the TCGA data portal  presence of pathologically confirmed biliary tract adenocarcinoma, (2) treatment by radical resection without preoperative radiation or chemotherapy, and (3) availability of paraffin-embedded tumor specimens. Postoperative pathological staging was based on the American Joint Committee on Cancer staging criteria, 8th edition. Patient data, including age, sex, date of diagnosis, recurrence, and death, were retrieved from the electronic medical records.9. The cut-off H score for high DKK1 expression was set at 80 using maximally selected rank statistics (quartile) calculated using R statistical programming, version 3.2.3 (https://www.r-project.org/), and high CD8 positivity was set at 32 (median), following previous reference articles25.Immunohistochemistry was performed on formalin-fixed, paraffin-embedded tissue sections. Whole tissue sections of representative tumor samples were used for antigen retrieval and incubated with human-specific antibodies against DKK1  and β-catenin . The intensity of DKK1 staining was scored as follows: 0, no staining; 1, weakly positive; 2, moderately positive; and 3, strongly positive. The proportion of DKK1-stained cells was calculated as the percentage of positive tumor cells. H scores, ranging from 0 to 300, were determined by multiplying the intensity score by the proportion of DKK1-stained cells. For the characterization of the inflammatory components, sections were incubated with primary antibodies: CD8  following the manufacturer’s instructions. ß-catenin protein immunoreaction was considered positive if more than 10% of tumor cells showed nuclear and cytoplasmic stainingCategorical variables were compared using the chi-square test and Fisher’s exact test, as needed. Overall survival (OS) was defined as the time from diagnosis to any-cause death or the last follow-up. Relapse-free survival (RFS) was calculated from the date of diagnosis to the date of the first distant or local disease recurrence or the last follow-up. Survival curves were generated using the Kaplan–Meier method and compared using the log-rank test by GraphPad Prism 8.0 . Cox proportional hazards regression models were used to identify the significance of prognostic factors. Survival rates and hazard ratios (HRs) were shown with their respective 95% confidence intervals (CIs). All statistical analyses were performed using R statistical programming (version 3.4.1) and the SPSS software package . A two-sided P-value of < 0.05 was considered statistically significant in all tests and models.This study was approved by the Institutional Research Ethics Board of Uijeongbu St. Mary’s Hospital of the Catholic University of Korea (UC15SISI0155) and adhered to the Declaration of Helsinki. Written informed consent was obtained from all subjects involved in the study. Patient anonymity was preserved.Neither DKK1 nor CTNNB1 expression was associated with CD8A gene expression , advanced TNM stage (P = 0.002), and BTC other than GBC (P = 0.040) . However, there was no association between ß-catenin positivity and any clinicopathological factor based on CD8 expression   than those with negative expression, but the difference was not significant , but not RFS (P = 0.2924) . Meanwhile, the overall 5-year RFS rate was 14.4% and the median RFS time was 18.2 months . Analysis of the correlation between marker expression and prognosis showed that TIL expression was a significant predictor of favorable OS and RFS, as expected , had poorly differentiated grade (P < 0.001), non-GBC primary site (P = 0.036), positive margin (P = 0.015), advanced stage (P < 0.001), lymphatic invasion status (P < 0.001), and perineural invasion status (P < 0.001) predicted poor RFS (Table P = 0.020), positive margin , advanced stage  and perineural invasion status  were independent prognostic factors of poor RFS in the multivariate analysis. A shorter OS was found to be significantly associated with older age (P = 0.006), poorly differentiated grade (P < 0.001), positive margin (P = 0.022), advanced stage (P < 0.001), lymphatic invasion status (P = 0.001), and perineural invasion status (P < 0.001) in the univariate analysis. Risk factors that were significantly associated with shortening OS included older age  and advanced stage  in the multivariate analysis. High CD8+ TIL level was associated with good prognostic values in both RFS  and OS . Although, positive β-catenin and DKK1 expression did not reveal statistical significance in RFS, these were correlated with poor prognostic values in OS .In the univariate analyses, patients who were older   and DKK1 expression  in the TIL-low BTC group 23. Wnt/β-catenin signaling is essential for T cell differentiation, effector functions, proliferation, and migration via the T-cell factor (TCF), which is the effector transcription factor of the Wnt signaling pathway41. As such, “hot tumors" present with more profound Wnt/β-catenin signaling and TCF modulation. Meanwhile, Wnt/β-catenin signaling and TCF modulation do not have a significant influence on the prognosis of “cold tumors” because of the insufficient substrates available for alteration in these tumors26. The differences in the resulting clinical changes in the protumoral and immunosuppressive status, according to β-catenin activation, were more prominent in the high TIL inflamed subgroup, whose clinical prognosis was comparable to that of the immune exclusive subgroup.The complex prognostic role of DKK1 remains unclear. Data from recent studies have demonstrated that DKK1 acts not only as a tumor suppressor protein, but also as an oncogene24. It has been widely accepted that Wnt/β-catenin signaling affects cancer immunosurveillance across various tumor types43. Tumor-intrinsic Wnt/β-catenin pathway activation impedes antitumor immunity via various mechanisms, including the release of immunosuppressive chemokines, tumor exclusion of dendritic cells, downregulation of innate immune sensors on the tumor cell surface, and suppression of dendritic cell maturation44. Interestingly, Wnt/β-catenin signaling is involved in T cell differentiation, effector function, and migration. The differentiation of naïve CD8+ T cells into CD8+ T effector cells is inhibited by the activation of TCF-1/β-catenin signaling43, in accordance with the findings of this study. The mechanisms discussed above indicate that the increased expression of β-catenin in tumors is inversely correlated with intratumoral T-cell infiltration. However, we did not find an inverse correlation between tumoral β-catenin or DKK1 expression and the CD8+ TILs. This was similar to the findings of Saleh et al., who reported that Wnt signaling- and β-catenin/TCF complex-associated genes were among the significantly upregulated genes in CD8+ TILs among patients with advanced colorectal cancer45. This suggests that the cytotoxic functions of CD8+ effector T cells could be compromised by Wnt/β-catenin signaling. β-catenin or DKK1 protein expression in patients with a high-density of CD8+ TILs in the intra-tumoral area was correlated with poor histological differentiation.Notably, we found no significant differences between the OS and RFS of patients with high β-catenin/DKK1 and high TIL expression and those with low TIL expression. These results suggest that despite the high CD8 TIL levels in the tumor tissues, the favorable clinical effect of CD8 T cells could be expected from low Wnt/β-catenin signaling activation. Several modulators are associated with T cell infiltration in the TME, and the Wnt/β-catenin signaling molecule is one of the best characterized factors46, a stratified molecular study found a difference in prognosis among these subtypes. There are several possible explanations for this result. First, the immune escape mechanisms vary according to the type of biliary malignancy. In general, BTC is constantly stimulated by intestinal microbes and toxic materials and possesses an immune tolerance ability that can suppress inappropriate inflammatory responses, in addition to an immune ability that protects against harmful stimuli, such as infection and tumor21. Therefore, the immunobiology, including the immune escape mechanism, depends on the location of exposure47. T-cell receptor clonality, the immune gene signature/RNA repertoire, and major histocompatibility complex class polymorphisms have been extensively investigated in various human cancers, including BTC48. Goeppert et al. reported that downregulated MHC 1 expression in cancer cells may be associated with a low density of anti-tumor inflammatory cells, such as TILs30. This mechanism might underlie the poor prognostic role of DKK1 and β-catenin in GBC and ECC, and the opposite result in ICC.Our data confirmed that the distribution of TILs and the roles of DKK1 and β-catenin differ among GBC, ECC, and ICC. Although several studies have shown similar prognoses among the three BTC subtypes using a traditional merged analysisThe present study has some limitations. First, this retrospective analysis was performed with a small sample size, which may be attributed to the complexity of the biological function of the molecules and the different cut-off values used to evaluate the significance of these molecules. However, considering the rarity of BTC, the sample size is adequate to derive significant findings and establish the prognostic impact of the expression of the β-catenin and DKK1 proteins. Second, it is difficult to draw widely applicable conclusions from the results, owing to insufficient numbers of patients in some subgroups classified on the basis of the tumor response patterns. Third, the association between the expression levels of the Wnt/β-catenin gene and DKK1 and distribution of CD8+ TIL were not confirmed by other methods. The amount of archival tissue available for this retrospective study was insufficient to conduct other procedures, such as whole-genome and RNA sequencing. Since the immune signatures and molecular profiling of ICC, ECC, and GBC are different, we focused on identifying the clinical implications at the protein level and conducted analyses using the immunohistochemistry method. To the best of our knowledge, no study has investigated the dynamicity and differential effects of the two molecules (β-catenin and DKK1) in BTC depending on the infiltration of CD8+ TILs. Further research is needed to validate the mechanism and prognostic function of DKK1 and β-catenin according to the heterogeneous immune status in BTC.β-catenin or DKK1 protein expression is associated with poor clinical outcomes in patients with resected BTC, whereas high CD8+ TIL levels are associated with good clinical outcomes. Notably, a high CD8+ TIL level with high β-catenin or DKK1 protein expression is associated with an unfavorable prognosis. These findings confirm the differential clinical implications of Wnt/β-catenin proteins according to TIL expression in BTC.Supplementary Figures.


Wnt/β-catenin is believed to regulate different sets of genes with different coactivators, cAMP response element-binding protein (CREB)-binding protein (CBP) or p300. However, the factors that determine which coactivators act on a particular promoter remain elusive. ICG-001 is a specific inhibitor for β-catenin/CBP but not for β-catenin/p300. By taking advantage of the action of ICG-001, we sought to investigate regulatory mechanisms underlying β-catenin coactivator usage in human pancreatic carcinoma PANC-1 cells through combinatorial analysis of chromatin immunoprecipitation-sequencing and RNA-sequencing. CBP and p300 preferentially bound to regions with the TCF motif alone and with both the TCF and AP-1 motifs, respectively. ICG-001 increased β-catenin binding to regions with both the TCF and AP-1 motifs, flanking the genes induced by ICG-001, concomitant with the increments of the p300 and AP-1 component c-JUN binding. Taken together, AP-1 possibly coordinates β-catenin coactivator usage in PANC-1 cells. These results would further our understanding of the canonical Wnt/β-catenin signaling divergence. Wnt; β-catenin; Coactivator CREB-binding protein; p300; AP-1. Wnt proAXIN1, AXIN2, RNF43, and ZNRF3 in colorectal cancer, hepatocellular carcinoma, pancreatic cancer, and adrenocortical carcinoma -binding protein (CBP) or its homologous protein p300, just as well as other basal transcription factors, to activate target gene expression . Though It is difficult to investigate the unique roles of CBP and p300 in Wnt/β-catenin signaling using a genetic approach, due to their complex interactomes . Small cIn this study, we conducted chromatin immunoprecipitation and massively parallel sequencing (ChIP-seq) and RNA-sequencing (RNA-seq) to reveal the genome-scale action of β-catenin and coactivators CBP and p300 upon ICG-001 treatment. We further identified that ICG-001 increases p300 binding to β-catenin-associated regions with an activator protein-1 (AP-1) binding DNA motif through the induction of the AP-1 transcription factor family members in PANC-1 cells.22.1de novo motif analysis with sequences of β-catenin-associated regions. The TCF7L2 (TCF) and FOS:JUN (AP-1) motifs were significantly enriched in β-catenin-associated regions under both conditions  using chromatin immunoprecipitation and massively parallel sequencing (ChIP-seq). PANC-1 cells were treated with Wnt/β-catenin activator CHIR-99021 along either with DMSO or ICG-001 (C + D or C + I). We identified 630 and 759 β-catenin-associated regions in cells treated with or without ICG-001, respectively A. Of thenditions E. In β-cnditions F. In con2.2We examined the involvement of JUN family transcription factors on the genomic binding of β-catenin using RNA-seq data. Among the family members, only the JUN gene was upregulated by ICG-001 as reported previously  Figure A. The tiTo examine the role of the AP-1 motif in β-catenin/TCF signaling, we focused on β-catenin-associated regions with a TCF motif. We examined the peak intensities of β-catenin, CBP, p300, and c-JUN on β-catenin-associated regions with or without AP-1 motifs in the presence or absence of ICG-001. The analysis showed that the CBP peak intensity was decreased by the ICG-001 treatment under any conditions as demonstrated by a previous biochemical assay  Figure E. The ICTo explore the effect of ICG-001 on the JUN gene transcription, we examined the binding of β-catenin, CBP, p300, c-JUN, and chromatin accessibility in the JUN gene. β-catenin, CBP, and p300 binding were found at the region downstream of the JUN gene, while p300 and c-JUN binding was found at the promoter of the JUN gene . Chromat2.3p < 0.05). In these differentially expressed genes, 664 and 1032 genes were down- and upregulated in cells treated with a combination of CHIR-99021 and ICG-001 (C + I), respectively, compared to cells treated with CHIR-99021 and DMSO (C + D). Gene ontology (GO) analysis showed that the top 5 GO terms enriched in downregulated genes were related to the cell cycle and GO terms enriched in upregulated genes were related to RNA biogenesis  PANC-1 cells. The effect of ICG-001 on transcriptions was confirmed by the suppression of BIRC5 and induction of EPHB2 as reported previously . The ind3In this study, we explored the effect of ICG-001 on the genomic distribution of β-catenin and its binding partners CBP, p300, and c-JUN by ChIP-seq analysis. As expected from previous reports, ICG-001 decreased common peaks of β-catenin and CBP while it increased common peaks of β-catenin and p300 . In addiAs the regulatory sequences of a gene are assumed to be enriched in the vicinity of the given gene, β-catenin peaks were classified based on the expression of neighboring genes. There are few peaks with the AP-1 motif flanking genes induced by CHIR-99021 and suppressed by ICG-001. On the other hand, many peaks with the AP-1 motif, especially peaks with both the TCF and AP-1 motifs, were enriched at regions flanking genes induced by ICG-001. Peak intensities of β-catenin, p300, and c-JUN at the β-catenin peaks near upregulated genes increased with ICG-001 but not at peaks near downregulated genes, whereas peak intensities of CBP at any β-catenin peaks decreased. At β-catenin peaks with TCF motif alone, CBP seems to be a major coactivator for β-catenin target gene expression, as the p300 and c-JUN peak intensities at peaks near downregulated genes were lower than those near upregulated genes. Therefore, ICG-001 suppressed CBP-mediated target gene expression, whereas ICG-001 induced different target genes through recruitment of p300 and c-JUN at β-catenin-associated regions.Herein, in addition to EPHB2, we identified HDAC9 as an ICG-001-c-JUN target. One of the problems with the therapeutic use of Wnt/β-catenin inhibitors is bone loss  because The functional interplay between β-catenin/TCF and AP-1 in the target gene expression has already been reported previously . Gene exIn summary, we propose that the AP-1 motif possibly determines the components of the β-catenin complex on the genome and the response to ICG-001 in the Wnt/β-catenin axis. Therefore, this property allows the specific expression inhibition of a part of β-catenin target genes with ICG-001. These findings lead to the elucidation of the gene expression switch mechanism by Wnt/β-catenin that controls different gene sets depending on cells and the development of therapeutics targeting the β-catenin target gene subset.3.1In this study, we were unable to examine the effect on the expression of genes distant from β-catenin binding regions as we analyzed the genes closest to these regions as putative targets. In future studies, investigating how cis-elements identified herein could influence the distant genes with information of comprehensive chromatin interactions would be essential.PANC-1 cells were purchased from ATCC  and maintained in D-MEM supplemented with 10% fetal bovine serum and 100 U/ml penicillin and 100 μg/ml streptomycin. Confluent cells were stimulated by replacing the culture medium with a warmed medium containing 3 μM of CHIR-99021  and 30 μM of ICG-001 (synthesized in house) or the same volume of DMSO and cultured for 24 hours for ChIP-seq, ATAC-seq, and RNA-seq.ChIP-seq and ATAC-seq were performed as described previously . BrieflyTotal RNA was purified using the RNeasy Mini Kit . The libraries were prepared using the TruSeq standard mRNA library kit  and sequenced using NovaSeq 6000 (Illumina). The sequence reads were mapped to the human genome (hg19) with TopHat2 . The traTotal RNA was purified with RNeasy Mini Kit . Complementary DNA was synthesized with ReverTra Ace qPCR RT Master Mix with gDNA Remover . PCR was performed with ThunderBird SYBR qPCR mix  and Thermal cycler Dice (Takara-bio).Whole-cell lysates were prepared with a lysis buffer ). The concentration of extracted proteins was measured with the QuickStart protein assay reagent (Bio-Rad). Twenty micrograms of proteins were separated in 10% SDS-PAGE gel and transferred on Immobilon-P PVDF membrane (MECK). Membranes were blotted with anti-c-JUN antibody  or anti-β-actin antibody  with appropriate secondary antibody conjugated with horseradish peroxidase. Signals were developed with ECL prime .LentiCRISPR v2 neo, psPAX2, and pCMV-VSV-G were obtained from Addgene. Sequences of guide RNAs were cloned into BsmBI sites of LentiCRISPR v2 neo vector. The lentivirus was rescued in 293T cells with psPAX2 and pCMV-VSV-G vectors. PANC-1 cells were infected with the virus and cloned under G418 selection. JUN KO PANC-1 clones were screened by western blotting for c-JUN.Tomomitsu Doi, Hironori Hojo: Conceived and designed the experiments; Wrote the paper.Shinsuke Ohba: Conceived and designed the experiments.Kunie Obayashi: Performed the experiments.Motoyoshi Endo, Toshimasa Ishizaki, Hiroyuki Kouji: Analyzed and interpreted the data.Akira Katoh: Contributed reagents, materials, analysis tools or data.10.13039/501100001691JSPS KAKENHI Grant Number JP21K07972.This work was supported by Data associated with this study has been deposited at GEO data base in NCBI.The authors declare no conflict of interest.No additional information is available for this paper.


Though numerous studies have focused on the cell wall disassembly of bananas during the ripening process, the modification of homogalacturonan (HG) during fruit development remains exclusive. To better understand the role of HGs in controlling banana fruit growth and ripening, RNA-Seq, qPCR, immunofluorescence labeling, and biochemical methods were employed to reveal their dynamic changes in banana peels during these processes. Most HG-modifying genes in banana peels showed a decline in expression during fruit development. Four polygalacturonase and three pectin acetylesterases showing higher expression levels at later developmental stages than earlier ones might be related to fruit expansion. Six out of the 10 top genes in the Core Enrichment Gene Set were HG degradation genes, and all were upregulated after softening, paralleled to the significant increase in HG degradation enzyme activities, decline in peel firmness, and the epitope levels of 2F4, CCRC-M38, JIM7, and LM18 antibodies. Most differentially expressed alpha-1,4-galacturonosyltransferases were upregulated by ethylene treatment, suggesting active HG biosynthesis during the fruit softening process. The epitope level of the CCRC-M38 antibody was positively correlated to the firmness of banana peel during fruit development and ripening. These results have provided new insights into the role of cell wall HGs in fruit development and ripening. Musa spp.) are one of the most important tropical fruits and food crops in the world. It is commonly harvested at the mature-green stage when the angularity on the fruit surface disappears. Banana is a typical climacteric fruit, and exogenous ethylene is usually used to artificially initiate fruit ripening prior to commercialization. During the ripening process, there is a burst of ethylene production coordinated with rapid softening-associated processes, including peel de-greening, peel, and pulp softening [Bananas  were significantly firmer than control ones and displayed a reduction in solubilization and depolymerization of polyuronides . HoweverIn the case of bananas, multiple studies have been performed to investigate the mechanism involved in banana fruit ripening at physiological, biochemical, and molecular levels during the past decades. Most studies have been focused on gene expression ,28,29,30Musa spp. AAA) at different developmental stages and the ripening process based on the whole-transcriptome gene expression analysis. In addition, an immunofluorescence labeling technique was employed to reveal the changes in the spatial and temporal distribution of different HG components in banana peels during developmental and ripening stages, besides the changes in pectin content and activities of key pectin degradation enzymes. The results will provide new insights into the role of cell wall HGs in banana fruit development and ripening.Though cell wall disassembly regard to banana softening has been extensively studied, little is known about cell wall construction during early fruit development. This study was to analyze the changes in HG-modifying genes in the peel of a Cavendish dwarf banana cultivar ‘Huanong Aijiao 1’ (The study was performed on fruits of 0 d (just emerging from the bunch), 35 d, 60 d, green matured , and yellow matured  fruits. The change in appearance is shown in To get an overview of molecular changes underlying banana fruit development, we have performed transcriptome profiling at its different developmental stages, as well as ripening. The numbers of differentially expressed genes (DEGs) between every two stages were shown in In total, there are 61 MaGAUTs, 64 MaPGs, 3 MaPGIs, 27 MaPMEs, 41 MaporPMEs, 47 MaPMEIs, 12 MaPAEs, and 18 MaPLs in banana Genome A.GAUT is the key gene family associated with the biosynthesis of HG. Nowadays, GAUT1, GAUT7, GAUT13/14 were proved to be involved in the biosynthesis of pectin HG. As shown in In total, 23 MaPGs showed differentially expressed levels during banana fruit development and 36 after ethylene treatment. Only four MaPGs  showed a higher expression level during the fruit developmental stages, while it was 16 for the ripening stage. The expression of two MaPGIs decreased with the development and was inhibited by ethylene treatment .All five MaPMEs differentially expressed during fruit development showed a decline in expression level. On the contrary, all four MaPMEs differentially expressed during fruit ripening were upregulated by ethylene treatment. Similar trends were observed with the expression of MaproPMEs, MaPLs, and MaPMEIs except for Ma08_g12230 (proPME) and Ma08_g12230 (PMEI) .Nine MaPAEs were differentially expressed during banana fruit development and ripening. Three of them  showed an increased expression level with the development of banana fruits, but a decreased one after ripening. On the contrary, the expression of Ma04_g18920 decreased with fruit development but increased after softening. Another three MaPAEs  were also induced by ethylene treatment, but the other two MaPAEs (Ma02_g20280 and Ma04_g18910) were downregulated during banana fruit development .To validate the results of the RNA-Seq analysis, the expression of 25 representative DEGs (one–six ones from each gene family) were analyzed by qPCR. The result indicated that it was consistent with the result from RNA-Seq .As shown in The pectin content in the peel of banana fruit at 0 d was 62.70 mg/g (dry weight). It decreased with the fruit development and reached the lowest level at harvest, which was significantly lower than that at 0 d. After ripening, it increased to 57.83 mg/g, which was at the same level as that at 35 d. The degree of pectin methylesterification (DM) of fruit peel at 0 d was the highest (59.07%) while the YM fruit showed the lowest one (47.60%), but no significant difference was observed with DM during fruit development and after ripening .N. It showed an increasing trend with the development of fruits and peaked at 60 d, followed by a decline thereafter. The peel firmness of GM fruits was at the same level as that of fruits at 0 d. However, the peel firmness of YM fruits was only 4.93 N, significantly lower than that of fruit peel at all the other stages.The changes in the firmness of fruit peel during development and ripening stages were also investigated. The rupture force of fruit peel at 0 d was 10.90 Ruthenium red (RR) was used to reveal the spatial and temporal changes of pectin in banana peels during fruit development after treatment of the section with NaOH. As shown in In the fruits at 0 d, the signal of banana PME antibody was nearly undetectable, with a relatively stronger one in the epidermic cells, cortex cells adjacent to it, and latex cells . No signThe JIM5 recognizes partially methyl-esterified HG (up to 40%), and this epitope was mainly present in the xylem vessel, the vascular bundles, and the epidermic cells of the peel at 0 d . The antThe JIM7 recognizing epitope is HG with a methylesterification degree of 40–80%. The signal of this antibody in the transverse section of banana peel at 0 d was also very weak, with a relatively higher level in the xylem vessel and the epidermic cells . The sigLM18 antibody recognizes lowly methyl-esterified HG, and its labeling pattern in the peel at 0 d was similar to that of JIM5, but with a relatively higher epitope level . The LM1No detectable signal was observed with CCRC-M34, CCRC-M130, and LM20 antibodies in banana peel (data not shown).p value of 0.012 during fruit development, 0.9611 with p value of 0.009 during fruit development and ripening stage. There was also a positive correlation between peel firmness and 2F4 intensity during fruit development .Because the epitope levels of 2F4, CCRC-M38, JIM7, and LM18 antibodies showed similar dynamic changes to peel firmness during banana fruit development and ripening. The correlation coefficient between them was analyzed. The coefficient between peel firmness and CCRC-M38 intensity was 0.9877 with Previous studies indicated that there were great differences in pectin content and the expression of HG-modifying genes between the peel and pulp during banana ripening ,14,42,43Fruit growth involves cell divisions and cell expansion resulting from a dynamic interplay between cell turgor pressure, cell wall biosynthesis, and degradation, as well as cell wall remodeling. It has been proposed that the cell wall component of fruit exocarp/peel controlled the expansion of the developing fruits and contributed to the final size and shape of the fruit ,45. HowePGIs, PMEs, proPMEs, and PLs, as well as most PGs, PMEIs, and PAEs in banana peels decreased during the fruit development. However, both biochemical and histological analyses revealed that the pectin content in banana peels decreased with fruit development, and there was a significant difference between banana peels at harvest and 0 d. This might be due to the down-regulation of GAUTs  in the meanwhile. On the other hand, four PGs  and three PAEs  showed higher expression level at later development stages than earlier ones. They might be related to the expansion of fruit and provide the flexible, extensible mechanical support that resists the strains resulting from the expansion of the underlying cortex tissue [Prunus persica) fruit [In the present study, the expression levels of all differentially expressed x tissue ,47. The a) fruit  and banaa) fruit ,48.The firmness of fruit peels increased significantly with the development of fruits and peaked at 60 d, followed by a decline at harvest. This decline in firmness of banana peel was suggested to be due to the significant increase in PL activity. Interestingly, the IF data revealed that the epitope levels of JIM7 and LM18 antibodies increased with the fruit development and peaked at harvest, while those of 2F4 and CCRC-M38 antibodies peaked at 60 d. In addition, there was a significantly positive correlation between peel firmness and the CCRC-M38/2F4 intensity during fruit development, suggesting that the content of fully de-esterified HG and unesterified/calcium ion cross-linked HG recognized by CCRC-M38/2F4 antibody, instead of pectin content and DM, was significantly correlated with the firmness of banana peel during fruit development. The increase in the antigen level recognized by the JIM7 antibody was also observed during the early development of tomato pericarp .Banana is a climacteric fruit. It is well known dramatic changes at physiological, biochemical, and molecular levels, including peel de-greening, fruit softening, and flavor change during the ripening stage, concomitantly with a burst of ethylene production and respiration, took place during the ripening stage of this type of fruits. Among them, the most apparent change is the reduction in fruit firmness. Cell wall disassembly is considered to be the major determinant of fruit softening during the ripening process in fleshy fruits ,51, whicMusa AAA cv. Brazilian were found to be upregulated after ripening via ethylene treatment [Over the last decades, the dynamic expression profiles of cell wall-modifying genes and corresponding changes in enzyme activities and cell wall composition revealed that pectin modification is primarily responsible for the progressive loss of firmness in ripening and withering fruits ,53,54. Ireatment . Howeverreatment ,15,38. Sreatment ,56,57,58MaGAUT7s, three MaGAUT1s, and two MaGAUT13/14s were differentially expressed during banana fruit ripening. Seven out of these 10 MaGAUTs were upregulated by ethylene treatment, suggesting there was still active biosynthesis of pectin HG during this process, which was in accordance with the observation in the ripening process of apple  [Nowadays, it is proved that GAUT1, 7, 13, and 14 are involved in the biosynthesis of HG ,59. In tmestica) .Consistent with the gene expression pattern, the activity of PG and PL in the peel of YM fruits was 2.25 and 6.60 times higher than that of GM fruits, respectively. Additionally, the PME activity also increased significantly from 13.09 to 21.10 in the present study. As a result, the fruit firmness decreased significantly after softening. Previous studies also found the activity of PG and/or PL in banana peels increased ,32,33, bPersea americana Mill.). In the present study, the firmness of banana peel decreased significantly after softening. However, both biochemical and histological analyses revealed that the YM fruits even contained higher levels of pectin than GM fruits, though a significant increase in activities of PME, PG, and PL was observed. This might be due to strong activity in HG biosynthesis taking place in the soft YM fruits. Similarly, the GalA content in the cell wall of apple also increased concomitant with a decrease in cell wall stiffness after being subjected to ultrasounds [Besides, at the molecular level, fruit cell walls also undergo extensive changes in structure and composition during fruit ripening, which results in the reduction in their mechanical strength. In the case of bananas, Duan et al.  suggesterasounds . These r2+ also could influence the formation of cross-linked pectin gels, and they both contribute to tissue firmness. In the present study, the HG components recognized by JIM7, CCRC-M38, and LM18 antibodies showed significantly lower levels in YM fruits than in GM fruits, and the intensity of 2F4 antibody recognizing un-esterified/Calcium ion cross-linked HG also decreased 20%. As mentioned above, the levels of these antigens showed the same trends as the firmness of banana peel during fruit development. Thus, the lower DM, lower levels of Ca2+ cross-linked HG, as well as unesterified or deesterified to partially methylesterified HG were contributed to the reduction in firmness of YM fruits.On the other hand, the pectin DM of YM fruit was lower than that of GM fruits, which might mainly result from the significant increase in PME activity. The removal of the methylesters from HG by PME during fruit development and ripening not only could facilitate PG action to increase cell wall porosity and decrease cell adhesion but also favor cell adhesion and the rigidity of the pectin network , and theMusa AAA, cv. Huanong Aijiao 1) at 0 d, 35 d, 60 d, and 85 d  were collected from the field at the same time when the daily average temperature is approximately 25 °C. The fruit peel was isolated and frozen completely in liquid N2 before storage at −80 °C till use. Additional GM fruit samples were kept at 18–22 °C and 100% of humidity after treatment with 500 μL/L of ethylene (40% v/w) to initiate ripening. Twenty-four hours later, the humidity was lowered to 80–90% while the temperature was kept the same. The fruit became soft, and the peel became yellow 6 d after ethylene treatment, namely YM fruits. These fruits were also sampled for analysis. Each treatment consisted of three biological replicates, each containing six fruit fingers from independent plants.The fruits of dwarf banana  according to the manufacturer’s instructions. The compression force is measured at the maximum peak of the force recorded on the graph and expressed in N.Fruit peel firmness was assayed using a texture analyzer | > 1 and a threshold of false discovery rate < 0.05 were used as the threshold to evaluate the significance of DEGs. GSEAPreranked was employed for gene set functional enrichment analysis [https://bigd.big.ac.cn/gsa/browse/CRA005631, accessed on 28 August 2021.RNA preparation, library preparation for RNA-Seq, and data analyses were carried out as described previously . In brieanalysis . Heatmapanalysis . All RNA−△△CT method. The primers used for qPCR are listed in Total RNA was extracted from the samples using the hot borate method described by Wan et al. , followew/v) polyvinylpyrrolidone followed incubation at 4 °C for 1 h with occasional stirring. The extracts were centrifuged at 15000 rpm for 30 min at 4 °C and the supernatant was collected to assay the activities of PME, PG, and PL.The extraction of the crude enzyme was adopted from the method described by Mirshekari et al.  with sli−1, at 30 °C and pH 6.5. PG activity was determined according to the modified method of Pathak and Sanwal [PME activity of the supernatant was determined using a modification of the method developed by Marcus and Schejter , exactlyd Sanwal : 100 μL d Sanwal . After td Sanwal . The actd Sanwal .v/v) at 70 °C for 30 min after homogenization. The pellet was crushed in liquid nitrogen and dried at 60 °C for 3 d. The determination of both pectin and methanol was carried out by the method described by Ma et al. [Measurement of pectin and methanol was based on the obtained alcohol-insoluble residue, which was prepared as described by Louvet et al.  with mina et al. . The DM The immuno-labeling of all antibodies was carried out exactly as described by Xu et al. , exceptfw/v)  in PBS buffer was used to stain de-methyl-esterified pectin. The sections were prepared, dewaxed in absolute ethanol, rehydrated in ethanol-PBS series, and washed in PBS for immuno-labeling fluorescence. RR was dropped onto the sections and kept at room temperature for 30 min. After rinsing in PBS 3 times, the sections were mounted in immersion oil  before observation with an Axio Imager D2 . To stain all pectin, the sections were first treated with 0.1 mol/L NaOH for 3 min before RR staining.RR  in the statistical program SPSS 19.0 for Windows . Three replicates were set for each treatment. Data are presented as the mean ± SE. Duncan’s multiple range tests are employed to evaluate multiple differences among means at a 5% probability level.We use the “cor.test” function in R (version 4) to calculate the correlation coefficient between banana fruit peel firmness and enzyme activity.Banana is one of the most important foods and fruit crops in the world. It is well-known plant cell walls play a very important role in fruit development and ripening. As a typical climacteric fruit, the disassembly of banana fruits during the ripening stage has been extensively studied. However, the associated information during banana fruit development and the organization of HG components in situ and their modification during the ripening process are unavailable.PMEs, proPMEs, PGIs, and PLs, most GAUTs, PGs, PMEIs, and PAEs in banana peel showed a decline in expression during the fruit development, concomitant to the relatively stable activities of HG degradation enzymes and pectin DM. Four MaPGs  and three MaPAEs  might be involved in the expansion of fruit peel during banana development. All the investigated HG-modifying gene families showed differential expression patterns after ethylene treatment except for the PGI gene family. Six out of 10 top genes in the Core Enrichment Gene Set belong to HG degradation genes, and all of them were upregulated after softening, paralleled to the significant increase in activities of HG degradation enzymes and decline in fruit firmness. Interestingly, active HG biosynthesis took place during the fruit ripening process. The lower DM, lower levels of Ca2+ cross-linked HG, as well as un-esterified or de-esterified to partially methyesterified HG might be related to the reduction in fruit firmness after softening. The content of fully de-esterified HG recognized by CCRC-M38 antibody, instead of total pectin content, was positively correlated with the firmness of banana peel during both development and ripening processes. These results suggested that the HG-modifying genes are collaborated in the expansion and growth of cells in banana peels during fruit development and played a key role in the disassembly of pectin polysaccharides in fruit ripening.In the present study, all differentially expressed