Escherichia coli sequence type 131 (ST131) has emerged globally as the most predominant extraintestinal pathogenic lineage within this clinically important species, and its association with fluoroquinolone and extended-spectrum cephalosporin resistance impacts significantly on treatment. The evolutionary histories of this lineage, and of important antimicrobial resistance elements within it, remain unclearly defined. This study of the largest worldwide collection (n = 215) of sequenced ST131 E. coli isolates to date demonstrates that the clonal expansion of two previously recognized antimicrobial-resistant clades, C1/H30R and C2/H30Rx, started around 25 years ago, consistent with the widespread introduction of fluoroquinolones and extended-spectrum cephalosporins in clinical medicine. These two clades appear to have emerged in the United States, with the expansion of the C2/H30Rx clade driven by the acquisition of a blaCTX-M-15-containing IncFII-like plasmid that has subsequently undergone extensive rearrangement. Several other evolutionary processes influencing the trajectory of this drug-resistant lineage are described, including sporadic acquisitions of CTX-M resistance plasmids and chromosomal integration of blaCTX-M within subclusters followed by vertical evolution. These processes are also occurring for another family of CTX-M gene variants more recently observed among ST131, the blaCTX-M-14/14-like group. The complexity of the evolutionary history of ST131 has important implications for antimicrobial resistance surveillance, epidemiological analysis, and control of emerging clinical lineages of E. coli. These data also highlight the global imperative to reduce specific antibiotic selection pressures and demonstrate the important and varied roles played by plasmids and other mobile genetic elements in the perpetuation of antimicrobial resistance within lineages. Escherichia coli, perennially a major bacterial pathogen, is becoming increasingly difficult to manage due to emerging resistance to all preferred antimicrobials. Resistance is concentrated within specific E. coli lineages, such as sequence type 131 (ST131). Clarification of the genetic basis for clonally associated resistance is key to devising intervention strategies. We used high-resolution genomic analysis of a large global collection of ST131 isolates to define the evolutionary history of extended-spectrum beta-lactamase production in ST131. We documented diverse contributory genetic processes, including stable chromosomal integrations of resistance genes, persistence and evolution of mobile resistance elements within sublineages, and sporadic acquisition of different resistance elements. Both global distribution and regional segregation were evident. The diversity of resistance element acquisition and propagation within ST131 indicates a need for control and surveillance strategies that target both bacterial strains and mobile genetic elements. Escherichia coli (ExPEC) represents a major clinical challenge and is commonly caused by the presence of extended-spectrum beta-lactamases (ESBLs). The majority of ESBL-associated E. coli infections are due to a recently emerged, globally distributed ExPEC clone, sequence type 131 (ST131)  , 3 or O11 , 3 or  (ST131)  and belo (ST131) , 6. It r (ST131) .blaCTX-M-15 beta-lactamase gene is the dominant ESBL gene in ST131, but other genetically divergent CTX-M genes also occur in this ST, particularly blaCTX-M-14/14-like variants, e.g., in Canada, China, and Spain  data to investigate the population structure of ST131. The first found that ST131 expansion in the United States has been driven by a single sublineage, CTX-M-15 , which i highest , 18. FurblaCTX-M-14/14-like-containing strains, alongside a subset of CTX-M plasmid sequences, to estimate the contribution of each potential route of dissemination to the worldwide prevalence of ST131.Here, we used a broader set of ST131 WGS data, including many more isolates from Asia and The 215 ST131 genome sequences analyzed included 67 strains from various locations in Southeast Asia, 33 from Oxford in the United Kingdom, 11 from a global resistance surveillance program at AstraZeneca, 8 from Canada, and 96 predominantly North American isolates previously reported by Price et al. , B (n = 51), and C (n = 139), with C containing two subclades, C1 (n = 57) and C2 (n = 82), characterized by the presence versus absence, respectively, of blaCTX-M-15 , equating to 1.00  mutation per genome per year.The estimated time to most recent common ancestor (TMRCA) for the whole genomic data set was ~130 years ago, when clade A diverged from clades B and C. Twenty-five years ago, clade C emerged out of the paraphyletic clade B, which was quickly followed by the split between subclades C1 and C2. The number of core SNVs separating the clades was approximately 250 for clade A versus clades B and C, 50 to 60 for clade B versus clades C1 and C2, and 10 to 30 for clade C1 versus clade C2. The evolutionary rate of ST131 was estimated in BEAST  at 2.46 × 10H30 and C2/H30Rx was strongly inferred as being in North America  with subsequent dissemination to Europe and Asia. Locations of more recent nodes are inferred with high confidence, as expected , and the B/C clades were inferred to originate from North America with ~88% confidence. The ancestral origin of C1/expected .blaCTX-M, including 74 isolates (34%) with blaCTX-M-15, 20 (9%) with blaCTX-M-14, 8 (4%) with blaCTX-M-27, and one each (0.4%) with blaCTX-M-19, blaCTX-M-24, and blaCTX-M-55 . blaCTX-M-14 and blaCTX-M-27 were also clustered within the two different clades A and C1 and absent from B and C2  ST131 isolates harbored CTX-M-55 . blaCTX-eviously , occurriB and C2 . OverallfimH variant was fimH30 , followed by fimH22  and fimH41 , whereas 23 isolates had novel fimH variants, and one was fimH null. As observed for blaCTX-M, fimH alleles were strongly associated with clade, with 21/25 (84%) isolates in clade A having fimH41, 23/51 (45%) in clade B having fimH22, and 122/139 (88%) in clade C having fimH30 .The most common gyrA and parC were also clade associated, with isolates in clades A and B typically having no or only single mutations in these genes’ quinolone resistance-determining regions (QRDRs)  . In contsistance  . In another isolate (JJ2547), the assembled contig with blaCTX-M-15 contained a series of N’s, suggesting possible uncertainty around the contig assembly or multiple locations of the gene. These five isolates were excluded from further analysis of flanking regions. In the 69 remaining isolates , blaCTX-M-15 was found downstream of a homologous tract of 48 bp preceded by an ISEcp1 right-end inverted repeat region (IRR-R) and upstream of a homologous tract of 46 bp followed by ORF477. This is consistent with the introduction of an ISEcp1-blaCTX-M-15-ORF477 unit within ST131 and subsequent rearrangement events affecting this structure.In four of the 74 blaCTX-M-15 was integrated into the chromosome of 8/65 (12%) isolates, with four unique integration events, one of which was stably present in a subcluster of five isolates with a common ancestor around 2002 and spread across two geographical regions  or flanked by likely plasmid-associated sequences in the contig assemblies (n = 7). In the remaining 30 isolates, the location of blaCTX-M-15 could not be defined due to limitations of the short-read assemblies. In all 57 isolates, the upstream sequence was either an intact or truncated ISEcp1 sequence, and in 51/57 (89%) isolates, the sequence downstream of ORF477 was either an intact or a truncated Tn2 structure  being nested within a complete or partial Tn2 transposon. In 40/57 (65%) isolates, IS26 repeat regions truncated either or both of these upstream and downstream contexts . Six isolates had plasmid-associated blaCTX-M-14 based on annotated flanking sequences/transformants, whereas for the rest (n = 14), the location of blaCTX-M-14 was uncertain due to limitations of the de novo assemblies.For Ecp1 was consistently located upstream of blaCTX-M-14, as with blaCTX-M-15, but at only a 43-bp distance, and the downstream flanking sequences were composed of either intact or truncated IS903B elements. In clade A, the genetic flanking sequences surrounding blaCTX-M-14 were consistent with the host strain subcluster and were homologous over the observed contig length within this subcluster . Specific IncF variants differed in frequency, with FII found in 199/215 isolates (93%), FIB in 155/215 (72%), FIA in 145/215 (67%), and FIC in 17/215 (8%). Specific IncF replicons and combinations thereof were clade associated . A numbeAY458016; pC15-1a; consistent with pMLST allele 2) was significantly associated with clade C2 . Within clade C2, a further 23 isolates had eight different FII_AY458016-like variants containing up to 12 SNVs among them; almost all of these variants were in isolates with FIA-FIB-FII replicon combinations , five had chromosomally integrated blaCTX-M-15 , one was blaCTX-M negative, and one contained deletions in blaCTX-M-15. There were only nine clade C2 isolates with FII_AY458016-like replicons but no blaCTX-M-15. The different FII replicon associated with blaCTX-M-15 in clade C2, FII_AJ851089, was also clade associated, being found predominantly in clades A  and C1  rather than B  and C2    see . OverallblaCTX-M plasmids , blaCTX-M-14 (n = 2), and blaCTX-M-27 (n = 1); one from clade B, containing blaCTX-M-55; three from clade C1, containing blaCTX-M-14 (n = 2) and blaCTX-M-24 (n = 1); 20 from clade C2, containing blaCTX-M-15; and two blaCTX-M-15 plasmids from non-ST131 isolates . BLASTn-based comparisons revealed that the clade A blaCTX-M-15 IncI plasmid  was circulating in a limited fashion . Although we did not have transformants or specific plasmid sequences for these, the blaCTX-M-15-containing contig assembled for JJ2591 was 88,693 bp long and very similar to the 11B00320_T assembly, whereas the AZ779845 blaCTX-M-15-containing contig was 32,228 bp long and likewise highly similar in structure  plasmids from clade C2 that contained IncFII_AY458016 identified a significant degree of homology -Ib-cr, a glucose-1-phosphatase-like-enzyme, a CAAX amino-terminal protease self-immunity protein, a hypothetical phage protein, and a pemI/pemK plasmid addiction system. This lack of gene conservation suggests that significant genetic exchange and rearrangement occur among these plasmids as they evolve within the subclade.Nineteen of 20 transformed homology   were identified in clade A, in host strains with a TMRCA within the last 15 years, and one in clade C1   . BLASTn-e, Laos) . 11B0197a_5108_T , suggestblaCTX-M-55 and blaCTX-M-24  apparently resulted from discrete plasmid acquisition and/or blaCTX-M transposition events within ST131 (see blaCTX-M evolution within established blaCTX-M-15 or blaCTX-M-14 plasmid backgrounds.The isolates containing n = 215) and most diverse collection of ST131 isolates to date establishes that the global emergence of drug-resistant clades  occurred approximately 25 years ago, most likely in a North American context and consistent with strong selection pressure exerted by the widespread introduction and use of fluoroquinolones and extended-spectrum cephalosporins. Interestingly, this appears to be at odds with the previous observation that ESBLs predominating in North America in the 1990s and early 2000s were mostly blaTEM or blaSHV variants , or did not test specifically for blaCTX-M variants.Our WGS analysis of the largest , supporting the hypothesis that both horizontal gene transfer and clonal expansion have contributed to the global dissemination of this sequence type. The estimated molecular evolutionary rate of ST131 (1.00 mutation per genome per year) is similar to previous estimates from ST131  and the blaCTX-M variants within specific ST131 clades is due to diverse mechanisms. These include (i) acquisition of a blaCTX-M-containing plasmid by a specific host strain subcluster, followed by evolution and spread across geographic regions ; (iii) horizontal transfer of common plasmid structures across clades . Despite this high degree of genetic plasticity, we also found clear structuring of blaCTX-M variants and plasmid content, with the near-complete absence of blaCTX-M in clade B and associations of blaCTX-M-14/14-like variants with clade A and clade C1/H30R, of blaCTX-M-15 with clade C2/H30Rx, and of specific combinations of IncF replicons with certain clades. This supports the hypothesis that some plasmid replicons are acquired and persist stably within clades. Although the evolutionary dynamics of plasmid-host combinations remain to be clearly elucidated, coevolution of host and plasmid in the case of C2/H30Rx appears to have ameliorated costs to the host and facilitated persistence of the replicon ; and  and rearrangement/recombination among IncFII-like plasmids, much of this associated with IS26 (pemI/pemK)  there are no geographic or major genotypic distinctions between clades C1 and C2 to explain why this would occur, (ii) there is a degree of homology in the flanking contexts around the gene throughout the clade, and (iii) flanking context/transformed plasmid structures also appear to be consistent within C2 subclusters.The almost ubiquitous presence of ith IS26  Fig. 3)blaCTX-M-mI/pemK) , whereasmids see . AlternablaCTX-M plasmids found in different clades and STs. This indicates that between-clade/ST transfer of these resistance plasmids occurs and that care is needed when inferring plasmid evolution by descent (blaCTX-M (as demonstrated here) is much more likely to represent ancestral plasmid acquisition and subsequent evolution by descent rather than multiple acquisition events but still needs to be interpreted with caution, as it may, for example, represent exposure to a common, global, plasmid reservoir.Our novel comparison of transformed, sequenced plasmids demonstrates that a substantial degree of similarity can exist among  descent . The obsde novo assemblies of these short-read data also must be interpreted cautiously, as these assembly methods are known to increase the number of SNVs compared with mapping-based approaches and may result in misinterpretations of genetic structures, particularly repetitive regions . This sin silico multilocus sequence typing (MLST) of de novo-assembled WGS data , in order to limit bias introduced by mapping to a more divergent reference. Repetitive regions  of the reference were identified using self-self BLASTn analysis with default settings; these regions were then masked prior to mapping and base calling. Single-nucleotide variants (SNVs) were determined across all mapped nonrepetitive sites using SAMtools (version 0.1.18) mpileup. mpileup was run twice to separate high-quality base calls from low-quality base calls: first, with options “-E -M0 -Q25 -q30 -m2 -D -S” and otherwise default settings, and second, with options “-B -M0 -Q0 -q0 -m2 -D -S” and otherwise default settings. Vcf files of annotated variant sites were created using GATK (v1.4.21). Base calls derived from these two Vcf files were then retained only if (i) the proportion of high-quality bases supporting the call was ≥90%, and ≥5 high-quality bases were required as a minimum; (ii) the root of the mean square mapping quality of reads covering a putative variable site was ≥30; (iii) the Phred scaled quality supporting a base call was ≥25; and (iv) reads spanning the putative variable site were made up of ≥35% high-quality bases. Core variable sites  derived from mapping to the SE15 reference were “padded” with invariant sites in a proportion consistent with the GC content and length of the reference genome , to generate a modified alignment of input sequences for our phylogenetic analyses (see below).Properly paired sequence reads were mapped using Stampy v1.0.17  to a fully sequenced De novo assemblies were generated using Velvet with the VelvetOptimiser wrapper (n = 211) (http://bioinformatics.net.au/software.velvetoptimiser.shtml) or A5-MiSeq . Using A5-MiSeq, assemblies for these four strains were generated with an appropriate median size of 5,143,908 bp and 269 contigs.n = 211)  (http://A5-MiSeq . The latde novo assemblies was used to identify: (i) blaCTX-M presence and variants (in-house reference gene database)   fimH presence and variant (http://pubmlst.org/plasmid/) ; otherwise, they were classified as unknown. IncFII_AY458016-like sequences were extracted, aligned, and visually inspected to confirm variant types using Geneious   ; (ii) geotation) , 44;  variant  and pMLSlasmid/) . GeneticZealand) .ExPEC bacteria are recombinogenic and contain recombination hot spots with higher-than-average recombination rates . Recombihttp://beast.bio.ed.ac.uk); adequate convergence of run statistics and mixing for each run and effective sample sizes (ESSs) for all parameters greater than 200 were required for an analysis to be considered adequate, in line with recommendations in the BEAST tutorials on the developers’ website (http://beast.bio.ed.ac.uk). We explored the application of several other models in BEAST incorporating the relaxed clock and variable population growth , but these either failed to converge, showed poor mixing, or had effective sample size (ESS) estimates of <200 and were therefore not considered robust.Using the modified alignment of ST131 sequences generated following the ClonalFrame analysis, mutation rate estimates across ST131 and a time-scaled phylogeny were calculated in BEAST . The mod8 MCMC steps. We subsequently reran the analysis including a fourth, unsampled deme, using the same model parameters, to determine whether this altered the outcome.We used the phylogeographic method BASTA  in the BblaCTX-M-containing plasmids from non-ST131 E. coli (one ST617/blaCTX-M-15 and one ST405/blaCTX-M-55) were also transformed and sequenced as an external comparison.Plasmid transformants were generated from 30 strains chosen on the basis of tree topology and association with CTX-M variants, aiming to transform at least one plasmid from each of the major CTX-M variant clusters. Two Plasmid DNA was extracted from subcultures of frozen stock grown overnight on blood agar, followed by selective culture of a single colony in lysogeny broth  with ceftriaxone at 1 µg/ml. DNA extraction was performed using the Qiagen plasmid minikit , in accordance with the manufacturer’s instructions, with the addition of Glycoblue coprecipitate  to the DNA eluates prior to isopropanol precipitation to enable better visualization of the DNA pellet. Plasmid DNA was redissolved in distilled water and then typically electroporated on the same day or stored in the refrigerator prior to electroporation within 24 h.E. coli  was used as the recipient cell strain for plasmid electroporation, because of its high transformation efficiency and the fact that the strain has been fully sequenced (NCBI RefSeq NC_010473.1)  . ElectroP = 0.003). Mapping was used to assess the reliability of our plasmid constructs and reflected the content present in each transformed and assembled resistance plasmid, with the exception of 8A16G_T.Sequencing was performed on the Illumina HiSeq or MiSeq sequencer, generating 150- or 300-base paired-end reads see , and assA single strain (P46212) from the data set was also sequenced using long-read technology (PacBio); the CTX-M-15 plasmid (pP46212) from this strain was assembled into a single, circularized contig as described elsewhere .de novo assembly for each ST131 isolate with each respective, concatenated plasmid reference sequence. Sequence identity of BLASTn hits across the plasmid reference was plotted using the heatmap.2 package in R, with a minimum threshold of 90% identity for plotting. For simplicity, values were averaged over 100-bp bins (script available at https://github.com/aesheppard/plasmid_comp).Plasmid content across the data set was investigated in a number of ways. First, the transformed plasmid sequences that we generated were used as references against which BLASTn-based comparisons for degree of presence/absence were made for the whole data set. We used default BLASTn settings to compare the https://github.com/aesheppard/plasmid_comp). The mean percent divergence for each plasmid sequence pair was then plotted against the time to most recent common ancestor (TMRCA) of the two host strains containing those transformed plasmid sequences  in Stata (SE) .Second, comparisons between each pair of transformed plasmid assemblies were undertaken, again using BLASTn with default settings. For the query sequence in each comparison, the percentage of sites contained in hits (counting overlapping hits only once) was identified from parsed blast output. For each pair, two percentage of homology statistics were generated, taking each member of the pair as a reference in turn, to account for differences in length  are listed in de novo assemblies for the transformed plasmid sequences are in http://modmedmicro.nsms.ox.ac.uk/stoesser-n-et-al/.The positions for called, variable sites across the data set .Sequencing data for the new isolates sequenced for this study have been deposited in the NCBI Short Read Archive . For all aligned, similarly colored regions, sequence homology is preserved; curly brackets cluster those isolates with identical flanking sequences. Flanking contexts not shown for isolates with known chromosomal integration or for blaCTX-M-negative/non-blaCTX-M-14/14-like isolates in the subclusters. Coloring of isolate names reflects geographic locations . Download Genetic contexts of clade A-associated Figure S1, PDF file, 0.2 MBFigure S2 blaCTX-M-14/14-like variants. Many contexts are limited by the extent of the assembled region around the blaCTX-M-15 gene (marked with “X”). For all aligned, similarly colored regions, sequence homology is preserved; curly brackets cluster those isolates with identical flanking sequences. Flanking contexts not shown for isolates with known chromosomal integration or for blaCTX-M-negative/non-blaCTX-M-14/14-like isolates in the subcluster . Download Genetic contexts of clade C1-associated Figure S2, PDF file, 0.2 MBFigure S3 x axis. Download Inc types identified in whole-isolate sequencing data, plotted with respect to ST131 host strain phylogeny. Blast match (%) denotes a composite score of percent matched length and percent homology to reference Inc sequence, with highest percent score/contig hit represented. Matches of <80% were excluded. Reference Inc sequences were downloaded from the PlasmidFinder database; those that were present (Blast match of ≥80%) in at least one isolate are represented on the Figure S3, PDF file, 0.5 MBFigure S4 BLASTn-based comparisons across the ST131 data set, using la_12107-3_T and la_5220-3_T as references. Color represents degree of presence/absence of corresponding reference sequence on an isolate-by-isolate basis per row. Rows/isolates arranged as in the Figure S4, PDF file, 2.3 MBText S1 E. coli genome. Download List of positions for called, variable sites across the data set, with respect to the reference SE15 Text S1, TXT file, 0.05 MBText S2 List of positions identified as being in recombinant regions through the ClonalFrame analysis. Download Text S2, TXT file, 4.5 MBText S3 de novo assemblies for the transformed plasmid sequences. Download Fasta file of contigs for the Text S3, TXT file, 3.4 MBTable S1 Details of newly sequenced ST131 strains included in the analysis.Table S1, XLSX file, 0.1 MBTable S2 blaCTX-M plasmid sequences.Details of transformed Table S2, XLSX file, 0.04 MB


Babesia bovis, a causative pathogen of the globally important cattle disease, bovine babesiosis. To date, two stable transfection systems have been developed for B. bovis, using selectable markers blasticidin-S deaminase (bsd) or human dihydrofolate reductase (hdhfr). In this work, we combine these two selectable markers in a sequential transfection system. Specifically, a parent transgenic B. bovis line which episomally expresses green fluorescent protein (GFP) and human dihydrofolate reductase (hDHFR), was transfected with a plasmid encoding a fusion protein consisting of red fluorescent protein (RFP) and blasticidin-S deaminase (BSD). Selection with WR99210 and blasticidin-S resulted in the emergence of parasites double positive for GFP and RFP. We then applied this method to complement gene function in a parasite line in which thioredoxin peroxidase-1 (Bbtpx-1) gene was knocked out using hDHFR as a selectable marker. A plasmid was constructed harboring both RFP-BSD and Bbtpx-1 expression cassettes, and transfected into a Bbtpx-1 knockout (KO) parasite. Transfectants were independently obtained by two transfection methods, episomal transfection and genome integration. Complementation of Bbtpx-1 resulted in full recovery of resistance to nitrosative stress, via the nitric oxide donor sodium nitroprusside, which was impaired in the Bbtpx-1 KO parasites. In conclusion, we developed a sequential transfection method in B. bovis and subsequently applied this technique in a gene complementation study. This method will enable broader genetic manipulation of Babesia toward enhancing our understanding of the biology of this parasite.Genetic manipulation is an essential technique to analyze gene function; however, limited methods are available for  Babesia bovis is an intraerythrocytic apicomplexan parasite, which is transmitted by ticks and causes the disease bovine babesiosis in cattle. This disease has a significant global economic and social burden [B. bovis was reported, but the function of more than 50% of the genes remains unassigned [Babesia parasites is the paucity of gene manipulation methodologies [Plasmodium spp. and Toxoplasma gondii, several genetic manipulation tools have been developed which are widely used to study gene functions, as well as applied to develop genetically defined attenuated vaccines [l burden –3, and nassigned , 5. A madologies . Applicadologies . For exavaccines .B. bovis began in the last decade by developing a transient transfection method using the rap-1 or ef-1α promoter and the terminator of the rap-1 genes of B. bovis controlling expression of luciferase as a reporter [gfp-bsd gene can be expressed and remain stable upon inoculation of transgenic parasites into calves [Bbtpx-1) gene locus by a homologous recombination approach [B. bovis Bbtpx-1 knock out (KO) clonal line which is able to grow in in vitro culture despite lacking expression of the Bbtpx-1 gene.The initial work for developing transfection methods of reporter –11. Subsreporter , 13. It o calves . Alternao calves . Applyinapproach . This stbsd and WR99210/hdhfr selection systems within a sequential transfection methodology for B. bovis. We show that the developed sequential transfection system is successfully adopted to complement Bbtpx-1 in a B. bovis Bbtpx-1 KO clonal line.In the present study, we combine blasticidin-S/Bbtpx-1 (BBOV_II004970) KO B. bovis lines were previously generated from the Texas strain of B. bovis [The GFP-expressing and B. bovis . The parB. bovis . WR99210ef-1α promoter. The rfp and bsd genes were amplified by PCR from the monomeric rfp expression plasmid  using the primer pairs 5’-AATTGATATCATGGCCTCCTCCGAGGAC-3’ and 5’-AATTGATATCGGCGCCGGTGGAGTGGCG-3’ (EcoRV sites are underlined), and the gfp-bsd expression plasmid [CGCCGATATCGAATTCCAGGCCAAGCCTTTGTCT-3’ and 5’-CATCCTGCAGGAATTTTATAAACGCATCTCATC-3’ (In-Fusion HD cloning sites are underlined), respectively. The amplified rfp gene was cloned into the EcoRV site of a plasmid containing the 5'-flanking region of the ef-1α gene (5'-ef-1α) and the 3'-flanking region of the rap-1 gene (3'-rap-1) [bsd gene into the EcoRI site by In-Fusion system . To construct the Bbtpx-1 expression plasmid pBtpx-1  and 3'-portion of Bbtpx-1 were independently amplified by PCR from B. bovis genomic DNA (gDNA) and inserted upstream and downstream of the RFP-BSD expression cassette in the XhoI and NotI site of the pBrfp-bsd plasmid, respectively. The Bbtpx-1 gene locus was amplified with the primers, 5’- CGGGCCCCCCCTCGAATTGCAAGGCAACCAATTTAC -3’ and 5’- TACCGTCGACCTCGATCGCATTTCGCATTAATTCAC -3’ (In-Fusion HD cloning sites are underlined). The 3'-portion of Bbtpx-1 was amplified with the primers, 5’- AGTTCTAGAGCGGCCTTCGTTAATGCCTTCAACTCG-3’ and 5’- ACCGCGGTGGCGGCCGAATCTGCAGCAACCATTAGC -3’ (In-Fusion cloning sites are underlined). The primers were designed based on genome sequence information available in PiroplasmaDB (http://piroplasmadb.org/piro/). Constructed plasmids were purified using Qiagen Plasmid Midi Kit  following the manufacturer’s instructions and inserted DNA sequences were confirmed by sequencing.The pBrfp-bsd plasmid  was desi plasmid  using pr'-rap-1) , 16; fol pBtpx-1 , the BbtB. bovis was transfected using a Nucleofector device  as described [escribed . Briefly-Bbtpx-1 probe was prepared by PCR amplification using primers 5’-AATTCTCGAGATTGCAAGGCAACCAATTTAC-3’ and 5’-AATTCTCGAGTCGCATTTCGCATTAATTCAC-3’. Hybridization and detection was carried out using AlkPhos Direct Labelling Reagents and CDP-Star Detection Reagent .Successful transfections were assessed by diagnostic PCR assays, sequencing of PCR products, and Southern blotting. IRBC were lysed with PBS containing 0.15% saponin (Wako) and DNA was extracted using a QIAamp DNA Mini Kit . PCR amplifications were performed using the primers Bbtpx-1 5’ F (5’-ACGCAGCATTCAGTTACTTCG-3’), Bbtpx-1 R (5’-AACTTCACCGTGCTTCTCGT-3’), dhfr F (5’-ATGGTTGGTTCGCTAAACTGC-3’), bsd F (5’-CAGGCCAAGCCTTTGTCT-3’), and Bbtpx-1 3’ R (5’-GTCATCGTTGGGTTTGACCT-3’); followed by sequence determination of PCR products. PCR reaction mixture was prepared according to the manufacture’s instruction of Tks Gflex DNA polymerase (Takara Bio). PCR condition was; one cycle of pre-denaturation at 94°C for 1 min, 35 cycles of denaturation at 94°C for 15 sec, annealing at 55°C for 20 sec, and elongation at 68°C for 1min (for PCR with Bbtpx-1 5’ F and Bbtpx-1 R) or 4 min (for PCR with dhfr F or bsd F and Bbtpx-1 3’ R), and one cycle of post-elongation at 68°C for 3 min. DNA sequences of PCR products were determined by primer-walking. For Southern blot analysis, 2.5 μg of the parasite gDNA was digested with XbaI, separated via agarose gel electrophoresis, and transferred onto a nylon membrane . The 5’Bbtpx-1 transcripts were detected by PCR from cDNA using primers as described [Parasite cultures were pelleted by centrifugation, supernatants discarded and total RNA extracted using the TRIzol reagent (Invitrogen) and purified using the SV Total RNA isolation system . Complementary DNA (cDNA) was prepared by RNase I treatment followed by reverse transcription using SuperScript III First Strand Synthesis System (Invitrogen). escribed . As a coescribed , 19. Thiescribed  and usedt-test and probability value of less than 5% (p < 0.05) was considered statistically significant.To evaluate the sensitivity of parasites against nitrosative stress or oxidative stress, parasites were maintained in the presence of a nitric oxide donor or super oxide donor, sodium nitroprusside , at concentrations of 0, 1, 5, 10, 20 and 50 μM; or paraquot (Wako) at concentrations of 0, 0.1, 0.5, 1, 5 and 10 μM, respectively [B. bovis, a plasmid expressing a RFP-BSD fusion protein . Parasite clones positive only for RFP fluorescence were selected for further expression and phenotype analyses.To attain genome integration of introduced DNA, we also transfected linearized versions of an expression plasmid construct . After mBbtpx-1 ORF region (Bbtpx-1 R) yielded identical 1.4 kb PCR products from wild type parasite and the parasite clone transfected with the linearized construct  and 3' downstream of the expected 3' recombination site (Bbtpx-1 3' R) amplified a 6.3 kb product from Bbtpx-1 KO parasite and the episomal transfectant, but not from wild type parasites or the parasite clone transfected with the linearized construct. Moreover, PCR amplification with a primer for bsd ORF region (bsd F) and Bbtpx-1 3' R primer amplified a 3.2 kb product from the parasites transfected with the linearized construct, but not from the other parasites  and onstruct . In contondition . Consistarasites . NucleotBbtpx-1 ORF region was inserted into the original Bbtpx-1 locus as expected , and comparable to that of wild type parasite at concentrations of 5, 10 and 20 μM  present during sequential or simultaneous transfections.Linearized pBbtpx-1 was designed to replace the GFP-hDHFR expression cassette with RFP-BSD, and BbTPx-1 expression cassettes by double crossover at the 5’ and 3’ UTRs of Bbtpx-1 locus, however the Southern blot probe detected additional weak bands only in the genome integrant parasites. Similar phenomenon was also observed in the first report which the GFP-BSD expression construct was inserted into the ef-1α locus [B. bovis in our hands. In addition, inter- and intrachromosomal segmental gene conversion were reported among ves multigene family members in B. bovis [Bbtpx-1 was integrated or converted into the genome as a result of transfection. To date, a limited number of promoter and terminator sequences have been available for B. bovis transfection [Plasmodium spp. and Toxoplasma gondii, may also be tested, which would provide advantages to avoid problems of undesirable integration.Furthermore, in the RFP positive and GFP negative parasites, the results of diagnostic PCR and Southern blot analysis were consistent to the expectation that the construct was inserted into the 1α locus . Until nB. bovis , so it isfection , 15. Thetpx-1 gene in either B. bovis or P. falciparum did not affect their growth under normal in vitro culture conditions [P. falciparum, the growth of tpx-1 KO line was suppressed in the presence of paraquat, an oxidative stress, or the nitrosative stress, SNP, in comparison with wild type parasites [B. bovis TPx-1 in resistance to nitrosative stress, but not to the oxidative stress caused by paraquat at concentrations up to 10 μM. This discrepancy between B. bovis and P. falciparum in the response of the tpx-1 KO line to oxidative stress may be explained by a presence of a functional compensation from other peroxide-reducing enzymes, such as catalases in B. bovis. For example, enzymatic activity of catalase has been reported in Babesia spp., but not in Plasmodium spp. [Thioredoxin peroxidases are a ubiquitous family of antioxidant enzymes . In publnditions , 20. In ium spp. .B. bovis. Double gene knockout approaches will accelerate the functional study of molecules encoded by multigene families and may allow future development of live attenuated vaccines upon the deletion of multiple virulence factor genes. Nonetheless, additional transfection methods need to be developed for further investigating the biology of Babesia parasites. In recent years, several transfection methodologies such as FLP/FRT-mediated conditional mutagenesis, a tetracycline-repressible transactivator system, and CRISPR/Cas9 genome editing system have been successfully used for the other apicomplexan parasites [bsd and WR99210/hdhfr selection systems developed in this study is ready to adapt to new techniques in transfection-based research on the biology of Babesia parasites.The double selection techniques described herein provide a platform to generate double gene knockouts in arasites –26. The


Mycobacterium tuberculosis antigens designed to boost BCG-induced immunity. Clinical trials with candidate malaria vaccines have demonstrated that adenoviral vector based priming immunisation, followed by MVA vector boost, induced high levels of immunity. We present the safety and immunogenicity results of the first clinical trial to evaluate this immunisation strategy in TB.MVA85A and AERAS-402 are two clinically advanced viral vectored TB vaccine candidates expressing In this phase 1, open-label trial, 40 healthy previously BCG-vaccinated participants were enrolled into three treatment groups and vaccinated with 1 or 2 doses of AERAS-402 followed by MVA85A; or 3 doses of AERAS-402.Most related adverse events (AEs) were mild and there were no vaccine related serious AEs. Boosting AERAS-402 with MVA85A significantly increased Ag85A-specific T-cell responses from day of vaccination. Two priming doses of AERAS-402 followed by MVA85A boost, resulted in a significantly higher AUC post-peak Ag85A response compared to three doses of AERAS-402 and historical data with MVA85A vaccination alone. The frequency of CD8+ T-cells producing IFN-γ, TNF-α and IL-2 was highest in the group receiving two priming doses of AERAS-402 followed by MVA85A.Vaccination with AERAS-402 followed by MVA85A was safe and increased the durability of antigen specific T-cell responses and the frequency and polyfunctionality of CD8+ T-cells, which may be important in protection against TB. Further clinical trials with adenoviral prime-MVA85A boost regimens are merited to optimise vaccination intervals, dose and route of immunisation and to evaluate this strategy in the target population in TB high burden countries.NCT01683773.ClinicalTrials.gov  Mycobacterium tuberculosis (M.tb) specific antigens, thus retaining the benefits of BCG against disseminated disease.Tuberculosis (TB) remains a major global public health burden, with an estimated 9.0 million incident cases and 1.5 million deaths in 2013 . A more M.tb antigen 85A, and induced potent Ag85A-specific CD4+ T-cell responses in BCG vaccinated adults [M.tb antigens Ag85A, Ag85B, and TB10.4. In adults, AERAS-402 induced a potent antigen-specific CD8+ T-cell response together with a less dominant CD4+ T cell response [M.tb, a balanced CD4+ and CD8+ cellular immune response may be necessary [MVA85A and AERAS-402 are two clinically advanced TB vaccine candidates. Both have been shown to boost immunity induced by prior BCG vaccination. MVA85A comprises the recombinant replication-deficient Modified Vaccinia virus Ankara expressing the immunodominant d adults . AERAS-4response . For optecessary . Preclinecessary . The aimThis was a phase 1 open-label, non-randomized clinical vaccine with three treatment groups. The trial was approved by the Medicines and Healthcare products Regulatory Agency , the Berkshire B Research Ethics Committee (reference12/SC/0283) and University Hospitals Birmingham NHS Foundation Trust Research and Development department (reference RRK4493). All participants provided written informed consent and the trial was conducted according to the principles of the Declaration of Helsinki and Good Clinical Practice. The CONSORT trial checklist and clinical trial protocol are available as Subjects were recruited from the Centre for Clinical Vaccinology and Tropical Medicine, University of Oxford and the National Institute for Health Research (NIHR)/Wellcome Trust Clinical Research Facility at Birmingham. Screening and follow up visits occurred at both sites; all enrolment/vaccination visits occurred at the Oxford site.2). Previous AERAS-402 clinical vaccine trials have included a Body Mass Index of > 33 kg/m2 as an exclusion factor; this was maintained in this study for consistency to allow comparison with previous clinical vaccine trials. Latent M.tb infection was excluded at screening by a negative T-SPOT.TB test (Oxford Immunotec).Participants were BCG-vaccinated, healthy adults aged 18 to 55 with no clinically significant abnormalities in baseline haematology or biochemistry, negative serological testing for HIV, hepatitis B and hepatitis C viruses and a body mass index between 18 and 33  was administered intramuscularly and MVA85A (1x108 plaque forming units) was administered intradermally in the deltoid region of the upper arm. Subjects in Group A received AERAS-402 at study day (D) 0 and D28 and MVA85A at D119. Subjects in Group B received AERAS-402 at D0 and MVA85A at D56. Subjects in Group C received AERAS-402 at D0, D28 and D119.AERAS-402  and volunteers in Group B were followed up until day 140 .The primary endpoint was safety as assessed by frequency and severity of vaccine-related local and systemic adverse events (AEs). Expected local AEs  and systemic AEs ) were solicited from subjects using a diary card for 7 days post-vaccination and at each follow-up visit. Haematology and biochemistry analysis was conducted at screening, and at D28, 42, 119 and 126 for subjects in Groups A and C, and at D14, 56 and 63 for Group B.The secondary endpoint was immunogenicity of vaccinations in all three groups. This was measured by the ex vivo interferon-gamma (IFN-γ) ELISpot assay performed on fresh peripheral blood mononuclear cells (PBMC) stimulated with pools of mycobacterial peptides, as well as flow cytometric estimation of T cell IFN-γ, TNF-α, and IL-2 production, as quantified by intracellular cytokine staining (ICS). Blood samples for immunological analysis were taken at each study visit for all subjects: Days 0 (first vaccination), 28,42,56,119,126,147 and 203 for volunteers in Groups A and C and Days 0,14,28,56,63,84 and 140 for volunteers in Group B.There were 21 protocol deviations during the trial period. 17 deviations were due to study visits which were out of specified time window as subjects could not attend on schedule due to unforeseen personal circumstances.Safety bloods were omitted in error for one subject in Group A at D126; these bloods were within normal range when added at D147 visit.Haematology safety bloods were collected in an incorrect vacutainer tube for one subject in Group C at D126 visit; these results were within normal range when repeated one week later.Immunology bloods were omitted in error for one subject in Group A at D119 visit; these bloods were collected 4 days later, and within the window period for that study visit.A lower dose of MVA85A (44 uL) was administered to one subject in Group B at D56 study visit as the subject had a vasovagal episode during vaccination.These listed protocol deviations were not deemed to have an adverse impact on the safety of the subjects involved or on the scientific data collected during the trial period and therefore all data from all subjects was included in the analysis.MVA85A was constructed as previously described  and prodPeripheral blood mononuclear cells (PBMC) were isolated from whole blood and interferon-gamma (IFN-γ) ELISpot assays were performed on freshly isolated PBMC from all subjects at 0, 28, 42, 56, 119, 126, 147 and 203 days post-vaccination (Groups A and C) and at 0, 14, 28, 56, 63, 84 and 140 days post-vaccination (Group B) as previously described  using th6 cells/well) were stimulated in 96-well round bottom cell culture plates with Ag85A, Ag85B, and TB10.4 peptide pools (1 μg/peptide/mL), peptides were pre-diluted in R10 medium containing CD107a-Alexa488 , GolgiStop and GolgiPlug . Dimethyl sulfoxide (Sigma) was used as a negative control and SEB (0.5 μg/mL) as the positive control. Cells were stimulated for 6–7 hours at 37°C and 5% CO2. After incubation, the cells were washed and stained with viability dye  before staining with fluorochrome-conjugated antibodies to surface markers CD4 , CD8 (Biolegend), CD14 and CD19 (BD), then permeabilized and stained for CD3 , IFN-γ, IL-2, CD154, TNF-α , IL-17A (Biolegend), and IL-22 (eBioscience). Cells were acquired on BD LSR flow cytometers (BD Biosciences). All sample analysis was performed with FlowJo software .Intracellular cytokine staining (ICS) was performed as previously described . PBMC weRecombinant Ag85A, Ag85B, and TB10.4, purified in-house at Aeras, were coated onto micro-well plates. The serum samples were diluted 1:100 and incubated with immobilized antigen for capture of antigen specific antibodies present in the serum samples. The captured antibodies were then probed by the addition of biotinylated anti-IgG antibodies and detected using a colorimetric substrate solution. Once the optimum color intensity was reached, the color development was stopped using a stop solution. The micro-well plates were read using an ELISA reader with SoftMax® Pro 5.4.1 data acquisition and analysis software.8 vp/mL) was added to each well. After incubation for neutralization, A549 cells were added at 1 x 104 cells/well, with a final ratio of 500 vp recombinant adenovirus/cell. After incubation at 37°C/10% CO2 for 24 to 26 hours, medium was discarded, PBS was added, and plates frozen at –35°C for a minimum of 12 hours. Plates were allowed to thaw at room temperature and Luciferase Steady-Lite Plus substrate was then added. After 15 minutes incubation, 50 μL of the lysate was transferred from each well into a B&W isoplate. Luminescence counts were recorded on a MicroBeta2. Data were imported into an Excel macro to calculate 90% inhibition titers. A response was defined as a titer >16, the lower limit of quantification for the serum neutralizing activity assay.The assay for Adenovirus 35 neutralizing activity was performed as previously described . BrieflySafety data were summarised by frequency and severity of adverse events using descriptive statistics. ELISpot data was analysed using GraphPad Prism. The Mann Whitney U-test and Area Under the Curve (AUC) analysis were used to detect differences between groups. The Wilcoxon matched-pairs signed rank test was used to detect differences between time points within the same group.The first volunteer was enrolled in October 2012 and the trial was completed in August 2014, within the expected 24 months trial duration.Fifty three subjects were assessed for eligibility of which 40 were enrolled (See The demographics of the enrolled subjects were comparable between the three groups .There were 908 adverse events (AEs) of which 708 (78%) were considered related to vaccination. Most AEs were mild or moderate .One unrelated Serious Adverse Event occurred in a subject 80 days post final vaccination in Group A. This subject was diagnosed with presumptive Dengue fever while travelling in an endemic region, hospitalised for 2 days for supportive treatment and made a full recovery.23 severe AEs were reported of which 12 were considered related to vaccination and occurred post AERAS-402 vaccination. Four subjects recorded transient severe pain (2 in Group A and 2 in Group C) and one subject recorded transient severe swelling at the vaccination site after AERAS-402.One subject reported feverishness and malaise on D1 post first AERAS-402 vaccination requiring one day absence from work; this subject also recorded a fever (39.3°C) on the day of vaccination which returned to 37.3°C within 6 hours. This same subject reported nausea and abdominal pain post the third AERAS-402 vaccination requiring one day absence from work, with resolution of symptoms by D2 post vaccination. Another subject reported severe headache on the evening of third AERAS-402 vaccination which resolved within 24 hours. These AEs was assigned possible causality due to temporal association with vaccination.21 laboratory abnormalities were detected of which 13  were considered related to AERAS-402 vaccination. There were no laboratory AEs related to MVA85A vaccination. One subject in Group C had a moderate increase in alanine transaminase and a severe increase in aspartate aminotransferase post second AERAS-402 vaccination. Both returned to baseline prior to third vaccination and were considered possibly related to vaccination due to a temporal association with vaccination. All related laboratory AEs were transient, asymptomatic and returned to baseline by the end of the trial. All related AEs (including laboratory AEs) are listed in vivo IFN-γ ELISpot responses to Ag85A, Ag85B and TB10.4 increased significantly after first vaccination with AERAS-402  analysis was performed on Groups A, B, C and subjects from a previous trial, TB022, who received MVA85A alone . Due to There was no difference between groups in their baseline responses to the adenovirus peptides. After first vaccination with AERAS-402, responses in all groups increased significantly  and Ag85B .AUC analysis for the expression of the CD4+ T-cell activation marker, CD154, was significantly higher in Group A than in Group C in both Ag85A- and Ag85B-stimulated cells .After MVA85A vaccination, CD4+ and CD8+ T cell responses to Ag85A were higher in Group B when compared with MVA85A alone , Group A had less Ag85A CD4+ polyfunctional positive T-cells than Group B. Groups A and B had more Ag85A-specific polyfunctional CD4+ T-cells than Group C. All three groups had less polyfunctional Ag85A-specific CD4+ T-cells than samples from TB022 .Polyfunctional CD8+ T-cells responses were only observed when MVA85A was preceded by AERAS-402 . Groups At plateau, , Group C had less polyfunctional Ag85A CD4+ T-cell response than all other groups . No polyAg85A-specific IgG responses increased significantly after vaccination with AERAS-402 and MVA85A . Group AAUC analyses for neutralising antibody responses show no significant differences between the three groups (data not shown).We report the first TB clinical vaccine trial to evaluate an adenoviral prime—MVA boost regimen. The safety profile of AERAS-402 and MVA85A were comparable to data reported previously , 16, 17.The vast majority of systemic AEs were mild or moderate in nature. There were 7 severe systemic AEs that were considered related to AERAS-402 vaccination, 4 of which were considered possibly related due to temporal association with vaccination. All of the vaccine related severe systemic AEs, apart from the transient rise in AST discussed below, resolved within 48 hours. The most objective measurement of systemic reactogenicity, pyrexia, was reported as severe on only one occasion throughout the study, a recorded fever of 39.3°C on the day of first AERAS-402 vaccination in a volunteer in Group C, which returned to normal within 6 hours. There was one severe laboratory AE, a transient elevation in AST post second AERAS-402 vaccination in Group C, which normalised prior to third vaccination. This AE was deemed possibly related to vaccination due to temporal association; the rise in AST may also have been attributable to excessive exercise prior to safety bloods measurement. There were no vaccination deferrals and there were no study withdrawals attributable to related AEs.vivo IFN-γ ELISpot responses showed that while the first AERAS-402 vaccination significantly increased antigen specific T-cell responses to Ag85A, Ag85B and TB10.4, homologous boosting at D28 and D119 did not increase these responses further. Boosting AERAS-402 with MVA85A induced strong and durable antigen specific T-cell responses to antigen 85A, with two priming doses of AERAS-402 resulting in a significantly higher AUC post-peak MVA85A vaccination than MVA85A alone. After one dose of AERAS-402, the AUC post-MVA85A response was not significantly higher than after MVA85A alone. Anti-vector adenoviral T cell responses were increased after AERAS-402 vaccination and remained above baseline for the duration of the trial. Anti-MVA CD8+ T cell responses were induced after vaccination with MVA85A, but returned to baseline by the end of the trial. Polyfunctional CD4+ and CD8+ T cells responses were comparable when MVA85A was preceded by one (Group B) or two (Group A) AERAS-402 vaccinations.Ex-Multiparameter flow cytometry showed a high frequency of cytokine producing CD8+ T-cells in Ag85A and Ag85B-stimulated PBMC after MVA85A boost in Groups A and B, with Group A showing a higher frequency post-boost. At peak response post-MVA85A, the frequency of polyfunctional CD4+ T-cells producing IFN-γ, TNF-α and IL-2, and expressing CD154, a marker of CD4+ T-cell activation, in response to stimulation with Ag85A, was highest in the MVA85A alone group, followed by Group B and then Group A. However, the frequency of CD8+ T-cells producing all 3 cytokines and upregulating the expression of CD107a, an indicator of CD8+ T-cell cytotoxicity, was highest in Group A, followed by Group B and barely detectable in the MVA85A alone group. The same trend is true for plateau responses, but with reduced frequencies.M.tb [Ag85A-specific IgG responses peaked four weeks after vaccination with MVA85A, but did not significantly differ between the groups receiving either one or two doses of AERAS-402 prime. However, responses were higher in both of the Adenovirus prime-MVA boost groups than either the MVA or Adenoviral alone groups. Although cell-mediated immunity is crucial in protection against TB, the role of antibody responses in protection against TB is of increasing interest; sera from TB contacts with high tuberculin-specific IgG titers has been shown to block proliferation of PBMC cultured with tuberculin . FurtherM.tb .We have shown that an AERAS-402 prime-MVA85A boost regimen increases durability of antigen specific T-cell responses, increases the frequency and polyfunctionality of CD8+ T-cells and increases antibody responses when compared with AERAS-402 or MVA85A vaccination alone. This improved cellular and humoral immunogenicity may be important in protection against TB. One of the challenges faced by the TB vaccine field is the low immunogenicity induced by candidate vaccines in the target population, compared to UK adults. This adenoviral prime-MVA boost strategy may increase the magnitude and durability of vaccine-induced responses and broaden the quality of the immune response in the target population. Further studies with adenoviral prime-MVA85A boost regimens are merited to optimise intervals between vaccinations, dose and route of immunisation, and also to evaluate this strategy in the target population in TB high burden countries.S1 CONSORT Checklist(DOC)Click here for additional data file.S1 Protocol(PDF)Click here for additional data file.


In addition, CD4+ Treg cells suppress the efficacy of CAR-T cell therapy, and different approaches to overcome this suppression are discussed in this review. Thus, next-generation CAR-T immunotherapy can be improved, based on our knowledge of T cell subsets functions, differentiation, proliferation, and signaling pathways to generate more active CAR-T cells against tumors.This review is focused on different subsets of T cells: CD4 and CD8, memory and effector functions, and their role in CAR-T therapy––a cellular adoptive immunotherapy with T cells expressing chimeric antigen receptor. The CAR-T cells recognize tumor antigens and induce cytotoxic activities against tumor cells. Recently, differences in T cell functions and the role of memory and effector T cells were shown to be important in CAR-T cell immunotherapy. The CD4 Cellular immunotherapy, such as CAR-T, a therapy with T cells expressing antibody-based chimeric antigen receptor targeting tumor antigen, is an effective therapy against different types of hematological malignancies and also against solid cancers ,2. CAR-TCAR combines a single chain variable fragment (scFv) of antibody that drives specificity against tumor antigens . The scFSCM) from a CD45RA+ T population with a high expression of CD62L, CD95, and CCR-7 were shown to be more persistent and more effective against tumors than T central memory cells . Th. Th+ T cTGF-beta . And eacTGF-beta .+ subsets. Th1 and Th2 CD4+ T cell subset cytokines were shown to drive different types of cytotoxicity generated by the second generation of CD28-containing CAR-T [Several reports demonstrated differential roles of different types of cytokines released by CD4ng CAR-T . Short-tng CAR-T . CAR-T cng CAR-T . IL-12 rng CAR-T . IL-12 rng CAR-T  suggesting CAR-T .+ cells from naive to effector or memory and central memory cells is shown in CM) or in the recently infected tissues––effector memory cells, T EM cells CD62L(+)) in the infused product [T cell differentiation and memory and effector T cells play a significant role in immunity against pathogenic agents . The difEM cells . During T regulatory cells differentiate into effector and memory cells. Naive conventional T cells and regulatory T cells (effector and memory subtypes) differ in their extracellular, intracellular, epigenetic, and genetic markers, transcription factors, and metabolic pathways (discussed below) .+ T cells are shown in SCM; T Central Memory cells, T CM; T effector memory cells, T EM; and T effector cells, T EFF. The different CD8+ markers upon cell differentiation—L-Selectin, CD45RO, CD45RA and CCR-7—are shown in + T cell differentiation, while memory function and proliferation are decreased , CD62L+ (L-Selectin+), CCR-7+  infection versus naïve T cells that are dependent on specific transcriptional profiles of the key effector genes Ifng, Gzmb, and Prf1 [The effector and memory T cell functions are regulated by genetic profiles of key effector genes, and also by epigenetic mechanisms such as chromatin state . For exaand Prf1 . The priand Prf1  The auth+ and CD8+ T cells based on their mitochondrial membrane demonstrated increased in vivo persistence and anti-tumor activity [+ and CD8+ T cells that depend on aerobic glycolysis with production of lactate from glucose-derived pyruvate, memory T cells have distinct metabolic pathways that depend on fatty acid oxidation [The naive and effector T cells differ in metabolic pathways. The quiescent naive T or memory cells have catabolic metabolism when nutrients are broken down to generate energy, while activated T cells have anabolic metabolism when nutrients are used to build molecular complexes and blocks to support cellular proliferation. During differentiation and activation, the metabolism is changed from oxidative phosphorylation (OXPHOS) to glycolysis . The maiactivity . In contxidation .+ cells were shown to be primarily glycolytic and expressed high levels of the glucose transporter Glut1 and active mTOR, while Treg cells were dependent on lipid metabolism and had a low level of Glut1 and a high level of AMP-activated protein kinase (AMPK) [Metabolic signaling varies depending on the state of T cell differentiation . Th1, The (AMPK) . Thus, tCAR-T therapy is very effective immunotherapy against hematological malignancies , althoug+ and CD4+ subsets expressed synergistic anti-tumor CAR-T activities [+ and CD4+ T cell subsets that were separately expanded in vitro and infused at a ratio of 1:1 [+ and CD8+ subsets in mouse experiments [+ T cells’ role in supporting and inducing CD8+ T cell memory functions [+ T CM subset or with second generation CD19-CAR-T using both CD8+ and CD4+ T CM subsets demonstrated the feasibility and safety of both approaches [+ and CD8+ T CM and second generation CAR-T cells demonstrated better persistence. Future clinical trials using CAR-T isolated from different T cell subsets will be important for understanding the detailed mechanisms of T cell functions in CAR-T immunotherapy.Several preclinical models demonstrated the advantage of different T cell subsets for effective CAR-T therapy: CD8(+)CD45RA(+)CCR7(+) CAR-T cells with closest to the T-memory stem cells phenotype cells produced greater anti-tumor activity of CAR-T cells ; both CDtivities . Recent o of 1:1 . The samo of 1:1 . These deriments , and CD4unctions . Anotherproaches , althougAnother aspect to consider is that the patient’s T cell profile is different from that of a healthy person and varies between the patients, suggesting that individualized T cell subset profiling and personalized immunotherapy are needed to effectively treat cancers. The development of next-generation sequencing, proteomics and metabolomics allow to create individualized immune profiles of patients and detect important T cell players that can improve CAR-T therapy .+ Treg cells infiltrated into solid tumors and decreased the efficacy of CAR-T therapy [+CD25+Foxp3+ Treg cells in melanoma cancer patients [+Foxp3+ cells had a negative impact on adoptive immunotherapy and immune responses [+ T cells on anti-tumor activity of CD28-CD3ζ-CAR-T cells demonstrated by other groups [in vivo [It has been shown that CD4 therapy . The aut therapy . The CD2 therapy . The admpatients . The levesponses  that is esponses . The resesponses  versus tr groups ,40,41, aAnother suppressing marker that is expressed in effector Treg cells is CTLA-4 (cytotoxic T-lymphocyte-associated protein 4), which is known to block immune response and decrease the efficacy of CAR-T in pre-clinical studies . To blocBefore starting CAR-T therapy, lymphodepletion in patients using fludarabine and/or cyclophosphamide decreases the number of circulating T cells and also Treg T cells . The lymEFF cells or suppress immunity and inflammation by promoting the lipid oxidation characteristic of Treg cell subsets [An alternative potential approach is to target T and CAR-T cell metabolism and thus increase effector cell functions, for example, by switching from catabolic to anabolic metabolism . In the  subsets . Recentl subsets . The 4-1 subsets . Thus, m+, CD28+,CD27+, CD127+, CCR-7+) and to up-regulation to stem cell markers such as Nanog, SOX-2, Oct-4, and LIN28A [+ T memory cell function and increase T cell anti-tumor activity [in vivo compared with CAR-T expanded with IL-2 [The modulation of cytokine cocktails can change the differentiation status of T cells . For exad LIN28A . Pre-tred LIN28A ,48. IL-1activity . CAR-T cith IL-2 . Culturiith IL-2 . Thus, mThis report shows the complexity of T cell differentiation, stem cell memory, memory and effector functions, their regulatory, intracellular, extracellular markers, cellular signaling, metabolism, cytokine-directed regulation of T cell differentiation and function that should be considered during cellular immunotherapy, including CAR-T therapy. Since CAR-T therapy includes withdrawal of T cells from cancer patients used for expansion, the ratio of CD4/CD8, and memory markers such as CD45RA, CD45RO and other markers such as CD26, CD95, CCR-7, and CD62L should be analyzed for a selection of different T cell subsets at different ratios to be studied in clinical trials for more effective and personalized therapy. The sorted CD4 or CD8 cell populations and T cell subsets with specific memory or stem cell memory markers and genetic, epigenetic, and metabolic profiles in combination with immune checkpoint inhibitors should be analyzed and used for regulating the efficacy of CAR-T therapy. The modulation of metabolic pathways of different T cell subsets with metabolic inhibitors will provide novel mechanisms of CAR-T immunotherapy. The genetic modifications of CAR constructs and the effects of the modification of inhibitory and stimulatory pathways on the efficacy on CAR-T cells can be studied further. Future studies with personalized T cell subsets patient profiles and the above discussed approaches will illuminate the key mechanisms of efficient CAR-T therapy against hematological malignancies and solid tumors.


Background: The dsrE3A-dsrE2B-tusA cluster from Metallosphaera cuprina encodes putative sulfur-trafficking proteins and is flanked by genes encoding a heterodisulfide reductase-like complex and dihydrolipoamide dehydrogenase.Results: DsrE3A and TusA react with tetrathionate, yielding protein Cys-S-thiosulfonates, triggering irreversible thiosulfate transfer from DsrE3A to TusA.Conclusion: DsrE3A and TusA are important players during dissimilatory sulfur and tetrathionate oxidation.Significance: Protein-mediated transfer of thiosulfonate is unprecedented in sulfur oxidizers. Escherichia coli, and DsrE is an active site subunit of the DsrEFH complex that is essential for sulfur trafficking in the phototrophic sulfur-oxidizing Allochromatium vinosum. In the acidothermophilic sulfur (S0)- and tetrathionate (S4O62−)-oxidizing Metallosphaera cuprina Ar-4, a dsrE3A-dsrE2B-tusA arrangement is situated immediately between genes encoding dihydrolipoamide dehydrogenase and a heterodisulfide reductase-like complex. In this study, the biochemical features and sulfur transferring abilities of the DsrE2B, DsrE3A, and TusA proteins were investigated. DsrE3A and TusA proved to react with tetrathionate but not with NaSH, glutathione persulfide, polysulfide, thiosulfate, or sulfite. The products were identified as protein-Cys-S-thiosulfonates. DsrE3A was also able to cleave the thiosulfate group from TusA-Cys18-S-thiosulfonate. DsrE2B did not react with any of the sulfur compounds tested. DsrE3A and TusA interacted physically with each other and formed a heterocomplex. The cysteine residue (Cys18) of TusA is crucial for this interaction. The single cysteine mutants DsrE3A-C93S and DsrE3A-C101S retained the ability to transfer the thiosulfonate group to TusA. TusA-C18S neither reacted with tetrathionate nor was it loaded with thiosulfate with DsrE3A-Cys-S-thiosulfonate as the donor. The transfer of thiosulfate, mediated by a DsrE-like protein and TusA, is unprecedented not only in M. cuprina but also in other sulfur-oxidizing prokaryotes. The results of this study provide new knowledge on oxidative microbial sulfur metabolism.Conserved clusters of genes encoding DsrE and TusA homologs occur in many archaeal and bacterial sulfur oxidizers. TusA has a well documented function as a sulfurtransferase in tRNA modification and molybdenum cofactor biosynthesis in  A-dsrE2) , 24. Not pathway , 46. Thospitalis , Sulfolotokodaii , Metalloa sedula , and M.  cuprina , as well cuprina , 30 and bacillus  and Thiolivibrio  Fig. 1)e.g. in Ay source . Additioon donor .M. cuprina /pLysRARE was used for overproduction of recombinant proteins. All E. coli strains were grown in Luria-Bertani medium at 37 °C. The antibiotics used were at 100 μg/ml for ampicillin and 25 μg/ml for chloramphenicol.Bacterial strains and plasmids used in this study are listed in  extract . Escheriof genes . E. coliPfu DNA polymerase using genomic DNA of M. cuprina Ar-4 as template. Primers are listed in The targeted genes were PCR-amplified with dsrE3A-C93S/C101S was constructed by introducing the C101S mutation to pET15b-dsrE3A-C93S. All of the genetic constructions were sequenced to exclude any PCR amplification errors.Replacements of cysteine residue by serine residue (site-directed mutations) were performed with gene splicing by overlap extension . PlasmidA molecular evolutionary analysis was performed using MEGA6 software . The phyE. coli BL21(DE3)/pLysRARE. Overnight precultures were used to inoculate fresh LB medium with a ratio of 1:60 (v/v). Synthesis of recombinant proteins in cells was induced by the addition of 0.1 mm IPTG when the culture reached an OD600 of 0.6–0.8 and was further incubated for 2.5 h at 37 °C before harvesting. Cells were pelleted at 3000 × g for 10 min, and were resuspended in buffer containing 50 mm Tris-HCl and 150 mm NaCl (pH 7.5). Cells were broken by sonification. Cellular lysate was centrifuged at 17,000 × g for 20 min at 4 °C. The supernatant was incubated in a water bath at 65 °C for 10 min, and the denatured proteins were removed by centrifugation at 17,000 × g for 20 min. The supernatant containing the recombinant protein was filtered with a 0.45-μm filter membrane  before being loaded to the gravity flow column for purification. His-tagged and Strep-tagged proteins were purified with TALON metal affinity resin (Clontech) and Strep-Tactin Superflow (IBA), respectively, according to protocols provided by the suppliers.Recombinant DsrE2B, DsrE3A, and TusA were produced in et al. . In a typical assay, each protein was incubated at 65 °C for 30 min with 5 mm substrate in buffer (pH 7.5) containing 50 mm Tris-HCl and 150 mm NaCl. In parallel, a reference test was run at identical conditions but without substrate. Excessive substrate was removed by using PD MiniTrap G-25 columns. Six substrates were tested in this study: sodium hydrosulfide (NaSH), glutathione persulfide (GSSH), polysulfide (Sn2−), thiosulfate (S2O32−), tetrathionate (S4O62−), and sulfite (SO32−). GSSH was synthesized according to the method of Rohwerder and Sand .The principle of 1,5-I-AEDANS gel assays has been described by Zheng et al. . Proteinand Sand  as speci  was incubated with equal amounts of thiosulfate acceptor proteins (TusA or DsrE3A) at 65 °C for 30 min. Thiosulfate transfer was evaluated by determination of cysteine modification, and visualization of cysteine modification was carried out as described above.For MALDI-TOF MS, the matrix was sinapinic acid in 50% acetonitrile and 0.1% trifluoroacetic acid solution. The buffer of protein samples was exchanged for 0.1% trifluoroacetic acid by using a PD MiniTrap G-25 column. About 10-pmol samples were detected in the positive linear mode with a Biflex III  or AB SCIEX TOF/TOF 5800 .During the Strep-tag pulldown assay, 10 nmol of Strep-tagged proteins and 30 nmol of non-Strep-tagged proteins were incubated together with 0.75 ml of Strep-Tactin Superflow on ice for 1 h. The mixture was then loaded to the gravity flow column to continue the pulldown assay. Reference tests were run in parallel under identical conditions, but only the non-Strep-tagged protein was incubated with Strep-Tactin Superflow.m acetic acid (pH 5.5) were covalently immobilized to the chip according to the protocol provided by the supplier, and the resonance units reached about 1500. PBST buffer (PBS containing 0.005% Tween 20 (pH 7.4)) was used as the running buffer with a flow rate of 30 μl/min. TusA proteins  in PBST buffer was injected for 2 min at a flow rate of 30 μl/min. Dissociation of protein-protein complexes on the sensor surface proceeded for 8 min by flowing PBST buffer over the sensor surface. A blank injection with PBST buffer was used as the control. The surface was regenerated with 5–60 μl of 10 or 20 mm NaOH. A control surface without ligand was used as a reference.A Biacore 3000 instrument  was equipped with a CM5 sensor chip  at 25 °C. DsrE3A (26 μg/ml) proteins (ligand) in 10 mTM purifier equipped with a HiLoad 16/60 Superdex 200 prep grade column . A solution of 50 mm Tris-HCl (pH 7.5) and 150 mm NaCl was used as the running buffer, and the flow rate was kept constant at 1.0 ml/min. Proteins were incubated with or without 25 μm Tris(2-carboxyethyl)phosphine (TCEP) at room temperature for 1 h before injection to the AKTATM purifier with a 0.5-ml sample loop for oligomerization analysis. The column used in these cases was a HiLoad 16/60 Superdex 75 prep grade column . 50 mm Tris-HCl (pH 7.5) and 150 mm KCl was used as the running buffer with a flow rate 0.5 ml/min. Blue dextran (2000 kDa), bovine catalase (240 kDa), bovine albumin (67 kDa), egg albumin (45 kDa), chymotrypsinogen A (25 kDa), equine myoglobin (17 kDa), and cytochrome c (12.3 kDa) were used for calibration.DsrE3A and TusA were incubated on ice for at least 1 h before being injected with a 2-ml sample loop to AKTAM. cuprina encodes four different DsrE-like proteins: Mcup_0681, Mcup_0682, Mcup_1706, and Mcup_1724. All of these proteins contain cysteine residues corresponding to the active cysteine residue of DsrE from A. vinosum (A), which had been demonstrated experimentally  and a gene for a protein with a sulfate transporter domain, as well as the gene for a potential sulfide:quinone oxidoreductase (Mcup_1723) and TusA (Mcup_1722). Mcup_0682 and a further tusA homolog (Mcup_0683) immediately precede the hdrC1B1AhyphdrC2B2 cluster (B). Mcup_0681 is transcribed in the opposite direction to Mcup_0682 and resides upstream of a genetic cluster that encodes a putative dihydrolipoamide dehydrogenase (Mcup_0680), a hypothetical protein (Mcup_0679), and a putative thioredoxin (Mcup_0678). Notably, a lipoamide-binding protein resembling protein H of the glycine cleavage system (Mcup_0662) and several proteins responsible for the biosynthesis of lipoamide-containing proteins (Mcup_0671–0673) are also encoded in the vicinity of these genes (B). In fact, related genes are also part of or reside in the immediate vicinity of all the tusA-dsrE-hdr genetic clusters in other genome-sequenced archaeal and bacterial sulfur oxidizers. As an example, the organization of the respective genes in Acidithiobacillus caldus is compared with that in M. cuprina in Fig. 2B.The genome of  vinosum A, which mentally . With th cluster B. Mcup_0se genes B. In facC). 1) The DsrE group consists of subunits of DsrEFH (prototype Alvin_1253). 2) The DsrE2 group consists of members that are genetically or functionally linked with Dsr or Hdr systems. Subgroup DsrE2A (prototype Alvin_2601) contains two strictly conserved cysteines. The prototype of subgroup DsrE2B is Mcup_0682. 3) The genes of the DsrE3 group are either immediately linked with genes for dihydrolipoamide dehydrogenase  or with genes for lipoamide-binding proteins . The proteins of group DsrE3A contain two conserved cysteine residues in a Cys-X7-Cys motif with the first cysteine corresponding to the established DsrE active site cysteine (A). 4) The members of the DsrE4 group are encoded downstream of sulfide:quinone oxidoreductase (prototype Mcup_1724). 5) Group DsrE5 is represented by Mcup_1706.Sequence alignments and phylogenetic analyses provided us with the basis for grouping DsrE homologs into the following five categories C. 1) Thecysteine A. 4) Thei.e. dsrE-tusA) were observed in sulfur-oxidizing species of the families Sulfolobaceae, Aquificaceae, Acidithiobacillaceae, Chromatiaceae, and Chlorobiaceae (dsrE gene might have been duplicated (as dsrE3A and dsrE2B) in members of the Sulfolobaceae and Aquificaceae. Genes encoding proteins or enzymes that are involved in reversible reduction of heterodisulfide bonds coupled with energy conservation (Hdr complex) or in sulfur oxidation (rhodanese (Rdh)-like protein) were found in conjunction with the dsrE-tusA cluster as shown in M. cuprina Ar-4 had 24% (89% coverage) and 41% (47% coverage) identity to the DsrE2A (AFE_2556) of A. ferrooxidans and DsrE2A (Alvin_2601) of A. vinosum, respectively. DsrE2B of M. cuprina Ar-4 had 34% (100% coverage) and 35% (86% coverage) identity to AFE_2556 and Alvin_2601, respectively. TusA (Mcup_0683) of M. cuprina Ar-4 has 39% (88% coverage) and 34% (91% coverage) identity to TusA (AFE_2557) of A. ferrooxidans and TusA (Alvin_2600) of A. vinosum, respectively.Similar genetic organizations (obiaceae . It appeE. coli BL21(DE3)/pLysRARE and purified by affinity chromatography. In all cases, the apparent molecular masses matched the theoretical masses for DsrE3A, DsrE2B, and TusA (His-tagged) at 17,697, 17,480, and 10,895 Da, respectively, with TusA Strep-tagged at 9,930 Da. In addition, six mutants  were produced, of which potentially important cysteine residues were replaced by serine residues.N-terminally His-tagged DsrE3A (Mcup_0681), DsrE2B (Mcup_0682), and TusA (Mcup_0683) as well as TusA carrying a carboxyl-terminal Strep-tag were produced in 99S were homodimers or homotrimers (data not shown). The oligomerization of DsrE3A and DsrE2B as trimers matches the structures deposited in the Protein Data Bank (PDB) for the corresponding proteins from Sulfolobus solfataricus P2 . TusA of M. cuprina occurred both as monomers and dimers. The mutant protein TusA-C18S occurred only in the monomeric state, suggesting that dimer formation depended on the presence of the Cys18 residue of wild-type TusA (data not shown).The purified DsrE3A molecules as well as all derivatives occurred in homotrimers as indicated by gel filtration. It was not clear whether DsrE2B and DsrE2B-Ci.e. persulfurated proteins would finally not be visible under UV light). The covalently attached protein-thiosulfonate groups are not reactive per se with 1,5-I-AEDANS. As evident from in vitro. DsrE3A and TusA were not modified by NaSH, GSSH, polysulfide, thiosulfate, or sulfite, but they reacted with tetrathionate. As shown in 18-S-thiosulfate to hydrolysis. Control experiments showed that the tetrathionate-treated proteins were a priori unable to react with 1,5-I-AEDANS, pointing to a modification of cysteines with sulfonate or S-thiosulfonate rather than sulfane groups. This conclusion was verified by an independent experimental approach. Mass changes arising after incubation with the different tested sulfur compounds were analyzed by MALDI-TOF mass spectrometry (A and B). The protein harbors two conserved cysteine residues, Cys93 and Cys101. Mutants DsrE3A-C93S (DsrE3A-Cys101) and DsrE3A-C101S (DsrE3A-Cys93) retained the ability to interact with tetrathionate, and each mutant protein covalently attached a group of 112 ± 4 Da, matching the molecular mass of thiosulfate , DsrE2B (Mcup_0682), and TusA (Mcup_0683) were incubated separately with six different sulfur-containing compounds: NaSH, GSSH, polysulfide, thiosulfate, tetrathionate, and sulfite. The untreated proteins and the proteins treated with sulfur-containing compounds were reacted with the fluorescent thiol-reactive reagent 1,5-I-AEDANS. Subsequent incubation with DTT was performed to cleave off possible covalently attached persulfides as 1,5-AEDANS-sulfide conjugates . A mass increase of 111 Da suggested that TusA also covalently attached a thiosulfate group. Because the mutant protein TusA-C18S lost the ability to react with the tetrathionate, it is deduced that the conserved cysteine residue of TusA played a key role in the reaction with tetrathionate . MALDI-TOF MS confirmed thiosulfate transfer from DsrE3A-Cys-S-thiosulfonate to TusA (B). Not only were the peaks representing TusA and DsrE3A-Cys-S-thiosulfonate observed, but a newly emergent peak matched the mass of TusA-Cys18-S-thiosulfonate, which was deduced to be a product resulting from the transfer of a thiosulfate group from DsrE3A-Cys-S-thiosulfonate to TusA.DsrE3A-Cys-ccessful A. MALDI- to TusA B. Not oni.e. DsrE3A-C93S or DsrE3A-C101S, retained its ability to transfer thiosulfate to TusA.Mutants of DsrE3A were also assayed. The results  showed t18-S-thiosulfonate was able to transfer its thiosulfate group to DsrE3A. The thiosulfate group of TusA-Cys18-S-thiosulfonate did not transfer to DsrE3A, but no trace of it was detected in the MALDI mass spectrum after incubation (A). Thus, we deduced that DsrE3A cleaved the TusA-Cys18-S-thiosulfonate and released free TusA. Additional experiments (B) showed that the double replacement mutant DsrE3A-C93S/C101S lost the ability to cleave the thiosulfate group from TusA-Cys18-S-thiosulfonate, suggesting that cysteine residues of DsrE3A played an important role during cleavage.We further determined whether TusA-Cyscubation A. Thus, eriments B showed S-thiosulfonate and TusA-Cys18-S-thiosulfonate were able to transfer their thiosulfate groups to DsrE2B. Thiosulfate transfer to DsrE2B was not observed (data not shown).We also tested whether DsrE3A-Cys-S-thiosulfonate and TusA-Cys18-S-thiosulfonate. In addition, DsrE3A cleaved TusA-Cys18-S-thiosulfonate. These results invoked the idea that DsrE3A and TusA might interact physically with each other. Pulldown assays were conducted for DsrE3A plus Strep-tagged TusA with Strep-Tactin Superflow gravity flow columns. In a control experiment, DsrE3A did not bind to the affinity matrix, whereas a considerable portion was retained on the column in the presence of Strep-tagged TusA and co-eluted with it (data not shown). DsrE3A did not interact with TusA-C18S, in which the active site cysteine was replaced by serine, showing that Cys18 of TusA is indispensable for the interaction. Analysis by gel permeation chromatography showed that DsrE3A and TusA formed a heterocomplex after they were incubated together  according to the fitted model was 0.1 nm. The very low value of DK demonstrated a strong interaction between DsrE3A and TusA.Moreover, surface plasmon resonance was applied to quantitatively evaluate the affinity between DsrE3A and TusA . DsrE3A M. cuprina Ar-4 have the ability to mobilize thiosulfate from tetrathionate. Moreover, thiosulfate transfer from DsrE3A-Cys-S-thiosulfonate to TusA was shown. To our knowledge, proteins that react with tetrathionate and form protein-Cys-S-thiosulfonates have thus far not been reported in sulfur oxidizers. Thus, DsrE3A and TusA from M. cuprina Ar-4 represent the first pair of proteins with such novel properties. Although both DsrE3A and TusA were able to react with tetrathionate, we observed that DsrE3A-Cys-S-thiosulfonate further transferred its thiosulfate group to TusA. This observation might imply that DsrE3A functions as a thiosulfate donor to TusA in vivo. Thiosulfonated TusA could then serve as the substrate for other enzymes such as the hdrC1B1AhyphdrC2B2-encoded proteins (This work demonstrated that proteins DsrE3A and TusA from the acidothermophilic archaeon proteins . We envihdr-like genes and the DsrE3A-encoding gene (Mcup_0681) with a lipoamide dehydrogenase-encoding gene, not only in M. cuprina but also in other sulfur-oxidizing archaea and bacteria (2B), opens the possibility of a functional linkage of these systems. This idea is corroborated by the tight interaction of archaeal TusA and DsrE3A proven in this work by several independent experimental approaches. Involvement of a lipoamide-binding protein as a potential sulfur carrier and linkage to lipoamide dehydrogenase could even result in transfer of some of the electrons arising from sulfane sulfur oxidation to NAD+.The close genomic linkage of the TusA-encoding gene (Mcup_0683) with bacteria  and 2B, 18-S-thiosulfonate. This reaction requires two electrons. In principle, such a mechanism resembles the reverse of the oxidative binding of thiosulfate to a cysteine of the SoxYZ protein, which occurs as the first step of thiosulfate oxidation catalyzed by the Sox system (93S and DsrE3A-C101S retained the ability to release thiosulfate from TusA-Cys18-S-thiosulfonate.In addition to the reaction with tetrathionate that leads to binding of a thiosulfonate group, we showed that DsrE3A is able to release thiosulfate from TusA-Cysx system . We envi18-S-thiosulfonate, we propose that TusA is involved in dissimilatory oxidation of tetrathionate in M. cuprina Ar-4 when grown with tetrathionate as energy source, a role distinct from the function of TusA as a sulfurtransferase in tRNA modification (E. coli. TusA of M. cuprina Ar-4 functions as a dissimilatory protein, just as has been reported for TusA from the purple sulfur bacterium A. vinosum (Our results demonstrated that TusA is involved in thiosulfate transfer. Based on the finding that TusA reacts with tetrathionate and that DsrE3A cleaves a thiosulfate group from TusA-Cysfication  and molyfication  in E. co vinosum .M. cuprina Ar-4 (A. ambivalens has been suggested to face toward the cytoplasm (As an alternative function, or even in addition to the model elaborated above, archaeal TusA could generate thiosulfate in the cytoplasm, which would be further oxidized by TQO . TQO oxiina Ar-4 , and theytoplasm . Tetrathytoplasm .


However, there are no studies on the effect of 2-Cl-IB-MECA and the molecular mechanism of action of A3AR in murine colitis models in vivo. Is it the same as that observed in vitro? The interaction between 2-CL-IB-MECA and A3AR in a murine colitis model and the signaling pathways associated with this interaction remain unclear. Here we demonstrate a role for the NF-κB signaling pathway and its effect on modifying the activity of proinflammatory factors in A3AR-mediated biological processes. Our results demonstrated that A3AR activation possessed marked effects on experimental colitis through the NF-κB signaling pathway.The role of the adenosine A3 receptor (A3AR) in experimental colitis is controversial. The A3AR agonist N Inflammatory bowel disease (IBD), a chronic and relapsing inflammation of the gastrointestinal tract, affects millions of people worldwide. The chronic mucosal inflammation in IBD is caused by hyperactivation of effector immune cells, which produce high levels of pro-inflammatory cytokines like tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1β), and interferon-gamma (IFN-γ), thereby resulting in colonic tissue damage. Nuclear factor-kappa B (NF-κB) was identified as one of the key regulators in this immunological setting12Dextran sulfate sodium (DSS) is commonly used in rodent models to chemically induce intestinal inflammation46Adenosine, a purine nucleoside, is released from metabolically active cells into extracellular space and plays an important role in various pathophysiological processes. Adenosine regulates many biological responses, including inflammation, by interaction with its receptors such as A1, A2A, A2B, and A38101112Adenosine A3 receptor (A3AR) belongs to the Gi/Gq-coupled receptor family. Stimulation of A3AR inhibits adenylate cyclase activity, activates phospholipase C, and triggers calcium mobilization, leading to modulation of signaling pathways such as WNT, PI3K/AKT, and NF-κB146-(3-iodobenzyl)adenosine-5'-N-methyluronamide(IB-MECA) was shown to ameliorate inflammation in two different mouse models of colitis, including DSS-induced colitis and spontaneous colitis found in IL-10-deficient miceInflammation can drive A3AR expression and induce up-regulation in peripheral blood mononuclear cells in rheumatoid arthritis and Crohn's diseaseThe effects of IB-MECA occur at high doses ranging from 1–3 mg/Kg body weight and may not necessarily be restricted to actions at A3AR as both high-affinity  and low-affinity (A2B and A3AR) receptors may be activated by oral A3AR drugs10111718196-(3-iodobenzyl)adenosine-5'-N-methylcarboxamide (2-Cl-IB-MECA) has been shown to be a potent A3AR agonist with a 2,500- and 1,400-fold selectivity for A3AR versus A1AR and A2AR, respectively21A synthetic, highly-selective agonist for A3AR was introduced for the treatment of inflammation. In vitro, 2-chloro-NThus, this study utilized the DSS colitis mouse model to investigate further the role of the selective A3AR agonist 2-Cl-IB-MECA in the development of murine colitis in vivo and the potential mechanism underlying its effects. We hypothesize that the A3AR agonist 2-Cl-IB-MECA attenuates the development of DSS colitis associated with the NF-κB signaling pathway.Oral administration of 2-Cl-IB-MECA significantly reduced clinical activity of DSS-induced murine colitis. No significant difference was observed between vehicle-treated mice with DSS colitis and mice with DSS colitis alone .Clinical activity was evaluated using the parameters of weight loss, stool consistency, and the guaiac fecal occult blood test. 2-Cl-IB-MECA reduced weight loss, diarrhea, and fecal occult blood . After DWe next evaluated the severity of acute murine colitis by blinded histological injury scoring in the distal colon. The histological examination of distal colons from DSS-induced colitis mice revealed inflammatory lesions that included ulceration, crypt damage, and infiltration of inflammatory cells consisting of macrophages, lymphocytes, and neutrophils.In contrast, the administration of 2-Cl-IB-MECA markedly reduced the mucosal damage. Representative examples of H&E histopathology for 2-Cl-IB-MECA treated mice and vehicle treated mice induced with DSS are shown in P < 0.01). No significant difference was observed between vehicle-treated mice with DSS colitis and mice with DSS colitis alone. However, 2-Cl-IB-MECA treatment significantly inhibited TNF-a and IL-1β expression (P < 0.05).We investigated whether 2-Cl-IB-MECA administration alters the production of pro-inflammatory cytokines, including TNF-a and IL-1β, in colon epithelia of DSS colitis mice. As shown in Given that the previous studies showed that 2-Cl-IB-MECA could exert its anti-inflammatory effects by blocking NF-κB activation in some inflammatory diseases in vitro and in vivo, we investigated this signaling pathway in the DSS-colitis murine model to confirm its role in vivo.To determine the involvement of NF-κB signaling pathway activity in the in vivo model of colitis, immunofluorescence was performed on colon sections of mice to verify the presence and distribution of IκB-α, p-IκB-α and NF-κB p65 in epithelia. The micrographs are shown in P < 0.01). No significant difference was observed between vehicle-treated mice with DSS colitis and mice with DSS colitis alone. Treatment with 2-Cl-IB-MECA suppressed the phosphorylation of IκB-α  and attenuated NF-κB p65 nuclear translocation , which was consistent with our IF results.To determine whether an A3AR agonist modulates DSS-induced NF-κB activation, we quantitatively estimated the expression levels of IκB-α, p-IκB-α and NF-κB p65 in murine colon epithelia by western blot analysis . As expeTo verify the presence and distribution of A3AR in colon tissue, IF was performed and the micrographs are shown in P > 0.05). These data may suggest that A3AR was down-regulated after induction of DSS colitis, and 2-Cl-IB-MECA treatment did not alter the expression of A3AR in colon epithelia, consistent with our IF results.The effects of DSS and 2-Cl-IB-MECA on the expression of A3AR were determined . ProteinRecent studies have demonstrated an important role for 2-Cl-IB-MECA in acting on A3AR with a variety of mechanisms in numerous diseases. For example, it can regulate the MEK1/2-ERK1/2 and PI3K/AKT signaling pathways in ameliorating myocardial ischemia/reperfusion injuryIn this study, we identified A3AR expressed on colonic epithelia and demonstrated the significant reduction in mRNA and protein expression in colonic epithelia after DSS-induced colitis, which was consistent with our previous study indicating that the expression of A3AR in crypt epithelial cells was reduced significantly in DSS colitisPharmacological studies in both in vitro and in vivo models have demonstrated that activation of A3AR suppresses pro-inflammatory cytokine production through the inhibition of NF-κB activation during inflammation142728In agreement with the anti-inflammatory role of A3AR, our studies found that 2-Cl-IB-MECA attenuated DSS colitis-induced NF-κB activation in murine colonic epithelia. Several lines of evidence suggest that NF-κB is a key modulator governing the molecular network leading to various cellular functions associated with IBD131In addition, 2-Cl-IB-MECA mediated a reduction of the expression of pro-inflammatory cytokines TNF-a and IL-1β. Upon induction of DSS-colitis, NF-κB was activated and translocated to the nucleus of colonic epithelial cells33In the present study, 2-Cl-IB-MECA significantly inhibited the NF-κB pathway in the colonic epithelia of DSS colitis mice. Inhibition of both NF-κB activation and IκBa phosphorylation was associated with the suppression of pro-inflammatory cytokines expression in colonic epithelia of DSS colitis mice. 2-Cl-IB-MECA also attenuated acute murine colitis induced by DSS administration. To the best of our knowledge, this is the first mechanistic determination of the anti-inflammatory effect of 2-Cl-IB-MECA in a murine DSS colitis model. Further studies are required to understand whether other molecular mechanisms are involved in the effects of 2-Cl-IB-MECA in the development of DSS colitis. This will help to clarify the anti-inflammatory mechanisms of A3AR activation, which were consistent with those described in previous studies16On the contrary, Ren T et al., by using female mice (3–4 months or 8–10 months of age) in a two-week DSS-induced colitis model, found that A3−/−AR protects against DSS-colitis. This was consistent with a novel hypothesis that A3AR activation contributes to the development of colitisIn conclusion, our findings indicate that A3AR is expressed in colonic epithelia and its selective activation has an anti-inflammatory activity through the inhibition of pro-inflammatory cytokine expression associated with the inhibition of NF-κB signaling pathways in murine DSS colitis in vivo. Therefore, the protective effects of 2-Cl-IB-MECA on murine colitis support its potential usefulness for the treatment in patients with IBD.Thirty specific pathogen-free mice  were breed from the Experimental Animal Center of Guangdong Medical College, Guangdong, China. All animal experiments were approved by the Animal Care Committee of Guangdong Medical College and performed in accordance with relevant guidelines and regulations. Mice were group-housed (a maximum of four mice/cage) under a controlled temperature (25°C) and a 12:12-h light-dark cycle and were allowed free access to standard mouse chow and water. DSS-water consumption, food intake, and body weight of the animals were monitored and recorded on a daily basis. Colitis in mice was induced by the administration of 5% DSS  in drinking water for 7 days. The drinking water was then replaced with normal saline solution for another 7 days. The selective A3AR agonist 2-Cl-IB-MECA (2-Cl-IB-MECA group) (1.0 mg/kg/day in 0.2 mL)  or 37.5% dimethyl sulfoxide (DMSO) (vehicle group) was administered to the mice intragastrically by a feeding needle once a day, starting at day 1 from colitis induction. The DSS group received a gavage of normal saline solution instead of the drug, while the control group was administered sterile drinking water for 14 days and received a gavage of sterile water instead of the drug.The mice were divided into four groups. In the first group, designated the normal control group (n = 6), colitis was not induced and no therapeutic intervention was performed. The second group, designated the DSS group (n = 8), received 5% DSS and normal saline solution orally. The third group, designated the vehicle group (n = 8), received 5% DSS and DMSO. The fourth group, designated the 2-Cl-IB-MECA group (n = 8), received 5% DSS and 2-Cl-IB-MECA (1.0 mg/kg/day) dissolved in DMSO. Typically, the experiments were carried out for 2 weeks, and on the 14th day after the induction of colitis. All experiments were performed under gaseous anesthesia and animals were euthanized by overdosage of anesthesia followed by cervical dislocation. The colon was removed and processed for histopathology or biochemical studies.The guaiac test  was used to detect fecal occult blood in the gastrointestinal tract, which is indicative of gastrointestinal bleeding associated with severe intestinal inflammation and tissue damage in the DSS colitis model. Fecal pellets from each mouse were tested for a positive guaiac test every day during the 2-week study, and data were analyzed in terms of the number of animals with a positive test result on each day.The distal colon was removed and then fixed overnight in 4% (w/v) paraformaldehyde in PBS. Paraffin sections  were processed for histopathological scoring. The histological score of haematoxylin and eosin (H&E)-stained sections of the colon was determined by a pathologist in a blinded fashion, according to a previously described methodHistopathological scoring was performed separately for inflammation, crypt damage, and ulcerationColonic MPO activity was used as an index of neutrophil infiltration into the injured/inflamed mucosa. Briefly, MPO activity was measured with an MPO enzyme-linked immunosorbent assay (ELISA) kit . The colonic samples  were thawed and homogenized on ice in 0.5 mL PBS buffer. The subsequent assay was performed according to the manufacturer's instructions. All experiments were performed in triplicate. Concentrations of MPO in the colon were expressed as ng/mL.IF staining was performed according to standard protocols by using 5-μm thick transverse-cut paraffin sections from colons of control or DSS colitis mice. Briefly, paraffin sections were rendered permeable by incubation in 0.1% Triton X-100 for 30 min; the sections were then blocked with 10% donkey serum albumin at room temperature (RT) for 30 min and incubated with primary antibodies  against A3AR (1:30 dilution), NF-κB p65 (1:50 dilution), nuclear factor-kappaB inhibitor alpha(IκB-α) (1:50 dilution), or phosphorylated-IκB-α (p-IκB-α) (1:50 dilution) overnight at 4°C. After rinsing three times with phosphate buffered saline (PBS), sections were incubated with secondary antibodies  for 1 h at room temperature in dark, then washed three times with PBS. Negative controls were prepared by omitting the primary antibodies. Finally, coverslips were mounted on slides by using fluorescent mounting medium with DAPI to counter-stain the nuclei. The staining was evaluated on a Leica inverted fluorescence microscope.The isolation of colonic epithelial cells was performed on the basis of previously described proceduresEqual amounts of protein (20–30 μg) were separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride membrane . The membrane was blocked for 1 h with 5% fat-free dried milk at RT, then incubated with primary antibodies (Santa Cruz Biotechnology) against A3AR (1:800 dilution), NF-κB p65 (1:600 dilution), IκB-α (1:400 dilution), p-IκB-α (1:400 dilution), Tubulin , or β-actin  at 4°C overnight. Membranes were washed three times with Tris-buffered saline with Tween-20 (TBS-T) and incubated with the corresponding secondary antibodies  for 1 h at room temperature. Signals were detected with an electrochemiluminescence (ECL) detection reagent . The images were obtained on Kodak film and quantified by Quantity One software .−ΔΔCT method. Data were expressed as the fold change in mRNA transcript levels relative to that of the normal control.For qRT-PCR experiments, total RNA from colon epithelia was extracted using RNAiso Plus by following the manufacturer's instructions . Total RNA (500 ng) was converted to cDNA, and PCR reactions were performed in triplicate using a SYBR Green PCR LightCycler®  in a 96-well format over 45 cycles with denaturation at 95°C for 10 s and annealing at 58°C for 20 s. The primers for mouse β-actin, A3AR, TNF-a, and IL-1β were purchased from Sangon Biotech . The sequences of these primers were listed in P < 0.05.Data are expressed as mean ± standard deviation (SD). Multiple groups were compared by a one-way analysis of variance (ANOVA) and post-hoc tests between individual groups. SPSS 16.0 software (SPSS Inc. USA) was used for analyses. Histopathology was analyzed by a nonparametric Mann–Whitney U-test. The chi-square test was used to analyze data from the guaiac test for the presence of occult blood in the stools or stool consistency (diarrhea). Differences were considered statistically significant at Y.Z. conceived the study. T.H.R., X.F. and Y.Z. designed the experiments. T.H.R., T.T., X.F., S.C.Y., H.W., W.Y.W., Y.M.Q., C.Y.Y., Y.T.H., J.C.Z., J.W.C. and Y.Z. performed the experiments. T.H.R., T.T. and X.F. analyzed the data. T.H.R., T.T., X.F. and Y.Z. wrote the paper.