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Dynamics of antibiotic resistance genes co-occurrence with pathogenic and non-pathogenic bacteria throughout wastewater treatment processes.

Wastewater treatment plants (WWTPs) are recognized hotspots for antibiotic resistance genes (ARGs) and pathogenic bacteria. Despite advancements in treatment technologies, the persistence of ARGs and pathogenic bacteria remains a concern. In this study, we analyzed the dynamic changes in ARGs and bacterial communities throughout the treatment processes within an anaerobic-anoxic-oxic (AAO) WWTP over one week by using HT-qPCR coupled with 16S rRNA gene amplicon sequencing. The connectedness index, based on network analysis, showed that the dynamics of ARGs and mobile genetic elements (MGEs) were more strongly associated with potentially pathogenic bacteria than with non-pathogenic bacteria, suggesting that ARG immigration and dissemination in the WWTP were likely driven by potentially pathogenic taxa. The AAO treatment significantly reduced ARGs in final effluent (EF) (∼64 %) and residual sludge (RS) (∼81 %); however, potential hosts of ARGs such as Comamonas testosteroni and Clostridioides difficile persisted with minimal changes in relative abundance and remained detectable in EF and RS. Notably, the abundance of ARGs was lower in RS than in EF, and source tracking analysis identified influent as the primary source of ARGs and potentially pathogenic taxa in EF, underscoring the greater health risks associated with effluent discharge.

Wastewater↗

Impaired T-cell differentiation in the thymus at the early stages of acute pathogenic chimeric simian-human immunodeficiency virus (SHIV) infection in contrast to less pathogenic SHIV infection.

One of the mechanisms by which HIV infection induces the depletion of CD4+ T cells has been suggested to be impairment of T-cell development in the thymus, although there is no direct evidence that this occurs. To examine this possibility, we compared T-cell maturation in the intrathymic progenitors between macaques infected with an acute pathogenic chimeric simian-human immunodeficiency virus (SHIV), which causes profound and irreversible CD4+ T-cell depletion, and macaques infected with a less pathogenic SHIV, which causes only a transient CD4+ T-cell decline. Within 27 days post-inoculation (dpi), the two virus infections caused similar increases in plasma viral loads and similar decreases in CD4+ T-cell counts. However, in the thymus, the acute pathogenic SHIV resulted in increased thymic involution, atrophy and the depletion of immature T cells including CD4(+)CD8(+) double-positive (DP) cells, whereas the less pathogenic SHIV did not have these effects. Ex vivo differentiation of CD3(-)CD4(-)CD8(-) triple-negative (TN) intrathymic progenitors to DP cells was assessed by a monkey-mouse xenogenic fetal thymus organ culture system. Differentiation was impaired in the TN intrathymic progenitors of the acute pathogenic SHIV-infected monkeys, while differentiation was not impaired in the TN intrathymic progenitors of the less pathogenic SHIV-infected monkeys. These differences suggest that dysfunction of thymic maturation makes an important contribution to the irreversible depletion of circulating CD4+ T cells in vivo.

Animals↗

Host plant-dependent phenotypic reversion of Ralstonia solanacearum from non-pathogenic to pathogenic forms via alterations in the phcA gene.

Ralstonia solanacearum is a plant pathogenic bacterium that undergoes a spontaneous phenotypic conversion (PC) from a wild-type pathogenic to a non-pathogenic form. PC is often associated with mutations in phcA, which is a key virulence regulatory gene. Until now, reversion to the wild-type pathogenic form has not been observed for PC variants and the biological significance of PC has been questioned. In this study, we characterized various alterations in phcA (eight IS element insertions, three tandem duplications, seven deletions and a base substitution) in 19 PC mutants from the model strain GMI1000. In five of these variants, reversion to the pathogenic form was observed in planta, while no reversion was ever noticed in vitro whatever culture media used. However, reversion was observed for a 64 bp tandem duplication in vitro in the presence of tomato root exudate. This is the first report showing a complete cycle of phenotypic conversion/reversion in a plant pathogenic bacterium.

Bacterial Proteins↗

Presence/absence polymorphism for alternative pathogenicity islands in Pseudomonas viridiflava, a pathogen of Arabidopsis.

The contribution of arms race dynamics to plant-pathogen coevolution has been called into question by the presence of balanced polymorphisms in resistance genes of Arabidopsis thaliana, but less is known about the pathogen side of the interaction. Here we investigate structural polymorphism in pathogenicity islands (PAIs) in Pseudomonas viridiflava, a prevalent bacterial pathogen of A. thaliana. PAIs encode the type III secretion system along with its effectors and are essential for pathogen recognition in plants. P. viridiflava harbors two structurally distinct and highly diverged PAI paralogs (T- and S-PAI) that are integrated in different chromosome locations in the P. viridiflava genome. Both PAIs are segregating as presence/absence polymorphisms such that only one PAI ([T-PAI, nablaS-PAI] and [nablaT-PAI, S-PAI]) is present in any individual cell. A worldwide population survey identified no isolate with neither or both PAI. T-PAI and S-PAI genotypes exhibit virulence differences and a host-specificity tradeoff. Orthologs of each PAI can be found in conserved syntenic locations in other Pseudomonas species, indicating vertical phylogenetic transmission in this genus. Molecular evolutionary analysis of PAI sequences also argues against "recent" horizontal transfer. Spikes in nucleotide divergence in flanking regions of PAI and nabla-PAI alleles suggest that the dual PAI polymorphism has been maintained in this species under some form of balancing selection. Virulence differences and host specificities are hypothesized to be responsible for the maintenance of the dual PAI system in this bacterial pathogen.

Arabidopsis↗

Prevalence of the "high-pathogenicity island" of Yersinia species among Escherichia coli strains that are pathogenic to humans.

The fyuA-irp gene cluster contributes to the virulence of highly pathogenic Yersinia (Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica 1B). The cluster encodes an iron uptake system mediated by the siderophore yersiniabactin and reveals features of a pathogenicity island. Two evolutionary lineages of this "high pathogenicity island" (HPI) can be distinguished on the basis of DNA sequence comparison: a Y. pestis group and a Y. enterocolitica group. In this study we demonstrate that the HPI of the Y. pestis evolutionary group is disseminated among species of the family Enterobacteriaceae which are pathogenic to humans. It prevails in enteroaggregative Escherichia coli and in E. coli blood culture isolates (93 and 80%, respectively), but is rarely found in enteropathogenic E. coli, enteroinvasive E. coli, and enterotoxigenic E. coli isolates. In contrast, the HPI was absent from enterohemorrhagic E. coli, Shigella, and Salmonella enterica strains investigated. Polypeptides encoded by the fyuA, irp1, and irp2 genes located on the HPI could be detected in E. coli strains pathogenic to humans. However, these E. coli strains showed a reduced sensitivity to the bacteriocin pesticin, whose uptake is mediated by the FyuA receptor. Escherichia strains do not possess the hms gene locus thought to be a part of the HPI of Y. pestis. Deletions of the juA-irp gene cluster affecting solely the fyuA part of the HPI were identified in 3% of the E. coli strains tested. These results suggest horizontal transfer of the HPI between Y. pestis and some pathogenic E. coli strains.

Bacterial Outer Membrane Proteins↗

Animal health and foodborne pathogens: enterohaemorrhagic O157:H7 strains and other pathogenic Escherichia coli virotypes (EPEC, ETEC, EIEC, EHEC).

The majority of interactions between microorganisms and animals are based on convenient relations for both of them. Symbiotic microorganisms, like intestinal microbiota, produce important vitamins for animals and protects them from putative pathogens. In general, for monogastric animals, the main contribution of intestinal microorganisms is to supply with growth factors the animal diet, and in some cases they are responsible for providing essential vitamins (e.g. vitamin K). Some particular and relatively few microbes like viruses, bacteria, fungi, protozoa and algae are responsible for animal illness. Because microorganisms are easily dispersed, display physiological diversity, and tolerate extreme conditions, they are ubiquitous and may contaminate and grow in many products, including food and raw materials. Foodborne diseases are caused by consumption of contaminated food or beverages. Many different disease-causing pathogens can contaminate food, so there are many different foodborne infections. In addition, poisonous chemicals and biological toxins can cause disease if they are present in food. To know how a particular disease is spreading is an important matter to take appropriate steps to stop it. For example Escherichia coli O157:H7 infections can spread through contaminated food (meat, vegetables, cheese, etc.), contaminated drinking water or juices, contaminated swimming water and from person to person. Among foodborne pathogens, the most frequently detected are bacteria, but also parasitic protozoa and worms, viruses, natural toxins and other pathogenic agents like prions are important agents for foodborne diseases. Particular pathogenic types of E. coli, classified by their specific pathogenic mechanisms (toxins, adhesins, invasiveness, etc.) are actually known as E. coli virotypes. Enterohaemorrhagic E. coli (EHEC), which constitute the main part of this review, were also named verotoxigenic E. coli (VTEC) or Shiga toxigenic E. coli (STEC). EHEC strains cause haemorrhagic colitis (HC), haemolytic uremic syndrome (HUS) and thrombotic thrombocytopaenic purpura (TP) in humans. They synthetize shigatoxins (verotoxins) which are potent cytotoxic substances, adherence factors and enterohaemolysin. EHEC are responsible for many outbreaks of bloody diarrhoea caused by contaminated foods: beef, milk, fruits, juice, water, etc. The most important serogroups among EHEC are O26, O111 and O157, being O157:H7 the most relevant serotype in foodborne outbreaks. The normal intestinal microflora of cattle was found to be the most relevant reservoir of EHEC strains.

Animals↗

Effect of naturally occurring intramammary infections by minor pathogens on new infections by major pathogens in cattle.

New mammary infections were recorded in 3 dairy herds during a lactation period by bacteriologic examination of milk samples at 3-week intervals. Influences of the infection status of quarters at the time of new infection and of microorganisms responsible for bacterial invasion were analyzed. The new infection rate in uninfected quarters was about 3 times the rate in quarters already harboring bacterial considered minor pathogens (coagulase-negative staphylococci and Corynebacterium bovis) or major pathogens (Staphylococcus aureus, streptococci). The frequency of new infections with major pathogens was almost halved by preexisting infections with minor pathogens (P = 0.05), mainly because of coagulase-negative staphylococci (P = 0.05) and, to a lesser extent, C bovis (P = 0.19). New infections by minor pathogens also were less frequent in quarters harboring a major pathogen (P less than 0.05), indicating that the competition or antagonism between mammary infections was a general phenomenon.

Animals↗

In vivo interference between pathogenic and non-pathogenic viruses.

The interference among viruses is a well-documented biological phenomenon, both in animals and tissue culture systems. In two of our previous in vivo experiments and in four independent animal experiments, which are described in this presentation, interferences were successfully used to influence the outcomes of viral diseases by using non-pathogenic viruses. In this study, four pathogenic viruses were studied in their natural hosts, and against these viruses, in different combinations, 15 non-pathogenic viruses were tested. There was great variation in mutual effects among pathogenic and non-pathogenic viruses. In our four experiments, the viruses were either simultaneously inoculated or the non-pathogenic viruses were preinoculated. Newcastle disease vaccine (Strain H) had remarkable effects in the development of mouse ascites-associated lymphoma virus. The 50% mortality rate in mice caused by a vaccine strain of rabies virus was reduced to 15% using avian encephalitis virus. The clinical manifestations of rabbit myxoma virus effects were significantly delayed by Newcastle disease vaccine (Strain H). The 72% mortality rate due to Rous sarcoma virus in chickens was decreased to 33.3% when the animals were preinoculated with avian bursa virus vaccine.

Animals↗

Prior infection of Manduca sexta with non-pathogenic Escherichia coli elicits immunity to pathogenic Photorhabdus luminescens: roles of immune-related proteins shown by RNA interference.

Prior infection of Manduca sexta caterpillars with the non-pathogenic bacterium Escherichia coli elicits effective immunity against subsequent infection by the usually lethal and highly virulent insect pathogen Photorhabdus luminescens TT01. Induction of this protective effect is associated with up-regulation of both microbial pattern recognition protein genes (hemolin, immulectin-2 and peptidoglycan recognition protein) and anti-bacterial effector genes (attacin, cecropin, lebocin, lysozyme and moricin). We used RNA interference to knock down over-transcription of members of both these sets of genes one at a time. Interfering with expression of individual recognition proteins had a drastic adverse effect on the E. coli elicited immunity. RNAi knock-down of immulectin-2 caused the greatest reduction in immunity, followed by hemolin and peptidoglycan recognition protein (PGRP) in that order, to the extent that knock-down of any one of these three proteins left the insects more susceptible to P. luminescens infection than insects that had not experienced prior infection with E. coli. Interfering with the expression of individual antibacterial effector proteins and peptides had a much less marked effect on immunity. Knock-down of attacin, cecropin or moricin caused treated insects to be more susceptible to P. luminescens infection than controls that had been pre-infected with E. coli but which had not received the specific RNAi reagents, but they were still less susceptible than insects that had not been pre-infected with E. coli. RNAi knock-down with expression of lebocin or lysozyme had no effect on E. coli-induced immunity to P. luminescens, indicating that these effectors are not involved in the response. By bleeding pre-infected caterpillars and growing the pathogen directly within cell-free insect haemolymph, we showed that at least part of the protection elicited by previous exposure to E. coli is due to the presence of factors within the blood plasma that inhibit the growth of P. luminescens. The production of these factors is inhibited by RNAi treatment with ds-RNA reagents that knock down hemolin, immulectin-2, and PGRP. These results demonstrate that the insect immune system can be effectively primed by prior infection with non-pathogenic bacteria against subsequent infection by a highly virulent pathogen. Given the continuous normal exposure of insects to environmental and symbiotic bacteria, we suggest that prior infection is likely to play a significant and underestimated role in determining the level of insect immunity found in nature.

Animals↗

Mycological assessment of sediments in Ligurian beaches in the Northwestern Mediterranean: pathogens and opportunistic pathogens.

Sediments of five Ligurian beaches in compliance with European Union bathing water regulations were studied based on the characteristics of the fungal assemblage during the tourism season. Among the 179 taxa of filamentous fungi isolated, 120 were opportunistic pathogens, such as Acremonium sp., and the genus Penicillium was also present as the pathogenic species P. citrinum. Furthermore, 5% of the total filamentous fungi belonged to the dermatophyte genus Microsporum, whose species can cause mycoses. Beach sediments showed elevated densities of opportunistic pathogens, of pathogenic filamentous fungi, and of yeasts during the tourism season. Although monitoring of beach sediments for microbiological contamination is not mandatory, and disease transmission from sediments has not yet been demonstrated, our study suggests that beach sediments may act as a reservoir of potential pathogens, including fungi. In addition, the mycoflora displayed high sensitivity to critical environmental situations in the beaches studied. Therefore, the fungal community can be a useful tool for assessing the quality of sandy beaches in terms of sanitary and environmental quality.

Analysis of Variance↗

DNA microarray for discrimination between pathogenic 0157:H7 EDL933 and non-pathogenic Escherichia coli strains.

The primary technique currently used to detect biological agents is based on immunoassays. Although sensitive and specific, currently employed immunoassays generally rely on the detection of a single epitope, and therefore often cannot discriminate subtle strain-specific differences. Since DNA microarrays can hybridize hundreds to thousands of genomic targets simultaneously and do not rely on phenotypic expression of these genetic features for identification purposes, they have enormous potential to provide inexpensive, flexible and specific strain-specific detection and identification of pathogens. In this study, pathogenic Escherichia coli O157:H7-specific genes, non-pathogenic K12-specific genes, common E. coli genes, and negative control genes were polymerase chain reaction-amplified and spotted onto the surface of treated glass slides. After labeled bacterial cDNA samples were hybridized with probes on the microarray, specific fluorescence patterns were obtained, enabling identification of pathogenic E. coli O157:H7 and non-pathogenic E. coli K12. To test the utility of this microarray device to detect genetically engineered bacteria, E. coli BL21 (a B strain derivative with antibiotic resistance gene, ampR) and E. coli JM107 (a K12 strain derivative lacking the gene ompT) were also employed. The array successfully confirmed the strain genotypes and demonstrated that antibiotic resistance can also be detected. The ability to assess multiple data points makes this array method more efficient and accurate than a typical immunoassay, which detects a single protein product.

Colony Count, Microbial↗

Three pathogen-inducible genes encoding lipid transfer protein from pepper are differentially activated by pathogens, abiotic, and environmental stresses.

The three cDNA clones, CALTPI, CALTPII, and CALTPIII, corresponding to pepper lipid transfer protein (LTP) genes were isolated from a pepper (Capsicum annuum) cDNA library from hypersensitive response (HR) lesions of leaves infected with Xanthomonas campestris pv. vesicatoria. The CALTP genes are well conserved in their coding region with 57-72% identity at the amino acid level, but display 72-83% identity at the nucleotide sequence level. The transcripts of the three CALTP genes differentially accumulated in pepper leaf, stem, and fruit tissues infected by X. campestris pv. vesicatoria, Phytophthora capsici and Colletotrichum gloeosporioides. The CALTP genes were also strongly induced in the systemic, upper leaves after immunization on lower leaves by either pathogenic or non-pathogenic bacteria. In situ hybridization results showed that the CALTPI mRNA was localized in phloem cells of vascular tissues in pepper leaf, stem and fruit tissues after pathogen infection. CALTPI and CALTPIII genes were predominantly expressed in various pepper tissues infected by pathogens, while infection by P. capsici and C. gloeosporioides did not induce the transcription of the CALTPII gene. Ethylene, methyl jasmonate and abscisic acid induced CALTPI and III gene expression in pepper leaves. Drought, high salinity, low temperature and wounding stresses also induced the expression of the CALTPI and CALTPIII genes in a similar manner. In contrast, only high salinity induced the CALTPII expression that was not generally affected by abiotic and other environmental stimuli. When compared with each other and with LTPs from other plants, CALTPI is more distantly related than CALTPII and CALTPIII sequences, indicating that the three pepper CALTP genes represent two different classes. These results thus show that CALTPI and CALTPIII genes, although different in sequence structure, are transcriptionally activated in pepper tissues by pathogen infection as well as abiotic and environmental stresses.

Journal Article↗

The broad host range pathogen Pseudomonas aeruginosa strain PA14 carries two pathogenicity islands harboring plant and animal virulence genes.

The ubiquitous bacterium Pseudomonas aeruginosa is the quintessential opportunistic pathogen. Certain isolates infect a broad range of host organisms, from plants to humans. The pathogenic promiscuity of particular variants may reflect an increased virulence gene repertoire beyond the core P. aeruginosa genome. We have identified and characterized two P. aeruginosa pathogenicity islands (PAPI-1 and PAPI-2) in the genome of PA14, a highly virulent clinical isolate. The 108-kb PAPI-1 and 11-kb PAPI-2, which are absent from the less virulent reference strain PAO1, exhibit highly modular structures, revealing their complex derivations from a wide array of bacterial species and mobile elements. Most of the genes within these islands that are homologous to known genes occur in other human and plant bacterial pathogens. For example, PAPI-1 carries a complete gene cluster predicted to encode a type IV group B pilus, a well known adhesin absent from strain PAO1. However, >80% of the PAPI-1 DNA sequence is unique, and 75 of its 115 predicted ORF products are unrelated to any known proteins or functional domains. Significantly, many PAPI-1 ORFs also occur in several P. aeruginosa cystic fibrosis isolates. Twenty-three PAPI ORFs were mutated, and 19 were found to be necessary for full plant or animal virulence, with 11 required for both. The large set of "extra" virulence functions encoded by both PAPIs may contribute to the increased promiscuity of highly virulent P. aeruginosa strains, by directing additional pathogenic functions.

Animals↗

Quinolone resistance in potentially pathogenic and non-pathogenic Escherichia coli strains isolated from healthy ruminants.

Quinolone resistance was studied in potentially pathogenic and non-pathogenic Escherichia coli strains from healthy ruminants. In cattle, 5.9% of the strains were resistant to nalidixic acid and 4.9% were resistant to enrofloxacin and ciprofloxacin, whereas in sheep and goats only 0.5% and 1.4%, respectively, of the strains were resistant to nalidixic acid and none to fluoroquinolones. Most of the strains resistant to quinolones were non-pathogenic strains isolated from cattle. However, the results of this study do not show that the potentially pathogenic E. coli strains isolated from healthy ruminants are more susceptible to quinolones than the non-pathogenic E. coli strains.

Animals↗

Determinants of pathogenicity in Xanthomonas campestris pv. vesicatoria are related to proteins involved in secretion in bacterial pathogens of animals.

One of the model systems investigated for studying plant bacterial pathogenesis is Xanthomonas campestris pv vesicatoria, the causal agent of bacterial spot disease of pepper and tomato. Genes necessary for both basic pathogenicity and the induction of the hypersensitive response in resistant plants (hrp genes) were previously isolated from X. c. pv. vesicatoria and characterized genetically. As a first step toward functional analysis, part of the hrp gene cluster, making up several loci, was sequenced. Here, we report the first indications of the function of hrp genes. Striking similarities to proteins from the mammalian pathogens Shigella flexneri, Yersinia enterocolitica, Y. pestis, and other bacteria were discovered. Proteins encoded by genes within the X. c. pv. vesicatoria loci hrpA, hrpB, and hrpC are similar to ATPases and to Yersinia Ysc and LcrD proteins, which are involved in secretion of Yop proteins, a particular class of essential pathogenicity factors produced by Yersinia species. This finding indicates, for the first time, that the fundamental determinants of pathogenicity may be conserved among bacterial pathogens of plants and animals. We hypothesize that hrp genes are involved in the secretion of molecules essential for the interaction of X. c. pv. vesicatoria with the plant.

Amino Acid Sequence↗

Genome-wide transcriptional analysis of the Arabidopsis thaliana interaction with the plant pathogen Pseudomonas syringae pv. tomato DC3000 and the human pathogen Escherichia coli O157:H7.

Pseudomonas syringae pv. tomato DC3000 (Pst) is a virulent pathogen that causes disease on tomato and Arabidopsis. The type III secretion system (TTSS) plays a key role in pathogenesis by translocating virulence effectors from the bacteria into the plant host cell, while the phytotoxin coronatine (COR) contributes to virulence and disease symptom development. Recent studies suggest that both the TTSS and COR are involved in the suppression of host basal defenses. However, little is known about the interplay between the host gene expression changes associated with basal defenses and the virulence activities of the TTSS and COR during infection. In this study, we used the Affymetrix full genome chip to determine the Arabidopsis transcriptome associated with basal defense to Pst DC3000 hrp mutants and the human pathogenic bacterium Escherichia coli O157:H7. We then used Pst DC3000 virulence mutants to characterize Arabidopsis transcriptional responses to the action of hrp-regulated virulence factors (e.g. TTSS and COR) during bacterial infection. Additionally, we used bacterial fliC mutants to assess the role of the pathogen-associated molecular pattern flagellin in induction of basal defense-associated transcriptional responses. In total, our global gene expression analysis identified 2800 Arabidopsis genes that are reproducibly regulated in response to bacterial pathogen inoculation. Regulation of these genes provides a molecular signature for Arabidopsis basal defense to plant and human pathogenic bacteria, and illustrates both common and distinct global virulence effects of the TTSS, COR, and possibly other hrp-regulated virulence factors during Pst DC3000 infection.

Abscisic Acid↗

A novel pathogenicity island integrated adjacent to the thrW tRNA gene of avian pathogenic Escherichia coli encodes a vacuolating autotransporter toxin.

We report the complete nucleotide sequence and genetic organization of the Vat-encoding pathogenicity island (PAI) of avian pathogenic Escherichia coli strain Ec222. The 22,139-bp PAI is situated adjacent to the 3' terminus of the thrW tRNA gene, has a G+C content of 41.2%, and includes a bacteriophage SfII integrase gene, mobile genetic elements, two open reading frames with products exhibiting sequence similarity to known proteins, and several other open reading frames of unknown function. The PAI encodes an autotransporter protein, Vat (vacuolating autotransporter toxin), which induces the formation of intracellular vacuoles resulting in cytotoxic effects similar to those caused by the VacA toxin from Helicobacter pylori. The predicted 148.3-kDa protein product possesses the three domains that are typical of serine protease autotransporters of Enterobacteriaceae: an N-terminal signal sequence of 55 amino acids, a 111.8-kDa passenger domain containing a modified serine protease site (ATSGSG), and a C-terminal outer membrane translocator of 30.5 kDa. Vat has 75% protein homology with the hemagglutinin Tsh, an autotransporter of avian pathogenic E. coli. A vat deletion mutant of Ec222 showed no virulence in respiratory and cellulitis infection models of disease in broiler chickens. We conclude that the newly described PAI and Vat may be involved in the pathogenicity of avian septicemic E. coli strain Ec222 and other avian pathogenic E. coli strains.

Amino Acid Sequence↗