Stenotrophomonas maltophilia, a member of γ-proteobacteria, is a ubiquitous environmental bacterium that is recognized as an opportunistic nosocomial pathogen. FecABCD system contributes to ferric citrate acquisition in Escherichia coli. FeoABC system, consisting of an inner membrane transporter (FeoB) and two cytoplasmic proteins (FeoA and FeoC), is a well-known ferrous iron transporter system in γ-proteobacteria. As revealed by the sequenced genome, S. maltophilia appears to be equipped with several iron acquisition systems; however, the understanding of these systems is limited. In this study, we aimed to elucidate the ferric citrate acquisition system of S. maltophilia.E. coli as a reference, and then revealed by transcriptome analysis of S. maltophilia KJ with and without 2,2′-dipyridyl (DIP) treatment. Function validation was carried out by deletion mutant construction and ferric citrate utilization assay. The bacterial adenylate cyclase two-hybrid system was used to verify intra-membrane protein–protein interaction.Candidate genes searching and function validation are the strategy for elucidating the genes involved in ferric citrate acquisition. The candidate genes responsible for ferric citrate acquisition were firstly selected using FecABCD of E. coli, were first considered; however, deletion mutant construction and functional validation ruled out their involvement in ferric citrate acquisition. FciA (Smlt1148), revealed by its upregulation in DIP-treated KJ cells, was the outer membrane receptor for ferric citrate uptake. The fciA gene is a member of the fciTABC operon, in which fciT, fciA, and fciC participated in ferric citrate acquisition. Uniquely, the Feo system of S. maltophilia is composed of a cytoplasmic protein FeoA, an inner membrane transporter FeoB, and a predicted inner membrane protein FeoI. The intra-membrane protein–protein interaction between FeoB and FeoI may extend the substrate profile of FeoB to ferric citrate. FeoABI system functioned as an inner membrane transporter of ferric citrate.Smlt2858 and Smlt2356, the homologues of FecA and FecC/D of S. maltophilia.The FciTABC and FeoABI systems contribute to ferric citrate acquisition in The online version contains supplementary material available at 10.1186/s12929-022-00809-y. Iron is an essential metal in almost all living organisms, and functions as a cofactor for proteins involved in redox chemistry and electron transport . TherefofecC/D/E in Escherichia coli [E. coli Fec system, ferric citrate is taken up by the outer membrane receptor (FecA) and is transported into the cytoplasm via the periplasmic protein FecB and inner membrane transport proteins FecC/D/E [E. coli and Pseudomonas aeruginosa, despite the high similarity in identity of their outer membrane receptors (FecAs). No FecB/C/D/E homologues have been found in the ferric citrate acquisition system in P. aeruginosa. In contrast, citrate-mediated iron uptake is compromised in feoB mutants of P. aeruginosa [, suggesting that FeoB is involved in the post-outer membrane transport of ferric citrate. However, the underlying mechanism remains unclear.Bacteria acquire citrate-mediated iron sources in two ways. Most bacteria can directly utilize exogenously supplied ferric citrate as an iron source, to fulfill their nutritional requirements under iron-depleted conditions . In addihia coli . In the FecC/D/E . The posruginosa , suggestfeo system, represented in Helicobacter pylori, is only composed of the feoB gene [feo systems of Leptospira biflexa and Campylobacter jejuni consist of feoA and feoB [Ferric iron is the major iron source for bacteria under aerobic conditions. However, under highly acidic and anaerobic conditions, ferrous iron transport systems are important for iron acquisition. Several bacterial ferrous iron transport systems have been described –14; howeeoB gene . In someand feoB , 21. In and feoB , 23.2+, and repress the transcription of almost all genes related to iron uptake. When the intracellular Fe2+ is too low to interact with Fur, the genes repressed by the Fur-Fe2+ complex are derepressed [fecIRABCDE cluster of E. coli and the fecIRA operon of P. aeruginosa [Given the lethal damage caused by excess cytoplasmic iron, iron-uptake systems must be controlled. Iron deplete conditions are generally considered as stimuli for the inducible expression of iron source acquisition systems . Two regepressed . A classepressed . In generuginosa , 27, 28.Stenotrophomonas maltophilia is ubiquitous in the environment, particularly in soil and plant rhizospheres [S. maltophilia should have evolved several iron acquisition systems for survival. However, the systems used by S. maltophilia to acquire iron have been poorly reported, except the FepA system for ferri-siderophore uptake [S. maltophilia is known to synthesize stenobactin, a catecholate siderophore, depending on the entCEBBFA gene cluster [S. maltophilia K279a genome [hemA, fecA, feoA, and feoB, respectively. However, little is known about the acquisition of hemin, ferric citrate, and ferrous iron in S. maltophilia. In this study, we aimed to elucidate the citrate-mediated iron acquisition system in S. maltophilia. We identified that the FciTABC and FeoABI, two previously unidentified systems, are responsible for ferric citrate utilization in S. maltophilia under iron-depleted conditions. This is distinct from the FecIRABCDE system of E. coli and the FecIRA/FeoB system of P. aeruginosa.ospheres . Furtherospheres . To inhae uptake  and the e uptake . S. malt cluster . FepA is cluster . We surva genome  and founThe primers used in this study are listed in Additional file S. maltophilia strains via conjugation. Integration of the deletion constructs into the chromosome was selected by resistance to norfloxacin (2.5 μg/ml) and tetracycline (30 μg/ml). The double cross-over tansconjugants were selected by 10% sucrose [In-frame deletion strains were constructed using double cross-over homologous recombination as described previously . In brie sucrose . The corlacZ promoter. The primers used and the resultant plasmids for complementation were summarized in Additional file Genes of interest were amplified by PCR with the primers as indicated and then cloned into pRK415 under the control of 5 CFU/μl followed by tenfold serial diluted. A 5 μl volume of each dilution was spotted onto the plates as indicated. After a 24-h incubation, the growth of bacterial cells was observed.The logarithmic phase bacterial strains tested were adjusted to a concentration of 2 × 10DNA-free RNA was isolated from mid-log phase KJ∆Fur cells as described previously . ReversefeoB and feoI genes were amplified by PCR using the primer sets of 25FeoB-F/R and 18FeoI-F/R , and transformants were grown in LB medium supplemented with ampicillin, kanamycin, and 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) for 16 h at 30 °C. The overnight cultures were harvested and assayed for β-galactosidase activity. β-galactosidase assays were performed using the method of Miller [. Experiments were independently repeated at least three times.The pUT18-derived and pKT25-derived plasmids as indicated were co-transformed into a f Miller . ExperimRNA was prepared from logarithmic phase bacterial cells grown in LB broth without or with additives as indicated and converted to cDNA by reverse transcription. qRT-PCR was performed by the ABI Prism 7000 Sequence Detection System according to the manufacturer’s protocols. All gene expressions were normalized with the internal control of 16S rRNA since . All expStenotrophomonas maltophilia can produce the catechol-type siderophore, stenobactin, under iron-depleted conditions [S. maltophilia KJ∆Ent [S. maltophilia can utilize ferric citrate as an iron source for growth, and its genome should contain the necessary genetic components for ferric citrate utilization.nditions . Sideropa KJ∆Ent , a stenoE. coli and P. aeruginosa [S. maltophilia K279a using E. coli FecA as a query. A homologue of FecA, Smlt2858, was identified. The Smlt2858 protein displayed 42% identity and 57% similarity to the FecA protein of E. coli, as well as 42% identity and 58% similarity to the FecA protein of P. aeruginosa  lacking dia Fig. A. Two pofecA and ferric citrate uptake, other TonB-dependent receptors were considered. It is presumably accepted that iron acquisition systems are upexpressed in an iron-depleted condition [S. maltophilia genomes.Given the failure to demonstrate the link between ondition . The comondition . There w∆fciA mutant. The deletion of fecA in KJ∆Ent∆FciA, yielding KJ∆Ent∆FciA∆FecA, did not further compromise growth in ferric citrate- and DIP-supplemented media . The fciT gene encodes a 134 amino acid (aa) protein, whereby the N-terminus (13–35 aa) is predicted to be a transmembrane domain and the C-terminus (36–134 aa) is distributed in the periplasmic space. The 239 aa FciB protein was predicted to be a periplasmic protein, and FciC a cytoplasmic protein with features of the Fe(II)/2-oxoglutatate (2-OG)-dependent oxygenase superfamily. We were interested in understanding whether similar gene clusters existed in E. coli and P. aeruginosa. Following a genomic search, ECK0794 and ECK0795 of E. coli were found to be homologues of fciA and fciC; and PA4514, PA4515, and PA4516 of P. aeruginosa were homologues of fciA, fciC, and fciB, respectively  . The fciA transcript levels, but not those of fciB and fciC, decreased significantly in KJ∆Ent∆FciT compared to those in KJ∆Ent   displayed better viability than that of KJ∆Ent∆FciT(pFciA) in ferric citrate-supplemented media  available on the NCBI database. Except for strains D457 and RA8, the remaining 11 strains contained the fciA gene. The FciA proteins of the 12 strains (including the KJ strain) shared identities ranging from 91 to 100% based on pairwise sequence identity scores. In addition, the presence of the fciA gene in 14 S. maltophilia clinical isolates was determined by colony PCR using primer sets of FciAc-F/R  was used for comparison , periplasmic ATP-binding protein (Smlt2354), periplasmic transport lipoprotein (Smlt2355), the FecC/D family inner membrane protein (Smlt2356), cytoplasmic protein (Smlt2357), and periplasmic protein (Smlt2358). It was, therefore, important to investigate the involvement of Smlt2356 in ferric citrate utilization; thus, we assessed the viability of KJ∆Ent∆2356 in ferric citrate- and DIP-supplemented media. KJ∆Ent∆2356 displayed viability comparable to that of KJ∆Ent ; whereas, the FeoC proteins characterized in other bacteria, such as E. coli, P. aeruginosa, and V. cholera, are cytoplasmic proteins [https://services.healthtech.dtu.dk/service.php?TMHMM-2.0), FeoI spans the inner membrane through a single transmembrane helix flanked by cytoplasmic- and periplasmic-orientated moieties at the N and C termini. The feoABI cluster appeared to have an operonic structure. To test this hypothesis, we performed RT-PCR and verified the presence of the feoABI operon , but no significant changes were observed in response to the challenge of DIP, citrate, and ferric citrate, either alone or combined  and P. aeruginosa FecA protein (FecAPa) share 63% identity and 73% similarity. Subsequently, fecA homologous genes have been annotated in many sequenced bacterial genomes based on their encoded protein sequences that are identical to FecAEc and FecAPa. However, the exact functions of these annotated FecA proteins have not yet been clearly elucidated. FecASm (Smlt2858) was the first candidate considered as the ferric citrate receptor in S. maltophilia, as it demonstrated the highest identity to FecAEc and FecAPa. Nevertheless, the functional investigation of FecASm did not reveal its role in ferric citrate acquisition  to utilize ferric citrate, but not ferrous iron, as the iron sources to support growth in an iron-depleted condition  system.Additional file 9: Table S1. Primers used for the construction of pEX18Tc-derived mutagenic plasmids, complementation plasmids, operon validation, and qRT-PCRAdditional file 10: Table S2. Bacterial strains and plasmids used in this studyAdditional file 11: Table S3. Transcriptomic analysis of TonB-dependent outer membrane receptor genes differentially expressed in S. maltophilia KJ with and without the DIP treatment


Leptospira strains show very large antigenic variation (serovars). Here, the rfb gene cluster is shown to be transferred horizontally within/among Leptospira species, discriminating serovar identity. Leptospira bacteria comprise numerous species, several of which cause serious disease to a broad range of hosts including humans. These spirochetes exhibit large intraspecific variation, resulting in complex tabulations of serogroups/serovars that crisscross the species classification. Serovar identity, linked to biological/clinical phenotypes, depends on the structure of surface-exposed LPS. Many LPS biosynthesis–encoding genes reside within the chromosomic rfb gene cluster. However, the genetic basis of intraspecies variability is not fully understood, constraining diagnostics/typing methods to cumbersome serologic procedures. We now show that the gene content of the rfb cluster strongly correlates with Leptospira serovar designation. Whole-genome sequencing of pathogenic L. noguchii, including strains of different serogroups, reveals that the rfb cluster undergoes extensive horizontal gene transfer. The rfb clusters from several Leptospira species disclose a univocal correspondence between gene composition and serovar identity. This work paves the way to genetic typing of Leptospira serovars, and to pinpointing specific genes within the distinct rfb clusters, encoding host-specific virulence traits. Further research shall unveil the molecular mechanism of rfb transfer among Leptospira strains and species. Leptospira species, the etiological agents of leptospirosis, infect a broad spectrum of hosts, with a global annual incidence of 1 million human cases and ∼60,000 deaths , which comprises the most important species causing human and animal diseases such as L. interrogans and L. borgpetersenii  of L. noguchii is currently available, data that would otherwise boost the power of comparative genomics analyses.tersenii . Many hunce 1940 , but it nce 1940 , baptizeL. noguchii WGS contribute with novel insights into leptospirosis? Comprising the largest reported genome among Leptospira spp.  is a common attribute within rminants . Probablrminants , differerminants . Despiterminants . A connerminants , but notL. noguchii strains (10 closed genomes and 2 drafts), we undertook an extensive comparative genomics approach, uncovering underlying reasons for Leptospira phenotypic complexity. We now reveal (i) the detailed genomic features and plasmid repertoire of L. noguchii and its phylogenetic structure; (ii) that the cluster comprising most of the LPS synthesis enzyme-encoding genes, known as rfb  that serovar identity is univocally linked to the presence/absence of specific genes within this rfb cluster.By sequencing the genomes of 12 n as rfb , exhibitL. noguchii, which allowed a comprehensive analysis of its genetic variability. Remarkably, after comparing with known serovars of different Leptospira species, it was possible to reveal serovar-specific genetic fingerprints encoded within a horizontally transferred gene cluster, paving the way toward genome-based serotyping and further molecular studies of the HGT mechanisms at play.In sum, this work constitutes the first report of complete genomes of L. noguchii strains (L. interrogans (∼4.6 Mb) and L. borgpetersenii (∼3.9 Mb). Most genomes reached a finished status, with three to eight contigs (Whole genomes from 12  strains  were seq contigs  correspoL. noguchii sv Panama strain CZ214 as reference) was >95% , consistent with previous determinations based on 16S rDNA sequence  compared with other Leptospira species (L. interrogans (26 in sv Copenhageni strain Fiocruz) or in the saprophyte Leptospira biflexa (8 in sv Patoc strain Ames) (The average nucleotide identity (ANI) among all sequenced strains in this study  .L. noguchii strains.ANI Table S1 Supplemental Data 1.Supplemental material.L. noguchii pangenome of the 12 strains sequenced in this study , uncovering a rich array of unique attributes that distinguish strains.Analysis of the is study  showed aO-antigen nucleotide sugar biosynthesis—a clear enrichment is observed in functions related to carbohydrate metabolism, and glycoside modification and synthesis. This KEGG mapping analysis was systematically extended to accessory genes present only in two strains, three, and further, confirming the importance of variations in carbohydrate-related metabolism as a main source of strain-specific genetic variability. Of note, a number of carbohydrate-related genes, including several that encode LPS biosynthesis enzymes, were present only in particular groups of strains. For example, UDP-glucuronate 4-epimerase  or 3-dese , among Functional mapping of strain-specific genes.Table S2 L. noguchii strains harbor a variable number of plasmids  revealed no core or even softcore genes, highlighting the extreme plasmid diversity in Leptospira. A network association analysis considering this extended set of plasmids revealed species-specific clustering of plasmid sequences, again uncovering cases of strong similarity/identity in the arrays of protein-encoding genes between different strains . A first general inquiry about the potential link between plasmid identity and environmental factors did not result in a clear association. Neither the geographical locations of strains, nor the infected host from which they were isolated, showed clear-cut connections with plasmids and their protein-encoding gene compositions. Plasmid-borne virulence factors and antibiotic resistance determinants were also explored, recognizing two genes encoding putative multidrug efflux proteins of the resistance–nodulation–division family  revealed this same cluster in plasmid p1 from three L. interrogans strains . Further work is needed to uncover the biological role of these proteins in Leptospira, especially considering that antibiotic resistance is not a usual feature in spirochetes. Functional analysis of clusters of orthologous genes (COG) showed a few categories to be absent from plasmidic genes in L. noguchii and other Leptospira species: (i) RNA processing and modification; (ii) chromatin structure and dynamics; (iii) carbohydrate metabolism and transport; (iv) nuclear structure; (v) cytoskeleton; and (vi) general function prediction. On the contrary, among the most represented COG categories were those related to (i) unknown function, (ii) replication and repair, (iii) transcription, and (iv) signal transduction. Surprisingly, the functional category linked to amino acid metabolism and transport was completely absent in L. noguchii plasmids, in stark contrast to other Leptospira species.A functional/biological analysis of n family , MtdA anL. noguchii to different hosts and geographic locations. To uncover such potential genotype/phenotype associations, the phylogenetic structure of L. noguchii was explored in greater detail by analyzing the genomes from 11 different geographic locations and nine types of hosts . This result confirms previous reports analyzing several different pathogenic Leptospira species  molecules to the lipid A moiety, subsequently incorporating heptoses and further modifications  among the different strains  revealed no variation in terms of differential presence of genes. The only difference concerned L. interrogans sv Weerasinghe, in which three genes coding for WaaA isoforms were found .A second component of LPS is the core oligosaccharide , whose bications . CompariO-antigen is the most variable part of the structure and most exposed to interact with the environment  and a sodium/sulfate symporter (SdcS) were consistently found, as in other pathogenic Leptospira species  is only observed within the rfb cluster, and not in the immediate surrounding segments . A detailed analysis by serogroup unveiled several important observations: (i) a few serovars belonging to the same serogroup showed indistinguishable patterns ; extreme relatedness had been previously reported for Copenhageni/Icterohaemorrhagiae  the four non-agglutinating L. noguchii strains showing an identical rfb composition (except for slight differences in IP1512017) did not share many genes with other serogroups, suggesting they perhaps belong to a new serogroup altogether.Very few genes were conserved in all the rrhagiae , where orfb genes were used to search orthologous protein sequences: Blast best hits’ accession numbers are listed.Table S6 rfb clusters. In this regard, Australis was the most variable serogroup , Hardjo (from L. borgpetersenii and L. interrogans), and Valbuzzi (from L. interrogans and L. kirschneri) . The gene presence/absence matrix calculated for the rfb clusters from this subgroup , irrespective of the species. Despite the previously stated constraint in terms of genomic fragmentation, a “pan-rfb” was created considering the genes resulting from the analysis in rfb against WGSs from different serovars, including more than one strain in each case, showed the same genetic pattern for several examples  compared with other relevant pathogens such as L. interrogans (4.6 Mb) and L. borgpetersenii (3.9 Mb), with a larger array of accessory genes that might be involved in L. noguchii’s remarkable adaptability. Indeed, ∼30% of the genes from the strains sequenced in this study correspond to accessory genome, with a clear enrichment in carbohydrate biochemistry pathways. Concerning the plasmid repertoire, some strains showed identical or nearly identical protein-encoding plasmidic genes . On the contrary, plasmid-borne gene variability seems larger in some Leptospira species , perhaps reflecting a varying contribution of horizontal plasmid acquisition as a source of adaptation. COG analyses revealed no variation in functional representation between plasmids from L. noguchii versus those from other species, except for the absence of genes related to amino acid metabolism and transport in L. noguchii plasmids.The genome architecture in terms of replicon content and its organization is overall similar to that found in other  species , exhibitic genes , suggestL. noguchii WGS data  and an O-polymerase (Wzy). This biosynthesis system is complex and includes several proteins highly conserved in Gram-negative and Gram-positive bacteria that possess glycan polymers on their cell surfaces, such as LPS O-antigen, spore coats, enterobacterial common antigen, and outer capsules  the characteristic low GC content within the rfb clusters, ostensibly distinct from its flanking DNA segments, (ii) signs of genomic rearrangements (inversions) in the cluster’s surroundings, and (iii) the fact that identical/nearly identical rfb clusters were found in different Leptospira species. A similar HGT scenario of genomic islands has been described in different classes of Proteobacteria, concerning LPS(O-antigen)- and capsule polysaccharide(K-antigen)–encoding loci . PacBio SMRT sequencing was performed with RSII technology . De novo assembly was performed with HGAP v.4 (https://github.com/marbl/canu) (https://github.com/rrwick/Unicycler) (https://github.com/rrwick/Trycycler) (Genomic DNA was extracted from 100 ml of a 10HGAP v.4  availablbl/canu) , Unicyclicycler) , or Trycycycler) . The polhttps://www.ncbi.nlm.nih.gov/genbank/) or from the Institut Pasteur Bacterial Isolate Genome Sequence Database (https://bigsdb.pasteur.fr/leptospira/). The metadata for all isolates, including for those sequenced in this study, are summarized in Table S4. Genomes from strains of known serovar used for the rfb cluster comparisons were downloaded from Patric (https://www.patricbrc.org), and their metadata are summarized in Table S5. Plasmid sequences from other Leptospira species were also obtained from GenBank, and their associated metadata are summarized in Table S3, along with the general features of plasmids from the strains sequenced in this study. Of note, plasmids p2 from strain “barbudensis” and p5 from “IP1712055” were not included in the network analysis , in GenBank plain text format with header CDS annotations as obtained from Prokka.L. interrogans str. 56601 and L. kirschneri str. 200702274 as outgroups) were annotated using Prokka version 1.13.7   .Leptospira genomes with assigned serovars and less than 500 contigs (Table S5) were downloaded, and their rfb clusters, lipid A, and core oligosaccharide biosynthesis–encoding clusters were analyzed. Location of sites and gene cluster sequence extraction were done with the bioinformatics tools included in Emboss 6.6.0  were performed by protein-level cross-matching and subsequent network associations. Briefly, a pairwise comparison of each rfb cluster with one another was conducted using Blastp. The network connection was thereafter established using the previously generated Blast files and NetworkX version 2.6.2  was performed using available tools at https://mev.tm4.org. Functional annotation was done using eggnog-mapper v2 (L. noguchii strains were obtained from the Prokka annotation and then represented using the online version of shinyCircos (https://venyao.xyz/shinycircos/)  .Supplemental Data 3.rfb clusters (listed in Table S6), in GenBank plain text format with header CDS annotations as obtained from Prokka.DNA sequences of PRJNA803166, specifically with the following accession numbers: L. noguchii strain barbudensis, CP091967–CP091970; L. noguchii strain 201601331, CP091962–CP091966; L. noguchii strain IP1512017, CP091957–CP091961; L. noguchii strain IP1605021, CP091953–CP091956; L. noguchii strain IP1611024, CP091947–CP091952; L. noguchii strain IP1703027, CP091943–CP091946; L. noguchii strain IP1705032, CP091940–CP091942; L. noguchii strain IP1709037, CP091936–CP091939; L. noguchii strain IP1712055, CP091928–CP091935; L. noguchii strain IP1804061, CP092112–CP092116; L. noguchii strain bajan, JAKNBP000000000; and L. noguchii strain 201102933, JAKNBO000000000.The genome sequences have been deposited in DDBJ/ENA/GenBank under the BioProject


Currently, algae arouse a growing interest in the pharmaceutical and cosmetic area due to the fact that they have a great diversity of bioactive compounds with the potential for pharmacological and nutraceutical applications. Due to lifestyle modifications brought on by rapid urbanization, diabetes mellitus, a metabolic illness, is the third largest cause of death globally. The hunt for an efficient natural-based antidiabetic therapy is crucial to battling diabetes and the associated consequences due to the unfavorable side effects of currently available antidiabetic medications. Finding the possible advantages of algae for the control of diabetes is crucial for the creation of natural drugs. Many of algae’s metabolic processes produce bioactive secondary metabolites, which give algae their diverse chemical and biological features. Numerous studies have demonstrated the antioxidant and antidiabetic benefits of algae, mostly by blocking carbohydrate hydrolyzing enzyme activity, such as α-amylase and α-glucosidase. Additionally, bioactive components from algae can lessen diabetic symptoms in vivo. Therefore, the current review concentrates on the role of various secondary bioactive substances found naturally in algae and their potential as antioxidants and antidiabetic materials, as well as the urgent need to apply these substances in the pharmaceutical industry. The prevalence of diabetes has increased rapidly over the past few years, mainly in low- to middle-income countries, and has become one of the major causes of premature death worldwide. According to International Diabetes Federation (IDF) , there aDiabetes mellitus is an extended metabolic disorder of several etiologies, characterized by chronic hyperglycemia with the ailment of carbohydrate, fat, and also protein metabolism, which typically results from an absolute or relative lack of insulin, impaired effectiveness of insulin action, or tissue insensitivity to insulin .Insulin is a hormone produced by the pancreatic β-cells that functions to maintain the strict control of blood glucose. This hormone enables the tissues and cells of the body to utilize glucose for energy. If insulin is absent or its action is impaired due to tissue insensitivity, cells, and tissues are unable to uptake glucose, which results in its accumulation in the blood, and consequently, diabetes symptoms occur .Symptoms of diabetes often go unobserved because they can be attributed to many other causes, and some patients fail to notice cautioning signs or practice definite indicative symptoms . However, and other specific categories of diabetes that have several causes, such as pancreatic or drug-related diseases, monogenic diabetic syndrome, and chemical inducers [There are various categories of diabetes: type 1 diabetes, type 2 diabetes, gestational diabetes mellitusinducers . The twoinducers ,7. In thinducers . As a reinducers . PatientAlternatively, type 2 affects about 95% of people diagnosed with diabetes mellitus and are 40 years and above in age . Fast foThe majority of diabetes types across the world may be correlated to modern diets, sedentary lifestyles, and obesity. The mortality associated with diabetes is mainly a result of the augmented danger of several complications of this disease. Even though diabetes is primarily defined by chronic hyperglycemia, many diabetic patients, particularly those with type 2, have elevated blood pressure (hypertension), chronic high levels of insulin (hyperinsulinemia), and abnormal levels of cholesterol, triglycerides, and/or other blood lipids (hyperlipidemia). In addition, lipoprotein abnormalities are some of the most prevalent problems associated with type 2 diabetes . These c2 are catalyzed by oxidase enzymes to produce chemical energy or oxidation of substrates. These enzymes are potential sources of reduced Cu2+ derivatives in biological settings; they also produce O2•− during catalysis [2 to O2•− [2•− radicals to form hydrogen peroxide (H2O2) and/or further react to form the hydroxyl secondary radical (•−OH) as another type of the ROS [Biochemical processes in the body may produce intermediate products called reactive oxygen species (ROS). Among ROS, harmful free radicals have one or more unpaired electrons that make them very reactive with other molecules. Excess ROS may lead to an imbalance in its metabolism and in the body’s ability to detoxify or counteract the harmful oxidant effects of the free radicals ,13. Thisatalysis ,18,19. T to O2•− ,21. Dism the ROS ,19. Alth the ROS  and diab the ROS . This re the ROS . As a re the ROS .The treatment of diabetes requires changes in lifestyle patterns and necessitates lifelong adaptations. In order to prevent, delay, or stop the microvascular and macrovascular effects of diabetes, the diabetic patient should be provided with the necessary tools to achieve the best management of glycemia, blood pressure, and lipidemia . CurrentSome synthetic inhibitors of these enzymes are used clinically to manage or remedy diabetes, such as sulfonylureas, biguanide, glycosidase inhibitors, insulin, aldose reductase inhibitor, thiazolidinediones, carbamoyl methyl benzoic acid gliclazide, metformin, acarbose, and voglibose . ContinuAntioxidants are constituents that delay or avoid the oxidation process by neutralizing the free radicals scavenging in body cells . The maiNatural antioxidants derived from natural sources can react rapidly with free radicals and impede or alleviate the extent of oxidative deterioration . FurtherAlgae represent a great group of diverse organisms from dissimilar phylogenetic groups and also several taxonomical divisions. Generally, algae may be identified as plant-like organisms that are regularly photosynthetic and aquatic but without real roots, stems, leaves, and vascular tissues, besides having elementary reproductive structures. They are distributed worldwide in seawater, freshwater, soil, and wastewater . AccordiThe term seaweed refers to the large visible macroalgae growing and attaching to rocks along the seashore. Seaweeds have been used widely for multiple applications, such as human food, animal feeds, and fertilizers . They caNatural products derived from different algal species, such as alkaloids, flavonoids, terpenoids, steroids, and phenols, have received considerable attention over the years due to their diverse pharmacological properties, including their antioxidant and antidiabetic functions . Among tLikewise, flavonoids have antimicrobial, antioxidant, or free radical scavengers and spasmolytic activity . Other bArthrospira platensis cyanobacterium (formerly Spirulina platensis) (Chlorella vulgaris (Chlorophyta)  . Moreover, the phytochemical components in the different solvents of Hydrodictyon reticulatum (Chlorophyta) microalgal extracts showed various antioxidant activities using different assays [Schizochytrium limacinum  could demonstrate a potential source of bioactive peptides with antioxidant activities. Therefore, S. limacinum demonstrates interesting properties for nutraceutical production or is used directly as a dietary intervention to prevent diseases associated with free radicals.The therapeutic potential of microalgae was documented by many studies to be associated with their biological compounds, such as alkaloids, carotenoids, terpenoids, steroids, phlorotannins, phenolic compounds, halogenated ketones, alkanes, cyclic polysulfides, in addition to being rich in proteins, vitamins, pigments, lipids, and essential minerals . Butanolatensis) a showed rophyta) b methanophyceae) c was recabolites . Recent e stress . Additioe stress  informedt assays . Moavenit assays  reportedIn the same context, seaweeds contain various inorganic and organic substances that can benefit human health . Marine Centroceras clavulatum (Rhodophyta) exhibited high antioxidant activity with various tested assays [Ulva lactuca (Chlorophyta)  a, showedSargassum polycystum and the acetone extract of Sargassum wightii . In addition, the antioxidant analysis showed that protein hydrolysate-ultrafiltrate fractions FSPH-UF derived from Fucus spiralis   b exhibitS. wightii fraction obtained by S. wightii ethyl acetate extraction showed potent antioxidant activity using DPPH, ABTS, and FRAP assays. Also, the methanolic extract of the brown algae Turbinaria decurrens contained a high concentration of polyphenols and exhibited a broad spectrum of antioxidant activity by showing potent radical scavenging activity using ABTS and DPPH free radicals assays, as well as a high ability to reduce copper ions [Results from the study by Vijayan et al.  clearly per ions .T. decurrens showed the highest antioxidant effect of the tested extracts by using DPPH, reducing power, and total antioxidant capacity assays. In addition, the ethanol extract of Taonia atomaria brown algae   d establiPadina boryana , which was strongly correlated with its antioxidant activity. The enzymatically degraded polysaccharide from Sargassum fusiforme brown algae possessed superior antioxidant activity on scavenging HO˙, O2⋅− and DPPH˙ radicals [P. pavonica extracted with ethanol had superior antioxidant activity in hydrogen atom transfer assays of DPPH and ORAC, and with the electron transfer assays of FRAP [Likewise, the results of the investigation of Abhishek et al.  demonstrradicals . Additio of FRAP .Laurencia dendroidea had the highest antioxidant activity when evaluated by DPPH radical scavenging assay, with a recorded IC50 value of 312.09 μg/mL [Jania rubens (Rhodophyta)  (Ahnfeltiopsis pygmaea (formerly Gymnogongrus pygmaeus) was a valuable source of antioxidants to cure oxidative-stress triggered diseases.Additionally, the ethyl acetate fraction from the red seaweed 09 μg/mL . Moreove09 μg/mL  indicatedophyta) e exhibitred alga f possessdophyta) g, obtainRegular consumption of functional foods appears to be associated with improved antioxidant enzymes and the suppressed production of pro-inflammatory cytokines, insulin sensitivity, and hypocholesterolemia functions, which are considered essential to preventing and controlling diabetes mellitus. There have been indications that microalgae could be used as antidiabetic foods/ingredients  exopolysaccharides extract compared to the other studied microalgae. The aqueous and methanolic extracts of Euglena cantabrica (Euglenozoa) microalgae displayed the highest antioxidant activity due to the presence of high phenolics content on these extracts [Fischerella BS1-EG (Cyanobacteria) demonstrated potential inhibition activity for α-glucosidase of 7.5%, indicating its antidiabetic effect. Also, fucoxanthin extracted from Phaeodactylum tricornutum (Bacillariophyta) showed strong inhibitory activity toward α-amylase in a concentration-dependent manner, with an IC50 value of 0.68 mmol/L and inhibitory activity against α-glucosidase, with an IC50 value of 4.75 mmol/L [Nannochloropsis oculata exhibited the highest level of α-amylase inhibition of 78.52% at a maximum concentration of 1000 μg/mL, with an IC50 value of 121.96 μg/mL. The same extract exhibited a significant inhibitory action on the α-glucosidase enzyme by 80.42% at the concentration of 1000 μg/mL with an IC50 value of 178.53 μg/mL [Gouda et al.  reportedi et al.  showed textracts . Resultsextracts  revealed5 mmol/L . Likewis53 μg/mL .A. platensis exhibited the maximum α-amylase enzyme inhibition activity of 96.46% with an IC50 value of 13.31 mg/mL compared to the pharmaceutical drug acarbose recorded 1.59 mg/mL. The same extract was also having a strong α-glucosidase inhibitory activity of 97.42% and an IC50 value of 9.56 mg/mL compared to acarbose’s IC50 value of 1.03 mg/mL. Moreover, the study by Priatni etal. [Porphyridium sp. (Rhodophyta) was the strongest among the studied microalgae in inhibiting the α-glucosidase enzyme activity (12.63%).Recently, Gheda et al.  recordedni etal.  showed tUlva reticulata (Chlorophyta) ethanol extract showed a maximum α-amylase and α-glucosidase inhibition activity of 89.1% and 79.55%, respectively. Moreover, different green (Chlorophyta) seaweed extracts, such as Ulva intestinalis   and α-glucosidase (IC50 289.7 μg/mL), and thus can prevent postprandial hyperglycemia [Spatoglossum asperum (Ochrophyta) showed a significant α-glucosidase inhibitory activity due to the presence of phytochemicals, such as flavonoids, tannins, and saponins [Seaweeds contain various inorganic and organic substances that can benefit human health . Due to tinalis) h, Chaetoha aerea i, and Clupestris j proved ase IC50 9.7 μg/mLsaponins .Hormophysa cuneiformis (Ochrophyta) was the most active, recording an inhibition activity of 53% for the α-glucosidase enzyme at the highest investigated concentration (1000 μg/mL) with an IC50 of 676.9 μg/mL [Fucus vesiculosus   [The methanolic extract of ntration 0 μg/mL wphyceae) k showed 6 μg/mL) .T. decurrens brown macroalgae presented a potent inhibitory activity (IC50 of 11 μg/mL) for α-glucosidase in vitro, as compared to using acarbose and metformin as antidiabetic drugs. Besides, the acetone extract of T. decurrens also showed the highest inhibitory effects for both carbohydrate hydrolyzing enzymes α-amylase (96.1%), with an IC50 value of 4.37 mg/mL and α-glucosidase (97.4%), with an IC50 value of 2.84 mg/mL, which was attributed to its total phenolic content capability [T. atomaria .Moreover, Tessema  reportedhyra sp. l on the 14 μg/mL . Similar14 μg/mL  revealedA. platensis cyanobacterium powder, showed antihyperglycemic activity by lowering the animal’s blood glucose level [A. platensis powder could reduce the adverse effect of hyperglycemia in alloxan-induced diabetic rats [A. platensis extract can aid in preventing diabetes or alleviate its adverse effects on blood parameters and the inflammatory phase [A. platensis methanol extract showed a significant reduction in elevated glucose levels, liver functions, renal functions, total bilirubin, and lipid profile. In addition, compared to the diabetic control group of rats, the same extract caused a regain in body weight loss, protein profile, albumin, hemoglobin, and HDL levels. The extract also improved the histological tissue damage due to diabetes induction in the liver and pancreatic tissues of the treated rats without causing any negative effects.Administration of cyanobacteria leads to regaining body weight in streptozotocin (STZ)-induced diabetic rats and also reverses the hepatic damage by renormalizing serum hepatic marker enzymes. Cyanobacteria show anti-hypoglycemic action through the potentiation of pancreatic insulin secretion from the intact β-cells of the islets. In a recent study, diabetic rabbits, when fed with se level . Moreovetic rats . Furtherry phase . In a stry phase , diabetiC. vulgaris green microalgae as an antioxidant supplement for diabetic rats boosted the recovery effect on their hematological parameters [P. tricornutum fucoxanthin extract. Likewise, Nasirian et al. [N. oculata microalgae was able to reverse the negative effect of STZ by reducing the lipid profile and glucose (except HDL-C), and by increasing insulin and HDL-C in diabetic rats. It also recovered the body weight loss in the diabetic rats. These effects were attributed to some components present in the microalgae powder, such as fibers content, lipid profile, and antioxidant pigments.Moreover, the effect of rameters . Also, Krameters  reportedn et al.  reportedU. lactuca ethyl acetate extract as an additive therapy during diabetes treatment. The extract could reverse the complications related to diabetes and was non-toxic to the important tissues. A promising protective effect of U. reticulata aqueous extracts against diabetic complications generated by STZ-mediated oxidative stress was reported in [U. reticulata for 45 days resulted in a significant reduction in fasting plasma glucose, thiobarbituric acid reactive substances (TBARS), and lipid hydroperoxides. Furthermore, using this extract elevated the activities of plasma insulin, vitamin E, and vitamin C and reduced glutathione (GSH) content when compared with the diabetic control group. A study by Labbaci and Boukortt [U. lactuca seaweed and its hydroethanolic extract mitigated insulin resistance, which plays a fundamental role in the pathogenesis of diabetes and helps regenerate damaged pancreatic β-cells. In addition, U. lactuca and its hydroethanolic extract may have anti-atherosclerotic effects by improving reverse cholesterol transport. Such results may have major therapeutic promise for helping to prevent the onset of complications in diabetic patients.Many studies documented the biological properties of green seaweeds. In a complementary study by Mohapatra et al. , the autorted in ,110. DiaBoukortt  indicateTurbinaria ornata exhibited antihyperlipidemic and antidiabetic activities through the reduction of blood glucose and other diabetic-boosted physiological changes [Padina boergesenii (Ochrophyta) was found to have a vital effect on the reduction of elevated blood glucose levels, kidney marker levels, and liver function lipid profile, in addition to its hepatoprotective activity [Polycladia myrica . In addition, the preprandial administration of the brown alga Ascophyllum nodosum extract was able to control the hyperglycemia of diabetic animals [Sargassum hystrix’s extracts at a dose of 300 mg/kg. This extract was able to lower the levels of preprandial and postprandial glucose levels of STZ-induced diabetic rats and reversed the weight loss of the rats, triglycerides, and cholesterol levels to their normal (control) levels. Moreover, this dose (300 mg/kg) had the best capability to prevent necrosis of the pancreas in diabetic rats [Sargassum oligocystum at a dose of 300 mg/kg had a healing effect on diabetic induced rats by reducing insulin resistance, decreasing glucose concentration and triglyceride, and regenerating of damaged pancreatic β-cells of the STZ-induced diabetic rats.Many researchers reported that most brown algae are rich in important secondary metabolites, which had in vivo antidiabetic activities . For exa changes . It was activity ,115. Likactivity  determina myrica m and Sir animals . The samtic rats . The dattic rats  mentioneT. decurrens acetone extract at 300 mg/kg dose exhibited antihyperglycemic activity against alloxan-induced diabetes by reducing the elevated blood glucose level, remarkably decreasing the liver and kidney functions, and reducing the hyperlipidemia related to diabetes. In addition, as compared to untreated diabetic rats, treatment with the same extract resulted in a recovery in body weight loss, total protein, albumin, and hemoglobin levels. Furthermore, treatment of rats with the same extract alleviated diabetes-related liver and pancreatic histopathological abnormalities.Recently, an in vivo investigation conducted by Abdel-Karim et al.  revealedGrateloupia lithophila methanolic extract, which could inhibit the diabetic enzymes α-amylase and α-glucosidase. Radhika and Priya [Acanthophora spicifera was able to reduce blood glucose levels and the hematological and biochemical parameters linked to diabetes and could also improve the loss in body weight. In the same manner, the red alga Gelidium amansii as a diet powder could control diabetes of STZ-induced diabetic rats via reducing their plasma glucose level, lipids, adipocytokines, and adipose tissue weight [Gracilaria opuntia against diabetes and the histological changes related to it was reported by Rayapu et al. [Gracilaria edulis possessed an inhibitory activity on the carbohydrate hydrolyzing enzymes [L. dendroidea could significantly suppress the glucose level of alloxan-diabetic mice, and the oral administration of the same extract was not toxic at a dose of 100 mg/kg of body weight, as determined by body weight changes and liver biochemical parameters.Concerning the biological activities of red seaweeds, Murugesan et al.  documentnd Priya  revealede weight . The effu et al. . Also, t enzymes . In vivo enzymes  revealedA. platensis powder, which showed antihyperglycemic activity by lowering their blood glucose levels. Similarly, the administration of A. platensis powder (400 mg/kg) could reduce the adverse effect of hyperglycemia in alloxan-induced diabetic rats [Arthrospira (Spirulina) brings its antihyperglycemic action may be through improving the pancreatic secretion of insulin from the β-cell islet or due to enhancing the transportation of blood glucose to the peripheral tissue. This was clearly demonstrated, according to the former study results, by the increased levels of insulin in the diabetic rats treated with Arthrospira.In a recent study , diabetitic rats . El-Baz tic rats  informedT. decurrens acetone extract administration (300 mg/kg body weight) in diabetic rats. These findings suggested that T. decurrens acetone extract might slow the absorption of dietary carbohydrates, hence preventing the rise in postprandial blood glucose levels. In a former study, extracts of some brown algae species exposed a beneficial effect in controlling postprandial glucose levels in diabetic obese rats [Through the same behavior, the increase in postprandial blood glucose level was significantly suppressed by ese rats .Sargassum ringgoldianum methanolic extract [P. boergesenii  aqueous extract [Sargassum longiotom “Selvaraj & Palanisamy” ethanolic extract [Hydroclathrus clathratus aqueous extract [T. ornata extract had a strong effect in lowering blood glucose levels in alloxan-induced diabetic rats, as established by the study of Husni et al. [S. wightii and U. lactuca showed remarkable effectiveness in lowering elevated glucose levels [Likewise,  extract  and P. b extract  demonstr extract  and with extract  showed ai et al. . In addie levels . In thise levels . Also, te levels .Alloxan-induced diabetes is accompanied by gradual body weight loss, which might be owing to increased muscle wasting or protein breakdown in the tissues . The ratSpirulina powder at different doses resulted in an increment in their body weights, and many studies reported similar findings. Pandey et al. [Limnospira maxima (formerly Spirulina maxima) showed a regain in their body weight, which may be well explained by either increased insulin secretion or increased food consumption. Likewise, oral administration of A. platensis aqueous extract to diabetic rats for 50 days led to an obvious restoring of their body weight, suggesting that this extract substantially improved the general health status and metabolic mechanisms by effective controlling or reversing gluconeogenesis [A. platensis powder at 400 mg/kg could significantly reduce the adverse effect of body weight loss in alloxan-inducted diabetic rats after witnessing a significant enhancement in body weight compared to diabetic control rats. Similarly, diabetic rabbits fed with A. platensis powder showed a noteworthy regain in their body weight loss [A. platensis methanol extract compared to the diabetic control (untreated) rats.Layam and Reddy  informedy et al.  stated togenesis . In addiogenesis  reportedght loss . Also, Gght loss  informedH. clathratus (brown seaweed) aqueous extract to restore body weight loss caused by diabetes induction in mice. The ethanol extract from different seaweeds, A. spicifera (Rhodophyta), Caulerpa scalpelliformis (Chlorophyta), and Padina tetrastomatica (Phaeophyceae), improved the loss of body weight of the experimental diabetic rats [S. wightii and U. lactuca demonstrated considerable activity in improving body weight loss in diabetic rats [T. decurrens acetone extract or standard Diabenor drug tended to reverse the loss of body weight due to diabetic-induced effects; meanwhile, the untreated diabetic rats had a significant decrease in body weight.In the same manner, Nagy  demonstrtic rats . Moreovetic rats . Likewistic rats  found thArthrospira might be due to the decreased level of blood glucose that automatically led to decreased Hb values [Arthrospira, which is a respectable source of iron, might contribute to raising the Hb levels. In a recent study, diabetic rabbits administrated with A. platensis powder showed increasing Hb levels [A. platensis extract.Many studies verified that the hemoglobin level is affected by the presence of glucose in the blood. The improvement in the level of Hb in animals supplemented by different doses of b values . Anotherb levels . Accordib levels , the Hb U. reticulata green seaweed in compensating for iron deficiency and boosting Hb levels. The treatment with ethanolic extract of Turbinaria conoides and methanolic extracts of S. wightii and T. conoides resulted in rising Hb levels, which was related to lowering blood glucose levels [P. boergesenii aqueous extract significantly increased Hb levels in diabetic rats [T. decurrens extract, which in turn, was directly proportional to glycosylated Hb level [In the same context, Banu and Mageswari  demonstre levels . Furthertic rats . LikewisHb level . Thus, iHb level  discoverAphanizomenon flos-aqua (Cyanobacteria) ethanolic extract and insulin-like protein [A. platensis methanolic extract exhibited a significant reduction. Similarly, the total bilirubin level in diabetic control rats was diminished following oral administration of various dosages of T. decurrens extract, as reported by Abdel-Karim et al. [Bilirubin concentrations may reflect the state of the liver and the type of damage it has received . Accordi protein . Bilirub protein . The totm et al. .A. platensis extract exhibited anti-inflammatory, antioxidant, membrane-stabilizing, and immune-correcting actions, and thus boosted hepatoprotective properties. These outcomes were in agreement with those mentioned by El-Baz et al. [A. platensis ethanolic extract could reduce AST and ALT levels. Also, Salem et al. [A. platensis powder and informed the notable reduction of hepatic enzyme activities.The levels of aminotransferase enzymes, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) in blood serum  are a prz et al. , who repm et al.  investigA. platensis powder showed a significant beneficial effect in reducing serum hepatic AST and ALT compared with the induced diabetic rats [A. platensis powder for the reduction of raised hepatic enzymes levels of diabetic rabbits. After administration of A. platensis extract as treatment, a decrease of the serum ALT and AST activities might trigger subsequent alleviation of the liver damage. The treatment with 15 mg/kg body weight dose of A. platensis extract was significantly effective in reduction of hepatic transaminase activities compared to the alloxan-diabetic control rats [Similarly, dietary supplementation of diabetic rats with tic rats ,144. In tic rats  documentrol rats .S. longiotom “Selvaraj & Palanisamy” nom. Inval. may prevent hepatic injury associated with diabetes. The extract could reduce the levels of serum glutamic-oxaloacetic transaminase (SGOT) and serum glutamic pyruvic transaminase (SGPT). These enzymes are enzyme markers that reflect the necrosis of the hepatocellular by liberating into the blood stream after damaging the cell membrane. Also, oral administration of H. clathratus aqueous extract resulted in a significant reduction in hepatic enzyme levels. [T. decurrens acetone extract could considerably compact the two parameters of ALT and AST levels in the blood serum, indicating liver function recovery.Furthermore, some studies investigated the effect of macroalgae as reducing agents for hepatic enzymes. Selvaraj and Kumar  suggeste levels. . Accordi levels. , amelior levels.  reportedThe most often used test for screening renal functions is the determination of blood urea. When combined with creatinine readings, urea levels can help in the differentiation of three kinds of azotemia . As the increased creatinine and urea levels represented a decrease in the glomerular filtration rate, the alloxan-induced diabetic rats demonstrated renal impairment. Thus, according to Kumar et al. , this waA. platensis extract exhibited a nephron-protective effect against diabetic-induced nephropathy. Likewise, Avdagić et al. [Arthrospira could decrease lipid peroxidation and elevate antioxidant levels, thus considerably modifying renal damage. El-Baz et al. [A. platensis ethanolic extract in reducing urea and creatinine levels to reach 31.00 and 0.96 mg/dl, respectively, when administrated to diabetic-induced rats. Also, Abbas et al. [Arthrospira on the decrement of urea and creatinine levels. As explained by Ripa et al. [A. platensis extract that improved the renal function via attenuation of the oxidative stress-mediated decline in kidney function. The same observations were informed by Gheda et al. [A. platensis methanolic extract.It has been reported that alloxan caused a considerable increase in serum urea and creatinine. In kidney tissue, alloxan increased the production of reactive oxygen species, increased protein carbonylation, and lipid peroxidation, and lowered intracellular antioxidant defense . As suggć et al.  reportedz et al.  reporteds et al.  reporteda et al. , this mia et al. , where tH. clathratus aqueous extract. Also, the water extract of P. boergesenii brown seaweed could efficiently lower the elevated levels of urea, uric acid, and creatinine in diabetic rat serum [T. decurrens extract had a considerable decreasing impact on blood urea and creatinine levels, notably at the dose of 300 mg/kg of body weight.Nagy  reportedat serum . Accordiat serum , The oraThe total protein concentration and percentage indicated by separate fractions may deviate significantly from normal values during disease . Total pA. platensis ethanolic extract at a dose of 15 mg/kg body weight was informed by Senthilkumar and John [A. platensis ethanolic extract at a dose of 15 mg/kg of body weight was reported to improve the decreased protein level related to STZ-induction in the diabetic rats [A. platensis powder at 15 mg/kg body weight dose was able to restore the reduced total protein levels as a reverse of diabetes effects. The same enhancement of the protein profile has been observed in the alloxan-induced diabetic rats treated with 15 mg/kg of A. platensis methanolic extract [A. platensis, besides its role in improving the hepatic function and/or its richness of proteins, as explained by Venkataraman [A. platensis is composed of numerous amino acids, so it may have become a direct source of protein for mice and produced several beneficial metabolic effects [The reduction of total protein content linked to diabetes may be a result of the decrease in the three major phases of protein secretion, intracellular transport and discharge, and/or due to increasing protein excretion . The roland John . A. plattic rats . Also, Stic rats  mentione extract . This imataraman . Further effects .P. boergesenii brown seaweed. Also, Abdel-Raouf et al. [H. cuneiformis brown alga in the promotion of total protein levels. The decrement of total protein in the induced diabetic rats was significantly improved upon the treatment with T. decurrens acetone extract at a dosage of 300 mg/kg of body weight, as mentioned by Abdel-Karim et al. [In the same direction, seaweeds were indorsed with the ability to mitigate the elevation in the total protein content related to diabetes disorders. The upgrading of total protein may be attributable to the marked change in the circulating amino acids level, hepatic amino acids uptake, and muscle output of different amino acid concentrations . This eff et al.  reportedm et al. .A. platensis ethanolic extract at 15 mg/kg of body weight could raise the reduced levels of albumin in diabetic rats [A. platensis methanolic extract, though the diabetic control rats exhibited a great reduction in the albumin levels. In the study conducted by Salem et al. [A. platensis powder (15 mg/kg) exhibited the same improvement effect on the albumin concentration. As previously explained for proteins, the enhancement of albumin levels may be induced due to the antioxidant activity and the stimulated immune effects of different algal extracts, which can improve disturbed liver functions [H. cuneiformis. In addition, Kumar et al. [P. boergesenii to treat the albumin reduction effect resulting from diabetes disorders. The same role was evidenced by Abdel-Karim et al. [T. decurrens acetone extract indicating amelioration of the adverse effects caused by diabetes induction.Hypoalbuminemia is a prevalent condition in diabetic animals that is usually linked to a decrease in total protein content . Diabetetic rats . Also, Gtic rats  informedm et al. , the treunctions . A signiunctions  after thr et al.  suggestem et al. , who detA. platensis powder of 15 mg/kg body weight dose and reversed the effect of the reduced HDL level as well. This hypolipidemic activity was possibly triggered by the existence of phenolic compounds in this powder or due to increasing the activity of lipoprotein lipase enzymes in the muscles while decreasing their activity in the adipose tissues [A. platensis methanolic extract in diabetic rats, which caused a significant decrease in serum lipid profile level.Diabetes frequently involves anomalous lipid metabolism in addition to faulty glucose metabolism, which is regarded as an additional metabolic condition in the diabetic complication series. The activation of lipoprotein lipase and lecithin acyl-cholesterol transferases enhanced the concentration of low-density lipoprotein cholesterol (LDL-C). The elevated levels of very-low-density lipoprotein cholesterol (VLDL-C) and triglycerides (TG) were followed by a decrease in high-density lipoprotein cholesterol (HDL-C) . Normall tissues . This al tissues . Similar tissues  through S. longiotom “Selvaraj & Palanisamy” nom. Inval. exerted a significant beneficial effect on the lipid profile by reducing TG, TC, and LDL levels and significantly increasing the HDL level in the experimental diabetic rats [H. clathratus caused a significant reduction in either the serum TG levels or TC and LDL-C levels in contrast to a significant elevation in HDL-C [S. wightii and U. lactuca resulted in the lowering of elevated levels of TG and TC in the diabetes-induced rats [P. boergesenii  was described by [Arthrospira powder at 200 mg/kg of body weight, except for the marked apoptosis of hepatocytes. This recovery effect may be a result of the antioxidant activity of the phenolic compounds present in the Arthrospira powder [A. platensis methanolic extract for 45 days. Therefore, according to studies conducted by El-Baky et al. [Arthrospira (Spirulina) supplementation could act as a potent anti-hepatotoxicity agent.The liver has a critical role in the excretion and removal of unwanted chemicals from the body. As a result of diabetes induction, characteristic histological changes frequently emerged that show liver disease modifications. The diabetic liver showed hydropic bulging, hepatocyte disarrangement, vacuolization of microvesicles with the removal of nuclei, granular disintegration, and necrosis of liver cells. Also, under diabetes conditions, Zhou et al.  and Abooribed by  to reverribed by  reporteda powder ,166. Thea powder , who shoy et al.  and Abdey et al. , ArthrosS. polycystum extract against damaged liver tissues was verified by Motshakeri et al. [H. clathratus aqueous extract against liver injury due to diabetes induction. Treatment with the ethyl acetate extracts of S. wightii and U. lactuca exhibited a great improvement in the hepatic morphological and physiological disorders caused by diabetes [T. conoides ethanolic extract, S. wightii methanolic extract, along with T. conoides and G. opuntia aqueous extracts.In the same direction, many studies mentioned the valuable curative role of macroalgal (seaweed) extracts for diabetes and its hepatic complications recovery. The hepatoprotective activity of i et al. . Likewisi et al.  reporteddiabetes . The necdiabetes . These eP. boergesenii aqueous extract had a protective effect on the injured livers of diabetic rats and reduced the exerted histopathological disorders. Similar findings on the recovery effect of T. decurrens acetone extract on the diabetic rats’ liver were described by Abdel-Karim et al. [Kumar et al.  reportedm et al.  after thThe pancreas is essential for the control of micronutrient metabolism. As a result, its tissues may be harmed intracellularly during diabetes induction. Due to the lack of visual inspection tools, there is insufficient information on the morphological changes of pancreatic islets with the progression of diabetes . MoreoveA. platensis powder showed hyperplasia in the β-cells of the pancreatic tissue and an increased number of Langerhans islets. A. platensis supplementation had a potent activity for free radical scavenging and reduced various indicators of toxicity, such as tissue damage in rats [A. platensis powder to reverse the pancreatic damage observed in diabetic animals. In addition, the histopathological investigation of the pancreas revealed that diabetic rats treated with 15 mg/kg of A. platensis extract were significantly improved. The histological architecture of the islets appeared with mild vacuolations compared to the diabetic controls [In the study by Abbas et al. , the aut in rats . The sam in rats , who repcontrols .H. clathratus aqueous extract was documented by Mir et al. [S. polycystum at 300 mg/kg dose was beneficial in alleviating the histological injuries in the pancreatic tissues in diabetic animals [P. boergesenii aqueous extract, which displayed a protective effect on pancreatic tissues, allowing for the restoration of the pancreas’ histological deformations [T. decurrens acetone extract at 300 mg/kg dosage had a beneficial impact on the wounded architecture of the pancreas and restored its normal cellular structure [Considering macroalgae, the antidiabetic activity of r et al.  in addit animals . These ormations . When cotructure .S. platensis methanolic extract [T. decurrens acetone extract [In addition, oral administration of normal rats with different doses of  extract , and T.  extract  did not The diverse anti-diabetic effects of bioactive algal components, which are only applicable to in vitro and in vivo treatments, are covered in this review. Numerous reports have been published on the various functions of the active components that have been extracted from algae. Together, these findings imply that active components derived from algae will be beneficial for the treatment of diabetes. As a result of this, significant anti-diabetic properties of the algal extracts may mark an intriguing advancement in the search for innovative functional uses in numerous industrial uses, including functional foods and pharmaceuticals. Algae-derived compounds proved their potent activity as a natural antioxidant agent and their ability to control postprandial blood glucose levels by inhibiting diabetes-linked enzymes. In addition, the algal extracts could improve the physiological parameters as well as reverse the histological damage related to diabetes mellitus in vivo. The finest possible nutrition and wellness are the goals of algae bioactive components. Despite these high expectations, no product lines have yet demonstrated that they are useful and commercially feasible. Although they are marketed as a functional food and medicine, their market performance falls short of expectations. However, consumer interest in employing natural bioactive compounds as medications has recently increased. Additionally, algae’s numerous biological processes have the potential to boost its value as a health-beneficial ingredient in pharmaceutical and functional food industries.


Streptococcus mutans genomes. Phage DNA is a major target of S. mutans spacers. S. mutans strains have also generated immunity against mobile DNA elements such as plasmids and integrative and conjugative elements. There may also be considerable immunity generated against bacterial DNA, although the relative contribution of self-targeting versus bona fide intra- or inter-species targeting needs to be investigated further. While there was clear evidence that these systems have acquired immunity against foreign DNA, there appeared to be minimal impact on horizontal gene transfer (HGT) constraints on a species-level. There was little or no impact on genome size, GC content and ‘openness’ of the pangenome when comparing between S. mutans strains with low or high CRISPR spacer loads. In summary, while there is evidence of CRISPR spacer acquisition against self and foreign DNA, CRISPR-Cas does not act as a barrier on the expansion of the S. mutans accessory genome.CRISPR-Cas is a bacterial immune system that restricts the acquisition of mobile DNA elements. These systems provide immunity against foreign DNA by encoding CRISPR spacers that help target DNA if it re-enters the cell. In this way, CRISPR spacers are a type of molecular tape recorder of foreign DNA encountered by the host microorganism. Here, we extracted ∼8,000 CRISPR spacers from a collection of over three hundred  Streptococcus mutans is a Gram positive bacteria that inhabits the oral cavity and has a strong association with causing dental caries (tooth decay)   across the species  . It has scovered . These oer (HGT) . The abi species . There adiseases .S. mutans strains from a collection of 477 genomes  domain compared with the UA159 Cas9 nuclease -Cas(CRISPR-associated protein) system in 95% of  genomes . CRISPR-ving RNA , are fou. mutans . A subst editing . It is l editing . Interfenuclease . Some pr strains . With ouS. mutans has a large pan-genome is in conflict with the high frequency of CRISPR-Cas systems across the species. These systems have primarily been considered as a phage defense mechanism. However, there is experimental evidence that CRISPR-Cas systems can limit other types of HGT  in laboratory settings including in S. mutans . For this study CRISPR spacers were isolated from 335 S. mutans genomes. Incomplete assemblies were evident in the other S. mutans genomes, likely because of Illumina based whole genome sequencing, leading to difficulty in identifying repeat regions .We have a collection of 477 Identification and data curation of the CRISPR systems found in our clinical isolates was conducted at the University of Florida HiPerGator cluster computer with the CRISPRCasFinder  version S. mutans. Phage DNA sequences were downloaded from NCBI . ICE sequences were downloaded from the ICE database ICEberg 2.0 (https://bioinfo-mml.sjtu.edu.cn/ICEberg2/download.html) (S. mutans plasmid sequences were compiled (available on the Github platform) , plasmids, and ad.html) . Plasmidad.html) . In addilatform) . ParametStreptococcus genus, annotations with Prokka were defined within Bacteria and Streptococci for better gene predictions. Multilocus sequence typing (MLST) was conducted with Roary , and other bacterial DNA. In total, 1913 spacers (23.7%) mapped to these targets  (S. mutans strains that carry CRISPR-Cas systems have phage targeting spacers (against M102AD or M102 or ɸAPCM01). The high frequency of CRISPR-Cas systems and anti-phage spacers would likely limit the efficacy of phage therapy in S. mutans.Isolation of  samples  and smHB plaque) . To undePlasmids. CRISPR spacers are also naturally acquired against plasmid DNA . When aligned to this region the CRISPR spacers qualitatively appear to have been generated to target the integrase and secretion system components instead of hypothetical regions  are another type of mobile genetic element that can transfer between bacterial species and strains . Sequencications . In the ed genes . Most of regions . The fun systems . PreventS. mutans has gained immunity against inter-species transfer of mobile genetic elements we next wanted to explore if CRISPR spacers have been acquired more generally against S. mutans strain DNA. To do this, we searched for CRISPR spacer hits within genes encoded by S. mutans across all the genomes included in this study. A total of sixty-three annotated genes are the target of CRISPR spacers , 22 reside in the core genome (>99% of genomes), 4 are soft core genes (95–99% of genomes), 17 are shell genes (15–95% of genomes), and 20 are cloud genes (<15% of genomes) (S. mutans core genes are over-represented given that core genes only account for 5% of the S. mutans pangenome. This is compared with cloud genes which make up 85% of the pangenome. Notable S. mutans gene CRISPR targets include the clp system (gtfC glucosyltransferase (spaP (fruA and lacF) . Several of the CRISPR spacers that target the S. mutans pangenome are likely targeting mobile DNA elements. For example, spacers against tyrosine recombinases , conjugal transfer protein traG, and the Int-Tn transposase from the ICE Tn916. To visualize the extent of CRISPR spacer acquisition against S. mutans genomes, spacers were mapped to two strains, UA159 and NN2025 (clpP (protein homeostasis), rexB (putative exonuclease) and a hypothetical protein.With the observation that  spacers . In addi spacers  are alsogenomes) . CRISPR p system , the gtfnsferase , the celse   and seved NN2025 . The numS. mutans strains  S. mutans genes . Our analysis shows that increased CRISPR spacer acquisition does not lead to reduced genome size in S. mutans.Analysis of edes HGT . Howeveraumannii . Here, wy others . For ourcomputed . We founS. mutans CRISPR spacers was 38.2% (S. mutans genome (36.8%). Phages that infect S. mutans, M102AD, M102, and SMHBZ8 have GC content of 39.6%, 39.21%, and 38.8% respectively (S. mutans genomes was consistent for strains with 1–20 (36.80%), 20–40 (36.82%) and 40–60 (36.79%) CRISPR spacers (t-test p <0.05). Although only nominally lower than the other groups, this might indicate a reduction in the acquisition of higher GC foreign DNA among strains with 60–80 CRISPR spacers.By limiting HGT CRISPR-Cas could also affect the GC content of host genomes by reducing the acquisition of higher or lower GC foreign DNA. The average GC content across all as 38.2%  which maectively . The GC  spacers . A signiS. mutans pangenome. In our analysis including 335 S. mutans genomes, the pangenome size was calculated to include 15,062 genes. There were 822 genes in the core genome (>99% of genomes), 496 soft core genes (95–99% of genomes), 982 shell genes (15–95% of genomes), and 12,762 cloud genes (<15% of genomes). Next, pangenome analysis was conducted on two groups, one with a low CRISPR spacer load  and another with a high CRISPR spacer load . For both groups we generated pie charts which show the percentage of genes located in the different gene clusters   . The lowS. mutans isolates. Although there is considerable ‘dark matter’ (undetected CRISPR spacer sequences), phage are the main target of S. mutans spacers. Isolates have also generated immunity against mobile DNA elements such as plasmids and ICEs. There may also be considerable immunity generated against bacterial DNA, although the relative contribution of self-targeting versus bona fide intra- or inter-species targeting needs to be investigated further. Collectively, CRISPR-Cas systems and acquired spacers are abundant across all S. mutans strains. While there is clear evidence that these systems have acquired immunity against foreign DNA, there appears to be minimal impact on HGT constraints on a species-level. There was little or minimal impact on genome size, GC content and ‘openness’ of the pangenome. There are several possible explanations for why there is a minimal impact on HGT and these include 1) the systems have specificity for phage infection, 2) self-targeting has led to a loss of activity, 3) the systems are highly regulated to only acquire immunity under certain conditions, 4) competence acquired DNA is single stranded and the CRISPR-Cas systems are less active against ssDNA, 5) spacer load may be an inaccurate measure of CRISPR-Cas activity and 6) target or spacer DNA has changed so that immunity no longer functions. A deeper understanding of the impact of these systems on the biology and evolution of S. mutans will likely require functional studies and/or model systems for interrogating spacer acquisition.Here, we have generated a comprehensive ‘memory’ of DNA that has attacked a diverse panel of


Legionella pneumophila is the causative agent of a severe type of pneumonia (lung infection) called Legionnaires’ disease. It is emerging as an antibiotic-resistant strain day by day. Hence, identifying novel drug targets and vaccine candidates is essential to fight against this pathogen. Here, attempts were taken through a subtractive genomics approach on the complete proteome of L. pneumophila to address the challenges of multidrug resistance. A total of 2,930 proteins from L. pneumophila proteome were investigated through diverse subtractive proteomics approaches, e.g., identification of human nonhomologous and pathogen-specific essential proteins, druggability and “anti-target” analysis, subcellular localization prediction, human microbiome nonhomology screening, and protein-protein interaction studies to find out effective drug and vaccine targets. Only three fulfilled these criteria and were proposed as novel drug targets against L. pneumophila. Furthermore, outer membrane protein TolB was identified as a potential vaccine target with a better antigenicity score. Antigenicity and transmembrane topology screening, allergenicity and toxicity assessment, population coverage analysis, and a molecular docking approach were adopted to generate the most potent epitopes. The final vaccine was constructed by the combination of highly immunogenic epitopes, along with suitable adjuvant and linkers. The designed vaccine construct showed higher binding interaction with different major histocompatibility complex (MHC) molecules and human immune TLR-2 receptors with minimum deformability at the molecular level. The present study aids the development of novel therapeutics and vaccine candidates for efficient treatment and prevention of L. pneumophila infections. However, further wet-lab-based phenotypic and genomic investigations and in vivo trials are highly recommended to validate our prediction experimentally.IMPORTANCELegionella pneumophila is a human pathogen distributed worldwide, causing Legionnaires’ disease (LD), a severe form of pneumonia and respiratory tract infection. L. pneumophila is emerging as an antibiotic-resistant strain, and controlling LD is now difficult. Hence, developing novel drugs and vaccines against L. pneumophila is a major research priority. Here, the complete proteome of L. pneumophila was considered for subtractive genomics approaches to address the challenge of antimicrobial resistance. Our subtractive proteomics approach identified three potential drug targets that are promising for future application. Furthermore, a possible vaccine candidate, “outer membrane protein TolB,” was proposed using reverse vaccinology analysis. The constructed vaccine candidate showed higher binding interaction with MHC molecules and human immune TLR-2 receptors at the molecular level. Overall, the present study aids in developing novel therapeutics and vaccine candidates for efficient treatment of the infections caused by L. pneumophila. Legionella pneumophila is a human pathogen distributed worldwide within freshwater and biofilms , a severe form of pneumonia and respiratory tract infection , the type II secretion system, and several outer membrane proteins (OMPs), i.e., outer membrane vesicles, peptidoglycan-associated lipoprotein, major secreted phospholipase (PlaA), major cell-associated phospholipase A/lysophospholipase A (PlaB), major OMP, PlaC, macrophage infectivity potentiator, Hsp60, and FeoB  to widely used antibiotics such as azithromycin, ceftriaxone, rifampicin, tigecycline, ciprofloxacin, moxifloxacin, doxycycline, erythromycin, levofloxacin, and clarithromycin , was attributed to macrolide resistance in the L. pneumophila pathogen  and fluoroquinolones  are the standard treatment for nfection , 21. Curnfection . A combinfection . The emenfection . Recent hromycin , 26. Deshromycin , 28. Horhromycin . Naturalhromycin . Antibioolerance 0 to 100%nous DNA . Horizonnous DNA . Even trnous DNA . L. pneuce genes . LpeA, apathogen . Researccrolides . Mutatiosistance . Therefosistance –38. Amonistance –. The maiistance –. Along wbstacles . Such vattenuate , 43. Morttenuate . Epitopettenuate –47. The tenuate –. This aptenuate ––52.L. pneumophila subsp. pneumophila (strain Philadelphia 1/ATCC 33152/DSM 7513). We particularly considered the key essential or survival proteins of the pathogen, which are nonhomologous to the host as well as host microbiota. We screened for outer membrane proteins (OMPs) and B- or T-cell epitopes. At the same time, we performed the human microbiome nonhomologous analysis. The highest scoring OMPs and epitopes will facilitate future in vitro and in vivo tests to produce drugs and suitable vaccine against intracellular L. pneumophila infection.In this study, we utilized an in-depth subtractive genomics and reverse vaccinology approach to identify novel therapeutic drug and vaccine targets in L. pneumophila was employed to identify novel drug and vaccine targets through subtractive genomics and reverse vaccinology approaches. The subtractive genomics approach is a continuous process where irrelevant or nontarget sequences are allowed to be subtracted from a vast pool of initially retrieved sequences. Here, a list of human “anti-target” proteins was used to cross-check with the screened drug target alongside other common procedures of subtractive genomics. Moreover, the human microbiome was also checked with the screened putative drug candidates since the unintentional interference of proteins available in the gut microflora may cause adverse consequences on host physiology. After successfully screening sequences, we added reverse vaccinology approaches to get an insight into the potential design of vaccine candidates. During the entire procedure, the study utilized up-to-date bioinformatics approaches to discover novel therapeutics against L. pneumophila. The overall workflow has been illustrated in L. pneumophila, including the number of proteins screened in each step, are summarized in Genome-wide proteome exploration of The whole proteome retrieved from UniProt (Proteome ID UP000000609) containing 2,930 proteins was analyzed in the CD-Hit tool using the threshold value 0.6 to eliminate paralogous and duplicate sequences showing >60% sequence identity. Paralogous protein sequences were removed, leaving 2,878 nonparalogous protein sequences.BLASTp screened nonparalogous protein sequences through Ensembl genome browser 92. A total of 868 proteins were found to be similar to the human genome, which was excluded, leaving a total of 2,010 nonhomologous proteins.L. pneumophila.Among the 2,010 nonhomologous proteins, only 125 protein sequences showed similarity in sequence identity with the essential proteins enlisted in the Database of Essential Genes (DEG) server from 48 bacterial strains. These 125 proteins are listed as essential proteins and are responsible for the survival of L. pneumophila were identified from the KEGG server, only 37 were detected as unique for the pathogen . The result from the KAAS server at KEGG revealed that 122 proteins among 125 had assigned KEGG Orthology (KO) identifiers. Of the 122 proteins, only 4 were involved in unique metabolic pathways . However, the study failed to predict the subcellular localization of the hypothetical protein (tr|Q5ZYU2) since these four servers generated different predictions.L. pneumophila, comprising of 13 cytoplasmic and 12 membrane proteins. Antigenicity analysis of membrane protein revealed that only one protein was nonantigenic, whereas the remaining 11 showed antigenicity scores of >0.4 according to the VaxiJen server (Only seven proteins showed similarity with the available drug targets  in the Dn server . FurtherProteins that are important for human cellular homeostasis and trigger hazardous side effects under the influence of a drug are termed “anti-targets” . To avoiL. pneumophila based on protein-protein interaction (PPI) study  interacted with proteins involved in cell wall biosynthesis pathways (tig)” had interactions with proteins that were mainly involved in binding with different rRNA and tRNA molecules (mviN)” interacted with proteins that were connected to the peptidoglycan biosynthesis pathway and transportation of lipid-linked peptidoglycan precursors (kup1),” presented a single interaction (tolB)” showed close interactions with other proteins  study . PPIs inpathways , whereasolecules . Moreoveecursors , where “eraction , and as proteins , mainly Q5ZV69), was modeled to determine three-dimensional (3D) structure through EasyModeller 4.0 . Complete results of template selection for homology modeling are provided in Table S6. The best structure generated for each target was optimized by the ModRefiner server. The refined protein TolB (Q5ZV69) model had a root mean square deviation (RMSD) of 0.1444 and a template modeling (TM) score of 0.9783 in the initial model. After refinement, the Protein Data Bank (PDB) structure of each protein was evaluated using PROCHECK and ERRAT. Psi-Phi pains in Ramachandran plots revealed that protein TolB (Q5ZV69) had 91.9% residues in the most favored regions, 7.8% in the additional allowed regions, and 0.3% in the generously allowed regions . According to ERRAT, the overall quality factor of this protein was 76.1084 . An ERRAT score of 50 is usually acceptable, and the value explains the statistics of nonbonded interaction molecules. This indicates that the modeled structure is reliable and stable.The selected single vaccine candidate, protein TolB (Legionella pneumophila (LEGPH) protein TolB (Q5ZV69) was selected based on PPI analysis with a total antigenicity score of 0.6740 for vaccine design against Legionella. The ProtParam tool was used to analyze the vaccine proteins’ physicochemical properties (see Table S7). The molecular weight (45.38 kDa) of protein TolB indicated its good antigenic potential. At the same time, the theoretical pI of 8.67 showed that the protein would have a net positive charge below the pI and vice versa. The extinction coefficient was 34,380 at 0.1% absorption, assuming all cysteine residues are reduced. The GRAVY (grand average of hydropathy) value −0.204 described the hydrophilic nature of the protein, whereas the aliphatic index 89.81 ensured the thermostability of the protein. The instability index (38.09) classified the protein as a stable one. In addition, various immunogenic epitopes from LEGPH protein TolB were identified as potential T-cell epitopes that can bind to various HLA-A and HLA-B cells with greater binding affinity. Epitopes that bind to the maximum number of HLA cells were selected.From the top membrane proteins, The best LEGPH protein TolB epitopes were ranked based on the topology of the transmembrane and the antigenicity score . All indToxinPred server predicted the relative toxicity of the top epitopes . Again, B-cell epitopes of the selected protein were generated using a total of six algorithms . For envMycobacterium tuberculosis HABA protein (AGV15514.1)—were used to construct vaccines. The adjuvants were added at the N-terminal end of the vaccine construct, which helps the vaccine to create a more robust immune response in the patient’s body. The EAAAK, GGGS, GPGPG, and KK linkers were used to connect the epitopes in the correct position. The PADRE sequence was conjugated with the vaccine construct to ensure the maximum major histocompatibility complex class II (MHC-II) allele coverage. Three constructs with 476 (V1), 561 (V2), and 590 (V3) amino acid residues were designed and used for analysis (Both clusters and singletons (which contain a single epitope) were used to construct the final vaccine molecules. Three adjuvants—beta-defensin, ribosomal protein L7/L12, and analysis . A schemEscherichia coliin vivo. The estimated extinction rate and an aliphatic index were 41,370 and 61.74, respectively. The protein’s computed GRAVY value was −0.545, while the instability index (33.51) classified the protein as stable. Constructed V1 was, in contrast, characterized by a 6.30% alpha-helix, a 24.78% sheet, and a 68.92% coil structure .The results revealed construct V1’s superiority due to a greater antigenicity score (0.603) and nonallergenic behavior . The finP value was 5.30e−11, which ensured better-quality modeling of the proposed vaccine. Of the residues, 81.4, 11.8, and 6.8% were in the favored, allowed, and outlier regions, respectively, prior to refinement. However, 88.4% of the residues were in the favored region after refining. A Ramachandran plot showed that 8.4%- and 3.2% of the residues were in the allowed and outlier regions (2YMU (chain A) from the PDB database was detected as the most suitable V1 template, and a single-domain 3D model was created through the Raptor X server . All 476 regions . Homolog regions .Docking’s study revealed that construct V1 bound in the groove of different HLAs with minimal binding energy, which was biologically significant . The preThe trajectory of the molecular dynamics (MD) simulation of the docked complex was analyzed to plot RMSD, root mean square fluctuation (RMSF), and radius of gyration (Rg) plots. RMSD plot  of the sE. coli K-12. The optimized codons were introduced into vector pET28a(+) and the restriction sites HindIII and BamHI. A 6,346-bp clone was created .The optimized codon adaptation index was 0.95, and the GC content of that sequence was 54.01%. These results showed better expression in L. pneumophila is one of the main causative agents showing high resistance against many antibiotics. Although clinical studies of antibiotic treatment against Legionnaires’ disease have been reported, the number of such cases is insufficient  was searched and downloaded from the NCBI protein database. Proteins involved in several common cellular systems emerged as homologous with the same functions between humans and bacteria during evolution , was analyzed to develop a potential and highly immunogenic vaccine candidate against L. pneumophila. Tol proteins are crucial for the survival and pathogenicity of many bacterial pathogens, such as Escherichia coli, Haemophilus ducreyi, Vibrio cholerae, and Salmonella enterica  in the future to lessen the disease caused by  effects . For exa effects , ebrotid effects , and tro effects  were witpathogen  and wereogen Q5ZV, was anaenterica . Proteinogen Q5ZV, was anaenterica . TolB isenterica , 73. Apa and pal . Tol-Pal and pal . It was  mutants . Therefo mutants , 78. In vaccines , 80. Thevaccines . Literatneumonia . Therefonea pigs , 84. Howgression . Althoug its use . Other a elegans , Drosophnogaster , wax motnogaster , and amonogaster , have beitations , 88. HenL. pneumophila and may help to reduce the rate of both mortality and morbidity caused by this pathogen. We mainly considered the key that is a must for survival of the pathogen, nonhomologous to the human, as well as human microbiota, and screened out B- and T-cell epitopes of OMPs. The highest scoring OMPs and epitopes will facilitate future wet lab-based experiments to develop drugs and suitable vaccine candidates against intracellular L. pneumophila infection. After intensive analysis, only three proteins were identified and proposed as novel therapeutic targets against L. pneumophila. Only OMP TolB was identified as a potential vaccine candidate with a better antigenicity score. However, we suggest further in vitro and in vivo laboratory trials to validate our prediction.In conclusion, by using a subtractive genomics and reverse vaccinology approach, we are trying to develop novel therapeutics against L. pneumophila subsp. pneumophila (strain Philadelphia 1/ATCC 33152/DSM 7513) (Proteome ID UP000000609), was retrieved from UniProt containing total 2930 proteins (http://weizhong-lab.ucsd.edu/cdhit-web-server/cgi-bin/index.cgi) (The entire proteome of proteins  which wedex.cgi) . The seqL. pneumophila subsp. pneumophila is to identify pathogen-specific proteins that are nonhomologous to the human. BLASTp analysis was carried out on nonparalogous proteins against the reference proteome of Homo sapiens using a threshold expectation value  of 10−3 through Ensembl (https://uswest.ensembl.org/Multi/Tools/Blast?db=core)  . Proteindb=core) .−10 and a minimum bit-score cutoff of 100 as parameters, but the hits with a E value of ≤10−100, an identity of ≥25%, and the same annotated function of the query were selected as essential proteins. These essential proteins were further subjected to metabolic pathway analysis.Nonhomologous proteins were subjected to the DEG . DEG conL. pneumophila subsp. pneumophila, respectively, all of the metabolic pathways present in the host (H. sapiens) and the pathogen were collected separately. A manual comparison was made to identify the metabolic pathways only present in the pathogen as unique to L. pneumophila subsp. pneumophila using the KEGG pathway database (L. pneumophila subsp. pneumophila were screened by BLASTp through KAAS (KEGG Automatic Annotation Server) (The Kyoto Encyclopedia of Genes and Genomes (KEGG) includes complete metabolic pathways in living organisms . Using tdatabase , whereas Server)  at KEGG  Server)  was usedL. pneumophila subsp. pneumophila proteins can be identified in five viable subcellular locations: cytoplasm, inner membrane, periplasm, outer membrane, and extracellular. Cytoplasmic proteins can be used as targets for medications, while surface membrane proteins can be used for both the medication and vaccine (http://www.psort.org/psortb/index.html) (http://cello.life.nctu.edu.tw/) (https://webs.iiitd.edu.in/raghava/pslpred/index.html)  , CELLO vedu.tw/) , and ngLedu.tw/)  were useex.html)  was usedL. pneumophila subsp. pneumophila drug targets were subjected to VaxiJen v2.0, selecting a threshold value of 0.4. Proteins that showed antigenicity prediction >0.4 in VaxiJen v2.0 were transferred for the next step as potential vaccine candidates.DrugBank Database 5.1.0  containsFor humans, the human ether-a-go-go-related gene (hERG), constitutive androstane receptor (CAR), pregnane X receptor (PXR), and P-glycoproteins (P-gp) are anti-targets or alternative drug targets for host protein candidates. Some receptive membranes are also classified as “anti-target.” They are adrenergic α1a, dopaminergic D2, muscarinic M1, and serotonergic 5-HT2C. A total of 210 human “anti-targets” have been identified in the literature , and thehttps://blast.ncbi.nlm.nih.gov/Blast.cgi) using an E value cutoff score of 1 against the data set present in Human Microbiome Project server (https://www.hmpdacc.org/hmp/) “43021 (BioProject)” ” . The Humroject)” . ProteinPPI analysis for selected shortlisted proteins were predicted using STRING . The dathttps://saves.mbi.ucla.edu/) to get the stereochemical quality of the model.Since no exact Protein Data Bank (PDB) structure was available for the selected proteins, a BLASTp analysis was carried out in the NCBI database for each protein, where PDB proteins were chosen as the database. The templates were chosen for homology modeling considering a sequence identity of ≥85 and a query cover of ≥30. EasyModeller 4.0 software generated the best 3D structure of each selected therapeutic protein . The besAccording to PPI analysis and antigenicity, the final vaccine target was selected among the membrane-associated proteins. The ProtParam  tool shoThe TMHMM server predicted transmembrane helices in the protein , while thttps://webs.iiitd.edu.in/raghava/toxinpred2/) (http:/tools.iedb.org/conservancy/) (The server ToxinPred (npred2/)  estimatervancy/) .Six different IEDB algorithms—the Kolaskar and Tongaonkar antigenicity scale , the KarMycobacterium tuberculosis HABA protein (AGV15514.1), have produced three separate vaccine molecules. Adjuvants interact with TLRs and cause immune activation by polarizing CTL reactions , the adjuvants, and the corresponding linkers. Three specific adjuvants, i.e., beta-defensin, ribosomal protein L7/L12, and eactions . The betepitopes , 137. ToAlgPred v.2.0  sever waThe RaptorX server generated a tertiary structure of the constructs based on the degree of similarity between the target protein and accessible template structure in the PDB database , 142. ReInflammations caused by bacterial antigen are involved with TLR-2 immune receptors present over the immune cells , 147. Th− ions were added to the system. The system underwent subsequent NVT and NPT ensemble MD simulations for 500 ps each time to maintain temperature and pressure. The system then underwent 100-ns MD simulation under equivalence at 300K and 1 bar. The simulation trajectory was then analyzed to plot the RMSD, RMSF, and Rg results. Finally, molecular interactions of the vaccine-TLR complex were analyzed using the PDBsum webserver to gain structural insights into the docked complex (Molecular dynamics simulation is an important step for analyzing the stability of a docked complex. Here, we evaluate the docked complex of TLR-2 and our V1 vaccine construct for stability. MD simulation was performed using the GROMACS 2020.1 package  using th complex .E. coli. The procedure was conducted by the JCAT server (A codon adaptation technique was used for greater expression of the vaccine protein in T server , while p