Purification and Characterization of a Novel Alginate Lyase from a Marine Streptomyces Species Isolated from Seaweed

Alginate, a natural polysaccharide derived from brown seaweed, is finding multiple applications in biomedicine via its transformation through chemical, physical, and, increasingly, enzymatic processes. In this study a novel alginate lyase, AlyDS44, was purified and characterized from a marine actinobacterium, Streptomyces luridiscabiei, which was isolated from decomposing seaweed. The purified enzyme had a specific activity of 108.6 U/mg, with a molecular weight of 28.6 kDa, and was composed of 260 amino acid residues. AlyDS44 is a bifunctional alginate lyase, active on both polyguluronate and polymannuronate, though it preferentially degrades polyguluronate. The optimal pH of this enzyme is 8.5 and the optimal temperature is 45 °C. It is a salt-tolerant alginate lyase with an optimal activity at 0.6 M NaCl. Metal ions Mn²⁺, Co²⁺, and Fe²⁺ increased the alginate degrading activity, but it was inhibited in the presence of Zn²⁺ and Cu²⁺. The highly conserved regions of its amino acid sequences indicated that AlyDS44 belongs to the polysaccharide lyase family 7. The main breakdown products of the enzyme on alginate were disaccharides, trisaccharides, and tetrasaccharides, which demonstrated that this enzyme acted as an endo-type alginate lyase. AlyDS44 is a novel enzyme, with the potential for efficient production of alginate oligosaccharides with low degrees of polymerization.     

Biochemical Characterization and Cold-Adaption Mechanism of a PL-17 Family Alginate Lyase Aly23 from Marine Bacterium Pseudoalteromonas sp. ASY5 and Its Application for Oligosaccharides Production

As an important enzyme involved in the marine carbon cycle, alginate lyase has received extensive attention because of its excellent degradation ability on brown algae, which is widely utilized for alginate oligosaccharide preparation or bioethanol production. In comparison with endo-type alginate lyases (PL-5, PL-7, and PL-18 families), limited studies have focused on PL-17 family alginate lyases, especially for those with special characteristics. In this study, a novel PL-17 family alginate lyase, Aly23, was identified and cloned from the marine bacterium Pseudoalteromonas carrageenovora ASY5. Aly23 exhibited maximum activity at 35 °C and retained 48.93% of its highest activity at 4 °C, representing an excellent cold-adaptation property. Comparative molecular dynamics analysis was implemented to explore the structural basis for the cold-adaptation property of Aly23. Aly23 had a high substrate preference for poly β-D-mannuronate and exhibited both endolytic and exolytic activities; its hydrolysis reaction mainly produced monosaccharides, disaccharides, and trisaccharides. Furthermore, the enzymatic hydrolyzed oligosaccharides displayed good antioxidant activities to reduce ferric and scavenge radicals, such as hydroxyl, ABTS⁺, and DPPH. Our work demonstrated that Aly23 is a promising cold-adapted biocatalyst for the preparation of natural antioxidants from brown algae.     

Hydrolysis of Fucoidan by Fucoidanase Isolated from the Marine Bacterium,Formosa algae

Intracellular fucoidanase was isolated from the marine bacterium, Formosa algae strain KMM 3553. The first appearance of fucoidan enzymatic hydrolysis products in a cell-free extract was detected after 4 h of bacterial growth, and maximal fucoidanase activity was observed after 12 h of growth. The fucoidanase displayed maximal activity in a wide range of pH values, from 6.5 to 9.1. The presence of Mg²⁺, Ca²⁺ and Ba²⁺ cations strongly activated the enzyme; however, Cu²⁺ and Zn²⁺ cations had inhibitory effects on the enzymatic activity. The enzymatic activity of fucoidanase was considerably reduced after prolonged (about 60 min) incubation of the enzyme solution at 45 °C. The fucoidanase catalyzed the hydrolysis of fucoidans from Fucus evanescens and Fucus vesiculosus, but not from Saccharina cichorioides. The fucoidanase also did not hydrolyze carrageenan. Desulfated fucoidan from F. evanescens was hydrolysed very weakly in contrast to deacetylated fucoidan, which was hydrolysed more actively compared to the native fucoidan from F. evanescens. Analysis of the structure of the enzymatic products showed that the marine bacteria, F. algae, synthesized an α-l-fucanase with an endo-type action that is specific for 1→4-bonds in a polysaccharide molecule built up of alternating three- and four-linked α-l-fucopyranose residues sulfated mainly at position 2.     

Cultivable Alginate Lyase-Excreting Bacteria Associated with the Arctic Brown Alga Laminaria

Although some alginate lyases have been isolated from marine bacteria, alginate lyases-excreting bacteria from the Arctic alga have not yet been investigated. Here, the diversity of the bacteria associated with the brown alga Laminaria from the Arctic Ocean was investigated for the first time. Sixty five strains belonging to nine genera were recovered from six Laminaria samples, in which Psychrobacter (33/65), Psychromonas (10/65) and Polaribacter (8/65) were the predominant groups. Moreover, 21 alginate lyase-excreting strains were further screened from these Laminaria-associated bacteria. These alginate lyase-excreting strains belong to five genera. Psychromonas (8/21), Psedoalteromonas (6/21) and Polaribacter (4/21) are the predominant genera, and Psychrobacter, Winogradskyella, Psychromonas and Polaribacter were first found to produce alginate lyases. The optimal temperatures for the growth and algiante lyase production of many strains were as low as 10–20 °C, indicating that they are psychrophilic bacteria. The alginate lyases produced by 11 strains showed the highest activity at 20–30 °C, indicating that these enzymes are cold-adapted enzymes. Some strians showed high levels of extracellular alginate lyase activity around 200 U/mL. These results suggest that these algiante lyase-excreting bacteria from the Arctic alga are good materials for studying bacterial cold-adapted alginate lyases.     

Stereochemical Trajectories of a Two-Component Regulatory System PmrA/B in a Colistin-Resistant Acinetobacter baumannii Clinical Isolate

Background:There is limited information on the 3D prediction and modeling of the colistin resistance-associated proteins PmrA/B TCS in Acinetobacter baumannii. We aimed to evaluate the stereochemical structure and domain characterization of PmrA/B in an A. baumannii isolate resistant to high-level colistin, using bioinformatics tools. Methods:The species of the isolate and its susceptibility to colistin were confirmed by PCR-sequencing and MIC assay, respectively. For 3D prediction of the PmrA/B, we used 16 template models with the highest quality (e-value <1 × 10−50). Results:Prediction of the PmrA structure revealed a monomeric non-redundant protein consisting of 28 α-helices and 22 β-sheets. The PmrA DNA-binding motif displayed three antiparallel α-helices, followed by three β-sheets, and was bond to the major groove of DNA by intermolecular van der Waals bonds through amino acids Lys, Asp, His, and Arg, respectively. Superimposition of the deduced PmrA 3D structure with the closely related PmrA protein model (GenBank no. {"type":"entrez-protein","attrs":{"text":"WP_071210493.1","term_id":"1098418292"}}WP_071210493.1) revealed no distortion in conformation, due to Glu→Lys substitution at position 218. Similarly, the PmrB protein structure displayed 24 α-helices and 13 β-sheets. In our case, His251 acted as a phosphate receptor in the HisKA domain. The amino acid substitutions were mainly observed at the putative N-terminus region of the protein. Furthermore, two substitutions (Lys21→Ser and Ser28→Arg) in the transmembrane domain were detected. Conclusion: TheDNA-binding motif of PmrA is highly conserved, though the N-terminal fragment of PmrB showed a high rate of base substitutions. This research provides valuable insights into the mechanism of colistin resistance in A. baumannii. Key Words: Acinetobacter baumannii, Amino acid substitution, Colistin, Mutation     

Effect of Extraction Temperature on Pressurized Liquid Extraction of Bioactive Compounds from Fucus vesiculosus

This study was aimed at investigating the effect of low polarity water (LPW) on the extraction of bioactive compounds from Fucus vesiculosus and to examine the influence of temperature on the extraction yield, total phenolic content, crude alginate, fucoidan content, and antioxidant activity. The extractions were performed at the temperature range of 120–200 °C with 10 °C increments, and the extraction yield increased linearly with the increasing extraction temperature, with the highest yields at 170–200 °C and with the maximum extraction yield (25.99 ± 2.22%) at 190 °C. The total phenolic content also increased with increasing temperature. The extracts showed a high antioxidant activity, measured with DPPH (2,2-Diphenyl-1-picrylhydrazyl) radicals scavenging and metal-chelating activities of 0.14 mg/mL and 1.39 mg/mL, respectively. The highest yield of alginate and crude fucoidan were found at 140 °C and 160 °C, respectively. The alginate and crude fucoidan contents of the extract were 2.13% and 22.3%, respectively. This study showed that the extraction of bioactive compounds from seaweed could be selectively maximized by controlling the polarity of an environmentally friendly solvent.     

OGG1 DNA Repair Gene Polymorphism As a Biomarker of Oxidative and Genotoxic DNA Damage

Background:Single nucleotide polymorphisms in OGG1 gene modulates DNA repair capacity and functions as one of the first lines of protective mechanisms against 8-OHdG mutagenicity. OGG1-Cys326 gene polymorphism may decrease DNA repair function, causing oxidative stress due to higher oxidative DNA damage. The main purpose of this study was to examine the link of oxidative and genotoxic DNA damage with DNA repair OGG1 gene polymorphism, in charcoal workers exposed to polyaromatic hydrocarbons. Methods:Urinary 8-OHdG excretion (a biomarker of oxidative DNA damage) was determined in both exposed and control populations. Genotyping of OGG1 DNA repair gene in the blood samples of subjects was carried out by PCR-RFLP method. Results:The 8-OHdG urinary concentration was significantly higher (p < 0.05) in the exposed (geometric mean 12.33 ± 3.78) than in the unexposed (geometric mean 7.36 ± 2.29) population. DNA damage, as measured by 8-OHdG and TM content, was found to be significantly higher in OGG1 homozygous mutants (mt/mt; 18.81 ± 3.34; 6.04 ± 0.52) as compared to wild-type genotypes (wt/wt; 10.34 ± 2.25; 5.19 ± 2.50) and heterozygous (wt/mt) mutants (12.82 ± 2.81; 6.04 ± 0.93) in the exposed group. Conclusion:We found a significant association of OGG1 heterozygous (wt/mt) and homozygous (mt/mt) variants with oxidative and genotoxic damage, suggesting that these polymorphisms may modulate the effects of PAH exposure in occupational workers. Key Words: 8-hydroxy-2’-deoxyguanosine, 1-hydroxypyrene, Polycyclic aromatic hydrocarbons     

Functional Characterization of a New GH107 Endo-α-(1,4)-Fucoidanase from the Marine Bacterium Formosa haliotis

Fucoidans from brown macroalgae are sulfated fucose-rich polysaccharides, that have several beneficial biological activities, including anti-inflammatory and anti-tumor effects. Controlled enzymatic depolymerization of the fucoidan backbone can help produce homogeneous, defined fucoidan products for structure-function research and pharmaceutical uses. However, only a few endo-fucoidanases have been described. This article reports the genome-based discovery, recombinant expression in Escherichia coli, stabilization, and functional characterization of a new bacterial endo-α-(1,4)-fucoidanase, Fhf1, from Formosa haliotis. Fhf1 catalyzes the cleavage of α-(1,4)-glycosidic linkages in fucoidans built of alternating α-(1,3)-/α-(1,4)-linked l-fucopyranosyl sulfated at C2. The native Fhf1 is 1120 amino acids long and belongs to glycoside hydrolase (GH) family 107. Deletion of the signal peptide and a 470 amino acid long C-terminal stretch led to the recombinant expression of a robust, minimized enzyme, Fhf1Δ470 (71 kDa). Fhf1Δ470 has optimal activity at pH 8, 37–40 °C, can tolerate up to 500 mM NaCl, and requires the presence of divalent cations, either Ca²⁺, Mn²⁺, Zn²⁺ or Ni²⁺, for maximal activity. This new enzyme has the potential to serve the need for controlled enzymatic fucoidan depolymerization to produce bioactive sulfated fucoidan oligomers.     

Expression and Characterization of a Novel Cold-Adapted and Stable β-Agarase Gene agaW1540 from the Deep-Sea Bacterium Shewanella sp. WPAGA9

The neoagaro-oligosaccharides, degraded from agarose by agarases, are important natural substances with many bioactivities. In this study, a novel agarase gene, agaW1540, from the genome of a deep-sea bacterium Shewanella sp. WPAGA9, was expressed, and the recombinant AgaW1540 (rAgaW1540) displayed the maximum activity under the optimal pH and temperature of 7.0 and 35 °C, respectively. rAgaW1540 retained 85.4% of its maximum activity at 0 °C and retained more than 92% of its maximum activity at the temperature range of 20–40 °C and the pH range of 4.0–9.0, respectively, indicating its extensive working temperature and pH values. The activity of rAgaW1540 was dramatically suppressed by Cu²⁺ and Zn²⁺, whereas Fe²⁺ displayed an intensification of enzymatic activity. The K_m and V_max of rAgaW1540 for agarose degradation were 15.7 mg/mL and 23.4 U/mg, respectively. rAgaW1540 retained 94.7%, 97.9%, and 42.4% of its maximum activity after incubation at 20 °C, 25 °C, and 30 °C for 60 min, respectively. Thin-layer chromatography and ion chromatography analyses verified that rAgaW1540 is an endo-acting β-agarase that degrades agarose into neoagarotetraose and neoagarohexaose as the main products. The wide variety of working conditions and stable activity at room temperatures make rAgaW1540an appropriate bio-tool for further industrial production of neoagaro-oligosaccharides.     

Eleganolone,a Diterpene from the French Marine Alga Bifurcaria bifurcata Inhibits Growth of the Human Pathogens Trypanosoma brucei and Plasmodium falciparum

Organic extracts of 20 species of French seaweed have been screened against Trypanosoma brucei rhodesiense trypomastigotes, the parasite responsible for sleeping sickness. These extracts have previously shown potent antiprotozoal activities in vitro against Plasmodium falciparum and Leishmania donovani. The selectivity of the extracts was also evaluated by testing cytotoxicity on a mammalian L6 cell line. The ethyl acetate extract of the brown seaweed, Bifurcaria bifurcata, showed strong trypanocidal activity with a mild selectivity index (IC₅₀ = 0.53 µg/mL; selectivity index (SI) = 11.6). Bio-guided fractionation led to the isolation of eleganolone, the main diterpenoid isolated from this species. Eleganolone contributes only mildly to the trypanocidal activity of the ethyl acetate extract (IC₅₀ = 45.0 µM, SI = 4.0). However, a selective activity against P. falciparum erythrocytic stages in vitro has been highlighted (IC₅₀ = 7.9 µM, SI = 21.6).     

In Vitro Prebiotic and Anti-Colon Cancer Activities of Agar-Derived Sugars from Red Seaweeds

Numerous health benefits of diets containing red seaweeds or agar-derived sugar mixtures produced by enzymatic or acid hydrolysis of agar have been reported. However, among various agar-derived sugars, the key components that confer health-beneficial effects, such as prebiotic and anti-colon cancer activities, remain unclear. Here, we prepared various agar-derived sugars by multiple enzymatic reactions using an endo-type and an exo-type of β-agarase and a neoagarobiose hydrolase and tested their in vitro prebiotic and anti-colon cancer activities. Among various agar-derived sugars, agarotriose exhibited prebiotic activity that was verified based on the fermentability of agarotriose by probiotic bifidobacteria. Furthermore, we demonstrated the anti-colon cancer activity of 3,6-anhydro-l-galactose, which significantly inhibited the proliferation of human colon cancer cells and induced their apoptosis. Our results provide crucial information regarding the key compounds derived from red seaweeds that confer beneficial health effects, including prebiotic and anti-colon cancer activities, to the host.     

Increased Osteogenic Potential of Pre-Osteoblasts on Three-Dimensional Printed Scaffolds Compared to Porous Scaffolds for Bone Regeneration

Background:One of the main challenges with conventional scaffold fabrication methods is the inability to control scaffold architecture. Recently, scaffolds with controlled shape and architecture have been fabricated using 3D-printing. Herein, we aimed to determine whether the much tighter control of microstructure of 3DP PLGA/β-TCP scaffolds is more effective in promoting osteogenesis than porous scaffolds produced by solvent casting/porogen leaching. Methods:Physical and mechanical properties of porous and 3DP scaffolds were studied. The response of pre-osteoblasts to the scaffolds was analyzed after 14 days. Results:The 3DP scaffolds had a smoother surface (R_a: 22 ± 3 µm) relative to the highly rough surface of porous scaffolds (R_a: 110 ± 15 µm). Water contact angle was 112 ± 4° on porous and 76 ± 6° on 3DP scaffolds. Porous and 3DP scaffolds had the pore size of 408 ± 90 and 315 ± 17 µm and porosity of 85 ± 5% and 39 ± 7%, respectively. Compressive strength of 3DP scaffolds (4.0 ± 0.3 MPa) was higher than porous scaffolds (1.7 ± 0.2 MPa). Collagenous matrix deposition was similar on both scaffolds. Cells proliferated from day 1 to day 14 by fourfold in porous and by 3.8-fold in 3DP scaffolds. ALP activity was 21-fold higher in 3DP scaffolds than porous scaffolds. Conclusion:The 3DP scaffolds show enhanced mechanical properties and ALP activity compared to porous scaffolds in vitro, suggesting that 3DP PLGA/β-TCP scaffolds are possibly more favorable for bone formation. Key Words: Alkaline phosphatase, β-tricalcium phosphate, Poly(lactic-co-glycolic) acid copolymer     

Clinical and Genetic Characteristics of Splicing Variant in CYP27A1 in an Iranian Family with Cerebrotendinous xanthomatosis

Background:CTX is a rare congenital lipid-storage disorder, leading to a progressive multisystem disease. CTX with autosomal recessive inheritance is caused by a defect in the CYP27A1 gene. Chronic diarrhea, tendon xanthomas, neurologic impairment, and bilateral cataracts are common symptoms of the disease. Methods:Three affected siblings with an initial diagnosis of non-syndromic intellectual disability were recruited for further molecular investigations. To identify the possible genetic cause(s), WES was performed on the proband. Sanger sequencing was applied to confirm the final variant. The clinical and molecular genetic features of the three siblings from the new CTX family and other patients with the same mutations, as previously reported, were analyzed. The CYP27A1 gene was also studied for the number of pathogenic variants and their location. Results:We found a homozygous splicing mutation, {"type":"entrez-nucleotide","attrs":{"text":"NM_000784","term_id":"1519312912"}}NM_000784: exon6: c.1184+1G>A, in CYP27A1 gene, which was confirmed by Sanger sequencing. Among the detected pathogenic variants, the splice site mutation had the highest prevalence, and the mutations were mostly found in exon 4. Conclusion:This study is the first to report the c.1184+1G>A mutation in Iran. Our findings highlight the other feature of the disease, which is the lack of relationship between phenotype and genotype. Due to nonspecific symptoms and delay in diagnosis, CYP27A1 genetic analysis should be the definitive method for CTX diagnosis. Key Words: Cerebrotendinous xanthomatosis, CYP27A1, Intellectual disability, Iran, Whole exome sequencing     

Fabrication and Characterization of Nanocomposite Hydrogel Based on Alginate/Nano-Hydroxyapatite Loaded with Linum usitatissimum Extract as a Bone Tissue Engineering Scaffold

In the current paper, we fabricated, characterized, and applied nanocomposite hydrogel based on alginate (Alg) and nano-hydroxyapatite (nHA) loaded with phenolic purified extracts from the aerial part of Linum usitatissimum (LOH) as the bone tissue engineering scaffold. nHA was synthesized based on the wet chemical technique/precipitation reaction and incorporated into Alg hydrogel as the filler via physical cross-linking. The characterizations (SEM, DLS, and Zeta potential) revealed that the synthesized nHA possess a plate-like shape with nanometric dimensions. The fabricated nanocomposite has a porous architecture with interconnected pores. The average pore size was in the range of 100–200 µm and the porosity range of 80–90%. The LOH release measurement showed that about 90% of the loaded drug was released within 12 h followed by a sustained release over 48 h. The in vitro assessments showed that the nanocomposite possesses significant antioxidant activity promoting bone regeneration. The hemolysis induction measurement showed that the nanocomposites were hemocompatible with negligible hemolysis induction. The cell viability/proliferation confirmed the biocompatibility of the nanocomposites, which induced proliferative effects in a dose-dependent manner. This study revealed the fabricated nanocomposites are bioactive and osteoactive applicable for bone tissue engineering applications.     

Recovery of Proteolytic and Collagenolytic Activities from Viscera By-products of Rayfish (Raja clavata)

The aim of this work was to study the recovery of proteolytic and collagenolytic activities from rayfish (Raja clavata) viscera wastes. Initially, different parts of the gastrointestinal tract by-products (stomach, duodenum section including pancreas, final intestine) were evaluated. The extracts from proximal intestine yielded the highest values of both enzymatic activities. Optimal conditions for protease activity quantification were established at pH = 6, T = 40 °C and incubation time ≤20 min. The mathematical equation used to model the joint effect of pH and temperature led to maximum activity at pH = 8.66 and 59.4 °C, respectively. Overcooled acetone was found to be best option for recovery of enzymatic activities in comparison with ethanol, PEG-4000, ammonium sulphate and ultrafiltration system. Finally, a simple and systematic protocol of partial purification and total recovery of proteases and collagenases was defined.     

A Novel Alginate Lyase: Identification,Characterization, and Potential Application in Alginate Trisaccharide Preparation

Alginate oligosaccharides (AOS) have many biological activities and significant applications in prebiotics, nutritional supplements, and plant growth development. Alginate lyases have unique advantages in the preparation of AOS. However, only a limited number of alginate lyases have been so far reported to have potentials in the preparation of AOS with specific degrees of polymerization. Here, an alginate-degrading strain Pseudoalteromonas arctica M9 was isolated from Sargassum, and five alginate lyases were predicted in its genome. These putative alginate lyases were expressed and their degradation products towards sodium alginate were analyzed. Among them, AlyM2 mainly generated trisaccharides, which accounted for 79.9% in the products. AlyM2 is a PL6 lyase with low sequence identity (≤28.3%) to the characterized alginate lyases and may adopt a distinct catalytic mechanism from the other PL6 alginate lyases based on sequence alignment. AlyM2 is a bifunctional endotype lyase, exhibiting the highest activity at 30 °C, pH 8.0, and 0.5 M NaCl. AlyM2 predominantly produces trisaccharides from homopolymeric M block (PM), homopolymeric G block (PG), or sodium alginate, with a trisaccharide production of 588.4 mg/g from sodium alginate, indicating its promising potential in preparing trisaccharides from these polysaccharides.     

Three New Asperentin Derivatives from the Algicolous Fungus Aspergillus sp. F00785

Three new asperentin-type compounds, 6-O-α-d-ribosylasperentin (1) and 6-O-α-d-ribosyl-8-O-methylasperentin (2) and 5-hydroxyl-6-O-methylasperentin (3), along with asperentin (4) and its known analogues (5–9), were isolated from a halotolerant Aspergillus sp. strain {"type":"entrez-nucleotide","attrs":{"text":"F00785","term_id":"707638","term_text":"F00785"}}F00785, an endotrophic fungus from marine alga. Their structures were determined using extensive NMR and HRESIMS spectroscopic analysis, including the X-ray crystallographic data for the assignment of the absolute configurations of compound 9. Compound 4 exhibited highly potent inhibitory activity against crop pathogens, Colletotrichum gleosporioides Penz. and Colletotrichum gleosporioides (Penz.) Sacc.     

Cloning,Expression and Characterization of Recombinant Exotoxin A-Flagellin Fusion Protein as a New Vaccine Candidate against Pseudomonas aeruginosa Infections

Background: Infections due to Pseudomonas aeruginosa are among the leading causes of morbidity and mortality in patients who suffer from impaired immune responses and chronic diseases such as cystic fibrosis. At present, aggressive antibiotic therapy is the only choice for management of P. aeruginosa infections, but emergence of highly resistant strains necessitated the development of novel alternative therapeutics including an effective vaccine. Several P. aeruginosa antigens have been tested for vaccine development, including lipopolysaccharide alone, polysaccharides alginate, extracellular proteins, exotoxin A (exo A) and killed whole cell. However, none of them are currently available clinically. Methods: In this research, recombinant exoA-flagellin (fliC) fusion protein as a cocktail antigen was expressed and purified and its antigenic characteristics were evaluated. Results: Expression of recombinant fusion protein by E. coli using pET22b vector resulted in production of exoA-fliC fusion protein in high concentration. Based on Western-blotting results, recombinant fusion protein showed a good antigenic interaction with sera from patients with various P. aeruginosa infections. Conclusion: These results suggested that recombinant exoA-fliC fusion protein can be produced in the laboratory, and tested as a candidate vaccine in P. aeruginosa infections. Key Words: Pseudomonas aeruginosa, Exotoxin A (exoA), Flagellin (fliC), Vaccines