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Polar and but not lateral flagellin proteins from Aeromonas hydrophila strain AH-1 (serotype O11) were found to be glycosylated. Top-down mass spectrometry studies of purified polar flagellins suggested the presence of a 403 Da glycan of mass. Bottom-up mass spectrometry studies showed the polar flagellin peptides to be modified with 403 Da glycans in O-linkage. The MS fragmentation pattern of this putative glycan was similar to that of pseudaminic acid derivative. Mutants lacking the biosynthesis of pseudaminic acid (pseB and pseI homologues) were unable to produce polar flagella but no changes were observed in lateral flagella by post-transcriptional regulation of the flagellin. Complementation was achieved by reintroduction of the wild-type pseB and pseI. We compared two pathogenic features (adhesion to eukaryotic cells and biofilm production) between the wild-type strain and two kinds of mutants: mutants lacking polar flagella glycosylation and lacking the O11-antigen lipopolysaccharide (LPS) but with unaltered polar flagella glycosylation. Results suggest that polar flagella glycosylation is extremely important for A. hydrophila AH-1 adhesion to Hep-2 cells and biofilm formation. In addition, we show the importance of the polar flagella glycosylation for immune stimulation of IL-8 production via toll-“like” receptor 5 (TLR5).

The repeat units of heteropolymeric O antigen are synthesized at the cytosolic side of the inner bacterial membrane via the Wzx/Wzy-dependent assembly pathway. After being translocated across the membrane by Wzx, each repeat unit is polymerized by Wzy to form a glycan chain. In this study, we demonstrate the need of the corresponding enzyme trans- ferring the initial HexNAc to undecaprenol phosphate (lipid carrier), together with the corre- sponding O-antigen polymerase (Wzy), to produce the Aeromonas hydrophila O:34- antigen. We suggest, the concerted action of WecA or P enzyme (UDP-HexNAc: polypre- nol-P HexNAc-1-P transferase) and Wzy is involved in the mechanism responsible for the A . hydrophila O-antigen polymerization.

Plesiomonas shigelloides is the unique member of the Enterobacteriaceae family able to produce polar flagella when grow in liquid medium and lateral flagella when grown in solid or semisolid media. In this study on P. shigelloides 302-73 strain, we found two different gene clusters, one exclusively for the lateral flagella biosynthesis and the other one containing the biosynthetic polar flagella genes with additional putative glycosylation genes. P. shigelloides is the first Enterobacteriaceae were a complete lateral flagella cluster leading to a lateral flagella production is described. We also show that both flagella in P. shigelloides 302-73 strain are glycosylated by a derivative of legionaminic acid (Leg), which explains the presence of Leg pathway genes between the two polar flagella regions in their biosynthetic gene cluster. It is the first bacterium reported with O-glycosylated Leg in both polar and lateral flagella. The flagella O-glycosylation is essential for bacterial flagella formation, either polar or lateral, because gene mutants on the biosynthesis of Leg are non-flagellated. Furthermore, the presence of the lateral flagella cluster and Leg O-flagella glycosylation genes are widely spread features among the P. shigelloides strains tested.

Erwinia amylovora, a Gram negative bacteria of the Enterobacteriaceae family, is the causal agent of fire blight, a devastating plant disease affecting a wide range of host species within Rosaceae and a major global threat to commercial apple and pear production. Among the limited number of control options currently available, prophylactic application of antibiotics during the bloom period appears the most effective. Pathogen cells enter plants through the nectarthodes of flowers and other natural openings, such as wounds, and are capable of rapid movement within plants and the establishment of systemic infections. Many virulence determinants of E. amylovora have been characterized, including the Type III secretion system (T3SS), the exopolysaccharide (EPS) amylovoran, biofilm formation, and motility. To successfully establish an infection, E. amylovora uses a complex regulatory network to sense the relevant environmental signals and coordinate the expression of early and late stage virulence factors involving two component signal transduction systems, bis-(3′-5′)-cyclic di-GMP (c-di-GMP) and quorum sensing. The LPS biosynthetic gene cluster is one of the relatively few genetic differences observed between Rubus- and Spiraeoideae-infecting genotypes of E. amylovora. Other differential factors, such as the presence and composition of an integrative conjugative element associated with the Hrp T3SS (hrp genes encoding the T3SS apparatus), have been recently described. In the present review, we present the recent findings on virulence factors research, focusing on their role in bacterial pathogenesis and indicating other virulence factors that deserve future research to characterize them.

A group of virulent Aeromonas hydrophila, A. sobria, and A. veronii biovar sobria strains isolated from humans and fish have been described, these strains classified to serotype O11 are serologically related by their lipopolysaccharide (LPS) O-antigen (O-polysaccharide), and the presence of an S-layer consisting of multiple copies of a crystalline surface array protein with a molecular weight of 52 kDa in the form of a crystalline surface array which lies peripheral to the cell wall. A. hydrophila strain AH-1 is one of them. We isolated the LPS from this strain and determined the structure of the O-polysaccharide, which was similar to that previously described for another strain of serotype O11. The genetics of the O11-antigen showed the genes (wbO11 cluster) in two sections separated by genes involved in biosynthesis and assembly of the S-layer. The O11-antigen LPS is an example of an ABC-2-transporter-dependent pathway for O-antigen heteropolysaccharide (disaccharide) assembly. The genes involved in the biosynthesis of the LPS core (waaO11 cluster) were also identified in three different chromosome regions being nearly identical to the ones described for A. hydrophila AH-3 (serotype O34). The genetic data and preliminary chemical analysis indicated that the LPS core for strain AH-1 is identical to the one for strain AH-3.

Polar flagellin proteins from Aeromonas hydrophila strain AH-3 (serotype O34) were found to be O-glycosylated with a heterogeneous glycan. Mutants unable to produce WecP or Gne enzymes showed altered motility, and the study of their polar flagellin glycosylation showed that the patterns of glycosylation differed from that observed with wild type polar flagellin. This suggested the involvement of a lipid carrier in glycosylation. A gene coding for an enzyme linking sugar to a lipid carrier was identified in strain AH-3 (WecX) and subsequent mutation abolished completely motility, flagella production by EM, and flagellin glycosylation. This is the first report of a lipid carrier involved in flagella O-glycosylation. A molecular model has been proposed. The results obtained suggested that the N -acetylhexosamines are N-acetylgalactosamines and that the heptasaccharide is completely independent of the O34-antigen lipopolysaccharide. Furthermore, by comparing the mutants with differing degrees of polar flagellin glycosylation, we established their importance in A. hydrophila flagella formation and motility.

Protein glycosylation had been considered as an eccentricity of a few bacteria. However, through advances in analytical methods and genome sequencing, it is now established that bacteria possess both N-linked and O-linked glycosylation pathways. Both glycosylation pathways can modify multiple proteins, flagellins from Archaea and Eubacteria being one of these. Flagella O-glycosylation has been demonstrated in many polar flagellins from Gram-negative bacteria and in only the Gram-positive genera Clostridium and Listeria. Furthermore, O-glycosylation has also been demonstrated in a limited number of lateral flagellins. In this work, we revised the current advances in flagellar glycosylation from Gram-negative bacteria, focusing on the structural diversity of glycans, the O-linked pathway and the biological function of flagella glycosylation.

Aeromonas hydrophila is the most common Aeromonas species causing infections in human and other animals such as amphibians, reptiles, fish and crustaceans. Pathogenesis of Aeromonas species have been reported to be associated with virulence factors such as lipopolysaccharides (LPS), bacterial toxins, bacterial secretion systems, flagella, and other surface molecules. Several mutant strains of A. hydrophila AH-3 were initially used to study their virulence in two animal species, Pacifastacus leniusculus (crayfish) and Tenebrio molitor larvae (mealworm). The AH-3 strains used in this study have mutations in genes involving the synthesis of flagella, LPS structures, secretion systems, and some other factors, which have been reported to be involved in A. hydrophila pathogenicity. Our study shows that the LPS (O-antigen and external core) is the most determinant A. hydrophila AH-3 virulence factor in both animals. Furthermore, we studied the immune responses of these hosts to infection of virulent or non-virulent strains of A. hydrophila AH-3. The AH-3 wild type (WT) containing the complete LPS core is highly virulent and this bacterium strongly stimulated the prophenoloxidase activating system resulting in melanization in both crayfish and mealworm. In contrast, the ΔwaaE mutant which has LPS without O-antigen and external core was non-virulent and lost ability to stimulate this system and melanization in these two animals. The high phenoloxidase activity found in WT infected crayfish appears to result from a low expression of pacifastin, a prophenoloxidase activating enzyme inhibitor, and this gene expression was not changed in the ΔwaaE mutant infected animal and consequently phenoloxidase activity was not altered as compared to non-infected animals. Therefore we show that the virulence factors of A. hydrophila are the same regardless whether an insect or a crustacean is infected and the O-antigen and external core is essential for activation of the proPO system and as virulence factors for this bacterium.

El género "Aeromonas" pertenece a la familia "Vibrionaceas". Dentro de este généro podemos diferenciar dos grupos: un primer grupo formado por microorganismos psicrófilos no mótiles en el que únicamente hallamos la especie "Aeromonas salmonicida" y un segundo grupo formado por microorganismos mesófilos mótiles en el que hallamos las especies "Aeromonas hydrophila", "Aeromonas sobria" y "Aeromonas caviae". Las cepas de "Aeromonas" mesófilas mótiles son microorganismos que se caracterizan por su elevada diversidad en cuanto al grado de virulencia. Estas diferencias se hallan estrechamente relacionadas con las estructuras moleculares presentes a nivel de la superficie bacteriana. La incidencia y variabilidad de este grupo hizo que obtuviesen a partir de muestras de agua diferentes bacteriófagos específicos para las estructuras superficiales bacterianas, los cuales pudiesen emplearse como marcadores biológicos que permitiesen diferenciar a un serotipo de los restantes dentro del grupo de "Aeromonas" mótiles. De este modo, se aislaron los bacteriófagos PM1, PM2 y PM3.El bacteriófago PM1 presenta doble cadena de DNA, capside poliédrica y una placa con fibras en la cola. Además, posee como receptor el antígeno O del lipopolisacárido (LPS) de las cepas de serotipo 0:34 y, en consecuencia, los mutantes espontáneos resistentes al citado bacteriófago carecen de antígeno 0 del LPS, aunque presentan sensibilidad a bacteriófagos de "Aeromonas" cuyo receptor es el núcleo del LPS. El bacteriófago PM2 presenta doble cadena de DNA, cápside poliédrica y una placa basal con espinas. Además, posee como receptor el núcleo del LPS de diferentes cepas de serotipo 0:34 y 0:11 (así como "A. salmonicida"); en consecuencia, los mutantes resistentes al mismo presentan alteraciones en los oligosacáridos del núcleo del LPS, careciendo en todos los casos de antígeno 0 del LPS. El bacteriófago PM3 presenta doble cadena de DNA y posee como receptor el flagelo monopolar de diferentes serotipos de "A. hydrophila" tanto con lámina S como sin lámina. Los mutantes resistentes a este bacteriófago presentan alteraciones en el flagelo o en la motilidad de dichas cepas bacterianas. Estos bacteriófagos han permitido la agrupación de diferentes cepas de "Aeromonas" mesófilas que luego han correspondido a serotipos distintos. Así, las cepas sensibles al bacteriófago PM1 pertenecen al serogrupo 0:34 y presentan las siguientes características:1.- Un perfil de proteínas de membrana externa tremendamente homogéneo.2.- Un LPS heterogéneo, químicamente formado por KDO, L-heptosas, glucosa y hesoxaminas, y carente en D-heptosas, galactosa, rhamnosa y pentosas.3.- Una DL(50) a 20ºC menos elevada que a 37°C, tanto en ratón como en pez, lo cual indica que son más virulentas a 20ºC que a 37°C. Este hecho se debe a la producción de antígeno 0 del LPS a 20ºC y a la práctica ausencia del mismo a 37°C. Todo ello implica al antigeno 0 del LPS en la virulencia de estas cepas.Uno de los serotipos más importantes dentro de las "Aeromonas" mesófilas a causa de su elevado grado de virulencia es el denominado serotipo 0:11. Este serotipo presenta:1. Un LPS homogéneo, formado químicamente por KDO y L-heptosas en el núcleo del LPS y glucosa, galactosa, mañosa y hexosaminas en el antígeno 0 del LPS. 2. Una lámina proteínica de estructura tetragonal, denominada lámina S, que recubre prácticamente la superficie celular haciendo que el LPS quede totalmente recubierto. Esta poca accesibilidad del antígeno 0 del LPS hace difícil encontrar marcadores biológicos (bacteriófagos) para este serotipo. 3. Todas las cepas de serotipo 0: 11 al igual que las de "A. salmonicida" poseen la propiedad de autoaglutinar. Dicha propiedad está ligada fundamentalmente a la presencia de lámina S y de flagelo.4. La DL(50) de este serotipo es dos logaritmos inferior a la existente en las cepas de serotipo 0:34 y además es muy similar en todos los mutantes isogénicos obtenidos para las diferentes estructuras superficiales. Una vez estudiados los serotipos 0:34 y 0:11 de las "Aeromonas" mesófilas se obtuvieron mutantes isogénicos, bien por resistencia a bacteriófagos específicos para las diferentes estructuras superficiales o bien mediante mutagénesis con un agente alquilante y contraselección con anticuerpos especificos frente a 1as estructuras superficiales a alterar.Con el uso de los citados mutantes se determinó que la vía clásica del complemento es la responsable en las "Aeromonas" pertenecientes al serotipo 0:34 y 0:11 de la activación del complemento y por consiguiente, de la actividad bactericida del suero. Además, se obeservó que la lámina S peresente en las cepas de serotipo 0:11 no activaba la acción del complemento (a diferencia de la lámina A de "A. salmonicida"), sino que el determinante a nivel de las estructuras superficiales de la resistencia a la actividad bactericida del suero en ambos serotipos son los oligosacáridos del núcleo del LPS. Finalmente se demostró que el LPS de todas las "Aeromonas" mesófilas es capaz de activar el complemento, y la razón de ser sensible o resistente a la actividad bactericida del suero se debe a la fijación o no de C3b pegado a la membrana y la consiguiente formación de los complejos C5b-9., The motile "Aeromonas" group is characterized by their high diversity degree of virulence. These differences are related with molecular structures located on the bacterial surface.Several bacteriophages of motile "Aeromonas" were isolated and characterized from water samples. Thus, it was obtained bacteriophages whose receptors are: the 0 antigen lipopolisaccharide (LPS) of 0:34 serotype strains, the core LPS of 0:34 and 0:11 serotype strains (as soon as "Aeromonas salmonicida" and the monopolar flagellum of motile "Aeromonas".Then, motile "Aeromnas" 0:34 and 0:11 isogenic mutants were obtained by resistance to the specific bacteriophages for different structures, or by mutagenesis with an alquilant agent and contraselection with specific antibodies.Using the different isogenic mutants it was possible to determinate: a) the complement activation pathway in the 0:34 and 0:11 serotypes motile "Aeromonas" is the classical pathway, b) the S-layer present on the 0:11 serotype strains not activate the complement action; but, in contrast, the A-layer present on the "Aeromonas salmonicida" strains active this action, c) the complement activation determinant at the structural bacterial surface are the core LPS oligosaccharides, and d) the LPS present at all motile "Aeromonas" strains activate the classical complement pathway; and the sensibility or resistance from the bactericidal action was due to the presence or absence of C3b linked on the membranes and the subsequence formation of C5b-9 complexes.

Marine microorganisms, including Aeromonas, are a source of compds. for drug development that have generated great expectations in the last decades. Aeromonas infections produce septicemia, and ulcerative and haemorrhagic diseases in fish. Among the pathogenic factors assocd. with Aeromonas, the lipopolysaccharides (LPS)​, a surface glyconconjugate unique to Gram-​neg. bacteria consisting of lipid A (lipid anchor of the mol.)​, core oligosaccharide and O-​specific polysaccharide (O antigen)​, are key elicitors of innate immune responses. The chem. structure of these three parts has been characterized in Aeromonas. Based on the high variability of repeated units of O-​polysaccharides, a total of 97 O-​serogroups have been described in Aeromonas species, of which four of them (O:11; O:16; O:18 and O:34) account for more than 60​% of the septicemia cases. The core of LPS is subdivided into two regions, the inner (highly conserved) and the outer core. The inner core of Aeromonas LPS is characterized by the presence of 3-​deoxy-​d-​manno-​oct-​2-​ulosonic (ketodeoxyoctonic) acid (Kdo) and l-​glycero-​d-​manno-​Heptoses (l,​d-​Hep)​, which are linked to the outer core, characterized by the presence of Glc, GlcN, Gal, and GalNAc (in Aeromonas salmonicida)​, d,​d-​Hep (in Aeromonas salmonicida)​, and l,​d-​Hep (in Aeromonas hydrophila)​. The biol. relevance of these differences in the distal part of the outer core among these species has not been fully assessed to date. The inner core is attached to the lipid A, a highly conserved structure that confers endotoxic properties to the LPS when the mol. is released in blood from lysed bacteria, thus inducing a major systemic inflammatory response known as septic or endotoxic shock. In Aeromonas salmonicida subsp. salmonicida the Lipid A components contain three major lipid A mols., differing in acylation patterns corresponding to tetra-​, penta- and hexa-​acylated lipid A species and comprising of 4'-​monophosphorylated β-​2-​amino-​2-​deoxy-​d-​glucopyranose-​(1→6)​-​2-​amino-​2-​deoxy-​d-​glucopyranose disaccharide. In the present review, we discuss the structure-​activity relationships of Aeromonas LPS, focusing on its role in bacterial pathogenesis and its possible applications.

Aeromonas hydrophila sodium-driven polar flagellum has a complex stator-motor. Consist of two sets of redundant and non-exchangeable proteins (PomA/PomB and PomA2/PomB2), which are homologs to other sodium-conducting polar flagellum stator motors; and also two essential proteins (MotX and MotY), that they interact with one of those two redundant pairs of proteins and form the T-ring. In this work, we described an essential protein for polar flagellum stability and rotation which is orthologs to Vibrio spp. FlgT and it is encoded outside of the A. hydrophila polar flagellum regions. The flgT was present in all mesophilic Aeromonas strains tested and also in the nonmotile Aeromonas salmonicida. The A. hydrophila 1flgT mutant is able to assemble the polar flagellum but is more unstable and released into the culture supernatant from the cell upon completion assembly. Presence of FlgT in purified polar hook-basal bodies (HBB) of wild-type strain was confirmed by Western blotting and electron microscopy observations showed an outer ring of the T-ring (H-ring) which is not present in the 1flgT mutant. Anchoring and motility of proton-driven lateral flagella was not affected in the 1flgT mutant and specific antibodies did not detect FlgT in purified lateral HBB of wild type strain.

Initial hydridization tests using Aeromonas salmonicida typical and atypical strains showed the possibility of different lipopolysaccharide (LPS) outer cores among these strains. By chemical structural analysis, LPS-core SDS-PAGE gel migration, and functional and comparative genomics we demonstrated that typical A. salmonicida (subsp. salmonicida) strains and atypical subsp. masoucida and probably smithia strains showed the same LPS outer core. A. salmonicida subsp. achromogenes strains show a similar LPS outer core but lack one of the most external residues (a galactose linked α1-6 to heptose), not affecting the O-antigen LPS linkage. A. salmonicida subsp. pectinolytica strains show a rather changed LPS outer core, which is identical to the LPS outer core from the majority of the A. hydrophila strains studied by genomic analyses. The LPS inner core in all tested A. salmonicida strains, typical and atypical, is well-conserved. Furthermore, the LPS inner core seems to be conserved in all the Aeromonas (psychrophilic or mesophilic) strains studied by genomic analyses.

We experimentally identified the activities of six predicted heptosyltransferases in Actinobacillus pleuropneumoniae genome serotype 5b strain L20 and serotype 3 strain JL03. The initial identification was based on a bioinformatic analysis of the amino acid similarity between these putative heptosyltrasferases with others of known function from enteric bacteria and Aeromonas. The putative functions of all the Actinobacillus pleuropneumoniae heptosyltrasferases were determined by using surrogate LPS acceptor molecules from well-defined A. hydrophyla AH-3 and A. salmonicida A450 mutants. Our results show that heptosyltransferases APL_0981 and APJL_1001 are responsible for the transfer of the terminal outer core D-glycero-D-manno-heptose (D,D-Hep) residue although they are not currently included in the CAZY glycosyltransferase 9 family. The WahF heptosyltransferase group signature sequence [S(T/S)(GA)XXH] differs from the heptosyltransferases consensus signature sequence [D(TS)(GA)XXH], because of the substitution of D(261) for S(261), being unique.

[spa] El género Aeromonas está constituido por bacilos gram negativos y comprende patógenos de animales poiquilotermos y oportunistas en humanos. La virulencia de Aeromonas hydrophila AH-1 es multifactorial, siendo el lipopolisacárido (LPS), el flagelo (polar y lateral) y la lámina S importantes factores de patogenicidad. La mutación del gen wbpL, implicado en la biosíntesis del antígeno O del LPS, da lugar a la pérdida de antígeno O, una bajada en el peso molecular de las flagelinas polares y la aparición de fimbrias en la superficie bacteriana. Se procedió a estudiar si las flagelinas polares y laterales de la cepa AH-1 se hallaban glicosiladas. Los análisis mediante LC-MS mostraron que las flagelinas laterales de la cepa AH-1 no se hallaban glicosiladas, mientras que las polares se hallaban glicosiladas con un glicano de 403 Da, derivado del ácido pseudamínico y otro glicano de 1060 Da, que no se encontraba en el mutante del gen wbpL. Este dato, junto con el hecho de que el mutante del gen rmlB que codifica para la biosíntesis de ramnosa (azúcar que compone el antígeno O) también pierde el glicano de 1060 Da, muestra la relación entre dicho glicano y el antígeno O del LPS. Asimismo se procedió a localizar los genes implicados en la síntesis de pseudamínico, adyacentes a la región 2 de flagelo polar; mutaciones tanto en alguno de estos genes (pseI, pseB) como en la glicosiltransferasa maf-1 abolía la presencia de flagelo polar pero no del lateral, demostrando así una regulación post-traduccional de las flagelinas polares mediante la glicosilación. También se observó la aparición de fimbrias. Se procedió a analizar las fimbrias que se inducían en los mutantes wbpL, pseI, pseB y maf-1. Se trata de fimbrias de tipo IV, denominadas MSHA por su homología a las descritas en Vibrio spp y ampliamente distribuidas en el género Aeromonas. La secuenciación de la agrupación mostró 22 ORF agrupadas en dos transcritos. Para analizar la inducción de las fimbrias en los mutantes se realizaron análisis de promotores, RT-PCR y detección mediante anticuerpos, que mostraron que la cepa salvaje AH-1 presenta las fimbrias pero no es capaz de ensamblarlas correctamente y las libera al exterior. La acumulación de azúcares activos en los mutantes parece ser el detonante para la expresión de estas fimbrias que juegan un papel fundamental en la formación de biofilms, favoreciendo la persistencia de la bacteria en el medio ambiente., [eng] The genus Aeromonas is composed of Gram-negative rod-shaped bacteria and comprises opportunistic and primary pathogens of poikilothermic and homeothermic animals, including humans. The lipopolysaccharide (LPS) and S layer are important virulence factor of Aeromonas hydrophila AH-1. Mutation of the gene wbpL, which is involved in the O antigen synthesis of the LPS, leads to loss of the O antigen, a lesser molecular weight of the polar flagella and the appearance of fimbriae on the bacterial surface. It was studied whether the polar and lateral flagella of the strain AH-1 appear glycosylated. The LC-MS analysis showed that the lateral flagella were not glycosylated, while polar flagella were carrying a 403 Da glycan, derived from pseudaminic acid, as well as a 1060 Da glycan that was not found in the wbpL mutant strain. This finding, together with the fact that a mutant of the rmlB-gene, which plays a role on ramnose-biosynthesis (a sugar of the O antigen), also loses the glycan of 1060 Da, shows the involvement of this glycan in the o antigen of the LPS. It was proceeded to identify the genes in pseudaminic-synthesis adjacent to region 2 of the polar flagellum genes. Mutations in some of these genes (pseI, pseB) as well as in glycosyltranferase maf-1 abolished the presence of polar but nor of lateral flagella and therefore showed a posttranslational regulation of the polar flagellins by glycosylation. As also fimbria formation was observed it was proceeded to analyse the fimbria that were induced in the mutants of wbpL, pseI, pseB and maf-1. They were identified as type IV fimbria, also called MSHA due to their homology to those observed in Vibrio ssp and broadly distributed in the genus Aeromonas. DNA-sequencing showed 22 ORF that were organized in 2 transcripts. To analyze the induction of the fimbria in above mentioned mutants, promoter analysis, RT-PCR and immunodetection was performed. This showed that wildtype AH-1 also expresses fimbria, but is not capable of assembling them correctly and therefore expulses them to the exterior. The accumulation of active sugars in the mutants seems to trigger the correct assembly of the fimbria that play an fundamental role in biofilm formation and therefore favor the persistence of the bacteria in the environment.

El género Aeromonas está constituido por bacilos gram negativos y comprende patógenos de animales poiquilotermos y oportunistas en humanos. La virulencia de Aeromonas hydrophila AH-1 es multifactorial, siendo el lipopolisacárido (LPS), el flagelo (polar y lateral) y la lámina S importantes factores de patogenicidad. La mutación del gen wbpL, implicado en la biosíntesis del antígeno O del LPS, da lugar a la pérdida de antígeno O, una bajada en el peso molecular de las flagelinas polares y la aparición de fimbrias en la superficie bacteriana. Se procedió a estudiar si las flagelinas polares y laterales de la cepa AH-1 se hallaban glicosiladas. Los análisis mediante LC-MS mostraron que las flagelinas laterales de la cepa AH-1 no se hallaban glicosiladas, mientras que las polares se hallaban glicosiladas con un glicano de 403 Da, derivado del ácido pseudamínico y otro glicano de 1060 Da, que no se encontraba en el mutante del gen wbpL. Este dato, junto con el hecho de que el mutante del gen rmlB que codifica para la biosíntesis de ramnosa (azúcar que compone el antígeno O) también pierde el glicano de 1060 Da, muestra la relación entre dicho glicano y el antígeno O del LPS. Asimismo se procedió a localizar los genes implicados en la síntesis de pseudamínico, adyacentes a la región 2 de flagelo polar; mutaciones tanto en alguno de estos genes (pseI, pseB) como en la glicosiltransferasa maf-1 abolía la presencia de flagelo polar pero no del lateral, demostrando así una regulación post-traduccional de las flagelinas polares mediante la glicosilación. También se observó la aparición de fimbrias. Se procedió a analizar las fimbrias que se inducían en los mutantes wbpL, pseI, pseB y maf-1. Se trata de fimbrias de tipo IV, denominadas MSHA por su homología a las descritas en Vibrio spp y ampliamente distribuidas en el género Aeromonas. La secuenciación de la agrupación mostró 22 ORF agrupadas en dos transcritos. Para analizar la inducción de las fimbrias en los mutantes se realizaron análisis de promotores, RT-PCR y detección mediante anticuerpos, que mostraron que la cepa salvaje AH-1 presenta las fimbrias pero no es capaz de ensamblarlas correctamente y las libera al exterior. La acumulación de azúcares activos en los mutantes parece ser el detonante para la expresión de estas fimbrias que juegan un papel fundamental en la formación de biofilms, favoreciendo la persistencia de la bacteria en el medio ambiente., The genus Aeromonas is composed of Gram-negative rod-shaped bacteria and comprises opportunistic and primary pathogens of poikilothermic and homeothermic animals, including humans. The lipopolysaccharide (LPS) and S layer are important virulence factor of Aeromonas hydrophila AH-1. Mutation of the gene wbpL, which is involved in the O antigen synthesis of the LPS, leads to loss of the O antigen, a lesser molecular weight of the polar flagella and the appearance of fimbriae on the bacterial surface. It was studied whether the polar and lateral flagella of the strain AH-1 appear glycosylated. The LC-MS analysis showed that the lateral flagella were not glycosylated, while polar flagella were carrying a 403 Da glycan, derived from pseudaminic acid, as well as a 1060 Da glycan that was not found in the wbpL mutant strain. This finding, together with the fact that a mutant of the rmlB-gene, which plays a role on ramnose-biosynthesis (a sugar of the O antigen), also loses the glycan of 1060 Da, shows the involvement of this glycan in the o antigen of the LPS. It was proceeded to identify the genes in pseudaminic-synthesis adjacent to region 2 of the polar flagellum genes. Mutations in some of these genes (pseI, pseB) as well as in glycosyltranferase maf-1 abolished the presence of polar but nor of lateral flagella and therefore showed a posttranslational regulation of the polar flagellins by glycosylation. As also fimbria formation was observed it was proceeded to analyse the fimbria that were induced in the mutants of wbpL, pseI, pseB and maf-1. They were identified as type IV fimbria, also called MSHA due to their homology to those observed in Vibrio ssp and broadly distributed in the genus Aeromonas. DNA-sequencing showed 22 ORF that were organized in 2 transcripts. To analyze the induction of the fimbria in above mentioned mutants, promoter analysis, RT-PCR and immunodetection was performed. This showed that wildtype AH-1 also expresses fimbria, but is not capable of assembling them correctly and therefore expulses them to the exterior. The accumulation of active sugars in the mutants seems to trigger the correct assembly of the fimbria that play an fundamental role in biofilm formation and therefore favor the persistence of the bacteria in the environment.

El género Aeromonas está constituido por bacilos Gram negativos y comprende patógenos de animales poiquilotermos y oportunistas en humanos. El lipopolisacárido (LPS) es un importante factor de virulencia de Aeromonas hydrophila, por lo que se planteó hallar los genes implicados en la biosíntesis del núcleo del LPS de la cepa AH-3. Con este fin, se aislaron tres mutantes por inserción del transposón mini-Tn5::Km1 con un fenotipo alterado del núcleo, lo que permitió la caracterización de tres regiones wa genómicas distintas. La construcción de mutantes en los genes en los que fue posible y la elucidación de la estructura química de su núcleo mediante ESI MS y el análisis de metilación de los monosacáridos, permitió, junto con el análisis fenotípico del LPS mediante SDS-PAGE y los datos obtenidos a partir de su secuencia y de ensayos de complementación, asignar funciones a cada uno de ellos. Las regiones 2 y 3 wa, constituidas por 4 y 2 genes, respectivamente, codifican las enzimas para la biosíntesis del núcleo interno. La región 1 wa contiene 7 genes que codifican la epimerasa HldD, la ligasa del antígeno O WaaL y las transferasas para la biosíntesis del núcleo externo y la incorporación de la L,D-HepIV. Además, se estudió la distribución de distintos genes del núcleo y se observó su elevada conservación entre diferentes serotipos de Aeromonas mesófilas.Por otro lado, se realizó el análisis y la caracterización funcional de algunos de los genes de la agrupación wb de la cepa AH-3 responsable de la biosíntesis del antígeno O:34, obtenida en un estudio anterior. Consta de 17 genes entre los que se incluyen genes para la biosíntesis de GDP-manosa y dTDP-6-desoxi-L talosa y para la transferencia de sus monosacáridos, para la modificación del antígeno O con acetilaciones, para el inicio de la síntesis de las subunidades O (wecA), su transporte (wzx) y su polimerización (wzy), y para la determinación de la longitud de la cadena (wzz). La región promotora del gen wzz, localizada en función de la determinación de su inicio de transcripción por 5'RACE, presentaba una regulación por temperatura, de manera que el nivel de transcripción/expresión del gen era menor a 37ºC que a 20ºC, según los ensayos de RT-PCR semicuantitativa y de la actividad β-galactosidasa de la fusión transcripcional de la región promotora con el gen indicador lacZ.Este hecho ofrece una explicación a la diferente producción del LPS que presenta la cepa AH-3 a ambas temperaturas., The genus Aeromonas is composed of Gram-negative rod-shaped bacteria and comprises opportunistic and primary pathogens of poikilothermic and homeothermic animals, including humans. The lipopolysaccharide (LPS) is an important virulence factor of Aeromonas hydrophila. In order to identify the genes involved in the LPS core biosynthesis a mini-Tn5::Km1 transposon mutagenesis was carried out in strain AH-3 and three different mutants with an altered core phenotype were isolated, which enabled the characterization of three different wa genomic regions. By means of mutant construction in each gene when possible and their LPS core structures elucidation by ESI-MS and monosaccharides methylation analysis, together with LPS phenotypic analysis by SDS-PAGE and data obtained from the sequence analysis and complementation assays, it was possible to assign functions to each gene. Regions 2 and 3 wa, containing 4 and 2 genes, respectively, encode inner core biosynthesis enzymes. Region 1 wa includes 7 genes encoding HldD epimerase, WaaL Oantigen ligase and transferases involved in outer core biosynthesis and L,D-HepIV incorporation. Moreover, the LPS core genes distribution study showed a high conservation between different mesophilic Aeromonas serotypes. Analysis and functional characterization of some of the 17 genes of the AH-3 wb gene cluster responsible for the O:34-antigen biosynthesis, which had been identified in a previous work, was also performed. It contains genes for activated monosaccharides GDP manose and dTDP-6-deoxy-L-talose production and for transfer, for O-antigen acetylation, for the O-unit biosynthesis initiation (wecA), transport (wzx) and polymerization (wzy) and to determine the O-antigen chain length (wzz). The wzz promoter region was localized due to its transcription start site determined by 5'RACE. Semi-quantitative RT-PCR and β-galactosidase fusion assays lead to the conclusion that the wzz gene transcription/expression level was much lower at 37ºC than at 20ºC, which explains the differential O:34-antigen production at both temperatures.

Aeromonas hydrophila es un bacilo gram-negativo, patógeno oportunista de animales y humanos. La patogénesis de A. hydrophila es multifactorial. Con objeto de identificar genes implicados en la virulencia de la cepa PPD134/91 de A. hydrophila, se realizaron experimentos de sustracción génica, que llevaron a la detección de 22 fragmentos de ADN que codificaban 19 putativos factores de virulencia, incluyendo un gen que codificaba una proteína de sistema de secreción de tipo III (T3SS). La creciente importancia del T3SS en la patogénesis de diversas bacterias, llevó a identificar y analizar la agrupación génica del T3SS de las cepas AH-1 y AH-3 de A. hydrophila, así como también el efector AexT en la cepa AH-3. La inactivación de los genes de T3SS aopB y aopD en A. hydrophila AH-1, y ascV en A. hydrophila AH-3 conlleva una disminución de la citotoxicidad, un incremento de la fagocitosis, y una reducción de la virulencia en diferentes modelos animales, demostrando estos resultados que el T3SS es necesario para la patogenicidad del género Aeromonas. El mutante en el gen aexT de la cepa AH-3 de A. hydrophila muestra una ligera reducción de la virulencia, ensayada con diferentes métodos. Por otro lado, la proteína AexT presentó actividad ADP-ribosiltransferasa, y se demostró su translocación vía T3SS al interior de células eucariotas, sistema que a su vez parece ser inducible in vitro en condiciones de depleción de calcio. Mediante el uso de diferentes sondas de ADN, se determinó la presencia del T3SS en cepas tanto clínicas como ambientales de diferentes especies del género Aeromonas: A. hydrophila, A. veronii, y A. caviae, y la codistribución de esta agrupación génica y del gen aexT. Finalmente, con el fin de analizar la activación transcripcional de la agrupación génica de T3SS y del efector AexT en A. hydrophila AH-3, los promotores predichos del operón aopN-aopD y el gen aexT se fusionaron con el gen reporter gfp (Green Fluorescence Protein). Además, se demostró que la expresión de ambos promotores depende de diferentes componentes bacterianos, como por ejemplo el sistema de dos componentes PhoP/PhoQ, el sistema de quórum sensing AhyI/AhyR, el sistema flagelar, el lipopolisacárido, la oxidoreductasa DsbA o el complejo enzimático piruvato deshidrogenasa., Aeromonas hydrophila is a gram-negative opportunistic pathogen of animals and humans and its pathogenesis is multifactorial. Genomic subtraction was used to identify putative virulence genes in A. hydrophila PPD134/91 and led to the identification of 22 unique DNA fragments encoding 19 putative virulence factors, including a gene encoding a type III secretion system (T3SS) protein. The increasing importance of the T3SS in the pathogenesis of several bacteria, led us to identify and further analyse the T3SS gene clusters in A. hydrophila AH-1 and AH-3. Insertional inactivation of the T3SS genes aopB and aopD in A. hydrophila AH-1 and ascV in A. hydrophila AH-3 led to decreased cytotoxicity, increased phagocytosis, and reduced virulence in several animal models. These results show that the T3SS is required for A. hydrophila pathogenicity. We also cloned and sequenced an ADP-ribosylating toxin (AexT) in A. hydrophila AH-3, and showed that this toxin is translocated via the T3SS, which also seems to be inducible in vitro under calcium depleted conditions. The A. hydrophila AH-3 aexT mutant showed a slight reduction in virulence assayed by several methods. By using several gene probes, we also determined the presence of the T3SS in clinical and environmental strains of different Aeromonas species: A. hydrophila, A. veronii, and A. caviae, and the co-distribution of this gene cluster and the gene encoding the toxin AexT. Finally, to study the transcriptional regulation of the T3SS gene cluster and the effector AexT in A. hydrophila AH-3, we isolated the predicted promoter regions of both operon aopN-aopD and gene aexT, which were fused with a green fluorescence protein reporter gene. Furthermore, we established that expression of the T3SS gene cluster and the effector AexT in A. hydrophila AH-3 depends on the expression of several bacterial components, such as the lipopolysaccharide, the polar flagellum, the two component regulatory system PhoP/PhoQ, the quorum sensing system based on AhyI/AhyR proteins, the pyruvate dehydrogenase complex and the periplasmic oxidoreductase DsbA.