Polar glycosylated and lateral non-glycosylated flagella from Aeromonas hydrophila strain AH-1 (serotype O11)

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

Sampling the conformational energy landscape of a hyperthermophilic protein by engineering key substitutions

Proteins exist as a dynamic ensemble of interconverting substates, which defines their conformational energy landscapes. Recent work has indicated that mutations that shift the balance between conformational substates (CSs) are one of the main mechanisms by which proteins evolve new functions. In the present study, we probe this assertion by examining phenotypic protein adaptation to extreme conditions, using the allosteric tetrameric lactate dehydrogenase (LDH) from the hyperthermophilic bacterium Thermus thermophilus (Tt) as a model enzyme. In the presence of fructose 1, 6 bis-phosphate (FBP), allosteric LDHs catalyze the conversion of pyruvate to lactate with concomitant oxidation of nicotinamide adenine dinucleotide, reduced form (NADH). The catalysis involves a structural transition between a low-affinity inactive 'T-state' and a high-affinity active 'R-state' with bound FBP. During this structural transition, two important residues undergo changes in their side chain conformations. These are R171 and H188, which are involved in substrate and FBP binding, respectively. We designed two mutants of Tt-LDH with one ('1-Mut') and five ('5-Mut') mutations distant from the active site and characterized their catalytic, dynamical, and structural properties. In 1-Mut Tt-LDH, without FBP, the KmPyr is reduced compared with that of the wild type, which is consistent with a complete shifting of the CS equilibrium of H188 to that observed in the R-state. By contrast, the CS populations of R171, kcat and protein stability are little changed. In 5-Mut Tt-LDH, without FBP, KmPyr approaches the values it has with FBP and becomes almost temperature independent, kcat increases substantially, and the CS populations of R171 shift toward those of the R-state. These changes are accompanied by a decrease in protein stability at higher temperature, which is consistent with an increased flexibility at lower temperature. Together, these results show that the thermal properties of an enzyme can be strongly modified by only a few or even a single mutation, which serve to alter the equilibrium and, hence, the relative populations of functionally important native-state CSs, without changing the nature of the CSs themselves. They also provide insights into the types of mutational pathways by which protein adaptation to temperature is achieved.</p

Functional genomics of the Aeromonas salmonicida lipopolysaccharide O-antigen and A-layer from typical and atypical strains

The A. salmonicida A450 LPS O-antigen, encoded by the wb salmo gene cluster, is exported through an ABC-2 trans porter-dependent pathway. It re presents the first example of an O-antigen LPS polysaccharide with three different monosaccharides in their repeating unit assembled by this pathway. Until now, only repeating units with one or two different monosaccharides have been described. F unctional genomic analysis of this wb salmo region is mostly in agreement with the LPS O-antigen structure of acetylated L -rhamnose (Rha), D -glucose (Glc), and 2-amino-2-deoxy- D -mannose (ManN). Between genes of the wb salmo we found the genes responsible for the biosynthe sis and assembly of the S-layer (named A-layer in these strains). Through comparative genomic analysis and in-frame deletions of some of the genes, we concluded that all the A. salmonicida typical and at ypical strains, other than A. salmonicida subsp. pectinolytica strains, shared the same wb salmo and presence of A-layer. A. salmonicida subsp. pectinolytica strains lack wb salmo and A-layer, two major virulence factors, and th is could be the reason they ar e the only ones not found as fish pathogens

Polar glycosylated and lateral non-glycosylated flagella from Aeromonas hydrophila strain AH-1 (serotype O11)

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

Inflammatory responses associated with acute coronary syndrome up-regulate IRAK-M and induce endotoxin tolerance in circulating monocytes

Acute coronary syndrome (ACS) groups different cardiac diseases whose development is associated with inflammation. Here we have analyzed the levels of inflammatory cytokines and of members of the TLR/IRAK pathway including IRAK-M in monocytes from ACS patients classified as either UA (unstable angina), STEMI (ST-elevation myocardial infarction) or NSTEMI (non-ST-elevation myocardial infarction). Circulating monocytes from all patients, but not from healthy individuals, showed high levels of pro-inflammatory cytokines, TNF-α and IL-6, as well as of IRAK-M and IL-10. TLR4 was also up-regulated, but IRAK-1, IRAK-4 and MyD88 levels were similar in patients and controls. Further, we investigated the consequences of cytokines/IRAK-M expression on the innate immune response to endotoxin. Ex vivo responses to LPS were markedly attenuated in patient monocytes compared to controls. Control monocytes cultured for 6 h in supplemented medium (10% serum from ACS patients) expressed IRAK-M, and LPS stimulation failed to induce TNF-α and IL-6 in these cultures. Pre-incubation of the serum with a blocking anti-TNF-α antibody reduced this endotoxin tolerance effect, suggesting that TNF-α controls this phenomenon, at least partially. We show for the first time that inflammatory responses associated with ACS induce an unresponsiveness state to endotoxin challenge in circulating monocytes, which correlates with expression of IRAK-M, TLR4 and IL-10. The magnitude of this response varies according to the clinical condition (UA, STEMI or NSTEMI), and is regulated by TNF-α