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The elucidation of the molecular details of antibiotic resistance will lead to improvements in extending the efficacy of current antimicrobials. In the current study, proteomic methodologies were applied to characterize functional outer membrane proteins (Omps) of E. coli K-12 responded to tetracycline and ampicillin resistance for understanding of universal pathways that form barriers for antimicrobial agents. For this purpose, E. coli K-12 expressional outer membrane proteome was characterized and identified with the use of 2-DE and MALDI-TOF/MS methods. Then, differential Omps due to tetracycline or ampcilin resistance were determined by comparison between tetracycline minimum inhibitory concentration (MIC)10, ampicillin MIC10, control0 and control10, showing 9 proteins with 11 spots for tetracycline and 8 protein with 9 spots for ampicillin, showing a difference in only 1 protein (decreased LamB in tetracyclin) between the two antibiotics. Among the proteins, 3 were known as antibiotic-resistant proteins, including TolC, OmpC and YhiU, while FimD precursor, LamB, Tsx, YfiO, OmpW, NlpB were first reported here to be antibiotic-resistance-related proteins. Our findings will be helpful for further understanding of antibiotic-resistant mechanism(s). This study also shows that the combination of Omp purification methods certainly contributes the sensitivity of Omp detection.

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Background: GAS virulence is dependent on a functional human fibrinolytic system.Results: Functional differences between different strains of GAS streptokinases have been found.Conclusion: Pressure on coinheritance of specific GAS streptokinases and M proteins is identifiable.Significance: Tissue tropism and virulence of GAS infections are reflected in the types of streptokinases and M proteins.

Background:The two-component CovRS system is a GAS virulence gene regulator. Results: A natural inactivating mutation has been found in CovS in a skin-invasive GAS strain, potentially affecting expression of key virulence genes. Conclusion: The mutation in CovS showed substantial effects on regulation of virulence genes. Significance: Expression of genes critical to GAS virulence can reveal reasons why specific strains of GAS are effective tissue specialists.

An intracellular mannanase was identified from the thermoacidophile Alicyclobacillus acidocaldarius Tc-12-31. This enzyme is particularly interesting, because it shows no significant sequence similarity to any known glycoside hydrolase. Gene cloning, biochemical characterization, and structural studies of this novel mannanase are reported in this paper. The gene consists of 963 bp and encodes a 320-amino acid protein, AaManA. Based on its substrate specificity and product profile, AaManA is classified as an endo-␤-1,4-mannanase that is capable of transglycosylation. Kinetic analysis studies revealed that the enzyme required at least five subsites for efficient hydrolysis. The crystal structure at 1.9 Å resolution showed that AaManA adopted a (␤/␣) 8 -barrel fold. Two catalytic residues were identified: Glu 151 at the C terminus of ␤-stand ␤4 and Glu 231 at the C terminus of ␤7. Based on the structure of the enzyme and evidence of its transglycosylation activity, AaManA is placed in clan GH-A. Superpositioning of its structure with that of other clan GH-A enzymes revealed that six of the eight GH-A key residues were functionally conserved in AaManA, with the exceptions being residues Thr 95 and Cys 150 . We propose a model of substrate binding in AaManA in which Glu 282 interacts with the axial OH-C(2) in ؊2 subsites. Based on sequence comparisons, the enzyme was assigned to a new glycoside hydrolase family (GH113) that belongs to clan GH-A.Mannans are polysaccharides found in plants and consist of a backbone of ␤-1,4-linked mannose and glucose units. Mannose residues often carry an ␣-galactosyl substitute at O-6, and the degree of substitution depends on the plant of origin (Fig. 1A) (1). Mannans are widely distributed in nature in parts of the hemicellulose fraction in hardwoods and softwoods (1), legume seeds (2), and beans of carob trees (3).Endo-␤-1,4-mannanases (mannanases; EC 3.2.1.78) are glycoside hydrolases that randomly cleave the ␤-1,4-linkage in mannans (Fig. 1, A and B) (4); these enzymes have been isolated from bacteria, fungi, plants, and some mollusks (5-8). Interest in these enzymes has been increasing due to their importance in hemicellulose hydrolysis and various other applications (5, 9, 10). During the last 2 decades, more than 80 sequences of the catalytic domains of mannanase have been reported (see the CAZy site on the World Wide Web) and classified into glycoside hydrolase (GH) 2 families 5 and 26, based on their sequence similarities (11). In recent years, crystallographic resolution of the structures of these enzymes has yielded information on their structure. At present, the tertiary structures of seven mannanases have been determined, of which five are from GH family 5 (12-16) and two are from family 26 (17, 18). All of these mannanases share a common (␤/␣) 8 barrel fold, a retaining reaction mechanism (Fig. 1C), and two conserved catalytic residues (two glutamate residues at the C termini of ␤4 and ␤7, respectively). Therefore, all of these enzymes have been assigned to the same GH ...

An unknown protein reacted with anti-human IgA, namely, IgA-like protein, has been reported in shrimp, but information regarding its identification is not available. In the present study, an affinity proteomic strategy was applied to identify the IgA-like protein of shrimp Litopenaeus vannamei. The protein of 75 kDa was isolated and confirmed by affinity chromatography and Western blotting with goat anti-human IgA, respectively, and then identified as hemocyanin, a member of IgSF, by mass spectrometry. Moreover, our results showed that human IgA and L. vannamei hemocyanin could separately react with goat anti-human IgA or rabbit anti-shrimp affinity hemocyanin (a-hemocyanin). Further evidences indicated that the recombinant protein of the Ig-like conserved domain could react with anti-human IgA. Interestingly, our results indicated that L. vannamei hemocyanin could aggregate with eight species of shrimp pathogenic bacteria and four types of animal erythrocytes directly. These results indicate that L. vannamei hemocyanin, an IgA-like protein, has dual function of reaction with anti-human IgA as an antigen and of activity binding to bacteria and animal erythrocytes as an agglutinin, suggesting its characteristic role as an IgSF molecule. In addition, our approach suggests that affinity proteomics based on heterogeneous antibody can speed up the identification of Fossman antigens.

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