Quantitative secretome analysis of polymyxin B resistance in Escherichia coli.

Bacterial resistance has become a serious threat to human health. In particular, the gradual development of resistance to polymyxins, the last line of defense for human infections, is a major issue. Secreted proteins contribute to the interactions between bacteria and the environment. In this study, we compared the secretomes of polymyxin B-sensitive and -resistant Escherichia coli strains by data-independent acquisition mass spectrometry. In total, 87 differentially expressed secreted proteins were identified in polymyxin B-resistant E. coli compared to the sensitive strain. A GO enrichment analysis indicated that the differentially expressed proteins were involved in biological processes, including bacterial-type flagellum-dependent cell motility, ion transport, carbohydrate derivative biosynthetic process, cellular response to stimulus, organelle organization, and cell wall organization or biogenesis. The differentially expressed secreted proteins in polymyxin B-resistant bacteria were enriched for multiple pathways, suggesting that the resistance phenotype depends on complex regulatory mechanisms. A potential biomarker or drug target (YebV) was found in polymyxin B-resistant E. coli. This work clarifies the secretome changes associated with the acquisition of polymyxin resistance and may contribute to drug development.

[Mass spectrometry identification and immune cross-reactivity of a minor shrimp allergen-sarcoplasmic calcium binding protein from Litopenaeus vannamei].

AIM: To identify sarcoplasmic calcium-binding protein (SCP) as a minor shrimp allergen by mass spectrometry, and to analyze the immune cross-reactivity among crustacean SCPs. METHODS: The M(r); 21 000 allergen from Litopenaeus vannamei was identified by MALDI-TOF/TOF-MS. BLAST and ClustalW were used to compare amino acid sequence identity of the allergen among crustaceans. The puritifed M(r); 21 000 allergen was injected subcutaneously in mice to produce the specific polyclonal antibodies to analyze immune cross-reactivity of the allergen with proteins from 8 other species of crustaceans by Western blotting. RESULTS: The M(r); 21 000 shrimp allergen was identified as SCP. Sequence comparison revealed that SCP had 81%-100% amino acid identity among crustaceans. Western blotting showed that the proteins with M(r); about 21 000, corresponding to SCP from Metapenaeus ensis, Penaeus monodon, Oratosquilla oratoria, Macrobrachium rosenbergii, Procambarus clarkii, Portunus pelagicus, Charybdis feriatus, Eriocheir sinensis were recognized by polyclonal antibodies against SCP of Litopenaeus vannamei. CONCLUSION: SCP is a minor shrimp allergen, and SCPs have a high sequence homology and strong immune cross-reactivity among crustaceans, which can be used as detective, diagnostic and safe immunotherapeutic agents for subjects with shrimp allergy.

[Identification, isolation, purification of allergens in shrimp and allergic analysis for the main allergens].

AIM: To identify the allergens in shrimp, and to isolate, purify and analyze the main allergen components. METHODS: The total shrimp proteins were extracted by PBS, the allergens were identified with 11 shrimp allergic patients' serum IgE by Western blot. Three main shrimp allergens 21,000, 36,000 and 80,000 were purified by ammonium sulfate precipitation, Sephadex G-50 chromatography and DEAE-exchange chromatography. Western blot and indirect ELISA were used to confirm and analyze allergenicity of the three main allergens. RESULTS: Western blot demonstrated that there were nine allergen components in shrimp proteins, and IgE binding to 21,000, 36,000 and 80,000 shrimp allergen by 5, 7, 5 (36.4%, 63.6%, 45.5%) of 11 shrimp allergic patients' sera. Results of indirect ELISA showed that the binding absorbency of allergic patients' sera IgE with the three main purified allergens were all higher than that with shrimp protein extraction. CONCLUSION: There are at least 9 allergens in shrimp; the 21,000, 36,000 and 80,000 proteins are the main allergen components; and the 36 000 protein is the primary main allergen, with the highest allergenicity and sensitization rate. In further investigation, we will use monoclonal antibody technique to verify weather the 21,000, 36,000, 80,000 allergens have common epitope, which will lay a foundation for shrimp allergy clinical diagnosis and shrimp allergen vaccine design.