ABSTRACT
Clostridium sordellii is a toxin-producing anaerobic bacillus that causes severe infections in humans and livestock. C. sordellii infections can be accompanied by a highly lethal toxic shock syndrome (TSS). Lethal toxin (TcsL) is an important mediator of TSS. We recently obtained a clinical strain of C. sordellii (DA-108) lacking the TcsL-encoding tcsL gene, which was not fatal in rodent models of infection, in contrast to a tcsL(+) reference strain (ATCC9714). Protein preparations derived from cell-free, stationary phase cultures obtained from ATCC9714 were lethal when injected into mice, while those obtained from DA-108 were not, a difference that was attributed to the unique presence of TcsL in the ATCC9714-derived proteins. We questioned whether there were other major differences between the extracellular proteomes of these two strains, apart from TcsL. Two-dimensional gel electrophoresis was conducted using crude cell-free supernatants from these strains and 14 differentially expressed proteins were subjected to mass spectrometric analysis. Nine of these 14 proteins were more highly expressed by DA-108 and 5 by ATCC9714. Twelve of the 14 proteins isolated from the 2-D gels were putatively identified by mass spectrometry. Several of these proteins were identical, possibly reflecting enzymatic cleavage, degradation, and/or post-translational modifications. Excluding identical sequences, only 5 unique proteins were identified. Four proteins (ferredoxin-nitrite reductase; formate acetyltransferase; Translation Elongation Factor G; and purine nucleoside phosphorylase) were over-expressed by DA-108 and 1 (N-acetylmuramoyl-l-alanine amidase) by ATCC9714. These results support the concept that TcsL is the major determinant of C. sordellii TSS during infection.
Subject(s)
Bacterial Proteins/analysis , Bacterial Proteins/metabolism , Clostridium sordellii/chemistry , Proteome/analysis , Virulence Factors/analysis , Bacterial Toxins/genetics , Clostridium sordellii/pathogenicity , Electrophoresis, Gel, Two-Dimensional , Mass SpectrometryABSTRACT
OBJECTIVES: To study 11 patients with melanoma-associated retinopathy (MAR) to clarify the reliability of various methods of diagnostic testing, to determine the underlying antigenic retinal proteins, and to study the clinical histories and types of associated melanomas. METHODS: Clinical data were obtained from patients with melanoma who developed marked visual problems. Testing included electroretinography, kinetic visual fields, comparative studies of Western blots, and indirect immunohistologic examination to detect antiretinal antibodies, as well as proteomic studies to identify underlying antigenic retinal proteins. RESULTS: Patients with MAR typically have rapid onset of photopsias, scotomata, and loss of central or paracentral vision. Ophthalmoscopy seldom shows significant changes early, but electroretinograms are abnormal. Results of Western blots and immunohistologic examination can show antiretinal antibodies but not always. Most patients (9 of 11) had a strong family history of autoimmune disorders. Any type of melanoma (cutaneous, choroidal, ciliary body, or choroidal nevi) may be associated with this paraneoplastic autoimmune reactivity. MAR may precede or follow the diagnosis of melanoma. Patients with MAR have the same antigenic retinal proteins that have been associated with cancer-associated retinopathy. In addition, 2 new antigenic retinal proteins, aldolase A and aldolase C, were found. CONCLUSIONS: There was a high prevalence of positive family histories of autoimmune disease in patients with MAR. To confirm the disorder, multiple clinical and serum diagnostic techniques (Western blot or indirect immunohistologic examination) are needed. Two newly observed antigenic retinal proteins, aldolase A and aldolase C, are associated with MAR.
Subject(s)
Autoantibodies/blood , Autoantigens/analysis , Autoimmune Diseases/immunology , Melanoma/immunology , Paraneoplastic Syndromes/immunology , Retinal Diseases/immunology , Adult , Aged , Aged, 80 and over , Autoimmune Diseases/etiology , Blotting, Western , Electrophoresis, Polyacrylamide Gel , Electroretinography , Female , Fluorescent Antibody Technique, Indirect , Fructose-Bisphosphate Aldolase/analysis , Humans , Male , Melanoma/pathology , Middle Aged , Paraneoplastic Syndromes/etiology , Retinal Diseases/etiology , Scotoma/etiology , Skin Neoplasms/immunology , Skin Neoplasms/pathology , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Uveal Neoplasms/immunology , Uveal Neoplasms/pathology , Visual FieldsABSTRACT
The zymogen granule (ZG) is the specialized organelle in pancreatic acinar cells for digestive enzyme storage and regulated secretion and has been a model for studying secretory granule functions. In an initial effort to comprehensively understand the functions of this organelle, we conducted a proteomic study to identify proteins from highly purified ZG membranes. By combining two-dimensional gel electrophoresis and two-dimensional LC with tandem mass spectrometry, 101 proteins were identified from purified ZG membranes including 28 known ZG proteins and 73 previously unknown proteins, including SNAP29, Rab27B, Rab11A, Rab6, Rap1, and myosin Vc. Moreover several hypothetical proteins were identified that represent potential novel proteins. The ZG localization of nine of these proteins was further confirmed by immunocytochemistry. To distinguish intrinsic membrane proteins from soluble and peripheral membrane proteins, a quantitative proteomic strategy was used to measure the enrichment of intrinsic membrane proteins through the purification process. The iTRAQ ratios correlated well with known or Transmembrane Hidden Markov Model-predicted soluble or membrane proteins. By combining subcellular fractionation with high resolution separation and comprehensive identification of proteins, we have begun to elucidate zymogen granule functions through proteomic and subsequent functional analysis of its membrane components.
