[PMC free article] [PubMed] [Google Scholar] 2

[PMC free article] [PubMed] [Google Scholar] 2. novel peptidoglycan structure (examined in recommendations 6, 10, and 21) and the presence of a disulfide-bond-cross-linked major outer membrane protein (MOMP) in the OM and cross-linked cysteine-rich proteins (CRPs) in the periplasm (examined in reference 12). The MOMP is the only chlamydial OM protein that has been well characterized. It is a porin (2, 30), is usually surface uncovered (7, 28), and may play a role in the attachment of to host cells (27, 28). Genes that potentially encode a family of proteins, referred to as polymorphic outer membrane proteins (Pomps), have been recognized in the genomes of species: nine Pomp genes (to serovars D and L2, 21 genes (to (15, 20, 26). However, only two Pomps have been shown to be produced in and (11, 16, 20, 22, 29). The functions of the Pomps are not known, nor is it known when Pomps are made during the developmental cycle. Several other OM proteins can be predicted from chlamydial genomic sequences (15, 26); however, none of these proteins have been experimentally recognized in OMs. The OM Abacavir sulfate of chlamydiae is usually poorly characterized, in part because of the difficulty of growing large quantities of chlamydiae but mainly because chlamydial OMs cannot be separated from IMs by density gradient centrifugation. Criteria that have been used to identify chlamydial OM proteins Abacavir sulfate include surface exposure, as detected by susceptibility to trypsin or reaction with antibodies following treatment of infectious elementary body (EBs) with these reagents, and insolubility in the poor anionic detergent sodium lauryl sarcosinate (Sarkosyl). Although these methodologies have been useful, they can yield deceptive results. For example, damage to EBs or contamination of EBs with osmotically fragile reticulate body during harvesting and purification can expose proteins that are not on the surface of EBs, and the failure to observe positive reactions will result if a surface-exposed protein lacks a trypsin-sensitive site or an immunodominant epitope. Insolubility in Sarkosyl is also subject to misinterpretation. The technique was originally developed by Filip et al. (9) to remove cytoplasmic membrane proteins from well-characterized integral OM proteins of and was adapted to chlamydial studies by Caldwell et al. (4) and Hatch et al. (14). Abacavir sulfate However, the reason for the differential solubility of IM and OM proteins in Sarkosyl is not known, and it is likely that some ACVRLK4 non-OM proteins fractionate in the Sarkosyl-insoluble portion and that some OM proteins are released from OMs by Sarkosyl. For these reasons we chose to identify LGV serovar L2 OM proteins on the basis of their reaction with 3-(trifluoromethyl)-3-(L2.EBs were harvested at 48 h after contamination of L929 cells, treated with 1% Nonidet P-40 (Sigma Chemical Co., St. Louis, Mo.) to eliminate osmotically fragile reticulate body, and purified by centrifugation for 30 min at 80,000 on a three-step gradient of 29, 34, and 40% Hypaque-76 (Nycomed Inc., Princeton, N.J.). Purified EBs from 2 108 cells were reacted with 25 Ci of [125I]TID, as previously explained (8), and incubated for 30 min at 37C in the presence or absence of 12 g of trypsin (type III from bovine pancreas; Sigma Chemical Co.) in 200 l of phosphate-buffered saline (pH 7.4), followed by the addition of 24 g of trypsin inhibitor (type II-O from chicken egg white; Sigma). After the EBs were washed once in trypsin inhibitor, one half of the EB preparation was solubilized by heating to 90C in Laemmli buffer (18) made up of 5% -mercaptoethanol and 10 mM dithiothreitol, and the other half was extracted with 500 l of 0.5% Sarkosyl (Sigma) in phosphate-buffered saline for 30 min at 37C. The Sarkosyl-insoluble portion was collected by centrifugation (14,500 for 20 min) and washed twice in 500 l of 0.5% Sarkosyl before it was solubilized in Laemmli buffer with reducing agents. Proteins in whole EBs and Abacavir sulfate the Sarkosyl-insoluble portion of EBs were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 7.5 to15% gradient gels, and incorporation of 125I into proteins was detected.