Elementary bodies (EB) of Chlamydia trachomatis serotypes C, E, and L2 were extrinsically radioiodinated, and whole-cell lysates of these serotypes were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Autoradiography of the polypeptide profiles identified a major surface protein with an apparent subunit molecular weight of 39,500 that was common to each C. trachomatis serotype. The abilities of nonionic (Triton X-100), dipolar ionic (Zwittergent TM-314), mild (sodium deoxycholate and sodium N-lauroyl sarcosine), and strongly anionic (SDS) detergents to extract this protein from intact EB of the L2 serotype were investigated by SDS-PAGE analysis of the soluble and insoluble fractions obtained after each detergent treatment. Only SDS readily extracted this protein from intact EB. Sarkosyl treatment selectively solubilized the majority of other EB proteins, leaving the 39,500-dalton protein associated with the Sarkosyl-insoluble fraction. Ultrastructural studies of the Sarkosyl-insoluble EB pellet showed it to consist of empty EB particles possessing an apparently intact outer membrane. No structural evidence for a peptidoglycan-like cell wall was found. Morphologically these chlamydial outer membrane complexes (COMC) resembled intact chlamydial EB outer membranes. The 39,500-dalton outer membrane protein was quantitatively extracted from COMC by treating them with 2% SDS at 60 degrees C. This protein accounted for 61% of the total COMC-associated protein, and its extraction resulted in a concomitant loss of the COMC membrane structure and morphology. The soluble extract obtained from SDS-treated COMC was adsorbed to a hydroxylapatite column and eluted with a linear sodium phosphate gradient. The 39,500-dalton protein was eluted from the column as a single peak at a phosphate concentration of approximately 0.3 M. The eluted protein was nearly homogeneous by SDS-PAGE and appeared free of contaminating carbohydrate, glycolipid, and nucleic acid. Hyperimmune mouse antiserum prepared against the 39,500-dalton protein from serotype L2 reacted with C. trachomatis serotypes Ba, E, D, K, L1, L2, and L3 by indirect immunofluorescence with EB but failed to react with serotypes A, B, C, F, G, H, I, and J, with the C. trachomatis mouse pneumonitis strain, or with the C. psittaci feline pneumonitis, guinea pig inclusion conjunctivitis, or 6BC strains. Thus, the 39,500-dalton major outer membrane protein is a serogroup antigen of C. trachomatis organisms.

Purification and partial characterization of the major outer membrane protein of Chlamydia trachomatis.

Molecular architecture and functioning of the outer membrane of Escherichia coli and other gram-negative bacteria

Publisher Summary This chapter attempts to synthesize the current knowledge of the outer membrane, emphasizing the structural and molecular basis of various functions. The chapter explores that all the bacterial cells except those of mycoplasma and L-forms are surrounded by cell wall. It is a common knowledge that a fundamental difference exists in the structure of cell wall between the Gram-positive and Gram-negative prokaryotes, and that in the latter the cell wall contains an outer membrane layer in addition to the underlying peptidoglycan layer. The outer membrane especially that of Escherichia coli and Salmonella pphimurium, has been studied extensively during the last several years, and at present it claims to understand its structure and function properly at the molecular level. The chapter highlights that together with the erythrocyte membrane, the outer membrane is one of the best studied biological membranes.

The Outer Membrane of Gram-negative Bacteria

Neisseria meningitidis serogroups B and C were originally subdivided into serotypes by the use of two different classification systems based upon type-specific bactericidal antibodies and immunoprecipitation in agar gels, respectively. The serotype specificities were later found to be associated with different major outer membrane proteins and with the lipopolysaccharide. Physiochemical characterization of the four to five major outer membrane proteins has resulted in designation of classes 1-5 on the basis of peptide mapping. The class 2 (41,000-dalton) or class 3 (38,000-dalton) protein is present in all meningococcal strains. They both are predominant proteins, show a useful degree of antigenic variation, and are epidemiologically relevant. A new serotyping nomenclature and scheme based on these two proteins and the lipopolysaccharide is therefore proposed. With use of this scheme, a meningococcal strain could be identified by serogroup to protein-serotype to lipopolysaccharide serotype, as in B:2a:L3. The class 1 (46,000-dalton) protein can be used to further define a meningococcal strain, as in B:2a:P1.2:L3.

Serotype antigens of Neisseria meningitidis and a proposed scheme for designation of serotypes.

Asymptomatic carriage of Neisseria meningitidis and Neisseria lactamica was studied in a total of 2,969 healthy infants and children in Danbury, Conn., between October 1971 and June 1975. The prevalence of N. meningitidis averaged 0.71% during the first four years of life and increased to 5.4% by 14--17 years. Rates of carriage of N. lactamica increased from 3.8% in three-month-old infants to a peak of 21.0% at 18 months and then declined to 1.8% by 14--17 years of age. Of the children who acquired N. lactamica, 66% developed fourfold or greater rises in titers of IgG antibody to groups A, B, and/or C meningococci as determined by immunofluorescence compared with only 5% of control children. Of new carriers of N. lactamica, 40% developed increased titers of bactericidal antibody to groups A, B, and/or C meningococci as compared with 7% of noncarriers. Carriage of N. lactamica may assist in the development of natural immunity to N. meningitidis by induction of cross-reactive antibodies.

Carriage of Neisseria meningitidis and Neisseria lactamica in Infants and Children

Meningococcal groups B and C have been subdivided into a series of serotypes based upon the antigenic specificity of protein serotype antigens (STA). The purpose of these studies was to obtain the STA by gentle methods and determine its anatomic location in the meningococcal cell. The STA was extracted from group B meningococcal strains by either 0.2 M LiCl or 0.2 M CaCl2 and isolated from the extracts by gel filtration on Sepharose 6B or by pelleting the STA by centrifugation at 100,000 g. The isolated STA was a lipoprotein-lipopolysaccharide complex with a mol wt of approximately 4 x 106 daltons. Antisera prepared against the type 2 STA were bactericidal only for homologous serotype strains. The STA proved to be a constituent of the outer membrane of the cell envelope. This was shown by SDS-polyacrylamide gel electrophoresis (PAGE) of the isolated outer membrane and of the purified STA. The type 2 STA complex contains three principal proteins, one of which is predominant with a mol wt of 41,000 daltons. The type 2 STA was dissociated by Triton X-100 and separated by sucrose gradient isodensity centrifugation into two peaks. The denser peak (ρ = 1.26 g/cm3) contained the majority of the 41,000 dalton major outer membrane protein as shown by SDS-PAGE. This peak also contained the type 2 antigenic determinant. Thus the major outer membrane protein, extracted as part of a lipoprotein-lipopolysaccharide complex, contains the type 2 STA determinant.

https://rupress.org/jem/article-pdf/140/1/87/1086390/87.pdf

O Emil C. Gotschlich

Papers

An outer membrane protein of neisseria meningitidis group b responsible for serotype specificity