Comparative proteome analysis ofChlamydia trachomatis serovar A, D and L2

Abstract

Chlamydia trachomatis represents a group of human pathogenic obligate intracellular and gram-negative bacteria. The genome of C. trachomatis D comprises 894 open reading frames (ORFs). In this study the global expression of genes in C. trachomatis A, D and L2, which are responsible for different chlamydial diseases, was investigated using a proteomics approach. Based on silver stained two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), gels with purified elementary bodies (EB) and auto-radiography of gels with 35S-labeled C. trachomatis proteins up to 700 protein spots were detectable within the range of the immobilized pH gradient (IPG) system used. Using mass spectrometry and N-terminal sequencing followed by database searching we identified 250 C. trachomatis proteins from purified EB of which 144 were derived from different genes representing 16% of the ORFs predicted from the C. trachomatis D genome and the 7.5 kb C. trachomatis plasmid. Important findings include identification of proteins from the type III secretion apparatus, enzymes from the central metabolism and confirmation of expression of 25 hypothetical ORFs and five polymorphic membrane proteins. Comparison of serovars generated novel data on genetic variability as indicated by electrophoretic variation and potentially important examples of serovar specific differences in protein abundance. The availability of the complete genome made it feasible to map and to identify proteins of C. trachomatis on a large scale and the integration of our data in a 2-D PAGE database will create a basis for post genomic research, important for the understanding of chlamydial development and pathogenesis.

Profiling of Human Antibody Responses toChlamydia trachomatisUrogenital Tract Infection Using Microplates Arrayed with 156 Chlamydial Fusion Proteins

“…Although the 156 proteins represent only a small fraction of proteins encoded by the entire chlamydial genome, they have provided us the opportunity to discover new antigens beyond MOMP. Third, in attempts to compare all chlamydial proteins, many previous studies have used purified chlamydial organisms as the source of antigens (8,41,44,46,47,55), which led to biased results. This is because chlamydial proteins are not equally represented in chlamydial organisms.…”

Chlamydia trachomatis: Genome sequence analysis of lymphogranuloma venereum isolates

“…Whilst fumC appears intact in strains UW-3 and Har-13 (CTA_0932 and CT855, respectively), the strain L2 ortholog, CTL0228, carries two frameshift mutations. Our findings further confirm the previous observations, in which a truncated FumC was detected in strain L2 during 2D gel/proteomic analysis (Shaw et al 2002). Like FumC, succinate dehydrogenase is also required for the metabolism of fumarate by facilitating the aerobic interconversion of fumarate and succinate.…”

Characterization of In Vitro Chlamydial Cultures in Low-Oxygen Atmospheres

“…To determine the visible effect of low oxygen tension on chlamydial inclusions, infected HeLa cells were cultured on coverslips at 4% and 20% O 2 . HeLa 229 cells (ATCC, Rockville, MD) cultured in 24-well trays (TPP, Trasadingen, Switzerland) were infected with either C. trachomatis serovar D/UW-3/CX (ATCC) or C. pneumoniae CWL029 (ATCC) as previously described (12,15) and cultured in the presence of cycloheximide in either 4% or 20% oxygen atmospheres. Low-oxygen atmospheres were achieved by placing trays with infected HeLa cells in an airtight custom-made box (40 by 30 by 16 cm [width by diameter by height]).…”