Journal: Infection and Immunity

Article Title: Infectivity Acts as In Vivo Selection for Maintenance of the Chlamydial Cryptic Plasmid ▿

doi: 10.1128/IAI.01105-10

Figure Lengend Snippet: Characteristics of C. muridarum strains used in this study

Article Snippet: Comparative genome hybridization was performed using a C. muridarum -specific DNA microarray (NimbleGen Systems, Inc., Madison, WI), the design of which was derived from the published and annotated sequence ( 24 ).

Techniques: Plasmid Preparation, Infection, Staining

Journal: Infection and Immunity

Article Title: Infectivity Acts as In Vivo Selection for Maintenance of the Chlamydial Cryptic Plasmid ▿

doi: 10.1128/IAI.01105-10

Figure Lengend Snippet: C. muridarum Nigg successfully competes with CM972 in vitro but not with CM3.1. (A) Competition with CM972. L929 fibroblasts were infected without centrifugation with C. muridarum Nigg, with CM972, or with a mixed inoculum and were incubated for 40 h before harvest. This process was repeated 5 times, using 1/10 of the prior passage volume as the inoculum each time. Samples from each passage were subsequently infected into L929 fibroblasts with centrifugation and incubated for 40 h before being fixed and stained with iodine to detect Nigg (iodine-positive) and CM972 (iodine-negative) inclusions. (B) Competition with CM3.1. L929 fibroblasts were infected (without centrifugation) with C. muridarum Nigg or CM3.1 or with a mixed inoculum for passage and analysis as described above. Data presented were obtained from the simultaneous counting and iodine scoring of 150 to 200 inclusions/well, with two replicates per passage. Each experiment was performed twice.

Article Snippet: Comparative genome hybridization was performed using a C. muridarum -specific DNA microarray (NimbleGen Systems, Inc., Madison, WI), the design of which was derived from the published and annotated sequence ( 24 ).

Techniques: In Vitro, Infection, Centrifugation, Incubation, Staining

Journal: Infection and Immunity

Article Title: Infectivity Acts as In Vivo Selection for Maintenance of the Chlamydial Cryptic Plasmid ▿

doi: 10.1128/IAI.01105-10

Figure Lengend Snippet: Comparative genome hybridization detects an SNP present in C. muridarum Nigg and its derivatives CM972 and CM3.1. (A) Graphic representation of data obtained from tiled-microarray analysis of the C. muridarum strain Nigg genome. A single nucleotide polymorphism was identified at 473155. (B) PCR products encompassing TC_0412 were generated and sequenced. The aligned sequence obtained from this analysis, representing the genomes from bp 473117 to bp 473214 and with the additional T insertion indicated, is presented.

Article Snippet: Comparative genome hybridization was performed using a C. muridarum -specific DNA microarray (NimbleGen Systems, Inc., Madison, WI), the design of which was derived from the published and annotated sequence ( 24 ).

Techniques: Hybridization, Microarray, Generated, Sequencing

Journal: Infection and Immunity

Article Title: Infectivity Acts as In Vivo Selection for Maintenance of the Chlamydial Cryptic Plasmid ▿

doi: 10.1128/IAI.01105-10

Figure Lengend Snippet: Comparative genomic hybridization detects a novel SNP in C. muridarum CM3.1. (A) Graphic representation of data obtained from a tiled microarray analysis of the C. muridarum CM3.1 genome. An SNP is identified at bp 276796. (The TC_0412 SNP was not detected in this analysis, because the C. muridarum Nigg isolate also carrying this mutation was used as the hybridization control). (B) Sequence comparison of TC_0236 with the most closely related members of pfam DUF 720 family. The nonconservative amino acid substitution Q119K is indicated.

Article Snippet: Comparative genome hybridization was performed using a C. muridarum -specific DNA microarray (NimbleGen Systems, Inc., Madison, WI), the design of which was derived from the published and annotated sequence ( 24 ).

Techniques: Hybridization, Microarray, Mutagenesis, Control, Sequencing, Comparison

Journal: Infection and Immunity

Article Title: Infectivity Acts as In Vivo Selection for Maintenance of the Chlamydial Cryptic Plasmid ▿

doi: 10.1128/IAI.01105-10

Figure Lengend Snippet: Cervicovaginal shedding of chlamydiae by mice infected with C. muridarum Nigg and CM972. (A) Groups of mice were infected with either Nigg or CM972, and cervicovaginal shedding over subsequent days was assessed by immunofluorescent staining using a mouse monoclonal antibody directed against chlamydial LPS (Nigg plus CM972) or a rabbit polyclonal antibody directed against pMoPn-encoded TCA08. Immunofluorescent staining with an anti-TCA08 antibody successfully discriminates between C. muridarum Nigg (open squares) and plasmid-deficient CM972 (open circles at 100) shed by infected mice. CM972 infection indicated by anti-LPS staining (closed circles) was less infectious than Nigg (closed squares) (P = 0.03 by two-way RM ANOVA). (B) Cervicovaginal shedding of chlamydiae by mice infected with C. muridarum Nigg and CM972 at ratio of 1:10. The total bacterial burden was detected by immunofluorescent staining with anti-LPS antibody. Results of shedding by parallel groups infected with Nigg or CM972 alone are indicated as controls. By day 3 of infection, mice infected with the mixed inoculum were shedding more chlamydiae than mice infected solely with CM972 (P = 0.04; two-way RM ANOVA with multiple comparisons), indicating that Nigg had quickly become predominant. (C) Cervicovaginal shedding of chlamydiae by mice infected with C. muridarum Nigg and CM972 at ratio of 1:100. The total bacterial burden was detected by immunofluorescent staining with anti-LPS antibody. Results of shedding by parallel groups infected with Nigg or CM972 alone are indicated as controls. Mice infected with 103 Nigg plus 105 CM972 shed chlamydiae in amounts similar to those seen with mice infected with wild-type Nigg by day 5 of infection, and levels were equivalent to those seen with mice infected with Nigg alone by day 9. Data represent the means ± standard deviations of the results for each group of 5 mice, and the experiment was performed twice.

Article Snippet: Comparative genome hybridization was performed using a C. muridarum -specific DNA microarray (NimbleGen Systems, Inc., Madison, WI), the design of which was derived from the published and annotated sequence ( 24 ).

Techniques: Infection, Staining, Plasmid Preparation

Journal: Infection and Immunity

Article Title: Infectivity Acts as In Vivo Selection for Maintenance of the Chlamydial Cryptic Plasmid ▿

doi: 10.1128/IAI.01105-10

Figure Lengend Snippet: Detection of C. muridarum Nigg expansion in vivo using anti-TCA08 antibody. Mixed-inoculum infections were compared with wild-type Nigg and CM972 infections using anti-TCA08 antibody to detect plasmid-containing bacteria. The results of infection with Nigg plus CM972 (1:10) were similar to the results of infection with Nigg alone (squares) on all days examined (P = 0.85). Plasmid-positive chlamydiae detected in mice infected with Nigg plus CM972 (1:100) became equal in number to those detected in mice infected with Nigg alone by day 7. Data represent the means ± standard deviations of the results for each group of 5 mice, and the experiment was performed twice.

Article Snippet: Comparative genome hybridization was performed using a C. muridarum -specific DNA microarray (NimbleGen Systems, Inc., Madison, WI), the design of which was derived from the published and annotated sequence ( 24 ).

Techniques: In Vivo, Plasmid Preparation, Bacteria, Infection