Journal: Frontiers in Cellular and Infection Microbiology

Article Title: CT295 Is Chlamydia trachomatis’ Phosphoglucomutase and a Type 3 Secretion Substrate

doi: 10.3389/fcimb.2022.866729

Figure Lengend Snippet: Host PGM1 does not accumulate in the inclusion lumen and is dispensable for C. trachomatis development. (A) – HeLa cells were transfected with the indicated Flag-tagged constructs, infected with C. trachomatis L2 and fixed 24 h later. Cells were permeabilized and stained for the inclusion membrane protein Cap1 (red) and Flag (green). DNA was stained with Hoechst (blue). The merge image is shown. An enlargement of the boxed area is displayed on the right. Only Flag-GlgA was consistently detected in the inclusion lumen (arrowheads). Bar is 10 μm. (B) – Two different siRNA were used to silence pgm1 expression and the effect on PGM1 level was measured by western blot. The arrowhead points to PGM1 (mw=61.5 kDa), higher bands are nonspecific. (C) – The effect of pgm1 silencing on bacterial progeny was measured 48 hpi. Dot plot distribution of the progeny for three independent experiments is shown. Gray bar, median. Statistical significance was calculated using two-way analysis of variance (ANOVA) with Dunnett’s multiple comparison test and was not significant (NS).

Article Snippet: C. trachomatis LGV serovar L2 strain 434/Bu (ATCC), the plasmid-less strain LGV L2 25667R ( ) and GFP-expressing L2 (L2 IncD GFP) ( ) were purified on density gradients as previously described ( ).

Techniques: Transfection, Construct, Infection, Staining, Membrane, Expressing, Western Blot, Comparison

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: CT295 Is Chlamydia trachomatis’ Phosphoglucomutase and a Type 3 Secretion Substrate

doi: 10.3389/fcimb.2022.866729

Figure Lengend Snippet: The chlamydial candidate PGMs CT295 and CT815 are related with other bacterial PGMs, stem from an ancient gene duplication event and are both monophyletic. (A) – Schematic representation of the two putative PGMs from C. trachomatis . PGMs are composed of four phosphoglucomutase domains depicted in blue. The PGM conserved motif sequence is in bold with the serine involved in the phophoryl-transfer in red. (B) – Phylogenetic analysis of chlamydial CT295 and CT815 homologs in eggNOG COG1109 (annotated as “phosphomannomutase”; 456 aligned positions) clustered at 80% amino acid identity over 90% query coverage (n = 2,829). Clades are colored by their affiliation to bacteria, eukaryota, and archaea in green, violet, and blue, respectively. Orange indicates the chlamydial CT295 clade (bacterial eggNOG ENOG4107QSU), and yellow indicates the chlamydial CT815 clade (bacterial eggNOG ENOG4107QJF). Maximum likelihood phylogenetic trees with best fit model LG+C60+G+F with 1,000 ultrafast bootstraps are shown. Bootstrap support for monophyly of chlamydial clades in the tree is ≥ 95%, and the SH-like approximate likelihood ratio is ≥ 80%. (C) – Phylogenetic analysis of chlamydial CT295 homologs (bacterial eggNOG ENOG4107QSU; n = 104) with its sister clade from COG1109 (575 aligned positions) used as outgroup (n = 93). Maximum likelihood phylogenetic tree based on LG+C60+G+F best tree using the posterior mean site frequency model and 100 non-parametric bootstraps. Bootstraps ≥ 70% are indicated by filled circles at splits in the tree. Scale bar indicates 0.2 substitution per position.

Article Snippet: C. trachomatis LGV serovar L2 strain 434/Bu (ATCC), the plasmid-less strain LGV L2 25667R ( ) and GFP-expressing L2 (L2 IncD GFP) ( ) were purified on density gradients as previously described ( ).

Techniques: Sequencing, Bacteria

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: CT295 Is Chlamydia trachomatis’ Phosphoglucomutase and a Type 3 Secretion Substrate

doi: 10.3389/fcimb.2022.866729

Figure Lengend Snippet: CT295 amino-terminal sequence is recognized by the T3S machinery of S. flexneri . (A) – The first 22 amino acids of CT295 were fused to the Cya reporter protein and expressed in a S . flexneri ipaB (constitutive T3S) or mxiD (defective T3S) strain. Exponential phase cultures expressing the reporter fusion protein were fractionated into supernatants (S) and pellets (P). Samples were resolved by SDS-PAGE, transferred to a PVDF membrane, and probed with anti-Cya (to detect the fusion protein), anti-IpaD ( Shigella secreted protein), or anti-CRP ( Shigella non-secreted protein) antibodies. (B) – Cells were infected with C. trachomatis L2 strain stably expressing mCherry with CT295-Flag (Tet-inducible promoter) for 40 h in the presence or not of 20 ng/ml anhydrotetracycline (aTc), before lysis and analysis by western blot. Upon induction, a weak band was detected upon aTc induction (arrow), the other bands correspond to background signal. The membrane was reprobed with antibodies against Chlamydia ’s major outer membrane protein (MOMP) to control for equal loading. (C, D) – Cells infected with C. trachomatis expressing CT295-Flag in the absence of presence of 20 nM aTc were fixed 24 hpi (C) or 40 hpi ( D , with aTc), permeabilized and stained with anti-Flag antibody. In panel (D) , cells infected with C. trachomatis L2 strain stably expressing GFP together with GlgA-Flag (endogenous promoter) were used for comparison. The white arrow points to GlgA-Flag detected in the cytoplasm of one infected cell. Bar is 10 μm. (E) – PGM1 deficient fibroblasts were transfected with Flag-PGM1 or infected with the indicated C. trachomatis L2 strain. Forty-eight hours later the cells were fixed, permeabilized and stained with the indicated antibodies. Nuclei were stained with Hoechst (blue). Bar is 20 μm.

Article Snippet: C. trachomatis LGV serovar L2 strain 434/Bu (ATCC), the plasmid-less strain LGV L2 25667R ( ) and GFP-expressing L2 (L2 IncD GFP) ( ) were purified on density gradients as previously described ( ).

Techniques: Sequencing, Expressing, SDS Page, Membrane, Infection, Stable Transfection, Lysis, Western Blot, Control, Staining, Comparison, Transfection

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: CT295 Is Chlamydia trachomatis’ Phosphoglucomutase and a Type 3 Secretion Substrate

doi: 10.3389/fcimb.2022.866729

Figure Lengend Snippet: The presence of a functional T3S signal in the PGMs of Chlamydiaceae correlates with glycogen storage in inclusions. (A) – Top panels: PAS staining of cells infected with the indicated strains. Bottom panels: C. caviae and C. pneumoniae infected cells were further stained with anti-mGroEL antibody followed with Alexa488-conjugated secondary antibodies to detect the inclusions (asterisks). Bar is 20 μm. (B) – Alignment of the first 24 amino acids of CT295 and its orthologs in the indicated Chlamydiaceae species. Amino acids that differ from the C. trachomatis sequence are shadowed. (C) – The first 22 amino acids of the indicated CT295 orthologs were fused to the Cya reporter protein and expressed in a S . flexneri ipaB (left) or mxiD (right) strains. Secretion was analyzed like in Figure 4A .

Article Snippet: C. trachomatis LGV serovar L2 strain 434/Bu (ATCC), the plasmid-less strain LGV L2 25667R ( ) and GFP-expressing L2 (L2 IncD GFP) ( ) were purified on density gradients as previously described ( ).

Techniques: Functional Assay, Staining, Infection, Sequencing

Journal: BMC Veterinary Research

Article Title: Genital tract lesions in sexually mature Göttingen minipigs during the initial stages of experimental vaginal infection with Chlamydia trachomatis serovar D

doi: 10.1186/s12917-016-0793-6

Figure Lengend Snippet: Experimental design. Ten sexually mature minipigs were intravaginally inoculated with C. trachomatis at oestrus. The pigs were euthanised and sampled on day 3, 5 and 7 post inoculation (PI). As controls for the histological stainings, genital organs from six age-matched, healthy, non-treated gilts were also included (not shown in the figure)

Article Snippet: C. trachomatis SvD (Trachoma type D strain UW-3/Cx, ATCC® VR-885TM) was propagated in HeLa cells as previously described [ ].

Techniques:

Journal: BMC Veterinary Research

Article Title: Genital tract lesions in sexually mature Göttingen minipigs during the initial stages of experimental vaginal infection with Chlamydia trachomatis serovar D

doi: 10.1186/s12917-016-0793-6

Figure Lengend Snippet: Detection of C. trachomatis in vaginal swabs. a cell culturing (shown as IFU/swab); b q-PCR (shown as ng of C. trachomatis /swab). Results are shown for single pigs

Article Snippet: C. trachomatis SvD (Trachoma type D strain UW-3/Cx, ATCC® VR-885TM) was propagated in HeLa cells as previously described [ ].

Techniques: Cell Culture

Journal: BMC Veterinary Research

Article Title: Genital tract lesions in sexually mature Göttingen minipigs during the initial stages of experimental vaginal infection with Chlamydia trachomatis serovar D

doi: 10.1186/s12917-016-0793-6

Figure Lengend Snippet: Detection of C. trachomatis by q-PCR in different parts of the genital tract at days 3, 5 and 7 PI

Article Snippet: C. trachomatis SvD (Trachoma type D strain UW-3/Cx, ATCC® VR-885TM) was propagated in HeLa cells as previously described [ ].

Techniques: