Complete deletion of the Chlamydia muridarum putative cytotoxin locus reveals contributions during invasion in tissue culture and oviduct pathology during murine genital tract infection (Figures1-4)

Figure 1. (A). Schematic of toxin genes tc037-0439 in relation to the Cm (WT) genome and their replacement with a blaM-gfp cassette in tox. The presence of chromosomal tc0437-0439 and gfp-specific signal was evaluated in DNA samples via qPCR where values were normalized to 16s content (ND = none detected). (C) Gene-specific mRNA levels for tarp, and toxin-flanking genes tc0436 and tc0440 were assessed during WT and tox samples via qRT-PCR analysis of whole-culture RNA. Values were normalized to rpoD expression levels and no statistically significant differences were found. (D). McCoy cells were equivalently infected with WT or tox Cm and cultures harvested at 24 hr for enumeration of progeny EBs (IFU counts). Figure 2. (A) A rifampin (rif) resistant isolate of tox lacking the tox locus (red) was co-cultured with rif-sensitive WT Cm where tox genes (green) are intact to allow lateral gene transfer. A recombinant strain where the tox deletion was repaired (toxRep) by transfer of genes from WT into the tox chromosome by was isolated by sequential culture with 15 ng rifampin and harvest of rif-resistant, non-fluorescent inclusions. Whole-culture material was harvested from McCoy cells equivalently infected with WT, mutant (tox) or repaired (toxRep) strains for 24 hrs. (B). DNA was probed with gene-specific primers via qPCR to establish relative abundance of tc0438. (C) McCoy cells were equivalently infected with indicated strains and cultures harvested at 24 hr for enumeration of progeny EBs (IFU counts). Figure 3. Absence of tox genes does not correlate with loss of immediate toxicity. McCoy monolayers were mock-treated or infected at an MOI of 500 with Ctr L2, WT Cm, or Cm where toxin genes were lacking (tox) or restored (toxRep). (A). Representative immunofluorescence images are show from 4 hr cultures stained with phalloidin for visualization. (B). Cell rounding was used as an indicator of immediate toxicity and was quantified by measuring circularity (4pi(area/perimeter2)) of cells. (C). Overt cellular toxicity was assessed via quantification of LDH release by infected monolayers. McCoy cells were equally infected for 4 hrs with Ctr L2 or Cm WT, tox, or toxRep Cm and LDH levels where measured in cell-free culture supernatants. Relative cytotoxicity was calculated by comparing spontaneous LDH release in mock infected cells to infection induced LDH release. Figure 4. Adherence of each strain to the host cell was evaluated by infecting McCoy cell monolayers untreated (A) or pretreated (B) with DEAE dextran. Two hours post infection; monolayers were thoroughly washed with HBSS, and the attached bacteria were quantified by assessing chromosome copy number via qPCR using 16s-specific primers. (C). Invasion was assessed by infecting McCoy cells at an MOI of 10 with Cm WT, tox, toxRep or tarp as a positive control.