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)

Description

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.

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Cell culture and organisms. These studies employed C. muridarum strain Nigg as the parent strain. Cm tox was generated from WT Cm via FRAEM. Reversal of the tox deletion was accomplished by lateral gene transfer via co-culture of toxRif with WT Cm at an initial 1:10 ratio in the absence of antibiotic selection for three 24hr passages. DNA copy number and gene expression. Relative gene copy number was determined form DNA extracted from 24 hrcultures. RNA was extracted from McCoy cultures infected at an MOI of 1 for 24 hr. Subsequent reverse-transcription PCR (RT-PCR) was accomplished using OneTaq RT-PCR kit (NEB). Tissue culture infectivity. McCoy cells were seeded onto 6 well plates (2 × 105 cells/well) 24 h prior to infection. Progeny EBs from 24 hr cultures were passaged onto fresh McCoy and enumerated by immunofluorescence of cultures methanol fixed and stained with Hsp60 antibodies at 24 hr. Invasion rates were tested by infection of McCoy monolayers on 12 mm glass coverslips at an MOI of 10 with respective strains. Adherence of respective strains to host cells was tested when mock or dextran-treated monolayers were infected in triplicate by rocking at 37°C for 1 hr with respective strains. Media were removed and monolayers were thoroughly washed with HBSS. Cytotoxicity assays. Replicate McCoy wells were mock infected or infected with Ctr L2 WT, Cm WT, Cm tox, or Cm toxRep at an MOI of 500. Polymerized actin was detected using ActinRed phalloidin (ThermoFisher). Coverslips were washed with PBS and mounted. Epi-fluorescence microscopy was used to image coverslips using a 40x objective. ImageJ was used to process images. Freehand tool was used to outline the border of the cells and the Measurement tool was used to measure circularity (4pi(area/perimeter2)). For this assay, 100 cells were measured for each triplicate for each strain. A LDH cytotoxicity assay was used to measure LDH release as an indicator of cytotoxicity. McCoy cells were seeded onto 96 well plates (2 × 104 cells/well) 24 h prior to infection. Replicated wells were then mock infected or infected with Ctr L2 WT, Cm WT, Cm Dtox, or Cm toxrep at an MOI of 500. Cultures were centrifuged at 900 x g for 30 min followed by an incubation at 37°C. Four h post infection, 50 µL aliquots were taken from supernatant of cell culture and placed onto 96 well plate. LDH release was quantified using the CyQUANT LDH Cytotoxicity Assay Kit (Invitrogen). Cytotoxicity was calculated by comparing its LDH release in mock infected cells to the maximum LDH release (% cytotoxicity = ((sample LDH release- spontaneous LDH release)/ (max LDH release-spontaneous LDH release) *100). This percentage was then normalized to the chromosome copy number of the inoculum assessed via qPCR using 16s-specific primers. (Relative cytotoxicity = % cytotoxicity / inoculum Ct values)

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