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Figures
- Fig. 1.
C. elegans feed on MAH. C. elegans were seeded onto NGM plates supplemented with FUdR (400 μM) and seeded with live MAH (C,D) or heat-killed MAH (B) each containing a fluorescent red marker. Worms were fed on E. coli OP50 with the fluorescent red marker for 24 h as a processing and image control (A). Worms were allowed to feed for 1 (A-C) or 5 days (D) at which time worms were collected, washed, and mounted on glass slides for microscopic observation. Images are representative of 20 worms visualized per experiment and independently repeated 5 times. All images are shown at 400× magnification; scale bars are 50 μm.
- Fig. 2.
MAH-td104 does not affect median or total lifespan of C. elegans. Worms were picked and incubated on NGM agar to remove external bacteria for 3 h. Thirty worms were individually placed onto NGM-FUdR (400 µM) plates and seeded with either 108 E. coli strain OP50 or MAH-td104. Worms were scored every 2 days for survival and worms that ruptured or crawled up the sides of the plate were censored and removed from the study. Kaplan–Meier statistics were used to construct and analyze growth characteristics. Data represents survival from one experiment and is representative of 2 independently completed experiments.
- Fig. 3.
MAH colonization persists after pulse-chase with E. coli strain OP50. C. elegans were placed onto NGM-FUdR (400 μg/ml) plates and seeded with 108 MAH-td104 and allowed to feed for 5 days. Worms were collected and moved to a new plate to remove extracellular bacteria. Worms were then transferred to a plate seeded with E. coli strain OP50, and allowed to feed for 24 h. Nematodes were collected and mounted onto glass slides for microscopic observation. Images are representative of 20 worms visualized per experiment and independently repeated 3 times. All images are shown at 400× magnification; scale bars are 50 μm.
- Fig. 4.
MAH colonize lumen of C. elegans intestinal tract. Worms were seeded onto NGM-FUdR (400 µM) plates for 5 days and samples were fixed and set into agarose blocks. Paraffin embedded samples were sectioned and acid-fast stained. Starved worms were seeded onto plates in the absence of bacteria (A) or onto plates containing MAH (B,C). Acid-fast positive bacilli within the intestinal space are indicated by arrows. Non-specific staining of fat deposits by carbol-fuchsin is indicated by asterisks. Images are representative of 10 worms sectioned and analyzed per treatment. All images are shown at 630× magnification; scale bars are 20 μm.
- Fig. 5.
Transmission electron microscopy of MAH-colonized C. elegans. Worms were seeded onto NGM-FUdR (400 µM) plates for 5 days and samples were fixed, processed, and visualized by transmission electron microscopy on an FEI Titan 80-200 microscope. Starved worms were seeded onto plates in the absence of bacteria (A,B) or onto plates seeded with 108 MAH (C-F). F illustrates a magnified view taken from E (dotted line) and shows disruption on the microvilli (bracket). Key: arrowheads, MAH; lumen, luminal space; mv, microvilli; am, apical epithelial membrane; bm, basal epithelial membrane. A-E, scale bar is 2 µm; F, scale bar is 500 nm.
- Fig. 6.
Binding of HEp-2 cells and colonization of C. elegans by MAH ΔGPL/4B2 mutant. (A) The MAH 4B2/ΔGPL mutant and the parental strain MAH A5 were used for HEp-2 binding assays. HEp-2 cells were infected at an MOI of 10:1 with each strain and binding was allowed to progress for 1 h at 4°C. Wells were lysed and quantified for percent of bound bacteria to the surface of epithelial cells during assay. Equivalent numbers of C. elegans were seeded onto NGM-FUdR (400 µM) plates containing 108 of each MAH strain and allowed to feed at 25°C for 5 days. Worms were collected, washed with levamisole (25 mM), treated with amikacin (200 µg/ml), and lysed for quantification of intestinal bacteria. (B) Worms were homogenized immediately after MAH feeding to determine intestinal binding ability after feeding. (C) Analysis for colonization using pulse-chase assays were conducted by transferring worms to NGM-FUdR plates seeded with E. coli strain OP50 for 24 h prior to homogenization and quantification. (D) The percent of each MAH strain remaining from the initial 5 days feeding after the pulse-chase was conducted was calculated by (MAH recovered−5 day feeding/MAH recovered−Pulse-Chase)×100. Data represent the mean±s.e.m. of 2 independent experiments each performed in triplicate (**P<0.01, ****P<0.0001 as determined by Student's t-test).
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