Establishing Caenorhabditis elegans as a model for Mycobacterium avium subspecies hominissuis infection and intestinal colonization

Abstract

The nematode Caenorhabditis elegans has become a model system for studying the disease interaction between pathogens and the host. To determine whether the transparent nematode could serve as a useful model for Mycobacterium avium subspecies hominissuis (MAH) infection of the intestinal tract, worms were fed MAH and assayed for the effects of the bacterial infection on the worm. It was observed during feeding that viable MAH increases in the intestinal lumen in a time dependent manner. Ingestion of MAH was deemed non-toxic to worms as MAH-fed populations have similar survival curves to those fed E. coli strain OP50. Pulse-chase analysis using E. coli strain OP50 revealed that MAH colonize the intestinal tract, as viable MAH remain within the intestine after the assay. Visualization of intestinal MAH using histology and transmission electron microscopy demonstrates that MAH localizes to the intestinal lumen, as well as establishes direct contact with intestinal epithelium. Bacterial colonization appears to have a detrimental effect on the microvilli of the intestinal epithelial cells. The MAH ΔGPL/4B2 strain with a mutation in glycopeptidolipid production is deficient in binding to human epithelial cells (HEp-2), as well as deficient in its ability to bind to and colonize the intestinal tract of C. elegans as efficiently as wild-type MAH. These data indicate the C. elegans may serve as a useful model system for MAH pathogenesis and in determining the mechanisms used by MAH during infection and colonization of the intestinal epithelium.

Establishment of a Host-to-Host Transmission Model for Mycobacterium avium subsp. hominissuis Using Caenorhabditis elegans and Identification of Colonization-Associated Genes

“…The nematodes were then homogenized and plated for quantification of CFU. Some of the nematodes were prepared for histopathology and electron microscopy as previously described (Everman et al, 2015 ). As shown in Figures 2A,B , infection was transmitted from one C. elegans to another and the pathogen can interact with the intestinal mucosa.…”

“…The transmission assay was performed by placing C. elegans , initially grown on a layer of E. coli OP50, on starvation media for 3 days (25°C) and then removing the nematode and placing it on a lawn of M. avium A5, M. avium 104, or mutants (1 × 10 5 bacteria) for 4 h (25°C). After the period of time, C. elegans were removed out of the plate, the bacteria bound to the outside of the nematode body cleared as previously described (Everman et al, 2015 ). C. elegans (20 worms) were then placed onto an agar plate without bacteria for 2 h at 25°C.…”

“…Fresh, uninfected C. elegans , maintained without eating for 3 days, were transposed to the plate that at this point contained bacteria excreted from the previously removed C. elegans . After an additional 2 h, the plates were washed in M9 saline (Everman et al, 2015 ) supplemented with 25 mM of levamisole hydrochloride (Sigma-Aldrich) for paralysis and prevention of elimination or uptake of bacteria during the washes. C. elegans were then exposed to amikacin sulfate (200 μg/ml) for 30 min to kill all bacteria bound to the outside of the nematode body (Everman et al, 2015 ), and subsequently washed twice in HBSS.…”

“…After an additional 2 h, the plates were washed in M9 saline (Everman et al, 2015 ) supplemented with 25 mM of levamisole hydrochloride (Sigma-Aldrich) for paralysis and prevention of elimination or uptake of bacteria during the washes. C. elegans were then exposed to amikacin sulfate (200 μg/ml) for 30 min to kill all bacteria bound to the outside of the nematode body (Everman et al, 2015 ), and subsequently washed twice in HBSS. To quantify bacteria ingested by C. elegans , suspensions of 10 nematodes were homogenized using a handheld motorized pot (VWR, Radnor, PA, USA) for 1 min in 0.1% triton X-100.…”

“…We have established the Caenorhabditis elegans as an experimental model of M. avium subsp. hominissuis ( M. avium ) colonization and infection (Everman et al, 2015 ). It is well-accepted that M. avium infects individuals by both respiratory and intestinal routes (Sangari et al, 2000 ; Babrak et al, 2015 ).…”