Cryptosporidium parvum multidrug resistance protein confers resistance to toxic gut microbial metabolite
Description
Cryptosporidium parvum subtypes differ in pathogenicity, but the genetic factors underlying these differences are largely unknown. Here, we show that two C. parvum isolates grow equally well in vitro but differ in pathogenicity in immunocompromised mice. Reduced oocyst shedding of the avirulent strain was restored by antibiotic treatment, suggesting susceptibility to colonization resistance imparted by the microbiota. To investigate the extent of genetic differences of the isolates, we comducted whole genome sequencing (WGS). To map significant associations between parasite genes and enhanced infectivity of the virulent strain, we performed genetic crosses followed by bulk segregant analysis (BSA) under permissive (antibiotic-treated) and restrictive (untreated) conditions. To evaluate the environmental differences between these conditions, we also profiled gut microbiota composition using 16S rRNA sequencing.
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Steps to reproduce
Purified oocysts (10^6) of each C. parvum AUB and IOWA-AZ were subjected to five freeze-thaw cycles, and the DNA was extracted from them using the QIAamp DNA minikit (Qiagen, United States). Libraries were constructed using automated protocols on the SciClone G3 NGSx workstation (Perkin Elmer) by the Genome Technology Access Center (GTAC) at Washington University. Whole genome sequencing (WGS) was acquired using Illumina protocols on the NovaSeq 6000 platform (Illumina, San Diego, CA, United States) to generate 150 bp reads. The sorted yellow oocysts and purified C. parvum oocysts of florescent tagged lines and from each mouse of 1st GKO + antibiotics, 3rd GKO + antibiotics, 1st GKO, and 3rd GKO were used for WGS. To increase the DNA extraction, the phenol-chloroform-isoamyl alcohol extraction method was used to extract whole genome DNA (wgDNA) from 106oocysts. Whole-genome amplification (WGA) was performed on the extracted wgDNA from 105 oocysts using a REPLI-g single cell kit (Qiagen, Cat. 150043) following the manufacturer-recommended. DNA products were sequenced on an Illumina NovaSeq 6000 using the 150-bp paired-end approach. To evaluate the effect of antibiotic treatment in mice, four GKO mice were given the antibiotic water for five days. Fecal samples were collected on 1 day before treatment, 5 days after, and 5 days after stopping treatment. DNA from cecal contents was isolated as described above. Each sample was amplified in triplicate with Golay-barcoded primers specific for the V4 region (F515/R806) and checked by gel electrophoresis. The final pooled samples, along with aliquots of the three sequencing primers, were sequenced using a paired-end 250bp Illumina protocol.
Institutions
- Washington University in St. Louis