Experiment
To mimic the genetic diversity in the human response to infection by Shiga toxin (STX)-producing E. coli (STEC), the capacity of an O157:H7 outbreak isolate to colonize mouse strains from the advanced recombinant inbred BXD panel was measured.
First the BXD parental strains C57BL/6 J (B6) and DBA/2 J (D2) were infected with either 8624 (Stx2a+) or TUV86-2, an Stx2a-negative isogenic mutant. Colonization levels were determined in an intact commensal flora (ICF) infection model. A significant difference in colonization levels between the parental B6 and D2 strains after infection with TUV86-2 but not with 8624 was found. The observation suggested that a host factor, that may have been masked by Stx2a, affects O157:H7 colonization in some genetic backgrounds.
Next the colonization levels of TUV86-2 were determined in 24 BXD strains in the ICF model. A total of 31 strains, 29 BXD strains and ancestral parental strains B6 and D2, with 321 mice total (each BXD strain n= 6-20; B6 and D2 n = 48) were used. The experiments included three mice per strain, with six to seven strains total. The parental B6 and D2 strains were included in all experiments as an internal control for colonization levels. Mice were weighed daily and colonization levels were reported as CFU per g feces on day one through day four post-infection.
General linear model analysis of covariates revealed that there were no differences associated with mouse age, source, or the seasonality of the experiment. The open-access web-based interval analysis on the GeneNetwork (GN) platform for complex trait analysis was used to identify QTL (NCBI Build 37).
Ten sets of analyses of the log CFU means, log CFU medians or corrected coefficient of log CFU/g feces for the following variables were done:
1) colonization one day post-infection;
2) colonization two days post-infection;
3) colonization 3 days post-infection;
4) colonization 4 days post-infection;
5) difference in colonization between day four and one post-infection;
6) difference in colonization between day four and two after infection;
7) difference in colonization between day four and three after infection;
8) difference in colonization between day three and one after infection;
9) difference in colonization between day three and two after infection; and,
10) difference in colonization between day two and one after infection, for 30 traits analyzed.
A highly significant QTL was linked with the log of colonization means from day 1 with an LRS=20.19, LOD=4.4, p<0.05. QTL Dcollq1 (differential colonization level QTL 1) mapped to proximal chromosome 9 in an interval of 14 Mb between 12.5-26.7 Mb. Linkage analysis revealed a peak at 15.69 Mb associated with genetic markers gnf09.010.169, rs13480073, and mCV24962297. [Table 1, mm9 B37].
Strain distribution patterns (SDP) of the BXD strains revealed that high colonization levels on day one post-infection were associated with the B6 allele. Low colonization levels in the BXD panel were associated with D2 alleles.
Linkage, polymorphism and co-citation analyses of the mapped region revealed 36 candidate genes within the QTL [Table 2]; five genes were identified that were most likely responsible for the differential colonization: Panx1, Bmper, Dnmt1, Pde4a and Acad8.
Several suggestive QTL were also mapped to Chromosomes 1, 4, 5, 7, 9, 10, 12, 13, 14, 15, 17, 18, 19 and X for the differing post infection traits - see Table 1.
Analysis Tools