key: cord-0312459-44w9105u
authors: Scoggin, Kristin; Lynch, Rachel; Gupta, Jyotsana; Nagarajan, Aravindh; Sheffield, Max; Elsaadi, Ahmed; Bowden, Christopher; Aminian, Manuchehr; Peterson, Amy; Adams, L. Garry; Kirby, Michael; Threadgill, David W.; Andrews-Polymenis, Helene
title: Genetic background influences survival of infections with Salmonella enterica serovar Typhimurium in the Collaborative Cross
date: 2022-02-07
journal: bioRxiv
DOI: 10.1101/2022.02.07.479341
sha: 3c16eecb27a26bacc1ac7ba7000d039bda435ca9
doc_id: 312459
cord_uid: 44w9105u

Salmonella infections typically cause self-limiting gastroenteritis, but in some individuals these bacteria can spread systemically and cause disseminated disease. Salmonella Typhimurium (STm), which causes severe systemic disease in most inbred mice, has been used as a model for disseminated disease. To screen for new infection phenotypes across a range of host genetics, we orally infected 32 Collaborative Cross (CC) mouse strains with STm and monitored their disease progression for seven days by telemetry. Our data revealed a broad range of phenotypes across CC strains in many parameters including survival, bacterial colonization, tissue damage, complete blood counts (CBC), and serum cytokines. Eighteen CC strains survived to day 7, while fourteen susceptible strains succumbed to infection before day 7. Several CC strains had sex differences in survival and colonization. Surviving strains had lower pre-infection baseline temperatures and were less active during their daily active period. Core body temperature disruptions were detected earlier after STm infection than activity disruptions, making temperature a better detector of illness. All CC strains had STm in spleen and liver, but susceptible strains were more highly colonized. Tissue damage was weakly negatively correlated to survival. We identified loci associated with survival on Chromosomes (Chr) 1, 2, 4, 7. Polymorphisms in Ncf2 and Slc11a1, known to reduce survival in mice after STm infections, are located in the Chr 1 interval, and the Chr 7 association overlaps with a previously identified QTL peak called Ses2. We identified two new genetic regions on Chr 2 and 4 associated with susceptibility to STm infection. Our data reveal the diversity of responses to STm infection across a range of host genetics and identified new candidate regions for survival of STm infection. Author Summary Salmonella Typhimurium (STm) infections typically cause self-limiting diarrheal symptoms, but in some individuals, the bacteria can spread throughout the body and cause life-threatening infection. We used a population of genetically different mice (Collaborative Cross) to identify their range of responses to STm infection. We identified a broad range of outcomes across these different mice, including a group of mice susceptible to lethal infection and a group that survived our 7 day study. We found that mice that survived STm infection had a cooler core body temperature before infection than susceptible mice, while remaining active. Thus, body temperature, rather than activity, appears to be a better predictor of poor outcomes after STm infection. We identified several regions of the mouse genome that are associated with outcome after STm infection. One of these regions, mouse Chromosome (Chr) 1 has genes that are already known to influence susceptibility to STm infection. Two other regions that we identified to influence survival after STm infection, located on mouse Chr 2 and 4, are novel and contain numerous genes of interest that may be linked to susceptibility. Our work defines the utility of exploring how host genetic diversity influences infection outcomes with bacterial pathogens.

into CC mice, our STm oral infections produced a much wider range of colonization in 150 critical organs (Fig. 1 ). Using sex-combined median data, the correlation between liver 151 and spleen colonization with survival time was R = -0.62 and -0.63, respectively (Supp. 152 Fig. 1 ). 153 All B6 mice met the euthanasia criteria between day 3 and 6 post-infection with a 154 median at day 4 and were classified as highly susceptible ( Fig. 2A) . B6 mice lost a 155 median of 20.73% of their body weight during infection (Fig. 2B) . CBA mice survived to 156 day 7 and were classified as resistant ( Fig. 2A) with a median loss of 7.92% of their CC042 has been previously attributed to a de novo mutation in integrin alpha (Itgal) 173 (41) . Susceptible strains were also colonized at least 10-fold higher than CBA control 174 mice, with the exception of CC032 (Fig. 2C, D) . CC032 mice were colonized in both 175 liver and spleen similarly to CBA with a median of 4.11 x10 3 and 3.96 x10 4 CFU/g, 176 respectively, yet succumbed to infection before day 7 (Fig. 2C, 2D) . 177 An additional 18 strains were categorized as resistant or tolerant. Resistance 178 was defined as surviving infection by preventing colonization or by clearing the bacteria. 179 Tolerance was defined as surviving infection while maintaining high bacterial burden. 180 Both resistant and tolerant strains had </= 2 mice meeting euthanasia criteria prior to 181 day 7. Of these 18 strains, all 6 mice lived to day 7 in 12 strains ( Fig. 2A) . Strains that 182 survived longer lost less weight than those that did not with a correlation of R = 0.71 183 across all strains ( Fig. 2B and Supp. Fig. 1 ). However, CC011 lost substantially more 184 weight than expected for a surviving strain. All the resistant and tolerant strains, with 185 two exceptions (CC045 and CC051), were equally colonized across systemic organs, 186 thus making these phenotypes difficult to differentiate.

One strain -CC051 -had 100-fold lower bacterial burden than CBA in both liver 188 and spleen with medians of 9.30 x10 1 and 2.92 x10 2 CFU/g, respectively, and it 189 therefore met our criteria for resistance (Fig. 2C, D) . Another strain -CC045 -had a 190 100-fold higher bacterial burden than CBA mice with 3.31 x10 5 and 4.83 x10 6 CFU/g, 191 respectively, making it the strongest candidate for tolerance (Fig. 2C, D) . Of the 192 remaining 16 surviving strains, eight were colonized in the liver to the same level as Sex-dependent variation in susceptibility is strain dependent 197 Sex has been shown to be an influencing factor in many phenotypes, including 198 response to infection (28, (42) (43) (44) . Across the CC population that we tested, sex did not Surviving mice have lower baseline body temperatures and stay on baseline circadian 216 pattern for activity longer Implantable telemetry devices tracked body temperature and activity levels at 218 one-minute intervals both before and throughout infections. Prior to infection, each 219 strain had unique diurnal pattern of baseline temperature and activity prior to infection 220 (Supp. Fig. 3 ). When strains were grouped by survival outcome, differences between 221 groups became apparent (Fig. 4) Table 1 and Supp. Fig. 4 ). Minimal damage was noted in the intestinal 258 sections examined (ileum, cecum, and colon, scores 0-1) and these were excluded from 259 further analyses (Supp. Fig. 5 ). To assess the relationship between survival and tissue 260 damage, linear regression was performed showing survival and combined spleen and liver pathology scores with R 2 = 0.1256, while the spleen pathology score alone was 262 0.1323 and the liver pathology score alone was 0.08382 ( Fig. 5B , C, D, all P = <0.001). 263 Thus, damage to the spleen appears to be driving the small but significant 264 correlation between histopathology scores and survival. Organs from susceptible strains 265 were not necessarily more damaged than those from surviving strains, perhaps Complete blood counts (CBC) were performed on pre-infection blood and on 272 blood collected at necropsy (S1 Table and S2 Table) . The differential counts for Table and S4 Table) . Susceptibility or survival was not significantly correlated to the 279 cytokine profile. The cytokine profile was influenced more by strain than infection status.

Highly STm susceptible CC042 mice had high levels of almost every cytokine, 281 suggesting that the mice were suffering a cytokine storm (S4 Table) .

A point mutation in Slc11a1 (also known as Nramp1, or Ity) is associated with 285 susceptibility to lethal systemic STm infection (2, 14, 16) . B6 is the only CC founder strain 286 to carry this mutation, and B6 is susceptible to systemic infection with STm. Only 5 CC 287 strains in the panel analyzed -CC021, CC031, CC042, CC045, and CC061 -carry the 288 Slc11a1 mutation (Fig. 2) . All of these strains were highly colonized in liver and spleen, 289 and, with the exception of one strain (CC045), succumbed to infection before day 7. Tlr4 also has a known point mutation linked to STm susceptibility, but none of the 302 CC founder strains carry a mutation in this gene, making it impossible to verify the effect 303 of this mutation on STm infection using CC mice (21, 22) . Table 1 ). We identified a 318 significant association on Chr 4 (Stq1 (Survival Time QTL)) and a significant association 319 on Chr 7 (Stq2). The heritability of survival time was 45.55. Although the slope of 320 survival percentages over time did not return significant temporal QTL, suggestive 321 associations overlapped with those from the other survival parameters (Supp. Fig. 7) .

Other parameters such as weight change, CFUs, and CBCs were also analyzed using 323 gQTL with no significant or suggestive associations detected, further supporting their 324 independence from STm infection survival.

Two suggestive associations on Chr 1 overlapped with the Slc11a1 and Ncf2 326 genes, suggesting that these genes underlie these associations. The significant association on Chr 6 contains four genes, three hypothetical genes and one annotated 328 gene, Cntn3, which is involved in nervous system development (Supp. Fig. 8A ).

However, no SNP differences were found in Cntn3 that were consistent with founder 330 haplotype effects. Although genes on Chr 2 and 4 have previously been shown to be 331 involved in STm infections, the QTL regions we identified do not overlap with previously 332 detected loci. The association on Chr 7 overlaps with a previously detected QTL region 333 called Ses2 (S. enteritidis susceptibility locus 1) which is linked to Salmonella clearance 334 in the spleen, but the causative gene in this region remains unknown (47, 48) .

Using founder effect plots ( Fig. 6 and Supp. Fig. 8 candidate genes with SNP differences. For percent of mice that survived to day 7, the 364 Chr 2 region had 503 genes, of these genes 51 had SNP differences matching 365 haplotype effects (Table 3 ). The Chr 4 region had 28 genes and Stq2 had 115 genes, 366 but neither region had SNP differences corresponding to haplotype differences. Table 3 : Candidate genes from percent survived to day 7 QTL associations that had SNP differences corresponding to haplotype differences. The impact the SNP difference is predicted to have on the resulting protein is shown.

Although CC strains have been extensively used to identify genes influencing 370 susceptibility to viral infection (34) (35) (36) (49) (50) (51) (52) (53) , host determinants influencing the (40, 41) . The phenotype of CC042 mice has been linked to a mutation in the integrin alpha L gene (Itgal, also known as Cd11a) resulting in a defect in T cell recruitment 392 (39, 41) . Finally, the remaining 18 CC strains we screened survived the full study period.

Two of these strains, CC001 and CC002, were previously shown to display resistance 394 to M. tuberculosis infection (37) . mutations that remain to be discovered.

Sex is well known to influence both innate and adaptive immune responses (54) 417 as well as infection outcome (28, 42, 43, 55, 56) . We identified several CC strains that 418 exhibited sex differences in survival after STm infection (CC013, CC042, CC030, and 419 CC027, Figure 2 ). In keeping with the general hypothesis that females have stronger We analyzed all of the parameters we collected to identify putative linked 482 genomic regions using gQTL (46) . Only survival time yielded significant, 0.85 or higher, associations. Intervals on Chr 1 contained Slc11a1 and Ncf2, known genes that have previously unknown role in immunity against bacterial pathogens. Further investigation 507 is needed to prove such a link. In both median survival and percent survived to day 508 seven QTL intervals for Chr 4 and 7, haplotype differences did not help us narrow the 509 gene list. RNA expression analysis, currently underway, should identify candidate genes 510 for these intervals. 

Quantitative detection of deviation from the baseline "off-pattern," using temperature 600 data was calculated on an individual basis. A temperature time series was filtered, a 601 definition of healthy variation was defined, then the time of first "off-pattern" was 602 calculated using that definition on post-inoculation data.

Each mouse time series was preprocessed using a moving median filter with a 604 one-day window. For a specific minute t, the median collection of temperature values 605 from [t-720, t+720] was used in calculating a median for the value t. After this 606 processing, healthy variation was defined as any temperature falling within the range of 607 minimum to maximum values during the pre-inoculation phase [T-5760,T] (5760 608 minutes = 4 days). This choice allowed for enough data to account for natural inter-day 609 variation due to potential factors such as inter-strain variation, epigenetic differences, 610 and sex differences, while avoiding bias due to observed acclimation time after transfer 611 to a new facility in some mice in the first few days of observation (Supp. Fig. 11A ). "memoryless" assumption were attempted but did not see any feasible agreement with 641 observed activity data (Supp. Fig. 11B ).

The second approach mentioned above produced a qualitatively cleaner signal, Mean and standard deviation shown by strains and individual mice shown by dots. 

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