A murine model of diarrhea, growth impairment and metabolic disturbances with Shigella flexneri infection and the role of zinc deficiency

Abstract

Shigella is one of the major enteric pathogens worldwide. We present a murine model of S. flexneri infection and investigate the role of zinc deficiency (ZD). C57BL/6 mice fed either standard chow (HC) or ZD diets were pretreated with an antibiotic cocktail and received S. flexneri strain 2457T orally. Antibiotic pre-treated ZD mice showed higher S. flexneri colonization than non-treated mice. ZD mice showed persistent colonization for at least 50 days post-infection (pi). S. flexneri-infected mice showed significant weight loss, diarrhea and increased levels of fecal MPO and LCN in both HC and ZD fed mice. S. flexneri preferentially colonized the colon, caused epithelial disruption and inflammatory cell infiltrate, and promoted cytokine production which correlated with weight loss and histopathological changes. Infection with S. flexneri ΔmxiG (critical for type 3 secretion system) did not cause weight loss or diarrhea, and had decreased stool shedding duration and tissue burden. Several biochemical changes related to energy, inflammation and gut-microbial metabolism were observed. Zinc supplementation increased weight gains and reduced intestinal inflammation and stool shedding in ZD infected mice. In conclusion, young antibiotic-treated mice provide a new model of oral S. flexneri infection, with ZD promoting prolonged infection outcomes.

Keywords: Shigellosis; intestinal microbiota; mouse model; urine metabolomics; zinc deficiency.

Figure 1.

Antibiotic treatment and zinc deficiency increased S. flexneri stool shedding.C57BL/6 mice were fed house chow (HC), protein deficient (PD) and zinc-deficient (ZD) defined diets for 2 weeks and submitted or not to antibiotic cocktail in the drinking water for 3 d before oral infection with 10⁸ CFU/mouse S. flexneri 2457T strain. DNA extracted from stools after infection were analyzed by ipaH gene qPCR. (a) Stool shedding using antibiotics (with Abx) across HC, PD and ZD diets. * P < 0.05 by two-way ANOVA, HC + S. flex vs ZD + S. flex, # P < 0.05 by two-way ANOVA, PD + S. flex vs ZD + S flex (N = 4/group); (b) Stool shedding without using antibiotics (without Abx) across HC, PD and ZD diets (N = 4/group). Antibiotic pre-treated nourished C57BL/6 mice were evaluated for bodyweight growth change and signs of diarrhea induced by oral S. flexneri 2457T infection (N = 8/group). Data were replicated at least two times. N = 8/group.

Figure 2.

S. flexneri orally administered in antibiotic pre-treated house chow-fed and zinc-deficient mice caused acute growth impairment, diarrhea and intestinal inflammation. Mice previously exposed to 2 weeks on house chow or zinc-deficient diet and pre-treated with antibiotic cocktail in the drinking water were challenged with S. flexneri 10⁸ CFU/mouse inoculum. Bodyweight growth change and signs of diarrhea were observed, and specimens were collected for protein extraction and measurement of biomarkers of intestinal inflammation – myeloperoxidase (MPO) and lipocalin (LCN) by ELISA kits (a). Growth impairment induced by S. flexneri on days 2–4 in nourished mice. * P < 0.05 by two-way ANOVA, HC vs HC + S. flex. (b) Representative pictures of diarrhea in nourished mice at day 2 pi. (c) Cecal contents MPO and LCN levels from day 7 pi in house chow-fed mice. * P = 0.005 for MPO and P = 0.0006 for LCN by Mann–Whitney test, HC vs HC + S. flex. (d) Growth impairment induced by S. flexneri on days 8–9 pi in zinc-deficient mice. * P < 0.05 by two-way ANOVA, ZD vs ZD + S. flex. (e) Representative pictures of diarrhea in nourished mice at day 7 pi. (f) Cecal contents MPO and LCN levels from day 7 pi in zinc-deficient mice. * P = 0.042 for MPO and P = 0.016 for LCN by Mann–Whitney test, ZD vs ZD + S. flex. Data were replicated at least two times. N = 8/group.

Figure 3.

S. flexneri 2457T preferentially colonized the colon and caused intestinal epithelial damage. S. flexneri infected nourished and zinc-deficient C57BL/6 mice were euthanized and intestinal specimens collected. S. flexneri target molecular quantification of DNA extracted from different intestinal sections from both nourished and zinc-deficient infected mice at day 3 pi. Colon sections from infected zinc-deficient mice were stained for H&E, as well as for S. flexneri specific and E-cadherin (epithelium marker) antibodies. (a) S. flexneri tissue burden levels across duodenum, jejunum, ileum and colon at day 3 pi. * P = 0.008 by Mann–Whitney test, HC + S. flex vs ZD + S. flex in the colon. (b) Representative picture of H&E staining of colon section from infected and uninfected house chow-fed and zinc-deficient mice. Arrows indicate epithelial damage and inflammatory cell infiltrate. (c) Analysis of histopathological scoring of colon from infected and uninfected house chow-fed mice. * P = 0.018 by Mann–Whitney test. (d) Analysis of histopathological scoring of colon from infected and uninfected zinc-deficient mice. (e) Representative picture of stained colon sections showing S. flexneri in red and E-cadherin in green, indicating by arrows S. flexneri present adhered to mucosa and epithelium, as well as in the lamina propria in house chow-fed mice (on top); and abundant extracellular S. flexneri in zinc-deficient mice (on bottom). Data were replicated at least two times. N = 4/group.

Figure 4.

Challenge with the S. flexneri ΔmxiG did not lead to weight loss, pathogen colonization and intestinal inflammation. Mice were infected by either a wild type or ΔmixG S. flexneri 2457T strain. Bodyweights changes, diarrhea, stool shedding, tissue burden and intestinal inflammation were analyzed. (a) Bodyweight growth change induced by S. flexneri ΔmixG in antibiotic pre-treated nourished C57BL/6 mice. * P < 0.05 by two-way ANOVA, HC + S. flex vs HC + S. flex ΔmixG. (b) S. flexneri stool shedding comparing wild type S. flexneri and S. flexneri ΔmixG until day 9 pi. * P = 0.0095 by Mann-Whitney test, HC + S. flex vs HC + S. flex ΔmixG. at day 3pi. (c) S. flexneri tissue burden of duodenum, jejunum, ileum and colon comparing wild-type S. flexneri and S. flexneri ΔmixG at day 3 pi. * P = 0.0286 by Mann–Whitney test. (d) Fecal MPO and LCN levels of mice infected by wild-type S. flexneri and S. flexneri ΔmixG at days 2 and 6 pi. * P < 0.05 by one-way ANOVA. Data were replicated at least two times. N = 4/group.

Figure 5.

S. flexneri triggered cytokine immune responses in both house chow and zinc-deficient mice. Total protein was extracted from intestinal sections for immune response markers analysis at day 15 pi. (a) Correlation between colonic TNF-α protein levels and colonic damage score at day 3 pi in house chow-fed mice, r = 0.640, P = 0.0285. (b) Correlation between colonic IL-1β protein levels and colonic damage score at day 3 pi in house chow-fed mice, r = 0.780, P = 0.004. (c) Correlation between colonic IL-10 protein levels and colonic damage score at day 3 pi in house chow-fed mice, r = 0.775, P = 0.004. (d) TNF-α and IL-10 protein levels from the cecal contents at day 15 pi in house chow-fed and zinc-deficient mice after S. flexneri infection. * P = 0.043 for TNF-α ZD vs ZD + S. flex and * P = 0.286 for IL-10 ZD vs ZD + S. flex by Mann–Whitney test. Data were replicated at least two times. N = 4–8/group.

Figure 6.

Zinc supplementation reduced S. flexneri stool shedding and intestinal inflammation, and improved bodyweight growth in zinc-deficient mice. S. flexneri infected zinc-deficient mice started to receive zinc treatment at day 33 pi and followed until day 48 pi (total of 15 d). Zinc sulfate was dissolved in water and given in the drinking water. Stool shedding, bodyweight growth and intestinal inflammation myeloperoxidase (MPO) and lipocalin-2 (LCN) cecal contents levels were analyzed. (a) S. flexneri stool shedding of infected zinc-deficient mice receiving zinc or not after 15 d. * P = 0.0295 by Mann–Whitney test. (b) S. flexneri burden in the cecal contents of infected zinc-deficient mice receiving zinc or not after 15 d. (c) Bodyweight growth of infected zinc-deficient mice receiving zinc or not after 15 d. * P < 0.0001 by two-way ANOVA at day 15 post-treatment. (d) MPO and LCN levels from cecal contents at day 15 post-treatment. * P < 0.05 by Mann–Whitney test, ZD + S. flex vs ZD + S. flex + Zn. Data were replicated at least two times. N = 8/group.

Figure 7.

Metabolic perturbations induced by S. flexneri infection. Heat map summarizing the significant urinary metabolic alterations induced by S.flexneri identified by OPLS-DA models. Results are presented as correlation coefficients (r). Red color indicates increased excretion and blue indicated decreased excretion of urinary metabolites in nourished mice infected at day 2 and day 6 versus diet and age-matched uninfected mice, in zinc-deficient infected mice at day 6 compared to diet and age-matched controls and in nourished mice infected with the S. flexneri ΔmixG strain versus mice infected with the wild type strain. Abbreviations: 2-OG, 2-oxoglutarate, 2-OIV, 2-oxoisovalerate, 2-OIC, 2-oxoisocaproate, 2-MOV, 3-methyl-2-oxovalerate, 2-PY, N-methyl-2-pyridone-5-carboxamide, 3-IS, 3indoxylsulfate, 3-UPA, 3-ureidopropionic acid, 4-HPA, 4-hydroxyphenylacetate, 4-PY, N-methyl-4-pyridone-3-carboxamide, BG, butyrylglycine, GAA guanidinoacetate, HG, hexanoylglycine, IVG, isovalerylglycine, m-HPPS, m-hydroxyphenylpropiobylsulfate, NAG, N-acetyl glutamine, NMND, N-methylnicotinamide, TMA, trimethylamine, TMAO, trimethylamine-N-oxide. Data were replicated at least two times. N = 8/group.