Lipocalin 2 regulates intestine bacterial survival by interplaying with siderophore in a weaned piglet model of Escherichia coli infection

Abstract

Iron is an essential nutrient that facilitates cell proliferation and growth, which plays a pivotal role in modulating the battle for survival between mammalian hosts and their pathogens. Pathogenic bacteria secrete siderophores to acquire iron from the host. However, lipocalin 2 (Lcn2), a siderophore-binding antimicrobial protein, binds to siderophores to prevent bacterial uptake of iron, which is critical for the control of systemic infection with Escherichia coli (E. coli). But few studies focus on the anti-infective response of Lcn2 in the intestines by inhibiting bacterial proliferation based on microbial iron metabolism. In this study, we showed that iron was sequestrated within cells in a piglet model of E. coli K88 infection. Siderophores was produced following E. coli K88 infection and siderophore-related genes expression was upregulated in iron-deficiency environment in vitro. Meanwhile, we found that Lcn2 expression was rapidly and robustly induced in jejunum by E. coli K88 infection and could be stimulated by IL-17 and IL-22. Furthermore, both Lcn2 induced in epithelial cells IPEC-1 and added exogenously as a recombinant protein could inhibit the growth of E. coli. We can conclude that Lcn2 is a crucial component of mucosal immune defense against intestinal infection with E. coli K88.

Keywords: Escherichia coli K88; intestinal infection; iron sequestration; lipocalin 2; siderophore.

Figure 1. Establishment of a piglet model of E.coli K88 challenge

Effects of challenge on average daily feed intake (ADFI), average daily gain (ADG) (A), E. coli populations in tissues (B), transcription levels of inflammatory cytokines in ileum (C) and jejunum (D) and morphology of duodenum and jejunum of piglets (E). mRNA levels of IL-1b, IL-6, TNF-a and IFN-g were determined by qRT-PCR. 18S rRNA was used as the housekeeping gene. mRNA expression ratio was normalized to the mean value of control group of 1. Error bars indicate SD. *p < 0.05.

Figure 2. The alteration of serum and tissue iron concentration in E. coli

K88-challenged piglets with decreased FPN mRNA and protein levels. Control and challenged piglets were analyzed for circulating serum iron levels (A), transferrin saturation (B), and nonheme iron content in liver (C) and spleen (D). The same piglets were analyzed for mRNA and protein expressions of iron metabolism related genes in liver (E), spleen (F) and duodenum (G). Error bars indicate SD. *p < 0.05, **p < 0.01.

Figure 3. Effects of low iron concentration on the expression of bacterial iron metabolism-related genes and siderophores

E. coli K12 grown in LB media at 37°C separately treated with 50 mM Fe³⁺ (FeCl₃), 100 mM Fe³⁺ (FeCl₃) and 200 mM 2,2-Dipyridyl (DP), then the expressions of bacterial siderophore-related genes entD (A), entS (B), fepA (C) and iron metabolism-related protein bfr (D) and ftnA(E) were analyzed. E. coli K88 grown in M9 media at 37°C separately treated with 10 mM Fe³⁺ (FeCl₃), 40 mM Fe³⁺ (FeCl₃), then the secretion level of bacterial siderophores was analyzed (F). Error bars indicate SD. *p < 0.05, **p < 0.01.

Figure 4. Effects of E. coli K88 challenge and IL-17/IL-22 on the Lcn2 expression

Control and challenged piglets were analyzed for Lcn2 (A), IL-17 (B) and IL-22 (C) mRNA levels in ileum, jejunum and duodenum. Epithelial cells IPEC-1 were analyzed for effects of IL-17 and IL-22 stimulation on the expression levels of Lcn2 mRNA (D). Error bars indicate SD. *p < 0.05, **p < 0.01.

Figure 5. The bacteriostatic effects of Lcn2

Lcn2 expression induced by IL-17 and IL-22 in transwell culturing monolayer epithelial cells IPEC-1(A). Effects of apical and basolateral cell culture supernatants of transwell culturing monolayer epithelial cells IPEC-1 on E. coli K12 and E. coli K88 proliferation by measuring OD₆₀₀ absorbance (B). Effects of additional 0.1 µg/ml, 0.5 µg/ml, and 1 µg/ml recombinant Lcn2 on E. coli K88 proliferation (C). Error bars indicate SD. *p < 0.05, **p < 0.01.