Small proline-rich protein 2A is a gut bactericidal protein deployed during helminth infection

Abstract

A diverse group of antimicrobial proteins (AMPs) helps protect the mammalian intestine from varied microbial challenges. We show that small proline-rich protein 2A (SPRR2A) is an intestinal antibacterial protein that is phylogenetically unrelated to previously discovered mammalian AMPs. In this study, SPRR2A was expressed in Paneth cells and goblet cells and selectively killed Gram-positive bacteria by disrupting their membranes. SPRR2A shaped intestinal microbiota composition, restricted bacterial association with the intestinal surface, and protected against Listeria monocytogenes infection. SPRR2A differed from other intestinal AMPs in that it was induced by type 2 cytokines produced during helminth infection. Moreover, SPRR2A protected against helminth-induced bacterial invasion of intestinal tissue. Thus, SPRR2A is a distinctive AMP triggered by type 2 immunity that protects the intestinal barrier during helminth infection.

Fig. 1.. SPRR2A is expressed in goblet cells and Paneth cells in the mouse intestine.

(A) Quantitative PCR (qPCR) analysis of Sprr2a expression in various organs of wild-type C57BL/6 mice. Values were normalized to 18S rRNA expression. n=3 mice per group. (B) qPCR analysis of Sprr2a expression in mouse small intestine (SI), small intestinal epithelial cells (IEC) acquired by laser capture microdissection, lamina propria lymphocytes (LPL), and intraepithelial lymphocytes (IEL). Values were normalized to 18S rRNA expression. (C) In situ hybridization of small intestine sections with anti-sense and sense Sprr2a RNA probes. Examples of positive signals are indicated by yellow arrowheads. Scale bar=50 μm. (D and E) Immunofluorescence detection of SPRR2A (red) in sections of paraffin-embedded mouse small intestine. Ulex europaeus agglutinin-I (UEA-I) (D) and anti-lysozyme antibody (E) were used to identify goblet cells (UEA-I) and Paneth cells (UEA-I and anti-lysozyme). Nuclei were detected with 4’,6-diamidino-2-phenylindole (DAPI). Sections from Sprr2a^−/− mice (generated as shown in fig. S2) were used as negative controls. Scale bars=50 μm. (F) Immunofluorescence detection of SPRR2A (red) in OCT-embedded frozen sections of mouse small-intestinal organoids. Scale bars=25 μm. (G) Immunoblot detection of SPRR2A protein in mouse colon, the colon mucus layer, and stool samples. (H) qPCR analysis of epithelial cell Sprr2a expression in germ-free (GF) wild-type (WT) and Myd88-deficient (Myd88^−/−) C57BL/6 mice with or without conventionalization (CV). Epithelial cells were harvested by laser capture microdissection. Values were normalized to Gapdh expression. n=3 mice per group. (I) qPCR analysis of Sprr2a expression in the small intestines of germ-free Swiss-Webster mice treated with lipopolysaccharide (LPS). PBS was administered as a vehicle control. Values were normalized to Gapdh expression. n=3-4 mice per group. Means ± SEM (error bars) are plotted. *P<0.05; **P<0.01; ***P<0.001; ns, not significant by two-tailed t test.

Fig. 2.. SPRR2A is a bactericidal protein that targets Gram-positive bacteria by membrane permeabilization.

(A) Recombinant SPRR2A was expressed in a baculovirus expression system and purified by size exclusion chromatography. SPRR2A was added to ~10⁴ CFU of log-phase bacteria for 2 hours, and surviving bacteria were enumerated by dilution plating. (B) SPRR2A was added to log-phase Listeria monocytogenes in the presence of anti-SPRR2A antibody and surviving bacteria were enumerated by dilution plating. (C) Transmission electron microscopy of L. monocytogenes after incubation with SPRR2A. Bovine serum albumin (BSA) was used as a negative control. Examples of cell surface damage and cytoplasmic leakage are indicated with red arrowheads. Scale bars=200 nm. (D) L. monocytogenes (left) and Lactobacillus reuteri (right) were treated with SPRR2A or BSA as control and propidium iodide (PI) uptake was measured over 1 hour. (E) Membranes displaying various lipids were incubated with 1 μg/ml of SPRR2A and detected with anti-SPRR2A antibody. (F) SPRR2A was incubated with liposomes having the indicated lipid compositions. After ultracentrifugation, the liposome-free supernatant (S) and the liposome pellet (P) were analyzed by SDS-PAGE and Coomassie blue staining. I, input; PC, phosphatidylcholine; PS, phosphatidylserine; CL, cardiolipin. (G) Transmission electron microscopy of PC:PS liposomes after treatment with 5 μM SPRR2A. Scale bars=100 nm. (H) Carboxyfluorescein (CF)-loaded liposomes with the indicated lipid compositions were treated with 10 μM SPRR2A. Dye efflux was monitored over time and is expressed as a percentage of maximal efflux in the presence of the detergent octyl glucoside (OG). (I) 5 μM SPRR2A was added to log-phase L. monocytogenes in the presence of increasing lipopolysaccharide (LPS) concentrations. Surviving bacteria were then enumerated by dilution plating. (J) CF-loaded PC:PS liposomes were treated with 10 μM SPRR2A in the presence or absence of 0.2 mg/ml of LPS, and dye efflux was monitored over time and is expressed as a percentage of maximal efflux in the presence of OG. (K) Quantification of the results in J; n=3. All assays were performed in triplicate and results are representative of at least two independent experiments. Means ± SEM (error bars) are plotted. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001; ns, not significant by two-tailed t test.

Fig. 3.. Mice lacking SPRR2A have an altered intestinal microbiota and are more susceptible to Listeria monocytogenes infection.

(A) Male wild-type (WT) and Sprr2a^−/−littermates were separated at weaning and caged by genotype for 6 weeks. Intestinal mucus-associated and lumenal microbial communities were characterized by 16S rRNA sequencing. Relative abundances of bacterial classes in wild-type and Sprr2a^−/− mice are shown. n=4 mice per group. (B) Relative abundances of bacterial genera in wild-type and Sprr2a^−/− mice. (C) qPCR analysis of specific bacterial groups in the intestinal mucus-associated and lumenal microbial communities of wild-type and Sprr2a^−/− littermates. Values for each bacterial group are expressed relative to total 16S rDNA levels. *P<0.05; **P<0.01; ***P<0.001; ns, not significant by two-tailed t test. (D) Fluorescence in situ hybridization (FISH) detection of bacteria in the small intestines of wild-type and Sprr2a^−/− mice. Scale bars=50 μM (E) 8-10-week-old wild-type (n=12) and Sprr2a^−/− mice (n=14) were treated with antibiotics and orally infected with 1×10⁹ CFU of log-phase L. monocytogenes. Liver, spleen and mesenteric lymph nodes (MLN) were collected after 24 hours and bacterial counts were determined by dilution plating. Results were pooled from three independent experiments. **P<0.01 by Mann–Whitney U test. Dotted line indicates limit of detection. (F) 8-10-week-old wild-type (n=9) and Sprr2a^−/− mice (n=8) were orally infected with 2.5×10⁹ CFU of log-phase Listeria monocytogenes. Survival rates were monitored over 10 days. Results were pooled from two independent experiments. *P<0.05 by the log-rank test. WT, wild-type; SFB, segmented filamentous bacteria; CFU, colony-forming units.

Fig. 4.. SPRR2A expression is induced by type 2 cytokines during helminth infection of the intestine.

(A) Wild-type C57BL/6 (top) and BALB/c (bottom) mice were orally infected with 200 infective L3 H. polygyrus larvae for 2 weeks, and gene expression in the proximal jejunum was analyzed by qPCR. All values were normalized to Gapdh expression. (B) Wild-type C57BL/6 or BALB/c mice were orally infected with 200 infective L3 H. polygyrus larvae for 2 weeks. Control littermates were treated with PBS. SPRR2A was detected by immunofluorescence in sections of proximal jejunum. Nuclei were detected with DAPI. Scale bars=100 μm. Representative images are shown for 3-4 mice per group. (C) qPCR analysis of Sprr2a expression in small intestinal organoids derived from wild-type C57BL/6 mice treated with the indicated cytokines. Values were normalized to Gapdh expression. (D) qPCR analysis of intestinal epithelial Sprr2a expression in wild-type BALB/c mice intraperitoneally injected with recombinant IL-13, IL-22, or vehicle. Intestinal epithelial cells were harvested by laser-capture microdissection. Values were normalized to Gapdh expression. n=3 mice per group. (E) Wild-type and Stat6^−/− mice were orally infected with 200 infective L3 H. polygyrus larvae, and Sprr2a expression in the proximal jejunum was analyzed by qPCR after 2 weeks. All values were normalized to Gapdh expression. Means ± SEM (error bars) are plotted. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001; ns, not significant by two-tailed t test.

Fig. 5.. SPRR2A protects against helminth-induced bacterial invasion of intestinal tissue.

(A) Wild-type and Sprr2a^−/− mice were orally infected with 200 infective L3 H. polygyrus larvae for two weeks, and helminth burden in the intestine (upper panel) and egg numbers in the stool (lower panel) were counted. Results were pooled from two independent experiments. (B) Detection of bacteria by fluorescence in situ hybridization in the proximal jejunum of wild-type and Sprr2a^−/− mice after H. polygyrus infection for 2 weeks. Nuclei were detected with DAPI. Results are representative of three independent experiments. (C) qPCR determination of total bacterial numbers in the proximal jejunal tissue, proximal jejunal lumen and mesenteric lymph nodes of wild-type and Sprr2a^−/− mice after H. polygyrus infection for 2 weeks. Values were normalized to tissue weight or length. Results are from three independent experiments. Means ± SEM (error bars) are plotted. **P<0.01; ***P<0.001; ****P<0.0001; ns, not significant by two-tailed t test. WT, wild-type; H.p., Heligmosomoides polygyrus.