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. 2015;6(6):392-7.
doi: 10.1080/19490976.2015.1107697.

Advancing the use of Lactobacillus acidophilus surface layer protein A for the treatment of intestinal disorders in humans

Bikash Sahay  1   2 , Yong Ge  1   2 , Natacha Colliou  1   2 , Mojgan Zadeh  1   2 , Chelsea Weiner  1   2 , Ashley Mila  1   2 , Jennifer L Owen  3 , Mansour Mohamadzadeh  1   2
Affiliations

Advancing the use of Lactobacillus acidophilus surface layer protein A for the treatment of intestinal disorders in humans

Bikash Sahay et al. Gut Microbes. 2015.

Erratum in

  • Addendum to: Lightfoot YL, Selle K, Yang T, Goh YJ, Sahay B, Zadeh M, Owen JL, Colliou N, Li E, Johannssen T, Lepenies B, Klaenhammer TR, Mohamadzadeh M. SIGNR3-dependent immune regulation by Lactobacillus acidophilus-Surface layer protein A in colitis. EMBO Journal 2015 Apr 1;34(7):881-95; PMID: 2566591; PMCID: PMC4388597.

Abstract

Intestinal immunity is subject to complex and fine-tuned regulation dictated by interactions of the resident microbial community and their gene products with host innate cells. Deterioration of this delicate process may result in devastating autoinflammatory diseases, including inflammatory bowel disease (IBD), which primarily comprises Crohn's disease (CD) and ulcerative colitis (UC). Efficacious interventions to regulate proinflammatory signals, which play critical roles in IBD, require further scientific investigation. We recently demonstrated that rebalancing intestinal immunity via the surface layer protein A (SlpA) from Lactobacillus acidophilus NCFM potentially represents a feasible therapeutic approach to restore intestinal homeostasis. To expand on these findings, we established a new method of purifying bacterial SlpA, a new SlpA-specific monoclonal antibody, and found no SlpA-associated toxicity in mice. Thus, these data may assist in our efforts to determine the immune regulatory efficacy of SlpA in humans.

Keywords: bacterial protein isolation; colonic inflammation; gut microbiota; intestinal immune regulation; surface layer protein A.

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Figures

Figure 1.
Figure 1.
L. acidophilus-SlpA isolation by NaCl. L. acidophilus-SlpA was isolated and purified, as described previously, with substitution of LiCl with NaCl. (A) SDS-PAGE containing 2.5 μg of LiCl− and NaCl−isolated SlpA stained with Coomassie blue to visualize the purified protein. (B) Mass spectrometry data analyzed on the Scaffold platform showed 97 unique spectra with 55 unique peptides with the possibility of 2 proteins (C). The predicted protein gi|58336516 (SlpA) shows 54% coverage whereas gi|362076610 (SlpB) reveals only 18% of coverage (highlighted portion, D). The regions of SlpB matching the generated peptides are common between SlpA and SlpB (shown in the red box, D), and no single unique peptide from SlpB was identified.
Figure 2.
Figure 2.
NaCl−purified SlpA is not overtly toxic to mice. (A–F) C57BL/6 mice were treated orally every other day with SlpA (0, 150, 300, 600 μg/100μL per mouse), for a total of 4 times. One week later, mice were sacrificed and a whole blood chemistry profile was generated for each mouse with a comprehensive metabolic chemistry panel, using a VetScan V2S analyzer. All animal experiments were performed under the guidelines of the Animal Welfare Act and the Public Health Policy on Humane Care, and with approval by the Institutional Animal Care and Use Committee (IACUC protocol 201406559) at the University of Florida. Data represent observations from 4 independent experiments (n = 5) and are shown as mean ± standard error of the mean.
Figure 3.
Figure 3.
Generated mAb BM1 recognizes L. acidophilus-SlpA. C57BL/6 mice were immunized once a week for 3 months with 100 μg of SlpA, and 300 μg of heat-killed Lactobacillus gasseri as adjuvant. Polyclonal sera were tested for recognition of isolated SlpA by Western Blot (WB), and splenic cells from SlpA-reactive mice were fused with Sp2/0 myeloma cells at a ratio of 7:1. Hybridomas were seeded on a semi-solid medium for clone selection and screening. Subsequently, clones were screened by ELISA for SlpA reactivity. Reactive clones were isotyped and all IgM secretors removed. Clone BM1 (IgG) was selected for its ability to recognize SlpA by WB (A), flow cytometry (B), confocal microscopy (C), and ELISA (D, E, F). (A). L. acidophilus-SlpA detection by WB with BM1. 100 ng of purified SlpA, 108 CFU L. acidophilus (L. a.), 108 CFU L. reuteri (L. r.), or 100 ng of BSA proteins were separated by SDS-PAGE, transferred onto a PVDF membrane, and detected by BM1. (B). L. acidophilus-SlpA detection with BM1 by flow cytometry. Carboxylated Dynabeads were coated with purified SlpA and the reactivity of the BM1 mAb confirmed by Canto II flow cytometry. Data were analyzed by FlowJo. Experiments were performed at least 3 times with similar trends. (C). L. acidophilus-SlpA detection with BM1 by confocal microscopy. RAW 264.7 cells were pulsed for 1 or 3 hrs with NaCl purified SlpA (10 μg/mL). Subsequently, cells were fixed and stained with BM1 mAb for detection by confocal microscopy. Cells were incubated with BM1 mAb overnight. Cells were washed and subsequently incubated with a secondary antibody (Alexa Fluor 488 anti-mouse IgG1, 1:100) for 4 hrs. Nuclei were stained with DAPI (15 min) and visualized by a Zeiss confocal microscope. (D). L. acidophilus-SlpA detection with BM1 by ELISA. ELISA plates were coated with 500 ng of purified SlpA overnight, and binding by BM1 was tested thereafter. (E). Two-fold serial dilutions of SlpA were coated on ELISA plate overnight, and binding of BM1 was tested. (F). Germ-free (GF) mice were orally treated with 109 CFU L. acidophilus, 150 μg of SlpA, or left untreated. Fecal pellets from these mice were used to coat ELISA plates; BSA was used as a negative control. BM1 mAb only bound to plates coated with feces derived from treated mice. All animal experiments were performed under the guidelines of the Animal Welfare Act and the Public Health Policy on Humane Care, and with approval by the Institutional Animal Care and Use Committee (IACUC protocol 201406559) at the University of Florida. Data represent observations from 4 independent experiments (n = 4) and are shown as mean ± standard error of the mean. ** denotes statistical significance p < 0.01, ***p < 0.001.
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