PACAP is a pathogen-inducible resident antimicrobial neuropeptide affording rapid and contextual molecular host defense of the brain - PubMed (original) (raw)

. 2021 Jan 5;118(1):e1917623117.

doi: 10.1073/pnas.1917623117.

Liana C Chan 3 4 5, Huiyuan Wang 3 4, Juelline Lieng 1, Mandy Hung 1, Yashes Srinivasan 1, Jennifer Wang 1, James A Waschek 6, Andrew L Ferguson 7, Kuo-Fen Lee 8, Nannette Y Yount 3 4, Michael R Yeaman 9 5 6, Gerard C L Wong 10 11 12

Affiliations

PACAP is a pathogen-inducible resident antimicrobial neuropeptide affording rapid and contextual molecular host defense of the brain

Ernest Y Lee et al. Proc Natl Acad Sci U S A. 2021.

Abstract

Defense of the central nervous system (CNS) against infection must be accomplished without generation of potentially injurious immune cell-mediated or off-target inflammation which could impair key functions. As the CNS is an immune-privileged compartment, inducible innate defense mechanisms endogenous to the CNS likely play an essential role in this regard. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide known to regulate neurodevelopment, emotion, and certain stress responses. While PACAP is known to interact with the immune system, its significance in direct defense of brain or other tissues is not established. Here, we show that our machine-learning classifier can screen for immune activity in neuropeptides, and correctly identified PACAP as an antimicrobial neuropeptide in agreement with previous experimental work. Furthermore, synchrotron X-ray scattering, antimicrobial assays, and mechanistic fingerprinting provided precise insights into how PACAP exerts antimicrobial activities vs. pathogens via multiple and synergistic mechanisms, including dysregulation of membrane integrity and energetics and activation of cell death pathways. Importantly, resident PACAP is selectively induced up to 50-fold in the brain in mouse models of Staphylococcus aureus or Candida albicans infection in vivo, without inducing immune cell infiltration. We show differential PACAP induction even in various tissues outside the CNS, and how these observed patterns of induction are consistent with the antimicrobial efficacy of PACAP measured in conditions simulating specific physiologic contexts of those tissues. Phylogenetic analysis of PACAP revealed close conservation of predicted antimicrobial properties spanning primitive invertebrates to modern mammals. Together, these findings substantiate our hypothesis that PACAP is an ancient neuro-endocrine-immune effector that defends the CNS against infection while minimizing potentially injurious neuroinflammation.

Keywords: antimicrobial peptides; host defense; innate immunity; neuroimmunology; neuropeptides.

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Conflict of interest statement

Competing interest statement: M.R.Y is founder and shareholder of NovaDigm Therapeutics, Inc., which pursues novel antiinfective agents and strategies; he holds patents in the area of antimicrobial peptides and related molecules.

Figures

Fig. 1.

Fig. 1.

PACAP is a GPCR-binding neuropeptide with strong physicochemical homology to human cathelicidin from innate immunity. (A) Screening a neuropeptide database (red) using the support vector machine-based MAPT for sequences with hidden membrane-restructuring activity revealed PACAP (purple) to be a hypothesized endogenous HDP. LL37 (green) is shown for comparison alongside sequences generated by a Monte Carlo–based directed search of sequence space (gray). (B) Comparison of the distribution of residue character along the lengths of PACAP and LL37 (blue = cationic, green = hydrophilic, white = hydrophobic, red = anionic). Helical wheel diagrams of (C) PACAP and (D) LL37 show similar distributions of hydrophobic (yellow) and cationic residues (teal). Anionic amino acids (red) and polar amino acids (purple) are also shown. (E) PACAP and LL37 exhibit similar lysine-to-arginine ratios and mean hydrophobicities, which suggests that PACAP (purple) can generate negative Gaussian membrane curvature similar to LL37 (green) and other ɑ-helical AMPs (gray). (F) Structural alignment of PACAP and LL37 yields an rmsd of 1.184 Å, demonstrating close structural similarity, despite (G) very low sequence homology. (H and I) An in silico mutational analysis of the entire PACAP sequence demonstrates that PACAP is locally optimized for membrane-remodeling activity, and that the majority of mutations reduce the microbicidal potential of the peptide.

Fig. 2.

Fig. 2.

PACAP preferentially induces NGC in microbial membranes to facilitate potent bactericidal and fungicidal activity. (A) PACAP recapitulates semiselective membrane remodeling activity of HDPs, inducing NGC in the bacterial model membrane PG/PE 20/80, but not in the eukaryotic-like PS/PC 20/80. A schematic of a transmembrane pore rich in NGC is shown. NGC is present everywhere along the surface highlighted in the dark shaded area spanned by the principal curvature C1 and C2. (B) PACAP was incubated with bacteria grown to midlog phase (1 × 106 CFU/mL). MBC assays were performed on S. aureus (SA113), E. coli (DH5ɑ), and P. aeruginosa (PA01), demonstrating strong antibacterial activity. (C) LDH-release assays with mouse macrophages demonstrate that PACAP is nontoxic to eukaryotic cells at bactericidal concentrations. Assays in B and C are representative of three independent experiments. (D_–_I) Radial diffusion assays show that PACAP exerts potent antibacterial and antifungal activity against MDR organisms, including MRSA (USA300), A. baumannii (HUMC-1), and C. albicans (CA 36082S). The diameter of induced clearing d is measured in millimeters. *P < 0.05, **P < 0.01. P values were calculated using a two-tailed Student’s t test.

Fig. 3.

Fig. 3.

PACAP is a multimodal HDP that kills bacteria and fungi via synergistic mechanisms. Mechanistic fingerprinting experiments were conducted using single-cell flow cytometry. SDS (a membrane detergent), PACAP or comparator HDP LL-37, or kinocidin congeners RP-1 and ɣ-RP-1 were incubated with HDP-sensitive or HDP-resistant strains of S. aureus (A_–_D), S. typhimurium (E_–_H), or C. albicans (I_–_L) in the presence of specific fluorescent reporters (Materials and Methods). Six mechanistic parameters were measured in distinct pH conditions mirroring bloodstream (pH 7.5) or abscess/phagolysosomal (pH 5.5) contexts: forward scatter (FSC; cell size), side scatter (SSC; intracellular refractivity indicative of nucleic acid condensation), cellular membrane energetics (ENR; transmembrane potential), cell membrane permeability (PRM), PS accessibility (PSA; membrane turnover indicating early-stage regulated cell death), or activation of CDP (indicative of late-stage regulated cell death) pathways.

Fig. 4.

Fig. 4.

PACAP is strongly and selectively induced in the brain and other tissues in response to infection. (A) Immunohistochemistry for PACAP in brain, spleen, and kidney was compared in control (uninfected) and hematogenously-infected mice inoculated with S. aureus (SA), E. coli (EC), or C. albicans (CA). Images represent at least five independent experiments. PACAP induction was quantified using image processing in ImageJ. Relative change in expression was calculated vs. control uninfected tissue. All images are shown at 100× magnification. (B) In the brain, PACAP exhibits ∼40- to 50-fold induction in response to S. aureus and C. albicans infection but not E. coli. (C) In the spleen, PACAP is strongly induced in the presence of all three organisms (∼10- to 25-fold). (D) In the kidney, PACAP is induced ∼80- to 100-fold in response to infection with S. aureus and C. albicans, similar to brain. In B_–_D, n = 5 for all conditions, *P < 0.05, **P < 0.01, ***P < 0.001 relative to control. All other comparisons not significant. P values were calculated using a two-tailed Student’s t test.

Fig. 5.

Fig. 5.

PACAP exerts context-dependent antimicrobial efficacy. Growth inhibition assays were conducted in context-dependent media mimicking brain infections, urinary tract infections, and abscesses (Materials and Methods). For each context, 1 or 10 µg/mL of PACAP simulating low or high expression levels were incubated with (A) S. aureus, (B) E. coli, or (C) C. albicans at pH 5.5 and pH 7.5. Growth inhibition was measured after incubation at 37 °C. PACAP inhibited the growth of all organisms in brain-, urine- and abscess-simulating media to varying degrees. PACAP exhibited the strongest bactericidal ability against S. aureus and C. albicans in brain medium and against E. coli in urine medium. n = 3 for all assay conditions. *P < 0.05, **P < 0.01. All other comparisons were not significant. P values were calculated using a two-tailed Student’s t test.

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