Diversity, Antimicrobial Action and Structure-Activity Relationship of Buffalo Cathelicidins

<div><p>Cathelicidins are an ancient class of antimicrobial peptides (AMPs) with broad spectrum bactericidal activities. In this study, we investigated the diversity and biological activity of cathelicidins of buffalo, a species known for its disease resistance. A series of new homologs of cathelicidin4 (<i>CATHL4</i>), which were structurally diverse in their antimicrobial domain, was identified in buffalo. AMPs of newly identified buffalo CATHL4s (buCATHL4s) displayed potent antimicrobial activity against selected Gram positive (G+) and Gram negative (G-) bacteria. These peptides were prompt to disrupt the membrane integrity of bacteria and induced specific changes such as blebing, budding, and pore like structure formation on bacterial membrane. The peptides assumed different secondary structure conformations in aqueous and membrane-mimicking environments. Simulation studies suggested that the amphipathic design of buCATHL4 was crucial for water permeation following membrane disruption. A great diversity, broad-spectrum antimicrobial action, and ability to induce an inflammatory response indicated the pleiotropic role of cathelicidins in innate immunity of buffalo. This study suggests short buffalo cathelicidin peptides with potent bactericidal properties and low cytotoxicity have potential translational applications for the development of novel antibiotics and antimicrobial peptidomimetics.</p></div

Repertoire of cathelicidin AMPs in buffalo.

<p>(a) Comparison of AAMP region of cattle and buffalo cathelicidins. (b) Phylogenetic relationship and consensus of amino acid residues of the 12 CATHL4 AMPs. The Neighbor-joining tree construction was based on complete nucleotide sequences of the CATHL4 variants (NCBI Accession ID: KJ173930—KJ173976).</p

Circular dichroism (CD) spectroscopy of selected peptides in sodium phosphate (10 mM) with or without sodium dodecyl sulphate (SDS).

<p>buCATHL4s assumed backbone orientation ranging from random coil to alpha helix in water. The helical propensity increased further in presence of SDS micelles.</p

Comparative Genomic Analysis of Buffalo (<i>Bubalus bubalis</i>) NOD1 and NOD2 Receptors and Their Functional Role in <i>In-Vitro</i> Cellular Immune Response

<div><p>Nucleotide binding and oligomerization domain (NOD)-like receptors (NLRs) are innate immune receptors that recognize bacterial cell wall components and initiate host immune response. Structure and function of NLRs have been well studied in human and mice, but little information exists on genetic composition and role of these receptors in innate immune system of water buffalo—a species known for its exceptional disease resistance. Here, a comparative study on the functional domains of NOD1 and NOD2 was performed across different species. The NOD mediated <i>in-vitro</i> cellular responses were studied in buffalo peripheral blood mononuclear cells, resident macrophages, mammary epithelial, and fibroblast cells. Buffalo NOD1 (buNOD1) and buNOD2 showed conserved domain architectures as found in other mammals. The domains of buNOD1 and buNOD2 showed analogy in secondary and tertiary conformations. Constitutive expressions of NODs were ubiquitous in different tissues. Following treatment with NOD agonists, peripheral lymphocytes showed an IFN-γ response along-with production of pro-inflammatory cytokines. Alveolar macrophages and mammary epithelial cells showed NOD mediated <i>in-vitro</i> immune response through NF-κB dependent pathway. Fibroblasts showed pro-inflammatory cytokine response following agonist treatment. Our study demonstrates that both immune and non-immune cells could generate NOD-mediated responses to pathogens though the type and magnitude of response depend on the cell types. The structural basis of ligand recognition by buffalo NODs and knowledge of immune response by different cell types could be useful for development of non-infective innate immune modulators and next generation anti-inflammatory compounds.</p></div

Binding of buCATH4B with model phospholipid membranes.

<p>(A) Single buCATH4B and 64 DPPC lipids at starting configuration (left) and at the end of 100 ns simulation (right). The peptide is modeled as stick; water molecules are shown as point; phosphate, oxygen, and nitrogen atoms are colored tan, red and blue, respectively. (B) Interaction of buCATH4B with phosphate groups present around 5 Å of the peptide (left), and orientation of buCATH4B (orange) on the DPPC membrane (right). (C) Four buCATH4Bs and 128 DPPC lipids, captured at starting configuration (left), at 100 ns (middle), and at the end of 200 ns simulation (right). Peptides are colored in orange and membrane in grey. Phosphate atoms are shown as grey spheres. (D) Eight buCATH4Bs and a 31 mixture of 96 POPC (grey) and 32 POPG (cyan) lipids. Peptides are colored in orange. Phosphate atoms of POPC and POPG are shown as grey and cyan spheres, respectively. (E) Order parameter (<i>Scd</i>) of the carbon atoms along the <i>sn-1</i> (upper) and <i>sn-2</i> (lower) acyl chain of phospholipids used in the simulation studies. Lower <i>Scd</i> value indicates more disordered lipid tails and <i>vice versa</i>.</p

Reduction level of <i>S</i>. <i>aureus</i> and <i>P</i>. <i>aeruginosa</i> biofilm growth by peptides at 1× and 2× MIC.

<p>Reduction level of <i>S</i>. <i>aureus</i> and <i>P</i>. <i>aeruginosa</i> biofilm growth by peptides at 1× and 2× MIC.</p

Cytotoxicity profiles of CATHL4 AMPs against RBC and fibroblast cells.

<p>(A) Haemolysis percentage of buffalo red blood cells following addition peptides at different molar concentrations. (B) Percentage of propidium iodide (PI) positive fibroblast cells following addition of peptides at different molar concentrations.</p

Peptide induced changes in membrane permeability of bacteria as assessed by SYTO9/PI dyes.

<p>Green fluorescence indicates live bacteria with intact plasma membrane. Red and yellow fluorescence indicates bacteria with altered permeability that had uptaken propidium iodide dye.</p

Time kill kinetics of buCATHL4 peptides against (A) <i>S</i>. <i>aureus</i> (B) <i>S</i>. <i>typhimurium</i> and (C) <i>P</i>. <i>aeruginosa</i> following addition of peptides at 2× and 4× MIC concentration.

<p>Time kill kinetics of buCATHL4 peptides against (A) <i>S</i>. <i>aureus</i> (B) <i>S</i>. <i>typhimurium</i> and (C) <i>P</i>. <i>aeruginosa</i> following addition of peptides at 2× and 4× MIC concentration.</p

Relative fold change in mRNA expressions of cytokines following addition of peptides.

<p>Muramyl di peptide (MDP) and PBS were positive and negative controls, respectively. Columns with asterisk (*) had significantly (p<0.05) higher expression level over negative control.</p