Antimicrobial Activity of Native and Synthetic Surfactant Protein B Peptides 1 Marnie A. Ryan,* Henry T. Akinbi,* Alicia G. Serrano, ‡ Jesus Perez-Gil, ‡ Huixing Wu, † Francis X. McCormack, † and Timothy E. Weaver 2 * Surfactant protein B (SP-B) is secreted into the airspaces with surfactant phospholipids where it reduces surface tension and prevents alveolar collapse at end expiration. SP-B is a member of the saposin-like family of proteins, several of which have antimicrobial properties. SP-B lyses negatively charged liposomes and was previously reported to inhibit the growth of Escherichia coli in vitro; however, a separate study indicated that elevated levels of SP-B in the airspaces of transgenic mice did not confer resistance to infection. The goal of this study was to assess the antimicrobial properties of native SP-B and synthetic peptides derived from the native peptide. Native SP-B aggregated and killed clinical isolates of Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, and group B streptococcus by increasing membrane permeability; however, SP-B also lysed RBC, indicating that the membranolytic activity was not selective for bacteria. Both the antimicrobial and hemolytic activities of native SP-B were inhibited by surfactant phospholipids, suggesting that endogenous SP-B may not play a significant role in alveolar host defense. Synthetic peptides derived from native SP-B were effective at killing both Gram-positive and Gram-negative bacteria at low peptide concentrations (0.15–5.0 M). The SP-B derivatives selectively lysed bacterial membranes and were more resistant to inhibition by phospholipids; furthermore, helix 1 (residues 7–22) retained significant antimicrobial activity in the presence of native surfactant. These results suggest that the role of endogenous SP-B in host defense may be limited; however, synthetic peptides derived from SP-B may be useful in the treatment of bacterial pneumonias. The Journal of Immunology, 2006, 176: 416 – 425. T he respiratory tree terminates in small sac-like structures (alveoli) that provide an extensive gas exchange surface composed of type I epithelial cells. Hydration of the gas exchange surface leads to elevated surface tension at the air/liquid interface, generating a high collapsing force at end expiration. Type II epithelial cells synthesize and secrete pulmonary surfac- tant, which forms a stable phospholipid-rich film at the air/liquid interface and prevents alveolar collapse and impaired gas ex- change. Dipalmitoylphosphatidylcholine (DPPC), 3 the main lipid component of surfactant, reduces surface tension to near zero as the surfactant film is compressed during exhalation. During inha- lation, surfactant phospholipids are inserted into the expanding surface film, a process facilitated by the hydrophobic peptides sur- factant protein B (SP-B) and SP-C. The importance of SP-B for surfactant function is underscored by the fact that deficiency of SP-B in both mice and humans results in lethal neonatal respira- tory distress syndrome (1, 2). In addition to its biophysical function, surfactant plays an im- portant role in maintaining the sterility of the gas exchange sur- face. The surface film serves as a physical barrier to inhaled patho- gens and the hydrophilic surfactant proteins SP-A and SP-D, associated with the large and small aggregate fractions of surfac- tant, respectively (3, 4), promote clearance of microorganisms from the distal airspaces. SP-A and SP-D opsonize, aggregate, and enhance phagocytosis of microbes by resident macrophages (5–7). SP-A and SP-D may also directly kill bacteria by permeabilizing the bacterial membrane (8). The role of SP-B in alveolar host defense is less clear. A synthetic peptide corresponding to SP-B was reported to inhibit the growth of Escherichia coli in vitro (9); however, bacterial burden was not increased in the lungs of SP-B heterozygous null (SP-B +/- ) mice nor was protection conferred by increased expression of SP-B in transgenic mice (10). SP-B is a member of the saposin-like family of proteins (SAPLIP), several members of which exhibit potent antimicrobial activity (11). SAPLIP family members NK-lysin, granulysin, and amoebapore all kill bacteria by permeabilizing bacterial mem- branes, but the mechanism of membrane permeabilization differs among the peptides. Positively charged amino acids located on the surface of NK-lysin and granulysin mediate interaction of the pep- tides with the negatively charged membranes of bacteria, resulting in membrane destabilization and/or permeabilization (12, 13). In contrast, amoebapore A is much more hydrophobic and permeabi- lizes bacterial membranes in a pH- and oligomerization-dependent manner (14). SP-B shares features with both types of SAPLIP antimicrobial peptides: it is very hydrophobic and forms oligomers similar to amoebapore but is also cationic (net positive charge of +7) and lyses negatively charged liposomes at neutral pH, similar to NK-lysin and granulysin (15, 16). We have previously mapped *Division of Pulmonary Biology, Cincinnati Children’s Hospital Medical Center, and University of Cincinnati College of Medicine, and † Division of Pulmonary and Crit- ical Care Medicine, Department of Internal Medicine, University of Cincinnati Col- lege of Medicine, Cincinnati, OH 45229; and ‡ Departamento de Bioquimica y Bio- logia Molecular I, Facultad Biologia, Universidad Complutense, Madrid, Spain Received for publication June 28, 2005. Accepted for publication September 29, 2005. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by National Institutes of Health Grant R37-HL56285 (to T.E.W.). J.P.-G was supported by a grant from the Spanish Ministry of Science and Education (BIO2003-09056). F.X.M. was supported by HL68861 and a Veterans Affairs Merit Award. 2 Address correspondence and reprint requests to Dr. Timothy E. Weaver, Cincinnati Children’s Hospital Medical Center, Division of Pulmonary Biology, 3333 Burnet Avenue, Cincinnati, OH 45229-3039. E-mail address: tim.weaver@cchmc.org 3 Abbreviations used in this paper: DPPC, dipalmitoylphosphatidylcholine; PG, phos- phatidylglycerol; SP-B, surfactant protein B; SAPLIP, saposin-like family of proteins; h, human; BALF, bronchoalveolar lavage fluid; N-term, N-terminal. The Journal of Immunology Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00