Antimicrobial mechanism of pore-forming protegrin peptides: 100 pores to kill E. coli.

Antimicrobial peptides (AMPs), important effector molecules of the innate immune system, also provide templates for designing novel antibiotics. Protegrin, an especially potent AMP found in porcine leukocytes, was recently shown to form octameric transmembrane pores. We have employed a combination of experiments and models spanning length scales from the atomistic to the cellular level in order to elucidate the microbicidal mechanism of protegrin. Comparison of the modeling and experimental data suggests that approximately 10-100 protegrin pores are necessary to explain the observed rates of potassium leakage and Escherichia coli death in exponential-phase bacteria. The kinetics of viability loss suggest that bacterial death results largely from uncontrolled ion exchange processes and decay of transmembrane potential. However, ion exchange processes alone cannot account for the experimentally observed cell swelling and osmotic lysis-a redundant "overkill" mechanism most likely to occur in locales with high protegrin concentrations. Although our study is limited to protegrin and E. coli, the timeline of events described herein is likely shared by other AMPs that act primarily by permeabilizing microbial membranes. This work provides many of the missing links in describing antimicrobial action, as well as providing a quantitative connection between several previous experimental and simulation studies of protegrin.

Herpes simplex virus downregulates secretory leukocyte protease inhibitor: a novel immune evasion mechanism.

Secretory leukocyte protease inhibitor (SLPI), an anti-inflammatory mediator of mucosal immunity, inhibits human immunodeficiency virus (HIV) and herpes simplex virus (HSV) in cell culture. Epidemiological studies demonstrate that higher concentrations of SLPI in mucosal secretions are associated with a reduced risk of HIV transmission. The current studies were designed to test the hypothesis that HSV triggers a loss of SLPI to evade innate immunity and that this response may contribute to the increased risk of HIV infection in the setting of HSV infection. Exposure of human cervical epithelial cells to HSV-1 or HSV-2, but not HIV or vesicular stomatitis virus, triggered a significant and sustained reduction in SLPI levels. The reduction persisted when cells were infected in the presence of acyclovir but not following infection with UV-inactivated virus, indicating that viral gene expression, but not replication, is required. Reverse transcriptase PCR studies demonstrated that the loss of SLPI is mediated by downregulation of gene expression. SLPI downregulation was associated with activation of NF-kappaB signaling pathways and upregulation of proinflammatory cytokines, consistent with the known inhibitor effects of SLPI on NF-kappaB pathways. The downregulation mapped to viral early-gene expression, as variants impaired in expression of the ICP4 or ICP0 immediate-early gene failed to downregulate SLPI or activate NF-kappaB. Together, these results identify a novel role for HSV immediate-early-gene expression in regulating mucosal immune responses.

Seminal plasma reduces the effectiveness of topical polyanionic microbicides.

The objective of this study was to test the activity of microbicides against herpes simplex virus type 2 (HSV-2) introduced in seminal plasma. We found that seminal plasma interfered with the activity of PRO 2000 and of cellulose sulfate, increasing by 100-fold the concentration of drug required to inhibit 90% of viral plaque formation. Seminal plasma competitively inhibited binding of the microbicides to the HSV-2 envelope. Most of the interference was found in a high molecular-weight fraction; tandem mass spectrometry identified the proteins as fibronectin-1 and lactoferrin. In a murine model, the interference translated in vivo into a loss in protection. We found that 2% PRO 2000 gel protected 100% of mice challenged intravaginally with HSV-2 introduced in PBS, whereas only 55% of mice were protected if virus was introduced in seminal plasma (P=.0007, log rank test). If these findings are reflective of what occurs in humans, modifications to microbicides to ensure that they retain activity in the presence of seminal plasma are indicated.

A comprehensive murine model to evaluate topical vaginal microbicides: mucosal inflammation and susceptibility to genital herpes as surrogate markers of safety.

A critical gap in microbicide development is the absence of surrogate safety markers. The objective of the present study was to develop a murine model to examine the mucosal response to microbicides and to assess the functional implication of observed changes. Mice received 14 daily intravaginal doses of nonoxynol-9, PRO 2000, or placebo gel. Nonoxynol-9 induced an inflammatory response characterized by increases in levels of cytokines and chemokines, recruitment of neutrophils and monocytes into the genital tract, and activation of the transcription factors NF- kappa B and activator protein-1. Minimal inflammation was observed in response to 2% PRO 2000. Nonoxynol-9-treated mice were significantly more susceptible to challenge with a low dose of herpes simplex virus type 2; the response of PRO 2000-treated mice was similar to the response to placebo. These findings suggest that PRO 2000 has little deleterious effect on mucosal immunity and, if validated by clinical experiences, support the inclusion of this model in the preclinical evaluation of future candidate microbicides.

PRO 2000 elicits a decline in genital tract immune mediators without compromising intrinsic antimicrobial activity.

OBJECTIVE: Vaginal microbicides should protect against infection without disrupting the mucosal environment or its mediators of host defense. The objective of this study was to examine the effect of 14 daily applications of 0.5% PRO 2000 or placebo gel on mediators of mucosal immunity and intrinsic antimicrobial activity. DESIGN AND METHODS: A randomized, prospective, double-blind, placebo-controlled study was conducted among 24 healthy, abstinent women. Levels of cytokines, chemokines, defensins, and other protective factors and intrinsic antimicrobial activity were determined in cervicovaginal lavage samples collected on study days 0, 7, 14, and 21. RESULTS: No increase in pro-inflammatory cytokines was observed. Rather cytokines and protective factors including interleukin (IL)-1 receptor antagonist, immunoglobulins and human beta-defensin 2 were lower in the drug compared with the placebo group. All of the mediators returned towards baseline on day 21. Women who were cycling had lower levels of most proteins on study days 7 and/or 14 compared with women on oral contraceptives; however, the magnitude of decline was greater in women who received PRO 2000 compared with placebo gel. The reduction in protective factors was not associated with a loss in the intrinsic anti-viral (HIV or herpes simplex virus) activity or anti-bacterial activity (Escherichia coli or Staphylococcus aureus). CONCLUSION: In contrast to experience with nonoxynol-9, PRO 2000 did not trigger an inflammatory response in cervicovaginal secretions. There was a modest reduction in mucosal immune mediators, but this loss was not associated with a reduction in intrinsic antimicrobial activity.

Human alpha- and beta-defensins block multiple steps in herpes simplex virus infection.

This study examined the ability of nine human defensins (HD) to protect against herpes simplex virus infection. Noncytotoxic concentrations of all six alpha-defensins (HNP1-4, HD5, and HD6) and human beta-defensin (hBD) 3 inhibited HSV infection. Two other beta-defensins, hBD1 and 2, lacked this protective activity. Synchronized assays revealed that HNP-4, HD6, and hBD3 acted primarily by preventing binding and entry, whereas HNP1-3 and HD5 also inhibited postentry events. Even when added several hours after entry, substantial reduction in viral gene expression ensued. Human cervical epithelial cells incubated with HNP-1 or HD5 accumulated the peptides intracellularly. Surface plasmon resonance studies revealed that HNPs 1, 2, 3, and HD5 bound HSV glycoprotein B (gB) with high affinity, but showed minimal binding to heparan sulfate, the receptor for attachment. In contrast, HNP-4 and HD6 bound heparan sulfate, but not gB. HBD3 bound both gB and heparan sulfate, but hBD1 and hBD2 bound neither. Admixture of HD5 with hydroxyethylcellulose significantly protected mice from a viral challenge lethal to controls receiving an inactive peptide or hydroxyethylcellulose alone. These findings demonstrate that HDs act at multiple steps in the HSV life cycle and support the development of defensins or defensin-like peptides as microbicides.