PTPN2 Is a Critical Regulator of Ileal Paneth Cell Viability and Function in Mice.

BACKGROUND & AIMS: Loss-of-function variants in the PTPN2 gene are associated with increased risk of inflammatory bowel disease. We recently showed that Ptpn2 is critical for intestinal epithelial cell (IEC) barrier maintenance, IEC-macrophage communication, and modulation of the gut microbiome in mice, restricting expansion of a small intestinal pathobiont associated with inflammatory bowel disease. Here, we aimed to identify how Ptpn2 loss affects ileal IEC subtypes and their function in vivo. METHODS: Constitutive Ptpn2 wild-type, heterozygous, and knockout (KO) mice, as well as mice with inducible deletion of Ptpn2 in IECs, were used in the study. Investigation was performed using imaging techniques, flow cytometry, enteroid culture, and analysis of gene and protein levels of IEC markers. RESULTS: Partial transcriptome analysis showed that expression of Paneth cell-associated antimicrobial peptides Lyz1, Pla2g2a, and Defa6 was down-regulated markedly in Ptpn2-KO mice compared with wild-type and heterozygous. In parallel, Paneth cell numbers were reduced, their endoplasmic reticulum architecture was disrupted, and the endoplasmic reticulum stress protein, C/EBP-homologous protein (CHOP), was increased in Ptpn2-KO mice. Despite reduced Paneth cell number, flow cytometry showed increased expression of the Paneth cell-stimulatory cytokines interleukin 22 and interferon γ+ in CD4+ T cells isolated from Ptpn2-KO ileum. Key findings in constitutive Ptpn2-KO mice were confirmed in epithelium-specific Ptpn2ΔIEC mice, which also showed impaired lysozyme protein levels in Paneth cells compared with Ptpn2fl/fl control mice. CONCLUSIONS: Constitutive Ptpn2 deficiency affects Paneth cell viability and compromises Paneth cell-specific antimicrobial peptide production. The observed effects may contribute to the increased susceptibility to intestinal infection and dysbiosis in these mice.

Inhibition of miR-146a Expression and Regulation of Endotoxin Tolerance by Rhesus Theta-Defensin-1.

Theta- (θ-) defensins are pleiotropic host defense peptides with antimicrobial- and immune-modulating activities. Immune stimulation of cells with lipopolysaccharide (LPS, endotoxin) activates proinflammatory gene expression and cytokine secretion, both of which are attenuated by rhesus theta-defensin-1 (RTD-1) inhibition of NF-κB and MAP kinase pathways. Endotoxin tolerance is a condition that ensues when cells have an extended primary exposure to low levels of LPS, resulting in resistance to a subsequent LPS challenge. Recognition of LPS by Toll-like receptor-4 (TLR4) activates NF-κB, elevating levels of microRNA-146a (miR-146a), which targets IRAK1 and TRAF6 transcripts to reduce their protein levels and inhibits TLR signaling on secondary LPS stimulation. Here, we report that RTD-1 suppressed the expression of miR-146a and stabilized the IRAK1 protein in immune-stimulated, monocytic THP-1 cells. Cells that had primary exposure to LPS became endotoxin-tolerant, as evident from their failure to secrete TNF-α upon secondary endotoxin challenge. However, cells incubated with RTD-1 during the primary LPS stimulation secreted TNF-α after secondary LPS stimulation in an RTD-1 dose-dependent manner. Consistent with this, compared to the control treatment, cells treated with RTD-1 during primary LPS stimulation had increased NF-κB activity after secondary LPS stimulation. These results show that RTD-1 suppresses endotoxin tolerance by inhibiting the NF-κB pathway and demonstrates a novel inflammatory role for RTD-1 that is mediated by the downregulation of miR-146a during the innate immune response.

Decreased Paneth cell α-defensins promote fibrosis in a choline-deficient L-amino acid-defined high-fat diet-induced mouse model of nonalcoholic steatohepatitis via disrupting intestinal microbiota.

Nonalcoholic steatohepatitis (NASH) is a chronic liver disease characterized by fibrosis that develops from fatty liver. Disruption of intestinal microbiota homeostasis, dysbiosis, is associated with fibrosis development in NASH. An antimicrobial peptide α-defensin secreted by Paneth cells in the small intestine is known to regulate composition of the intestinal microbiota. However, involvement of α-defensin in NASH remains unknown. Here, we show that in diet-induced NASH model mice, decrease of fecal α-defensin along with dysbiosis occurs before NASH onset. When α-defensin levels in the intestinal lumen are restored by intravenous administration of R-Spondin1 to induce Paneth cell regeneration or by oral administration of α-defensins, liver fibrosis is ameliorated with dissolving dysbiosis. Furthermore, R-Spondin1 and α-defensin improved liver pathologies together with different features in the intestinal microbiota. These results indicate that decreased α-defensin secretion induces liver fibrosis through dysbiosis, further suggesting Paneth cell α-defensin as a potential therapeutic target for NASH.

A host-directed macrocyclic peptide therapeutic for MDR gram negative bacterial infections.

The emergence of infections by carbapenem resistant Enterobacteriaceae (CRE) pathogens has created an urgent public health threat, as carbapenems are among the drugs of last resort for infections caused by a growing fraction of multi-drug resistant (MDR) bacteria. There is global consensus that new preventive and therapeutic strategies are urgently needed to combat the growing problem of MDR bacterial infections. Here, we report on the efficacy of a novel macrocyclic peptide, minimized theta-defensin (MTD)-12813 in CRE sepsis. MTD12813 is a theta-defensin inspired cyclic peptide that is highly effective against CRE pathogens K. pneumoniae and E. coli in vivo. In mouse septicemia models, single dose administration of MTD12813 significantly enhanced survival by promoting rapid host-mediated bacterial clearance and by modulating pathologic cytokine responses, restoring immune homeostasis, and preventing lethal septic shock. The peptide lacks direct antibacterial activity in the presence of mouse serum or in peritoneal fluid, further evidence for its indirect antibacterial mode of action. MTD12813 is highly stable in biological matrices, resistant to bacterial proteases, and nontoxic to mice at dose levels 100 times the therapeutic dose level, properties which support further development of the peptide as a first in class anti-infective therapeutic.

Anti-Inflammatory Effects of RTD-1 in a Murine Model of Chronic Pseudomonas aeruginosa Lung Infection: Inhibition of NF-κB, Inflammasome Gene Expression, and Pro-IL-1ß Biosynthesis.

Vicious cycles of chronic airway obstruction, lung infections with Pseudomonas aeruginosa, and neutrophil-dominated inflammation contribute to morbidity and mortality in cystic fibrosis (CF) patients. Rhesus theta defensin-1 (RTD-1) is an antimicrobial macrocyclic peptide with immunomodulatory properties. Our objective was to investigate the anti-inflammatory effect of RTD-1 in a murine model of chronic P. aeruginosa lung infection. Mice received nebulized RTD-1 daily for 6 days. Bacterial burden, leukocyte counts, and cytokine concentrations were evaluated. Microarray analysis was performed on bronchoalveolar lavage fluid (BALF) cells and lung tissue homogenates. In vitro effects of RTD-1 in THP-1 cells were assessed using quantitative reverse transcription PCR, enzyme-linked immunosorbent assays, immunoblots, confocal microscopy, enzymatic activity assays, and NF-κB-reporter assays. RTD-1 significantly reduced lung white blood cell counts on days 3 (-54.95%; p = 0.0003) and 7 (-31.71%; p = 0.0097). Microarray analysis of lung tissue homogenates and BALF cells revealed that RTD-1 significantly reduced proinflammatory gene expression, particularly inflammasome-related genes (nod-like receptor protein 3, Mediterranean fever gene, interleukin (IL)-1α, and IL-1ß) relative to the control. In vitro studies demonstrated NF-κB activation was reduced two-fold (p ≤ 0.0001) by RTD-1 treatment. Immunoblots revealed that RTD-1 treatment inhibited proIL-1ß biosynthesis. Additionally, RTD-1 treatment was associated with a reduction in caspase-1 activation (FC = -1.79; p = 0.0052). RTD-1 exhibited potent anti-inflammatory activity in chronically infected mice. Importantly, RTD-1 inhibits inflammasome activity, which is possibly a downstream effect of NF-κB modulation. These findings support that this immunomodulatory peptide may be a promising therapeutic for CF-associated lung disease.

p300 Serine 89: A Critical Signaling Integrator and Its Effects on Intestinal Homeostasis and Repair.

Differential usage of Kat3 coactivators, CBP and p300, by ß-catenin is a fundamental regulatory mechanism in stem cell maintenance and initiation of differentiation and repair. Based upon our earlier pharmacologic studies, p300 serine 89 (S89) is critical for controlling differential coactivator usage by ß-catenin via post-translational phosphorylation in stem/progenitor populations, and appears to be a target for a number of kinase cascades. To further investigate mechanisms of signal integration effected by this domain, we generated p300 S89A knock-in mice. We show that S89A mice are extremely sensitive to intestinal insult resulting in colitis, which is known to significantly increase the risk of developing colorectal cancer. We demonstrate cell intrinsic differences, and microbiome compositional differences and differential immune responses, in intestine of S89A versus wild type mice. Genomic and proteomic analyses reveal pathway differences, including lipid metabolism, oxidative stress response, mitochondrial function and oxidative phosphorylation. The diverse effects on fundamental processes including epithelial differentiation, metabolism, immune response and microbiome colonization, all brought about by a single amino acid modification S89A, highlights the critical role of this region in p300 as a signaling nexus and the rationale for conservation of this residue and surrounding region for hundreds of million years of vertebrate evolution.

Host Defense Peptides as Templates for Antifungal Drug Development.

Current treatment for invasive fungal diseases is limited to three classes of antifungal drugs: azoles, polyenes, and echinocandins. The most recently introduced antifungal class, the echinocandins, was first approved nearly 30 years ago. The limited antifungal drug portfolio is rapidly losing its clinical utility due to the inexorable rise in the incidence of invasive fungal infections and the emergence of multidrug resistant (MDR) fungal pathogens. New antifungal therapeutic agents and novel approaches are desperately needed. Here, we detail attempts to exploit the antifungal and immunoregulatory properties of host defense peptides (HDPs) in the design and evaluation of new antifungal therapeutics and discuss historical limitations and recent advances in this quest.

RTD-1 therapeutically normalizes synovial gene signatures in rat autoimmune arthritis and suppresses proinflammatory mediators in RA synovial fibroblasts.

Rhesus theta defensin-1 (RTD-1), a macrocyclic immunomodulatory host defense peptide from Old World monkeys, is therapeutic in pristane-induced arthritis (PIA) in rats, a model of rheumatoid arthritis (RA). RNA-sequence (RNA-Seq) analysis was used to interrogate the changes in gene expression in PIA rats, which identified 617 differentially expressed genes (DEGs) in PIA synovial tissue of diseased rats. Upstream regulator analysis showed upregulation of gene expression pathways regulated by TNF, IL1B, IL6, proinflammatory cytokines, and matrix metalloproteases (MMPs) involved in RA. In contrast, ligand-dependent nuclear receptors like the liver X-receptors NR1H2 and NR1H3 and peroxisome proliferator-activated receptor gamma (PPARG) were downregulated in arthritic synovia. Daily RTD-1 treatment of PIA rats for 1-5 days following disease presentation modulated 340 of the 617 disease genes, and synovial gene expression in PIA rats treated 5 days with RTD-1 closely resembled the gene signature of naive synovium. Systemic RTD-1 inhibited proinflammatory upstream regulators such as TNF, IL1, and IL6 and activated antiarthritic ligand-dependent nuclear receptor pathways, including PPARG, NR1H2, and NR1H3, that were suppressed in untreated PIA rats. RTD-1 also inhibited proinflammatory responses in IL-1ß-stimulated human RA fibroblast-like synoviocytes (FLS) in vitro and diminished expression of human orthologs of disease genes that are induced in rat PIA synovium. Thus, the antiarthritic mechanisms of systemic RTD-1 include homeostatic regulation of arthritogenic gene networks in a manner that correlates temporally with clinical resolution of rat PIA.

Rhesus Theta Defensin 1 Promotes Long Term Survival in Systemic Candidiasis by Host Directed Mechanisms.

Invasive candidiasis is an increasingly frequent cause of serious and often fatal infections in hospitalized and immunosuppressed patients. Mortality rates associated with these infections have risen sharply due to the emergence of multidrug resistant (MDR) strains of C. albicans and other Candida spp., highlighting the urgent need of new antifungal therapies. Rhesus theta (θ) defensin-1 (RTD-1), a natural macrocyclic antimicrobial peptide, was recently shown to be rapidly fungicidal against clinical isolates of MDR C. albicans in vitro. Here we found that RTD-1 was rapidly fungicidal against blastospores of fluconazole/caspofungin resistant C. albicans strains, and was active against established C. albicans biofilms in vitro. In vivo, systemic administration of RTD-1, initiated at the time of infection or 24 h post-infection, promoted long term survival in candidemic mice whether infected with drug-sensitive or MDR strains of C. albicans. RTD-1 induced an early (4 h post treatment) increase in neutrophils in naive and infected mice. In vivo efficacy was associated with fungal clearance, restoration of dysregulated inflammatory cytokines including TNF-α, IL-1ß, IL-6, IL-10, and IL-17, and homeostatic reduction in numbers of circulating neutrophils and monocytes. Because these effects occurred using peptide doses that produced maximal plasma concentrations (Cmax) of less than 1% of RTD-1 levels required for in vitro antifungal activity in 50% mouse serum, while inducing a transient neutrophilia, we suggest that RTD-1 mediates its antifungal effects in vivo by host directed mechanisms rather than direct fungicidal activity. Results of this study suggest that θ-defensins represent a new class of host-directed compounds for treatment of disseminated candidiasis.

Essential role of IFN-γ in T cell-associated intestinal inflammation.

Paneth cells contribute to small intestinal homeostasis by secreting antimicrobial peptides and constituting the intestinal stem cell (ISC) niche. Certain T cell-mediated enteropathies are characterized by extensive Paneth cell depletion coincident with mucosal destruction and dysbiosis. In this study, mechanisms of intestinal crypt injury have been investigated by characterizing responses of mouse intestinal organoids (enteroids) in coculture with mouse T lymphocytes. Activated T cells induced enteroid damage, reduced Paneth cell and Lgr5+ ISC mRNA levels, and induced Paneth cell death through a caspase-3/7-dependent mechanism. IFN-γ mediated these effects, because IFN-γ receptor-null enteroids were unaffected by activated T cells. In mice, administration of IFN-γ induced enteropathy with crypt hyperplasia, villus shortening, Paneth cell depletion, and modified ISC marker expression. IFN-γ exacerbated radiation enteritis, which was ameliorated by treatment with a selective JAK1/2 inhibitor. Thus, IFN-γ induced Paneth cell death and impaired regeneration of small intestinal epithelium in vivo, suggesting that IFN-γ may be a useful target for treating defective mucosal regeneration in enteric inflammation.

Fungicidal Potency and Mechanisms of θ-Defensins against Multidrug-Resistant Candida Species.

Systemic candidiasis is a growing health care concern that is becoming even more challenging due to the growing frequency of infections caused by multidrug-resistant (MDR) Candida species. Thus, there is an urgent need for new therapeutic approaches to candidiasis, including strategies bioinspired by insights into natural host defense against fungal pathogens. The antifungal properties of θ-defensins, macrocyclic peptides expressed in tissues of Old World monkeys, were investigated against a panel of drug-sensitive and drug-resistant clinical isolates of Candida albicans and non-albicans Candida species. Rhesus θ-defensin 1 (RTD-1), the prototype θ-defensin, was rapidly and potently fungicidal against drug-sensitive and MDR C. albicans strains. Fungal killing occurred by cell permeabilization that was temporally correlated with ATP release and intracellular accumulation of reactive oxygen species (ROS). Killing by RTD-1 was compared with that by histatin 5 (Hst 5), an extensively characterized anticandidal peptide expressed in human saliva. RTD-1 killed C. albicans much more rapidly and at a >200-fold lower concentration than that of Hst 5. Unlike Hst 5, the anticandidal activity of RTD-1 was independent of mitochondrial ATP production. Moreover, RTD-1 was completely resistant to Candida proteases for 2 h under conditions that rapidly and completely degraded Hst 5. MICs and minimum fungicidal concentrations (MFCs) of 14 natural θ-defensins isoforms against drug-resistant C. albicans isolates identified peptides that are more active than amphotericin B and/or caspofungin against fluconazole-resistant organisms, including MDR Candida auris. These results point to the potential of macrocyclic θ-defensins as structural templates for the design of antifungal therapeutics.

Macrocyclic θ-defensins suppress tumor necrosis factor-α (TNF-α) shedding by inhibition of TNF-α-converting enzyme.

Theta-defensins (θ-defensins) are macrocyclic peptides expressed exclusively in granulocytes and selected epithelia of Old World monkeys. They contribute to anti-pathogen host defense responses by directly killing a diverse range of microbes. Of note, θ-defensins also modulate microbe-induced inflammation by affecting the production of soluble tumor necrosis factor (sTNF) and other proinflammatory cytokines. Here, we report that natural rhesus macaque θ-defensin (RTD) isoforms regulate sTNF cellular release by inhibiting TNF-α-converting enzyme (TACE; also known as adisintegrin and metalloprotease 17; ADAM17), the primary pro-TNF sheddase. Dose-dependent inhibition of cellular TACE activity by RTDs occurred when leukocytes were stimulated with live Escherichia coli cells as well as numerous Toll-like receptor agonists. Moreover, the relative inhibitory potencies of the RTD isoforms strongly correlated with their suppression of TNF release by stimulated blood leukocytes and THP-1 monocytes. RTD isoforms also inhibited ADAM10, a sheddase closely related to TACE. TACE inhibition was abrogated by introducing a single opening in the RTD-1 backbone, demonstrating that the intact macrocycle is required for enzyme inhibition. Enzymologic analyses showed that RTD-1 is a fast binding, reversible, non-competitive inhibitor of TACE. We conclude that θ-defensin-mediated inhibition of pro-TNF proteolysis by TACE represents a rapid mechanism for the regulation of sTNF and TNF-dependent inflammatory pathways. Molecules with structural and functional features mimicking those of θ-defensins may have clinical utility as TACE inhibitors for managing TNF-driven diseases.

Suppression and resolution of autoimmune arthritis by rhesus θ-defensin-1, an immunomodulatory macrocyclic peptide.

θ-defensins constitute a family of macrocyclic peptides expressed exclusively in Old World monkeys. The peptides are pleiotropic effectors of innate immunity, possessing broad spectrum antimicrobial activities and immunoregulatory properties. Here we report that rhesus θ-defensin 1 (RTD-1) is highly effective in arresting and reversing joint disease in a rodent model of rheumatoid arthritis (RA). Parenteral RTD-1 treatment of DA/OlaHsd rats with established pristane-induced arthritis (PIA) rapidly suppressed joint disease progression, restored limb mobility, and preserved normal joint architecture. RTD-1 significantly reduced joint IL-1ß levels compared with controls. RTD-1 dose-dependently inhibited fibroblast-like synoviocyte (FLS) invasiveness and FLS IL-6 production. Consistent with the inhibition of FLS invasiveness, RTD-1 was a potent inhibitor of arthritogenic proteases including ADAMs 17 and 10 which activate TNFα, and inhibited matrix metalloproteases, and cathepsin K. RTD-1 was non-toxic, non-immunogenic, and effective when administered as infrequently as once every five days. Thus θ-defensins, which are absent in humans, have potential as retroevolutionary biologics for the treatment of RA.

A Requirement for Metamorphic Interconversion in the Antimicrobial Activity of Chemokine XCL1.

Chemokines make up a superfamily of ∼50 small secreted proteins (8-12 kDa) involved in a host of physiological processes and disease states, with several previously shown to have direct antimicrobial activity comparable to that of defensins in efficacy. XCL1 is a unique metamorphic protein that interconverts between the canonical chemokine fold and a novel all-ß-sheet dimer. Phylogenetic analysis suggests that, within the chemokine family, XCL1 is most closely related to CCL20, which exhibits antibacterial activity. The in vitro antimicrobial activity of WT-XCL1 and structural variants was quantified using a radial diffusion assay (RDA) and in solution bactericidal assays against Gram-positive and Gram-negative species of bacteria. Comparisons of WT-XCL1 with variants that limit metamorphic interconversion showed a loss of antimicrobial activity when restricted to the conserved chemokine fold. These results suggest that metamorphic folding of XCL1 is required for potent antimicrobial activity.

Salmonella Mitigates Oxidative Stress and Thrives in the Inflamed Gut by Evading Calprotectin-Mediated Manganese Sequestration.

Neutrophils hinder bacterial growth by a variety of antimicrobial mechanisms, including the production of reactive oxygen species and the secretion of proteins that sequester nutrients essential to microbes. A major player in this process is calprotectin, a host protein that exerts antimicrobial activity by chelating zinc and manganese. Here we show that the intestinal pathogen Salmonella enterica serovar Typhimurium uses specialized metal transporters to evade calprotectin sequestration of manganese, allowing the bacteria to outcompete commensals and thrive in the inflamed gut. The pathogen's ability to acquire manganese in turn promotes function of SodA and KatN, enzymes that use the metal as a cofactor to detoxify reactive oxygen species. This manganese-dependent SodA activity allows the bacteria to evade neutrophil killing mediated by calprotectin and reactive oxygen species. Thus, manganese acquisition enables S. Typhimurium to overcome host antimicrobial defenses and support its competitive growth in the intestine.

Defensins Potentiate a Neutralizing Antibody Response to Enteric Viral Infection.

α-defensins are abundant antimicrobial peptides with broad, potent antibacterial, antifungal, and antiviral activities in vitro. Although their contribution to host defense against bacteria in vivo has been demonstrated, comparable studies of their antiviral activity in vivo are lacking. Using a mouse model deficient in activated α-defensins in the small intestine, we show that Paneth cell α-defensins protect mice from oral infection by a pathogenic virus, mouse adenovirus 1 (MAdV-1). Survival differences between mouse genotypes are lost upon parenteral MAdV-1 infection, strongly implicating a role for intestinal defenses in attenuating pathogenesis. Although differences in α-defensin expression impact the composition of the ileal commensal bacterial population, depletion studies using broad-spectrum antibiotics revealed no effect of the microbiota on α-defensin-dependent viral pathogenesis. Moreover, despite the sensitivity of MAdV-1 infection to α-defensin neutralization in cell culture, we observed no barrier effect due to Paneth cell α-defensin activation on the kinetics and magnitude of MAdV-1 dissemination to the brain. Rather, a protective neutralizing antibody response was delayed in the absence of α-defensins. This effect was specific to oral viral infection, because antibody responses to parenteral or mucosal ovalbumin exposure were not affected by α-defensin deficiency. Thus, α-defensins play an important role as adjuvants in antiviral immunity in vivo that is distinct from their direct antiviral activity observed in cell culture.

Rhesus macaque θ-defensin RTD-1 inhibits proinflammatory cytokine secretion and gene expression by inhibiting the activation of NF-κB and MAPK pathways.

θ-Defensins are pleiotropic, macrocyclic peptides that are expressed uniquely in Old World monkeys. The peptides are potent, broad-spectrum microbicides that also modulate inflammatory responses in vitro and in animal models of viral infection and polymicrobial sepsis. θ-Defensins suppress proinflammatory cytokine secretion by leukocytes stimulated with diverse Toll-like receptor (TLR) ligands. Studies were performed to delineate anti-inflammatory mechanisms of rhesus θ-defensin 1 (RTD-1), the most abundant θ-defensin isoform in macaque granulocytes. RTD-1 reduced the secretion of tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, and IL-8 in lipopolysaccharide (LPS)-stimulated human blood monocytes and THP-1 macrophages, and this was accompanied by inhibition of nuclear factor κB (NF-κB) activation and mitogen-activated protein kinase (MAPK) pathways. Peptide inhibition of NF-κB activation occurred following stimulation of extracellular (TLRs 1/2 and 4) and intracellular (TLR9) receptors. Although RTD-1 did not inhibit MAPK in unstimulated cells, it induced phosphorylation of Akt in otherwise untreated monocytes and THP-1 cells. In the latter, this occurred within 10 min of RTD-1 treatment and produced a sustained elevation of phosphorylated Akt (pAkt) for at least 4 h. pAkt is a negative regulator of MAPK and NF-κB activation. RTD-1 inhibited IκBα degradation and p38 MAPK phosphorylation, and stimulated Akt phosphorylation in LPS-treated human primary monocytes and THP-1 macrophages. Specific inhibition of phosphatidylinositol 3-kinase (PI3K) blocked RTD-1-stimulated Akt phosphorylation and reversed the suppression of NF-κB activation by the peptide. These studies indicate that the anti-inflammatory properties of θ-defensins are mediated by activation of the PI3K/Akt pathway and suppression of proinflammatory signals in immune-stimulated cells.

Microbicidal effects of α- and θ-defensins against antibiotic-resistant Staphylococcus aureus and Pseudomonas aeruginosa.

Antibiotic-resistant bacterial pathogens threaten public health. Because many antibiotics target specific bacterial enzymes or reactions, corresponding genes may mutate under selection and lead to antibiotic resistance. Accordingly, antimicrobials that selectively target overall microbial cell integrity may offer alternative approaches to therapeutic design. Naturally occurring mammalian α- and θ-defensins are potent, non-toxic microbicides that may be useful for treating infections by antibiotic-resistant pathogens because certain defensin peptides disrupt bacterial, but not mammalian, cell membranes. To test this concept, clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA), including vancomycin heteroresistant strains, and ciprofloxacin-resistant Pseudomonas aeruginosa (Cip(R)-PA) were tested for sensitivity to α-defensins Crp-4, RMAD-4 and HNPs 1-3, and to RTD-1, macaque θ-defensin-1. In vitro, 3 µM Crp-4, RMAD-4 and RTD-1 reduced MRSA cell survival by 99%, regardless of vancomycin susceptibility. For PA clinical isolates that differ in fluoroquinolone resistance and virulence phenotype, peptide efficacy was independent of strain ciprofloxacin resistance, site of isolation or virulence factor expression. Thus, Crp-4, RMAD-4 and RTD-1 are effective in vitro antimicrobials against clinical isolates of MRSA and Cip(R)-PA, perhaps providing templates for development of α- and θ-defensin-based microbicides against antibiotic resistant or virulent infectious agents.

Differential Susceptibility of Bacteria to Mouse Paneth Cell α-Defensins under Anaerobic Conditions.

Small intestinal Paneth cells secrete α-defensin peptides, termed cryptdins (Crps) in mice, into the intestinal lumen, where they confer immunity to oral infections and define the composition of the ileal microbiota. In these studies, facultative bacteria maintained under aerobic or anaerobic conditions displayed differential sensitivities to mouse α-defensins under in vitro assay conditions. Regardless of oxygenation, Crps 2 and 3 had robust and similar bactericidal activities against S. Typhimurium and S. flexneri, but Crp4 activity against S. flexneri was attenuated in the absence of oxygen. Anaerobic bacteria varied in their susceptibility to Crps 2-4, with Crp4 showing less activity than Crps 2 and 3 against Enterococcus faecalis, and Bacteroides fragilis in anaerobic assays, but Fusobacterium necrophorum was killed only by Crp4 and not by Crps 2 and 3. The influence of anaerobiosis in modulating Crp bactericidal activities in vitro suggests that α-defensin effects on the enteric microbiota may be subject to regulation by local oxygen tension.

Hydrophobic determinants of α-defensin bactericidal activity.

Mammalian α-defensins are approximately 4- to 5-kDa broad-spectrum antimicrobial peptides and abundant granule constituents of neutrophils and small intestinal Paneth cells. The bactericidal activities of amphipathic α-defensins depend in part on electropositive charge and on hydrophobic amino acids that enable membrane disruption by interactions with phospholipid acyl chains. Alignment of α-defensin primary structures identified conserved hydrophobic residues in the loop formed by the Cys(III)-Cys(V) disulfide bond, and we have studied their role by testing the effects of mutagenesis on bactericidal activities. Mouse α-defensin 4 (Crp-4) and rhesus myeloid α-defensin 4 (RMAD-4) were selected for these studies, because they are highly bactericidal in vitro and have the same overall electropositive charge. Elimination of hydrophobicity by site-directed mutagenesis at those positions in Crp-4 attenuated bactericidal activity markedly. In contrast to native Crp-4, the (I23/F25/L26/G)-Crp-4 variant lacked bactericidal activity against Salmonella enterica serovar Typhimurium and did not permeabilize Escherichia coli ML35 cells as a result of removing aliphatic side chains by Gly substitutions. Ala replacements in (I23/F25/L26/A)-Crp-4 restored activity, evidence that hydrophobicity contributed by Ala methyl R-groups was sufficient for activity. In macaques, neutrophil α-defensin RMAD-6 is identical to RMAD-4, except for a F28S difference, and (F28S)-RMAD-4 mutagenesis attenuated RMAD-4 bactericidal activity and E. coli permeabilization. Interestingly, (R31/32D)-Crp-4 lacks activity in these assays despite the presence of the Ile23, Phe25, and Leu26 hydrophobic patch. We infer that electrostatic interactions between cationic α-defensin residues and negative charge on bacteria precede interactions between critical hydrophobic residue positions that mediate membrane disruption and bacterial cell killing.