Glutathione reductase facilitates host defense by sustaining phagocytic oxidative burst and promoting the development of neutrophil extracellular traps

Glutathione reductase (Gsr) catalyzes the reduction of glutathione disulfide to glutathione, which plays an important role in the bactericidal function of phagocytes. Because Gsr has been implicated in the oxidative burst in human neutrophils and is abundantly expressed in the lymphoid system, we hypothesized that Gsr-deficient mice would exhibit marked defects during the immune response against bacterial challenge. We report in this study that Gsr-null mice exhibited enhanced susceptibility to Escherichia coli challenge, indicated by dramatically increased bacterial burden, cytokine storm, striking histological abnormalities, and substantially elevated mortality. Additionally, Gsr-null mice exhibited elevated sensitivity to Staphylococcus aureus. Examination of the bactericidal functions of the neutrophils from Gsr-deficient mice in vitro revealed impaired phagocytosis and defective bacterial killing activities. Although Gsr catalyzes the regeneration of glutathione, a major cellular antioxidant, Gsr-deficient neutrophils paradoxically produced far less reactive oxygen species upon activation both ex vivo and in vivo. Unlike wild-type neutrophils that exhibited a sustained oxidative burst upon stimulation with phorbol ester and fMLP, Gsr-deficient neutrophils displayed a very transient oxidative burst that abruptly ceased shortly after stimulation. Likewise, Gsr-deficient neutrophils also exhibited an attenuated oxidative burst upon encountering E. coli. Biochemical analysis revealed that the hexose monophosphate shunt was compromised in Gsr-deficient neutrophils. Moreover, Gsr-deficient neutrophils displayed a marked impairment in the formation of neutrophil extracellular traps, a bactericidal mechanism that operates after neutrophil death. Thus, Gsr-mediated redox regulation is crucial for bacterial clearance during host defense against massive bacterial challenge.     

Olfactomedin 4 Inhibits Cathepsin C-Mediated Protease Activities, Thereby Modulating Neutrophil Killing of Staphylococcus aureus and Escherichia coli in Mice

Neutrophils kill bacteria generally through oxidative and nonoxidative mechanisms. Whereas much research has focused on the enzymes essential for neutrophil killing, little is known about the regulatory molecules responsible for such killing. In this study, we investigated the role of olfactomedin 4 (OLFM4), an olfactomedin-related glycoprotein, in neutrophil bactericidal capability and host innate immunity. Neutrophils from OLFM4(-/-) mice have increased intracellular killing of Staphylococcus aureus and Escherichia coli in vitro. The OLFM4(-/-) mice have enhanced in vivo bacterial clearance and are more resistant to sepsis when challenged with S. aureus or E. coli by i.p. injection. OLFM4 was found to interact with cathepsin C, a cysteine protease that plays an important role in bacterial killing and immune regulation. We demonstrated that OLFM4 inhibited cathepsin C activity in vitro and in vivo. The cathepsin C activity in neutrophils from OLFM4(-/-) mice was significantly higher than that in neutrophils from wild-type littermate mice. The activities of three serine proteases (neutrophil elastase, cathepsin G, and proteinase 3), which require cathepsin C activity for processing and maturity, were also significantly higher in OLFM4(-/-) neutrophils. The bacterial killing and clearance capabilities observed in OLFM4(-/-) mice that were enhanced relative to wild-type mice were significantly compromised by the additional loss of cathepsin C in mice with OLFM4 and cathepsin C double deficiency. These results indicate that OLFM4 is an important negative regulator of neutrophil bactericidal activity by restricting cathepsin C activity and its downstream granule-associated serine proteases.     

The function of OmpA in Escherichia coli

Outer membrane protein A (OmpA) is a major protein in the Escherichia coli outer membrane. In this study, the function of OmpA in E. coli stress survival was examined. An E. coli K1 ompA-deletion mutant was significantly more sensitive than that of its parent strain to sodium dodecyl sulfate (SDS), cholate, acidic environment, high osmolarity, and pooled human serum. A number of amino acid changes at the extracellular loops of OmpA did not affect the viability of E. coli, while short peptide insertions in the periplasmic turns of the OmpA beta-barrel decreased E. coli resistance to environmental stresses. Moreover, ompA mutants were found to survive much better within brain microvascular endothelial cells than the wild-type strain, supporting that OmpA is a major target in mammalian host cell defense. These results indicated that OmpA plays a vital structural role in E. coli, and suggested that a perfect beta-barrel structure of OmpA is important for outer membrane stability. Based on these results and the published OmpA structural analyses, I propose that OmpA is composed of three functional domains including a hydrophilic extracellular mass, a beta-barrel transmembrane structure, and a peptidoglycan binding domain.     

Identification of formyl peptides from Listeria monocytogenes and Staphylococcus aureus as potent chemoattractants for mouse neutrophils

The prototypic formyl peptide N-formyl-Met-Leu-Phe (fMLF) is a major chemoattractant found in Escherichia coli culture supernatants and a potent agonist at human formyl peptide receptor (FPR) 1. Consistent with this, fMLF induces bactericidal functions in human neutrophils at nanomolar concentrations. However, it is a much less potent agonist for mouse FPR (mFPR) 1 and mouse neutrophils, requiring micromolar concentrations for cell activation. To determine whether other bacteria produce more potent agonists for mFPR1, we examined formyl peptides from Listeria monocytogenes and Staphylococcus aureus for their abilities to activate mouse neutrophils. A pentapeptide (N-formyl-Met-Ile-Val-Ile-Leu (fMIVIL)) from L. monocytogenes and a tetrapeptide (N-formyl-Met-Ile-Phe-Leu (fMIFL)) from S. aureus were found to induce mouse neutrophil chemotaxis at 1-10 nM and superoxide production at 10-100 nM, similar to the potency of fMLF on human neutrophils. Using transfected cell lines expressing mFPR1 and mFPR2, which are major forms of FPRs in mouse neutrophils, we found that mFPR1 is responsible for the high potency of fMIVIL and fMIFL. In comparison, activation of mFPR2 requires micromolar concentrations of the two peptides. Genetic deletion of mfpr1 resulted in abrogation of neutrophil superoxide production and degranulation in response to fMIVIL and fMIFL, further demonstrating that mFPR1 is the primary receptor for detection of these formyl peptides. In conclusion, the formyl peptides from L. monocytogenes and S. aureus are approximately 100-fold more potent than fMLF in activating mouse neutrophils. The ability of mFPR1 to detect bacterially derived formyl peptides indicates that this important host defense mechanism is conserved in mice.     

The effects of Escherichia coli capsule, O-antigen, host neutrophils, and complement in a rat model of Gram-negative pneumonia

Gram-negative enteric bacilli are agents of life-threatening pneumonia. The role of the bacterial capsule and O-antigen moiety of lipopolysaccharide in the pathogenesis of Gram-negative pneumonia was assessed. In a rat model of pneumonia the LD(50) of a wild-type extraintestinal pathogenic Escherichia coli strain (CP9) was significantly less than its isogenic derivatives deficient in capsule (CP9.137), O-antigen (CP921) or both capsule and O-antigen (CP923) (P< or =0.003). Studies using complement depleted or neutropenic animals established that both neutrophils and complement are important for the pulmonary clearance of E. coli. Data from these studies also support that capsule and O-antigen serve, at least in part, to counter the complement and neutrophil components of the pulmonary host defense response. Lastly, the contribution of E. coli versus neutrophils in causing lung injury was examined. Findings suggest that E. coli virulence factors and/or non-neutrophil host factors are more important mediators of lung injury than neutrophils. These findings extend our understanding of Gram-negative pneumonia and have treatment implications.     

An antimicrobial cathelicidin peptide, human CAP18/LL-37, suppresses neutrophil apoptosis via the activation of formyl-peptide receptor-like 1 and P2X7

Peptide antibiotics possess the potent antimicrobial activities against invading microorganisms and contribute to the innate host defense. An antibacterial cathelicidin, human cationic antibacterial protein of 18 kDa/LL-37, not only exhibits potent bactericidal activities against Gram-negative and Gram-positive bacteria, but also functions as a chemoattractant for immune cells, including neutrophils. During bacterial infections, the life span of neutrophils is regulated by various pathogen- and host-derived substances. In this study, to further evaluate the role of LL-37 in innate immunity, we investigated the action of LL-37 on neutrophil apoptosis. Neutrophil apoptosis was assessed using human blood neutrophils based on the morphological changes. Of note, LL-37 dose dependently (0.01-5 microg/ml) suppressed neutrophil apoptosis, accompanied with the phosphorylation of ERK-1/2, expression of Bcl-x(L) (an antiapoptotic protein), and inhibition of caspase 3 activity. Interestingly, LL-37-induced suppression of neutrophil apoptosis was attenuated by the antagonists for formyl-peptide receptor-like 1 (FPRL1) and P2X7 nucleotide receptor. Of importance, the agonists for FPRL1 and P2X7 apparently suppressed neutrophil apoptosis. Collectively, these observations indicate that LL-37 cannot only kill bacteria, but also modulate (suppress) neutrophil apoptosis via the activation of FPRL1 and P2X7 in bacterial infections. Suppression of neutrophil apoptosis results in the prolongation of their life span, and may be advantageous for host defense against bacterial invasion.     

Myeloperoxidase plays critical roles in killing Klebsiella pneumoniae and inactivating neutrophil elastase: effects on host defense

Activated neutrophils use myeloperoxidase (MPO) to generate an array of potent toxic oxidants. In the current studies we used genetically altered mice deficient in MPO to investigate the role of the enzyme in host defense against the Gram-negative bacterium Klebsiella pneumoniae, an important human pathogen. For comparison, we used mice deficient in the antimicrobial molecule, neutrophil elastase (NE). When challenged i.p., mice deficient in either MPO or NE were markedly more susceptible to bacterial infection and death. In vitro studies suggested that MPO impairs the morphology of bacteria in a distinctive way. Of importance, our in vitro studies found that MPO mediated oxidative inactivation of NE, an enzyme that has been widely implicated in the pathogenesis of various tissue-destructive diseases. This pathway of oxidative inactivation may be physiologically relevant, because activated neutrophils isolated from MPO-deficient mice exhibited increased elastase activity. Our observations provide strong evidence that MPO, like NE, is a key player in the killing of K. pneumoniae bacteria. They also suggest that MPO may modulate NE to protect the host from the tissue-degrading activity of this proteinase.     

Sap transporter mediated import and subsequent degradation of antimicrobial peptides in Haemophilus

Antimicrobial peptides (AMPs) contribute to host innate immune defense and are a critical component to control bacterial infection. Nontypeable Haemophilus influenzae (NTHI) is a commensal inhabitant of the human nasopharyngeal mucosa, yet is commonly associated with opportunistic infections of the upper and lower respiratory tracts. An important aspect of NTHI virulence is the ability to avert bactericidal effects of host-derived antimicrobial peptides (AMPs). The Sap (sensitivity to antimicrobial peptides) ABC transporter equips NTHI to resist AMPs, although the mechanism of this resistance has remained undefined. We previously determined that the periplasmic binding protein SapA bound AMPs and was required for NTHI virulence in vivo. We now demonstrate, by antibody-mediated neutralization of AMP in vivo, that SapA functions to directly counter AMP lethality during NTHI infection. We hypothesized that SapA would deliver AMPs to the Sap inner membrane complex for transport into the bacterial cytoplasm. We observed that AMPs localize to the bacterial cytoplasm of the parental NTHI strain and were susceptible to cytoplasmic peptidase activity. In striking contrast, AMPs accumulated in the periplasm of bacteria lacking a functional Sap permease complex. These data support a mechanism of Sap mediated import of AMPs, a novel strategy to reduce periplasmic and inner membrane accumulation of these host defense peptides.     

Functional interaction of human neutrophil peptide-1 with the cell wall precursor lipid II

Defensins constitute a major class of cationic antimicrobial peptides in mammals and vertebrates, acting as effectors of innate immunity against infectious microorganisms. It is generally accepted that defensins are bactericidal by disrupting the anionic microbial membrane. Here, we provide evidence that membrane activity of human α-defensins does not correlate with antibacterial killing. We further show that the α-defensin human neutrophil peptide-1 (HNP1) binds to the cell wall precursor lipid II and that reduction of lipid II levels in the bacterial membrane significantly reduces bacterial killing. The interaction between defensins and lipid II suggests the inhibition of cell wall synthesis as a novel antibacterial mechanism of this important class of host defense peptides.     

Interactions between Mytilus haemocytes and different strains of Escherichia coli and Vibrio cholerae O1 El Tor: role of kinase-mediated signalling

Marine bivalves accumulate large amounts of bacteria from the environment (mainly Vibrionaceae and coliforms). Although persistence of different bacteria in bivalve tissues largely depends on their sensitivity to the bactericidal activity of circulating haemocytes and haemolymph soluble factors, the mechanisms involved in bacteria-host cell interactions in these invertebrates are largely unknown. In the mussel Mytilus, differences in interactions between haemocytes and different Escherichia coli and Vibrio cholerae strains [E. coli MG155, a wild-type strain carrying type 1 fimbriae, and its unfimbriated derivative, AAEC072 Deltafim; V. cholerae O1 El Tor biotype strain N16961, carrying the mannose-sensitive haemagglutinin (MSHA), and its MSHA mutant] lead to differences in bactericidal activity in the presence of serum. Here we show that different bacteria induced distinct patterns of phosphorylation of mitogen-activated protein kinases (MAPKs), in particular of the stress-activated MAPKs involved in the immune response. Differences in phosphorylation of PKC-like proteins were also observed. The results support the hypothesis that, like in mammalian host cells, different bacteria can modulate the signalling pathways of mussel haemocytes. The lower anti-bacterial activity towards the mutant E. coli strain and wild-type V. cholerae compared with wild E. coli may result from a reduced capacity of activating MAPKs. Moreover, the mutant V. cholerae strain that was the most resistant to the haemocyte bactericidal activity induced downregulation of cell signalling and showed the strongest effect on lysosomal membrane stability, evaluated as a marker of bivalve cell stress. These data suggest that certain bacteria could evade the bactericidal activity of mussel haemocytes through disruption of the host signalling pathways.     

Neutrophil elastase mediates innate host protection against Pseudomonas aeruginosa

According to the widely accepted view, neutrophil elastase (NE), a neutrophil-specific serine protease, is a major contributor to Pseudomonas aeruginosa infection-associated host tissue inflammation and damage, which in severe cases can lead to death. Herein, we provide for the first time compelling evidence that the host rather employs NE to protect itself against P. aeruginosa infection. Using a clinically relevant model of pneumonia, targeted deficiency in NE increased the susceptibility of mice to P. aeruginosa. We found that NE was required for maximal intracellular killing of P. aeruginosa by neutrophils. In investigating the mechanism of NE-mediated killing of P. aeruginosa, we found that NE degraded the major outer membrane protein F, a protein with important functions, including porin activity, maintenance of structural integrity, and sensing of host immune system activation. Consistent with this, the use of an isogenic mutant deficient in outer membrane protein F negated the role of NE in host defense against P. aeruginosa infection.     

Human neutrophil defensins increase neutrophil uptake of influenza A virus and bacteria and modify virus-induced respiratory burst responses

Human neutrophil peptides (HNPs) are released from granules of neutrophils in response to various activating stimuli and they participate in the killing of bacteria and the stimulation of various inflammatory responses. HNPs also inhibit infectivity of enveloped viruses, including influenza A virus (IAV). In this study, we demonstrate that HNPs increase the uptake of IAV and bacteria by neutrophils. The dimeric HNPs also induced aggregation of IAV and bacterial particles, which may, in part, explain their ability to increase uptake. HNPs did not increase neutrophil respiratory burst responses to IAV. We have recently demonstrated direct interactions of HNPs with surfactant protein D (SP-D), another important effector of innate immunity and antimicrobial host defense. Although HNPs did not alter SP-D-dependent uptake of IAV, they counteracted the ability of SP-D to increase IAV-induced neutrophil H2O2 generation. Our studies reveal previously unappreciated functional effects of HNPs, expand our understanding of the antiviral properties of HNPs, and suggest important interactions between collectins and HNPs in the host response to viruses and bacteria.     

Bactericidal/permeability-increasing protein has endotoxin-neutralizing activity

Neutrophil granules contain proteins important in host defense against bacterial pathogens. Granule proteins released from activated neutrophils facilitate opsonization, phagocytosis, tissue digestion, and antimicrobial activity. Three similar, if not identical, neutrophil proteins, bactericidal/permeability-increasing protein (BPI), 57,000 m.w. cationic antimicrobial protein, and bactericidal protein have been described that specifically kill gram negative bacteria. Since LPS is a structure common to all gram-negative bacteria, we investigated whether the microbicidal protein BPI affects biologic activity of LPS in vitro. Human neutrophils can be activated both in vitro and in vivo by LPS. Upon stimulation, surface expression of CR1 and CR3 increases markedly. Using flow microfluorimetry, we analyzed surface expression of CR1 and CR3 as a measure of neutrophil stimulation in response to LPS. CR up-regulation on neutrophils was TNF independent, suggesting direct LPS stimulation of neutrophils in this system. Purified BPI completely inhibited CR up-regulation on neutrophils stimulated with both rough and smooth LPS chemotypes at 1.8 to 3.6 nM (100 to 200 ng/ml). By comparison, the polypeptide antibiotic polymyxin B completely inhibited the same dose of LPS at 0.4 nM. The inhibitory activity of BPI appeared to be specific for LPS because neutrophil stimulation by formylated peptide or TNF was unaffected. The specificity of BPI for LPS was further demonstrated by inhibition of LPS activity in the limulus amebocyte lysate assay. Therefore, the role of BPI in infection may not be limited to its microbicidal activity, but it may also regulate the neutrophil response to LPS.     

Cathepsin G and neutrophil elastase contribute to lung-protective immunity against mycobacterial infections in mice

The neutrophil serine proteases cathepsin G (CG) and neutrophil elastase (NE) are involved in immune-regulatory processes and exert antibacterial activity against various pathogens. To date, their role and their therapeutic potential in pulmonary host defense against mycobacterial infections are poorly defined. In this work, we studied the roles of CG and NE in the pulmonary resistance against Mycobacterium bovis bacillus Calmette-Guérin (BCG). CG-deficient mice and even more pronounced CG/NE-deficient mice showed significantly impaired pathogen elimination to infection with M. bovis BCG in comparison to wild-type mice. Moreover, granuloma formation was more pronounced in M. bovis BCG-infected CG/NE-deficient mice in comparison to CG-deficient and wild-type mice. A close examination of professional phagocyte subsets revealed that exclusively neutrophils shuttled CG and NE into the bronchoalveolar space of M. bovis BCG-infected mice. Accordingly, chimeric wild-type mice with a CG/NE-deficient hematopoietic system displayed significantly increased lung bacterial loads in response to M. bovis BCG infection. Therapeutically applied human CG/NE encapsulated in liposomes colocalized with mycobacteria in alveolar macrophages, as assessed by laser scanning and electron microscopy. Importantly, therapy with CG/NE-loaded liposomes significantly reduced mycobacterial loads in the lungs of mice. Together, neutrophil-derived CG and NE critically contribute to deceleration of pathogen replication during the early phase of antimycobacterial responses. In addition, to our knowledge, we show for the first time that liposomal encapsulated CG/NE exhibit therapeutic potential against pulmonary mycobacterial infections. These findings may be relevant for novel adjuvant approaches in the treatment of tuberculosis in humans.     

Compromised host defense on Pseudomonas aeruginosa biofilms: characterization of neutrophil and biofilm interactions

Pseudomonas aeruginosa is an opportunistic pathogen that forms biofilms on tissues and other surfaces. We characterized the interaction of purified human neutrophils with P. aeruginosa, growing in biofilms, with regard to morphology, oxygen consumption, phagocytosis, and degranulation. Scanning electron and confocal laser microscopy indicated that the neutrophils retained a round, unpolarized, unstimulated morphology when exposed to P. aeruginosa PAO1 biofilms. However, transmission electron microscopy demonstrated that neutrophils, although rounded on their dorsal side, were phagocytically active with moderate membrane rearrangement on their bacteria-adjacent surfaces. The settled neutrophils lacked pseudopodia, were impaired in motility, and were enveloped by a cloud of planktonic bacteria released from the biofilms. The oxygen consumption of the biofilm/neutrophil system increased 6- and 8-fold over that of the biofilm alone or unstimulated neutrophils in suspension, respectively. H(2)O(2) accumulation was transient, reaching a maximal measured value of 1 micro M. Following contact, stimulated degranulation was 20-40% (myeloperoxidase, beta-glucuronidase) and 40-80% (lactoferrin) of maximal when compared with formylmethionylleucylphenylalanine plus cytochalasin B stimulation. In summary, after neutrophils settle on P. aeruginosa biofilms, they become phagocytically engorged, partially degranulated, immobilized, and rounded. The settling also causes an increase in oxygen consumption of the system, apparently resulting from a combination of a bacterial respiration and escape response and the neutrophil respiratory burst but with little increase in the soluble concentration of H(2)O(2). Thus, host defense becomes compromised as biofilm bacteria escape while neutrophils remain immobilized with a diminished oxidative potential.     

Attenuated virulence of Streptococcus agalactiae deficient in D-alanyl-lipoteichoic acid is due to an increased susceptibility to defensins and phagocytic cells

D-alanylation of lipoteichoic acid (LTA), allows Gram-positive bacteria to modulate their surface charge, regulate ligand binding and control the electromechanical properties of the cell wall. In this study, the role of D-alanyl LTA in the virulence of the extracellular pathogen Streptococcus agalactiae was investigated. We demonstrate that a DltA- isogenic mutant displays an increased susceptibility to host defence peptides such as human defensins and animal-derived cationic peptides. Accordingly, the mutant strain is more susceptible to killing by mice bone marrow-derived macrophages and human neutrophils than the wild-type strain. In addition, the virulence of the DltA- mutant is severely impaired in mouse and neonatal rat models. This mutant was eliminated more rapidly than the wild-type strain from the lung of three-week-old mice inoculated intranasally and, consequently, is unable to induce a pneumonia. Finally, after intravenous injection of three-week-old mice, the survival of the DltA- mutant is markedly reduced in the blood in comparison to that of the wild-type strain. We hypothesize that the decreased virulence of the DltA- mutant is a consequence of its increased susceptibility to cationic antimicrobial peptides and to killing by phagocytes. These results demonstrate that the D-alanylation of LTA contributes to the virulence of S. agalactiae.     

Functional whole-colony screening method to identify antimicrobial peptides

A high throughput method for screening cDNA libraries has been developed to identify putative antimicrobial peptides (AMPs). It is based on a rapid dye inclusion assay for assessing antagonism of bacterial viability. Colonies are grown on a membrane on a permissive medium until full colony size is reached. The membrane, supporting the array of colonies, is transferred onto an inductive medium containing a vital dye. Upon expression of any antagonizing peptides, the cell membrane becomes compromised allowing dye infusion to permit visual identification of deleterious peptides. Our approach was validated by screening a synthetic oligonucleotide library expressed in Escherichia coli. A random oligonucleotide library, containing inserts of up to 75 nucleotides in length was constructed and expressed in E. coli. From a potential pool of 100000 peptides, in a single round of screening, three were found to be antimicrobial: L1, L3, and L8. Peptide L1 was shown to have a concentration-dependent bactericidal effect against Gram-negative E. coli and moderate biostatic activity against the Gram-positive bacteria Listeria monocytogenes. L8 was found to have bacteriostatic, and possibly bactericidal effect against E. coli, Pseudomonas aeruginosa and Salmonella typhimurium. These results validated this high throughput AMP identification assay based on filter bound colony array libraries and vital dye inclusion.     

Sensitivity of bacteria exposed to subinhibitory concentrations of antibiotics to the action of serum and liver phagocytes

Incubation of E.coli and S. aureus with subinhibitory concentration (1/5 MIC) of cefamandole modified bacterial morphology and resistance to host defence mechanisms. In fact, cefamandole induced filamentous forms of E. coli, when added to the perfusing medium of the isolated rat liver system, were phagocytized at a much fortes rate then control bacteria, but appeared less sensitive to serum bactericidal activity. In contrast, S.aureus, after exposure to the antibiotic, was more sensitive to the bactericidal activity of serum then controls, while treated and untreated cells were phagocytized at the some rate. The data suggest that even at low doses some antibiotics may alter bacterial structure and increase their susceptibility to host factors.     

Role of NADPH oxidase versus neutrophil proteases in antimicrobial host defense

NADPH oxidase is a crucial enzyme in mediating antimicrobial host defense and in regulating inflammation. Patients with chronic granulomatous disease, an inherited disorder of NADPH oxidase in which phagocytes are defective in generation of reactive oxidant intermediates (ROIs), suffer from life-threatening bacterial and fungal infections. The mechanisms by which NADPH oxidase mediate host defense are unclear. In addition to ROI generation, neutrophil NADPH oxidase activation is linked to the release of sequestered proteases that are posited to be critical effectors of host defense. To definitively determine the contribution of NADPH oxidase versus neutrophil serine proteases, we evaluated susceptibility to fungal and bacterial infection in mice with engineered disruptions of these pathways. NADPH oxidase-deficient mice (p47(phox-/-)) were highly susceptible to pulmonary infection with Aspergillus fumigatus. In contrast, double knockout neutrophil elastase (NE)(-/-)×cathepsin G (CG)(-/-) mice and lysosomal cysteine protease cathepsin C/dipeptidyl peptidase I (DPPI)-deficient mice that are defective in neutrophil serine protease activation demonstrated no impairment in antifungal host defense. In separate studies of systemic Burkholderia cepacia infection, uniform fatality occurred in p47(phox-/-) mice, whereas NE(-/-)×CG(-/-) mice cleared infection. Together, these results show a critical role for NADPH oxidase in antimicrobial host defense against A. fumigatus and B. cepacia, whereas the proteases we evaluated were dispensable. Our results indicate that NADPH oxidase dependent pathways separate from neutrophil serine protease activation are required for host defense against specific pathogens.