Insights into the Adolescent Cystic Fibrosis Airway Microbiome Using Shotgun Metagenomics.

Cystic fibrosis (CF) is an inherited genetic disorder which manifests primarily in airway disease. Recent advances in molecular technologies have unearthed the diverse polymicrobial nature of the CF airway. Numerous studies have characterised the genus-level composition of this airway community using targeted 16S rDNA sequencing. Here, we employed whole-genome shotgun metagenomics to provide a more comprehensive understanding of the early CF airway microbiome. We collected 48 sputum samples from 11 adolescents and children with CF over a 12-month period and performed shotgun metagenomics on the Illumina NextSeq platform. We carried out functional and taxonomic analysis of the lung microbiome at the species and strain levels. Correlations between microbial diversity measures and independent demographic and clinical variables were performed. Shotgun metagenomics detected a greater diversity of bacteria than culture-based methods. A large proportion of the top 25 most-dominant species were anaerobes. Samples dominated by Staphylococcus aureus and Prevotella melaninogenica had significantly higher microbiome diversity, while no CF pathogen was associated with reduced microbial diversity. There was a diverse resistome present in all samples in this study, with 57.8% agreement between shotgun metagenomics and culture-based methods for detection of resistance. Pathogenic sequence types (STs) of S. aureus, Pseudomonas aeruginosa, Haemophilus influenzae and Stenotrophomonas maltophilia were observed to persist in young CF patients, while STs of S. aureus were both persistent and shared between patients. This study provides new insight into the temporal changes in strain level composition of the microbiome and the landscape of the resistome in young people with CF. Shotgun metagenomics could provide a very useful one-stop assay for detecting pathogens, emergence of resistance and conversion to persistent colonisation in early CF disease.

Aspergillus fumigatus Supernatants Disrupt Bronchial Epithelial Monolayers: Potential Role for Enhanced Invasion in Cystic Fibrosis.

Aspergillus fumigatus is the most commonly isolated fungus in chronic lung diseases, with a prevalence of up to 60% in cystic fibrosis patients. Despite this, the impact of A. fumigatus colonisation on lung epithelia has not been thoroughly explored. We investigated the influence of A. fumigatus supernatants and the secondary metabolite, gliotoxin, on human bronchial epithelial cells (HBE) and CF bronchial epithelial (CFBE) cells. CFBE (F508del CFBE41o-) and HBE (16HBE14o-) trans-epithelial electrical resistance (TEER) was measured following exposure to A. fumigatus reference and clinical isolates, a gliotoxin-deficient mutant (ΔgliG) and pure gliotoxin. The impact on tight junction (TJ) proteins, zonula occludens-1 (ZO-1) and junctional adhesion molecule-A (JAM-A) were determined by western blot analysis and confocal microscopy. A. fumigatus conidia and supernatants caused significant disruption to CFBE and HBE TJs within 24 h. Supernatants from later cultures (72 h) caused the greatest disruption while ΔgliG mutant supernatants caused no disruption to TJ integrity. The ZO-1 and JAM-A distribution in epithelial monolayers were altered by A. fumigatus supernatants but not by ΔgliG supernatants, suggesting that gliotoxin is involved in this process. The fact that ΔgliG conidia were still capable of disrupting epithelial monolayers indicates that direct cell-cell contact also plays a role, independently of gliotoxin production. Gliotoxin is capable of disrupting TJ integrity which has the potential to contribute to airway damage, and enhance microbial invasion and sensitisation in CF.

Reducing human DNA bias in cystic fibrosis airway specimens for microbiome analysis.

Next generation sequencing (NGS) has transformed our understanding of airway microbiology, however there are methodology limitations that require consideration. The presence of high concentrations of human DNA in clinical specimens can significantly impact sequencing of the microbiome, especially in low biomass samples. Here we compared three different methods (0.025% saponin, NEBNext Microbiome DNA enrichment kit, QIAamp DNA microbiome kit) for the reduction of human DNA from six CF sputum samples and determined the impact on the microbiome detected using 16S rRNA gene sequencing. Human DNA in undepleted CF sputum accounted for 94.3% of the total DNA. Saponin, the NEBNext kit and the QIAamp kit reduced human DNA levels by an average of 38.7%, 61.8% and 94.8%, respectively. None of the depletion methods reduced total bacterial DNA concentrations. QIAamp depletion did not influence taxa richness or alpha diversity however alterations to the core genera were noted following depletion. While all methods reduced human DNA in the CF sputum samples, the QIAamp DNA microbiome kit reduced Human DNA levels significantly while leaving bacterial DNA levels unchanged. Human DNA depletion in low biomass, human DNA-dense CF sputum samples is vital for improving bacterial resolution in the CF airway microbiome.

Population genomics confirms acquisition of drug-resistant Aspergillus fumigatus infection by humans from the environment.

Infections caused by the fungal pathogen Aspergillus fumigatus are increasingly resistant to first-line azole antifungal drugs. However, despite its clinical importance, little is known about how susceptible patients acquire infection from drug-resistant genotypes in the environment. Here, we present a population genomic analysis of 218 A. fumigatus isolates from across the UK and Ireland (comprising 153 clinical isolates from 143 patients and 65 environmental isolates). First, phylogenomic analysis shows strong genetic structuring into two clades (A and B) with little interclade recombination and the majority of environmental azole resistance found within clade A. Second, we show occurrences where azole-resistant isolates of near-identical genotypes were obtained from both environmental and clinical sources, indicating with high confidence the infection of patients with resistant isolates transmitted from the environment. Third, genome-wide scans identified selective sweeps across multiple regions indicating a polygenic basis to the trait in some genetic backgrounds. These signatures of positive selection are seen for loci containing the canonical genes encoding fungicide resistance in the ergosterol biosynthetic pathway, while other regions under selection have no defined function. Lastly, pan-genome analysis identified genes linked to azole resistance and previously unknown resistance mechanisms. Understanding the environmental drivers and genetic basis of evolving fungal drug resistance needs urgent attention, especially in light of increasing numbers of patients with severe viral respiratory tract infections who are susceptible to opportunistic fungal superinfections.

Polymicrobial Interactions in the Cystic Fibrosis Airway Microbiome Impact the Antimicrobial Susceptibility of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is one of the most dominant pathogens in cystic fibrosis (CF) airway disease and contributes to significant inflammation, airway damage, and poorer disease outcomes. The CF airway is now known to be host to a complex community of microorganisms, and polymicrobial interactions have been shown to play an important role in shaping P. aeruginosa pathogenicity and resistance. P. aeruginosa can cause chronic infections that once established are almost impossible to eradicate with antibiotics. CF patients that develop chronic P. aeruginosa infection have poorer lung function, higher morbidity, and a reduced life expectancy. P. aeruginosa adapts to the CF airway and quickly develops resistance to several antibiotics. A perplexing phenomenon is the disparity between in vitro antimicrobial sensitivity testing and clinical response. Considering the CF airway is host to a diverse community of microorganisms or 'microbiome' and that these microorganisms are known to interact, the antimicrobial resistance and progression of P. aeruginosa infection is likely influenced by these microbial relationships. This review combines the literature to date on interactions between P. aeruginosa and other airway microorganisms and the influence of these interactions on P. aeruginosa tolerance to antimicrobials.

Early Interleukin-22 and Neutrophil Proteins Are Correlated to Future Lung Damage in Children With Cystic Fibrosis.

Cystic Fibrosis (CF) lung damage begins early in life. Lung function decline is associated with pulmonary infections, neutrophil infiltration and inflammation. In CF, neutrophils have an altered phenotype. In this pilot study, we aimed to determine if signals of dysfunctional neutrophil responses were evident early in life and whether these signals may be associated with lung damage in later childhood. We examined the pulmonary protein profiles of 14 clinical stable infants and pre-school children with CF employing the aptamer-based affinity platform, SOMAscan®. High resolution computed tomography (HRCT) was performed on all children after age 6 years and Brody score calculated. A Spearman's rank order correlation analysis and Benjamini-Hochberg adjustment was used to correlate protein concentrations in early life to Brody scores in later childhood. Early life concentrations of azurocidin and myeloperoxidase, were positively correlated with Brody score after age 6 (p = 0.0041 and p = 0.0182, respectively). Four other neutrophil associated proteins; Complement C3 (p = 0.0026), X-ray repair CCP 6 (p = 0.0059), C3a anaphylatoxin des Arginine (p = 0.0129) and cytokine receptor common subunit gamma (p = 0.0214) were all negatively correlated with Brody scores. Interestingly, patients with more severe lung damage after age 6 had significantly lower levels of IL-22 in early years of life (p = 0.0243). IL-22 has scarcely been reported to have implications in CF. Identification of early biomarkers that may predict more severe disease progression is particularly important for the future development of early therapeutic interventions in CF disease. We recommend further corroboration of these findings in prospective validation studies.

The Lung Microbiome in Young Children with Cystic Fibrosis: A Prospective Cohort Study.

The cystic fibrosis (CF) lung harbours a diverse microbiome and reduced diversity in the CF lung has been associated with advancing age, increased inflammation and poorer lung function. Data suggest that the window for intervention is early in CF, yet there is a paucity of studies on the lung microbiome in children with CF. The objective of this study was to thoroughly characterise the lower airway microbiome in pre-school children with CF. Bronchoalveolar lavage (BAL) samples were collected annually from children attending the three clinical centres. Clinical and demographic data were collated on all subjects alongside BAL inflammatory markers. 16S rRNA gene sequencing was performed on the Illumina MiSeq platform. Bioinformatics and data analysis were performed using Qiime and R project software. Data on 292 sequenced BALs from 101 children with CF and 51 without CF show the CF lung microbiome, while broadly similar to that in non-CF children, is distinct. Alpha diversity between the two cohorts was indistinguishable at this early age. The CF diagnosis explained only 1.1% of the variation between the cohort microbiomes. However, several key genera were significantly differentially abundant between the groups. While the non-CF lung microbiome diversity increased with age, diversity reduced in CF with age. Pseudomonas and Staphylococcus were more abundant with age, while genera such as Streptococcus, Porphyromonas and Veillonella were less abundant with age. There was a negative correlation between alpha diversity and interleukin-8 and neutrophil elastase in the CF population. Neither current flucloxacillin or azithromycin prophylaxis, nor previous oral or IV antibiotic exposure, was correlated with microbiome diversity. Consecutive annual BAL samples over 5 years from a subgroup of children demonstrated diverse patterns of development in the first years of life.

The prevalence of Aspergillus fumigatus in early cystic fibrosis disease is underestimated by culture-based diagnostic methods.

Aspergillus fumigatus is the most common fungus infecting/colonising people with cystic fibrosis (CF) and can negatively impact clinical status. Diagnostic laboratories rely on culture to detect A. fumigatus which is known to be less sensitive than molecular approaches. Therefore, A. fumigatus colonisation in the CF population may be underestimated. Sputum (n = 60) from 25 children with CF were collected and A. fumigatus was detected using routine culture (CM1), enhanced culture (CM2) and ITS1 qPCR. The prevalence of A. fumigatus in this young CF population was 68% by qPCR and only 16% by CM1. CM1, CM2 and qPCR detected A. fumigatus in 8%, 22% and 53% of samples, respectively. qPCR had a 94.2% and 77.4% increased odds of detecting A. fumigatus over CM1 and CM2, respectively. Molecular methods proved superior for detecting A. fumigatus in CF sputum. A. fumigatus is likely more prevalent in early CF disease than is currently reported.

Aspergillus fumigatus Inhibits Pseudomonas aeruginosa in Co-culture: Implications of a Mutually Antagonistic Relationship on Virulence and Inflammation in the CF Airway.

Many cystic fibrosis (CF) airway infections are considered to be polymicrobial and microbe-microbe interactions may play an important role in disease pathology. Pseudomonas aeruginosa and Aspergillus fumigatus are the most prevalent bacterial and fungal pathogens isolated from the CF airway, respectively. We have previously shown that patients co-colonized with these pathogens had comparable outcomes to those chronically colonized with P. aeruginosa. Our objective was to examine the interactions between A. fumigatus and P. aeruginosa, specifically the effects of co-colonization on biofilm formation, virulence and host pro-inflammatory responses. Our findings suggest that co-infections of A. fumigatus and P. aeruginosa in the Galleria mellonella acute infection model showed that pre-exposure of larvae to sub-lethal inocula of A. fumigatus increased the mortality caused by subsequent P. aeruginosa infection. Co-infection of human bronchial epithelial cells (CFBE41o-) with both pathogens did not enhance IL-6 and IL-8 production beyond the levels observed following single infections. In addition, both pathogens stimulated cytokine secretion via the same two mitogen-activated protein kinases (MAPKs) signaling pathways, ERK and p38. Mixed species biofilms showed overall reduced biofilm development with crystal violet staining. Quantification by species-specific qPCR revealed that both pathogens had mutually antagonistic effects on each other. A. fumigatus supernatants showed strong anti-Pseudomonal activity and gliotoxin was the main active agent. Gliotoxin resulted in varying levels of anti-biofilm activity toward other bacteria commonly found in the CF airways. Gliotoxin produced by A. fumigatus colonizing the CF airways may have a significant impact on the CF airway microbiome composition with potential clinical implications.

Co-colonisation with Aspergillus fumigatus and Pseudomonas aeruginosa is associated with poorer health in cystic fibrosis patients: an Irish registry analysis.

BACKGROUND: Pulmonary infection is the main cause of death in cystic fibrosis (CF). Aspergillus fumigatus (AF) and Pseudomonas aeruginosa (PA) are the most prevalent fungal and bacterial pathogens isolated from the CF airway, respectively. Our aim was to determine the effect of different colonisation profiles of AF and PA on the clinical status of patients with CF. METHODS: A retrospective analysis of data from the Cystic Fibrosis Registry of Ireland from 2013 was performed to determine the effect of intermittent and persistent colonisation with AF or PA or co-colonisation with both microorganisms on clinical outcome measures in patients with CF. Key outcomes measured included forced expiratory volume in one second (FEV1), number of hospitalisations, respiratory exacerbations and antimicrobials prescribed, and complications of CF, including CF related diabetes (CFRD) and allergic bronchopulmonary aspergillosis (ABPA). RESULTS: The prevalence of AF and PA colonisation were 11% (5% persistent, 6% intermittent) and 31% (19% persistent, 12% intermittent) in the Irish CF population, respectively. Co-colonisation with both pathogens was associated with a 13.8% reduction in FEV1 (p = 0.016), higher levels of exacerbations (p = 0.042), hospitalisations (p = 0.023) and antimicrobial usage (p = 0.014) compared to non-colonised patients and these clinical outcomes were comparable to those persistently colonised with PA. Intermittent and persistent AF colonisation were not associated with poorer clinical outcomes or ABPA. Patients with persistent PA had a higher prevalence of CFRD diagnosis (p = 0.012). CONCLUSIONS: CF patients co-colonised with AF and PA had poor clinical outcomes comparable to patients persistently colonised with PA, emphasising the clinical significance of co-colonisation with these microorganisms.

The basophil surface marker CD203c identifies Aspergillus species sensitization in patients with cystic fibrosis.

BACKGROUND: Colonization by Aspergillus fumigatus in patients with cystic fibrosis (CF) can cause A fumigatus sensitization and/or allergic bronchopulmonary aspergillosis (ABPA), which affects pulmonary function and clinical outcomes. Recent studies show that specific allergens upregulate the surface-expressed basophil marker CD203c in sensitized subjects, a response that can be readily measured by using flow cytometry. OBJECTIVE: We sought to identify A fumigatus sensitization in patients with CF by using the basophil activation test (BAT). METHODS: Patients with CF attending Beaumont Hospital were screened for study inclusion. BAT was used to identify A fumigatus sensitization. Serologic (total and A fumigatus-specific IgE), pulmonary function, and body mass index measurements were performed. RESULTS: The BAT discriminates A fumigatus-sensitized from nonsensitized patients with CF. Persistent isolation of A fumigatus in sputum is a significant risk factor for A fumigatus sensitization. Levels of the A fumigatus-stimulated basophil activation marker CD203c inversely correlated with pulmonary function and body mass index in A fumigatus-sensitized but not nonsensitized patients with CF. Total and A fumigatus-specific IgE, but not IgG, levels are increased in A fumigatus-sensitized patients with CF and ABPA when compared with those in A fumigatus-sensitized and nonsensitized patients with CF without ABPA. Itraconazole treatment did not affect A fumigatus sensitization. CONCLUSION: Combining the BAT with routine serologic testing allows classification of patients with CF into 3 groups: nonsensitized, A fumigatus-sensitized, and ABPA. Accurate and prompt identification of A fumigatus-associated clinical status might allow early and targeted therapeutic intervention, potentially improving clinical outcomes.

The microbial community of the cystic fibrosis airway is disrupted in early life.

BACKGROUND: Molecular techniques have uncovered vast numbers of organisms in the cystic fibrosis (CF) airways, the clinical significance of which is yet to be determined. The aim of this study was to describe and compare the microbial communities of the lower airway of clinically stable children with CF and children without CF. METHODS: Bronchoalveolar lavage (BAL) fluid and paired oropharyngeal swabs from clinically stable children with CF (n = 13) and BAL from children without CF (n = 9) were collected. DNA was isolated, the 16S rRNA regions amplified, fragmented, biotinylated and hybridised to a 16S rRNA microarray. Patient medical and demographic information was recorded and standard microbiological culture was performed. RESULTS: A diverse bacterial community was detected in the lower airways of children with CF and children without CF. The airway microbiome of clinically stable children with CF and children without CF were significantly different as measured by Shannon's Diversity Indices (p = 0.001; t test) and Principle coordinate analysis (p = 0.01; Adonis test). Overall the CF airway microbial community was more variable and had a less even distribution than the microbial community in the airways of children without CF. We highlighted several bacteria of interest, particularly Prevotella veroralis, CW040 and a Corynebacterium, which were of significantly differential abundance between the CF and non-CF lower airways. Both Pseudomonas aeruginosa and Streptococcus pneumoniae culture abundance were found to be associated with CF airway microbial community structure. The CF upper and lower airways were found to have a broadly similar microbial milieu. CONCLUSION: The microbial communities in the lower airways of stable children with CF and children without CF show significant differences in overall diversity. These discrepancies indicate a disruption of the airway microflora occurring early in life in children with CF.

The effect of Aspergillus fumigatus infection on vitamin D receptor expression in cystic fibrosis.

RATIONALE: Aspergillus fumigatus (A. fumigatus) in cystic fibrosis (CF) is increasingly recognized. Although allergic bronchopulmonary aspergillosis (ABPA) leads to deterioration of pulmonary function, the effect of A. fumigatus colonization in the absence of ABPA remains unclear. OBJECTIVES: To address this, we examined individuals with CF with A. fumigatus who were ABPA negative to identify the effects of itraconazole therapy on Aspergillus-induced lung inflammation. METHODS: The effect of A. fumigatus on nuclear vitamin D receptor (VDR) expression was investigated using qRT-PCR and Western blotting. IL-5 and IL-13 levels were quantified by ELISA. The effect of itraconazole was assessed by a combination of high-resolution computed tomography, lung function test, and microbiological analysis. MEASUREMENTS AND MAIN RESULTS: We demonstrate that A. fumigatus down-regulates VDR in macrophages and airway epithelial cells and that the fungal metabolite gliotoxin (Gt) is the main causative agent. Gt overcame the positive effect of 1,25-OH vitamin D(3) on VDR expression in vitro, resulting in increased IL-5 and IL-13 production. In vivo, A. fumigatus positivity was associated with increased Gt in CF bronchoalveolar lavage fluid and increased bronchoalveolar lavage fluid levels of IL-5 and IL-13. After airway eradication of A. fumigatus with itraconazole, we observed decreased Gt, IL-5 and IL-13, improved respiratory symptoms, and diminished high-resolution computed tomography mosaic pattern consistent with sustained pulmonary function. CONCLUSIONS: This study provides a rationale for the therapeutic effect of itraconazole and implied that the therapeutic potential of vitamin D supplementation in preventing ABPA is only feasible with concurrent elimination of A. fumigatus to permit VDR expression and its positive functional consequences.

Dose-dependent cellular and humoral responses in Galleria mellonella larvae following beta-glucan inoculation.

Galleria mellonella larvae were inoculated with different doses of beta-glucan by injection into the haemocoel. Those larvae that had received high doses of beta-glucan (15, 30 or 60microg/larva) demonstrated increased survival following infection with the yeast Candida albicans. High concentrations of glucan induced an increase in haemocyte density and a reduction in yeast proliferation within the haemocoel. Proteomic analysis of glucan-treated larvae revealed increased expression of a variety of peptides some of which may possess antimicrobial properties. Analysis of expression profiles revealed that low doses of beta-glucan (3.75microg/larva) triggered the increased expression of certain peptides (e.g. hemolin) while high dose inoculation was required before the increased expression of others (e.g. archaemetzincin) was evident. These results indicate that low doses of beta-glucan induce a limited immune response while high doses induce an immune response that has the potential to curtail the threat within the haemocoel but also withstand a subsequent infection. Immune priming gives insects the ability to withstand a potentially lethal infection if exposed to a low level of the pathogen 24-48h previously. Immune priming has resource implications and this work indicates that a graded immune response is initiated depending upon the amount of the immune priming agent encountered.

Translocation of proteins homologous to human neutrophil p47phox and p67phox to the cell membrane in activated hemocytes of Galleria mellonella.

Activation of the superoxide forming respiratory burst oxidase of human neutrophils, crucial in host defence, requires the cytosolic proteins p47phox and p67phox which translocate to the plasma membrane upon cell stimulation and activate flavocytochrome b558, the redox centre of this enzyme system. We have previously demonstrated the presence of proteins (67 and 47kDa) in hemocytes of the insect Galleria mellonella homologous to proteins of the superoxide-forming NADPH oxidase complex of neutrophils. The work presented here illustrates for the first time translocation of homologous hemocyte proteins, 67 and 47kDa from the cytosol to the plasma membrane upon phorbol 12-myristate 13 acetate (PMA) activation. In hemocytes, gliotoxin (GT), the fungal secondary metabolite significantly suppressed PMA-induced superoxide generation in a concentration dependent manner and reduced translocation to basel nonstimulated levels. Primarily these results correlate translocation of hemocyte 47 and 67kDa proteins with PMA induced oxidase activity. Collectively results presented here, demonstrate further cellular and functional similarities between phagocytes of insects and mammals and further justify the use of insects in place of mammals for modelling the innate immune response to microbial pathogens.

Susceptibility of larvae of Galleria mellonella to infection by Aspergillus fumigatus is dependent upon stage of conidial germination.

The ability of conidia of the human pathogenic fungus Aspergillus fumigatus to kill larvae of the insect Galleria mellonella was investigated. Conidia at different stages of the germination process displayed variations in their virulence as measured using the Galleria infection model. Non-germinating ('resting') conidia were avirulent except when an inoculation density of 1 x 10(7) conidia per insect was used. Conidia that had been induced to commence the germination process by pre-culturing in growth medium for 3 h were capable of killing larvae at densities of 1 x 10(6) and 1 x 10(7) per insect. An inoculation density of 1 x 10(5) conidia per insect remained avirulent. Conidia in the outgrowth phase of germination (characterised as the formation of a germ tube) were the most virulent and were capable of killing 100% of larvae after 5 or 24 h when 1 x 10(7) or 1 x 10(6) conidia, that had been allowed to germinate for 24 h, were used. Examination of the response of insect haemocytes to conidia at different stages of the germination process established that haemocytes could engulf non-germinating conidia and those in the early stages of the germination process but that conidia, which had reached the outgrowth stages of germination were not phagocytosed. The results presented here indicate that haemocytes of G. mellonella are capable of phagocytosing A. fumigatus conidia less than 3.0 microm in diameter but that conidia greater than this are too large to be engulfed. The virulence of A. fumigatus in G. mellonella larvae can be ascertained within 60-90 h if infection densities of 1 x 10(6) or 1 x 10(7) activated conidia (pre-incubated for 2-3 h) per insect are employed.

Superoxide production in Galleria mellonella hemocytes: identification of proteins homologous to the NADPH oxidase complex of human neutrophils.

The insect immune response has a number of structural and functional similarities to the innate immune response of mammals. The objective of the work presented here was to establish the mechanism by which insect hemocytes produce superoxide and to ascertain whether the proteins involved in superoxide production are similar to those involved in the NADPH oxidase-induced superoxide production in human neutrophils. Hemocytes of the greater wax moth (Galleria mellonella) were shown to be capable of phagocytosing bacterial and fungal cells. The kinetics of phagocytosis and microbial killing were similar in the insect hemocytes and human neutrophils. Superoxide production and microbial killing by both cell types were inhibited in the presence of the NADPH oxidase inhibitor diphenyleneiodonium chloride. Immunoblotting of G. mellonella hemocytes with antibodies raised against human neutrophil phox proteins revealed the presence of proteins homologous to gp91phox, p67phox, p47phox, and the GTP-binding protein rac 2. A protein equivalent to p40phox was not detected in insect hemocytes. Immunofluorescence analysis localized insect 47-kDa and 67-kDa proteins throughout the cytosol and in the perinuclear region. Hemocyte 67-kDa and 47-kDa proteins were immunoprecipitated and analyzed by matrix-assisted laser desorption ionization--time of flight analysis. The results revealed that the hemocyte 67-kDa and 47-kDa proteins contained peptides matching those of p67phox and p47phox of human neutrophils. The results presented here indicate that insect hemocytes phagocytose and kill bacterial and fungal cells by a mechanism similar to the mechanism used by human neutrophils via the production of superoxide. We identified proteins homologous to a number of proteins essential for superoxide production in human neutrophils and demonstrated that significant regions of the 67-kDa and 47-kDa insect proteins are identical to regions of the p67phox and p47phox proteins of neutrophils.