Reconstructing the Qo Site of Plasmodium falciparum bc 1 Complex in the Yeast Enzyme

The bc ₁ complex of the mitochondrial respiratory chain is essential for Plasmodium falciparum proliferation, the causative agent of human malaria. Therefore, this enzyme is an attractive target for antimalarials. However, biochemical investigations of the parasite enzyme needed for the study of new drugs are challenging. In order to facilitate the study of new compounds targeting the enzyme, we are modifying the inhibitor binding sites of the yeast Saccharomyces cerevisiae to generate a complex that mimics the P. falciparum enzyme. In this study we focused on its Q_o pocket, the site of atovaquone binding which is a leading antimalarial drug used in treatment and causal prophylaxis. We constructed and studied a series of mutants with modified Q_o sites where yeast residues have been replaced by P. falciparum equivalents, or, for comparison, by human equivalents. Mitochondria were prepared from the yeast Plasmodium-like and human-like Q_o mutants. We measured the bc ₁ complex sensitivity to atovaquone, azoxystrobin, a Q_o site targeting fungicide active against P. falciparum and RCQ06, a quinolone-derivative inhibitor of P. falciparum bc ₁ complex.The data obtained highlighted variations in the Q_o site that could explain the differences in inhibitor sensitivity between yeast, plasmodial and human enzymes. We showed that the yeast Plasmodium-like Q_o mutants could be useful and easy-to-use tools for the study of that class of antimalarials.     

Crystal Structures of YkuI and Its Complex with Second Messenger Cyclic Di-GMP Suggest Catalytic Mechanism of Phosphodiester Bond Cleavage by EAL Domains

Cyclic di-GMP (c-di-GMP) is a ubiquitous bacterial second messenger that is involved in the regulation of cell surface-associated traits and the persistence of infections. Omnipresent GGDEF and EAL domains, which occur in various combinations with regulatory domains, catalyze c-di-GMP synthesis and degradation, respectively. The crystal structure of full-length YkuI from Bacillus subtilis, composed of an EAL domain and a C-terminal PAS-like domain, has been determined in its native form and in complex with c-di-GMP and Ca²⁺. The EAL domain exhibits a triose-phosphate isomerase-barrel fold with one antiparallel β-strand. The complex with c-di-GMP-Ca²⁺ defines the active site of the putative phosphodiesterase located at the C-terminal end of the β-barrel. The EAL motif is part of the active site with Glu-33 of the motif being involved in cation coordination. The structure of the complex allows the proposal of a phosphodiesterase mechanism, in which the divalent cation and the general base Glu-209 activate a catalytic water molecule for nucleophilic in-line attack on the phosphorus. The C-terminal domain closely resembles the PAS-fold. Its pocket-like structure could accommodate a yet unknown ligand. YkuI forms a tight dimer via EAL-EAL and trans EAL-PAS-like domain association. The possible regulatory significance of the EAL-EAL interface and a mechanism for signal transduction between sensory and catalytic domains of c-di-GMP-specific phosphodiesterases are discussed.The dinucleotide cyclic di-GMP (c-di-GMP) was discovered about 20 years ago when it was found to regulate the activity of cellulase synthase in Acetobacter xylinum (1). However, its prominent role as a global second messenger has been realized only upon the recent recognition of the omnipresence of genes coding for domains that catalyze c-di-GMP biosynthesis and degradation in eubacteria (2). GGDEF domains catalyze the condensation of two GTP molecules to the cyclic 2-fold symmetric dinucleotide (diguanylate cyclase activity (3-6)), whereas EAL domains are involved in its degradation to yield the linear dinucleotide pGpG (phosphodiesterase (PDE)⁴ A activity) (3, 7-9). Recently, also HD-GYP domains have been implicated in c-di-GMP-specific PDE activity (10). All the domains have been named according to their sequence signature motifs. They are typically found in combinations with various other, mostly sensory or regulatory, domains. It is thought that the balance between antagonistic diguanylate cyclase and PDE-A activities determines the cellular level of c-di-GMP and, thus, affects a variety of physiological processes in bacteria.It has been shown that, in general, c-di-GMP regulates cell surface-associated traits and community behavior such as biofilm formation (for reviews see Refs. 11-12), and its relevance to the virulence of pathogenic bacteria has been demonstrated (11, 13, 14). In particular, the dinucleotide has been proposed to orchestrate the switch between acute and persistent phase of infection.The best characterized diguanylate cyclase is PleD from Caulobacter crescentus with a Rec-Rec-GGDEF domain architecture (Rec indicates response regulator receiver domain). The structure of its GGDEF domain revealed a single GTP-binding site and suggested that dimerization is the prerequisite for enzymatic activity (4). This has been corroborated recently by crystallography showing directly that modification of the first Rec domain, mimicking phosphorylation by the cognate kinase, induces formation of a tightly packed dimer (15). Additionally, an upper limit of c-di-GMP levels in the cell seems to be ensured by potent allosteric product inhibition of the PleD cyclase (4, 15, 16). Recently, the crystal structure of another diguanylate cyclase, WspR from Pseudomonas aeruginosa with a Rec-GGDEF domain architecture, has been determined (17), which showed a tetrameric quaternary structure and active and feedback inhibition sites that are very similar to those in PleD.For EAL domains, it has been demonstrated that genetic knock-out results in phenotypes that are in line with the paradigm that an elevated cellular c-di-GMP concentration corresponds to a sessile and a low concentration to a motile bacterial life style (13, 18, 19). Only recently, EAL-mediated PDE-A activity has been measured in vitro (7-9, 20-22).The Bacillus subtilis YkuI protein was targeted for structure determination by the Midwest Center for Structural Genomics as a member of the large sequence family that contains EAL (Pfam number PF00563) domains. Here we report the crystal structure of YkuI showing the fold of the N-terminal EAL domain and the C-terminal PAS-like domain. Co-crystallization with c-di-GMP revealed the substrate binding mode and allows the proposal of a catalytic mechanism. The PAS-like domain most probably has regulatory function, which is discussed. Recently, another EAL structure has been deposited in the Protein Data Bank by the Midwest Center for Structural Genomics, the EAL domain of a GGDEF-EAL protein from Thiobacillus denitrificans (tdEAL; PDB code 2r6o). Comparison of the two structures suggests a possible regulatory mechanism.     

Reaction Mechanism of Superoxide Generation during Ubiquinol Oxidation by the Cytochrome bc1 Complex

In addition to its main functions of electron transfer and proton translocation, the cytochrome bc₁ complex (bc₁) also catalyzes superoxide anion (O₂^˙̄) generation upon oxidation of ubiquinol in the presence of molecular oxygen. The reaction mechanism of superoxide generation by bc₁ remains elusive. The maximum O₂^˙̄ generation activity is observed when the complex is inhibited by antimycin A or inactivated by heat treatment or proteinase K digestion. The fact that the cytochrome bc₁ complex with less structural integrity has higher O₂^˙̄-generating activity encouraged us to speculate that O₂^˙̄ is generated inside the complex, perhaps in the hydrophobic environment of the Q_P pocket through bifurcated oxidation of ubiquinol by transferring its two electrons to a high potential electron acceptor, iron-sulfur cluster, and a low potential heme b_L or molecular oxygen. If this speculation is correct, then one should see more O₂^˙̄ generation upon oxidation of ubiquinol by a high potential oxidant, such as cytochrome c or ferricyanide, in the presence of phospholipid vesicles or detergent micelles than in the hydrophilic conditions, and this is indeed the case. The protein subunits, at least those surrounding the Q_P pocket, may play a role either in preventing the release of O₂^˙̄ from its production site to aqueous environments or in preventing O₂ from getting access to the hydrophobic Q_P pocket and might not directly participate in superoxide production.     

Mechanism of Allosteric Inhibition of N-Acetyl-L-glutamate Synthase by L-Arginine

N-Acetylglutamate synthase (NAGS) catalyzes the first committed step in l-arginine biosynthesis in plants and micro-organisms and is subject to feedback inhibition by l-arginine. This study compares the crystal structures of NAGS from Neisseria gonorrhoeae (ngNAGS) in the inactive T-state with l-arginine bound and in the active R-state complexed with CoA and l-glutamate. Under all of the conditions examined, the enzyme consists of two stacked trimers. Each monomer has two domains: an amino acid kinase (AAK) domain with an AAK-like fold but lacking kinase activity and an N-acetyltransferase (NAT) domain homologous to other GCN5-related transferases. Binding of l-arginine to the AAK domain induces a global conformational change that increases the diameter of the hexamer by ∼10 Å and decreases its height by ∼20Å_. AAK dimers move 5Å outward along their 2-fold axes, and their tilt relative to the plane of the hexamer decreases by ∼4°. The NAT domains rotate ∼109° relative to AAK domains enabling new interdomain interactions. Interactions between AAK and NAT domains on different subunits also change. Local motions of several loops at the l-arginine-binding site enable the protein to close around the bound ligand, whereas several loops at the NAT active site become disordered, markedly reducing enzymatic specific activity.l-Arginine biosynthesis in most micro-organisms and plants involves the initial acetylation of l-glutamate by N-acetylglutamate synthase (NAGS, EC 2.3.1.1)² to produce N-acetylglutamate (NAG). NAG is then converted by NAG kinase (NAGK, EC 2.7.2.8) to NAG-phosphate and subsequently to N-acetylornithine (1, 2). Two alternative reactions are used to remove the acetyl group from acetylornithine. The linear pathway uses N-acetylornithine deacetylase (EC 3.5.1.16) to catalyze the metal-dependent hydrolysis of the acetyl group to form l-ornithine and acetate, whereas the acetyl recycling pathway transfers the acetyl group from N-acetylornithine to l-glutamate, producing l-ornithine and NAG. This reaction is catalyzed by ornithine acetyltransferase (EC 2.3.1.35).In the linear pathway, NAGS is the only target of feedback inhibition by l-arginine. In contrast, in the acetyl cycling pathway l-arginine may inhibit NAGS and NAGK or ornithine acetyltransferase (3). Structure determinations of l-arginine-insensitive (4) and l-arginine-sensitive NAGKs (5) provided insights into the structural basis of l-arginine inhibition of NAGK. l-Arginine-insensitive Escherichia coli (ec) NAGK is a homodimer (4), whereas l-arginine-sensitive NAGKs from Thermotoga maritima (tm) and Pseudomonas aeruginosa (pa) are hexamers formed by pair-wise interlacing of the N-terminal helices of three ecNAGK-like dimers, to create a second type of dimer interface. l-Arginine binding to a site close to the C terminus induces global conformational changes that expands the ring by ∼8 Å and decreases the tilt of the ecNAGK-like dimers relative to the plane of the ring by ∼6°. The inhibition mechanism was proposed to involve the enlargement of an active site located close to the l-arginine-binding site.Because of the sequence similarity between NAGK and NAGS, it was speculated that they may have similar l-arginine-binding sites and hexameric ring structures (5). However, our recent structural determination of NAGS from Neisseria gonorrhoeae (ng) revealed the active site to be located in the NAT domain, >25 Å away from the proposed l-arginine-binding site (6). Therefore, the allosteric mechanism of NAGS is likely to be different from that of l-arginine-sensitive NAGKs. Here we compare the structures of ngNAGS in the inactive T-state with l-arginine bound and in the R-state complexed with CoA and l-glutamate and determine the structural basis for the allosteric inhibition of NAGS by l-arginine.     

Nature of Inhibition of Mitochondrial Respiratory Complex I by 6-Hydroxydopamine

Abstract: The catecholaminergic neurotoxin 6-hydroxydopamine causes parkinsonian symptoms in animals and it has been proposed that reactive oxygen species and oxidative stress, enhanced by iron, may play a key role in its toxicity. The present results demonstrate that 6-hydroxydopamine reversibly inhibits complex I (NADH dehydrogenase) of brain mitochondrial respiratory chain in isolated mitochondria. 6-Hydroxydopamine itself, rather than its oxidative products, was responsible for the inhibition. Iron(III) did not enhance inhibition but decreased it by stimulating the nonenzyme oxidation of 6-hydroxydopamine. Inhibition was potentiated to some extent by calcium ion. Desferrioxamine protected complex I activity against the inhibition, but it was not due to its chelator or antioxidative properties. Desferrioxamine was also shown to activate NADH dehydrogenase in the absence of 6-hydroxydopamine. Activation of mitochondrial respiration by desferrioxamine may contribute to the enhanced neuron survival in the presence of desferrioxamine in some neurodegenerative conditions.     

Inhibition of PbGP43 Expression May Suggest that gp43 is a Virulence Factor in Paracoccidioides brasiliensis

Glycoprotein gp43 is an immunodominant diagnostic antigen for paracoccidioidomycosis caused by Paracoccidioides brasiliensis. It is abundantly secreted in isolates such as Pb339. It is structurally related to beta-1,3-exoglucanases, however inactive. Its function in fungal biology is unknown, but it elicits humoral, innate and protective cellular immune responses; it binds to extracellular matrix-associated proteins. In this study we applied an antisense RNA (aRNA) technology and Agrobacterium tumefaciens-mediated transformation to generate mitotically stable PbGP43 mutants (PbGP43 aRNA) derived from wild type Pb339 to study its role in P. brasiliensis biology and during infection. Control PbEV was transformed with empty vector. Growth curve, cell vitality and morphology of PbGP43 aRNA mutants were indistinguishable from those of controls. PbGP43 expression was reduced 80–85% in mutants 1 and 2, as determined by real time PCR, correlating with a massive decrease in gp43 expression. This was shown by immunoblotting of culture supernatants revealed with anti-gp43 mouse monoclonal and rabbit polyclonal antibodies, and also by affinity-ligand assays of extracellular molecules with laminin and fibronectin. In vitro, there was significantly increased TNF-α production and reduced yeast recovery when PbGP43 aRNA1 was exposed to IFN-γ-stimulated macrophages, suggesting reduced binding/uptake and/or increased killing. In vivo, fungal burden in lungs of BALB/c mice infected with silenced mutant was negligible and associated with decreased lung ΙΛ−10 and IL-6. Therefore, our results correlated low gp43 expression with lower pathogenicity in mice, but that will be definitely proven when PbGP43 knockouts become available. This is the first study of gp43 using genetically modified P. brasiliensis.     

Resistance to the Novel Fungicide Pyrimorph in Phytophthora capsici: Risk Assessment and Detection of Point Mutations in CesA3 That Confer Resistance

Pyrimorph is a novel fungicide with high activity against the plant pathogen Phytophthora capsici. We investigated the risk that P. capsici can develop resistance to pyrimorph. The baseline sensitivities of 226 P. capsici isolates, tested by mycelial growth inhibition, showed a unimodal distribution with a mean EC₅₀ value of 1.4261 (±0.4002) µg/ml. Twelve pyrimorph-resistant mutants were obtained by repeated exposure to pyrimorph in vitro with a frequency of approximately 1×10⁻⁴. The resistance factors of the mutants ranged from 10.67 to 56.02. Pyrimorph resistance of the mutants was stable after 10 transfers on pyrimorph-free medium. Fitness in sporulation, cystospore germination, and pathogenicity in the pyrimorph-resistant mutants was similar to or less than that in the parental wild-type isolates. On detached pepper leaves and pepper plants treated with the recommended maximum dose of pyrimorph, however, virulence was greater for mutants with a high level of pyrimorph resistance than for the wild type. The results suggest that the risk of P. capsici developing resistance to pyrimorph is low to moderate. Among mutants with a high level of pyrimorph resistance, EC₅₀ values for pyrimorph and CAA fungicides flumorph, dimethomorph, and mandipropamid were positively correlated. This indicated that point mutations in cellulose synthase 3 (CesA3) may confer resistance to pyrimorph. Comparison of CesA3 in isolates with a high level of pyrimorph resistance and parental isolates showed that an amino acid change from glutamine to lysine at position 1077 resulted in stable, high resistance in the mutants. Based on the point mutations, an allele-specific PCR method was developed to detect pyrimorph resistance in P. capsici populations.     

Lack of Cytochrome c in Mouse Fibroblasts Disrupts Assembly/Stability of Respiratory Complexes I and IV

Cytochrome c (cyt c) is a heme-containing protein that participates in electron transport in the respiratory chain and as a signaling molecule in the apoptotic cascade. Here we addressed the effect of removing mammalian cyt c on the integrity of the respiratory complexes in mammalian cells. Mitochondria from cyt c knockout mouse cells lacked fully assembled complexes I and IV and had reduced levels of complex III. A redox-deficient mutant of cyt c was unable to rescue the levels of complexes I and IV. We found that cyt c is associated with both complex IV and respiratory supercomplexes, providing a potential mechanism for the requirement for cyt c in the assembly/stability of complex IV.The mitochondrial electron transport chain consists of four multisubunit complexes, namely, NADH-ubiquinone oxidoreductase (complex I),² succinate-ubiquinone oxidoreductase (complex II), ubiquinone-cytochrome c oxidoreductase (complex III), and cytochrome c oxidase (complex IV, COX). Cytochrome c (cyt c) shuttles electrons from oxidative phosphorylation complex III to complex IV. Electrons are transferred from reduced cyt c sequentially to the Cu_A site, heme a, heme a₃, and Cu_B binuclear center in the complex IV before being finally transferred to molecular oxygen to generate water (1). Respiratory complexes are assembled into supercomplexes (also called respirasomes). These contain complex I bound to dimeric complex III and a variable copy number of complex IV (2).In Saccharomyces cerevisiae, cyt c is encoded by two genes: CYC1 and CYC7. Mutagenesis studies in yeast have shown that cyt c is required for the assembly of COX (3, 4). In yeast lacking both the cyt c genes (CYC1 and CYC7), COX assembly was absent. It was also shown that cyt c is only structurally required for COX assembly, because a catalytic mutant of cyt c (W65S) was sufficient to bring about near normal levels of COX. However, because yeast lacks complex I, they could not analyze the role of cyt c in the assembly/stability of complex I. Mammals possess two different isoforms of cyt c encoded on different chromosomes: the somatic (cyt cS)- and testis (cyt cT)-specific isoforms. In mouse, the cDNAs bear 74% homology, whereas the proteins possess 86% identity with most dissimilarity in the C terminus.Cardiolipin (CL) is an anionic phospholipid present almost exclusively in the mitochondrial membranes and constitutes 25% of its total phospholipids (5). Work from several laboratories showed that CL is essential for the membrane anchorage of the respiratory supercomplexes. CL has two main roles in the mitochondrial structure and function, namely, stabilization of mitochondrial membranes and specific interactions with proteins. CL deficiency results in inefficient energy transformation by oxidative phosphorylation, swelling of mitochondria, decreased ATP/oxygen ratio, and reduced membrane potential (6, 7). In accordance, in S. cerevisiae lacking CL synthase, the supercomplex comprising complexes III and IV is unstable (8). Assembly mutants of COX had significantly reduced CL synthase activity, whereas assembly mutants of respiratory complex III and complex V showed less inhibition (9). Subsequently, the proton gradient across the inner mitochondrial membrane was found to be important for CL formation and that CL synthase was stimulated by alkaline pH at the matrix side (10). In this study, we investigated the role of cyt c depletion on CL levels by examining its content and composition in cyt c null cells.Here we aimed to answer the following questions: What is the role of cyt c in the assembly and maintenance of the different respiratory complexes in mammals? Are there changes in the content/composition of lipids in the cyt c-ablated cells? Analysis of mouse fibroblasts revealed that cyt c is essential for the assembly/stability of COX, and a catalytically mutant form of cyt c cannot rescue the COX defect in the cyt c null cells. CL and triacylglycerols showed significant differences in the cyt c null cells, both in content and composition.     

Mutations in CYC1, Encoding Cytochrome c1 Subunit of Respiratory Chain Complex III,Cause Insulin-Responsive Hyperglycemia

Many individuals with abnormalities of mitochondrial respiratory chain complex III remain genetically undefined. Here, we report mutations (c.288G>T [p.Trp96Cys] and c.643C>T [p.Leu215Phe]) in CYC1, encoding the cytochrome c₁ subunit of complex III, in two unrelated children presenting with recurrent episodes of ketoacidosis and insulin-responsive hyperglycemia. Cytochrome c₁, the heme-containing component of complex III, mediates the transfer of electrons from the Rieske iron-sulfur protein to cytochrome c. Cytochrome c₁ is present at reduced levels in the skeletal muscle and skin fibroblasts of affected individuals. Moreover, studies on yeast mutants and affected individuals’ fibroblasts have shown that exogenous expression of wild-type CYC1 rescues complex III activity, demonstrating the deleterious effect of each mutation on cytochrome c₁ stability and complex III activity.     

Structure of the Avian Mitochondrial Cytochrome bc 1 Complex

There are now four structures of vertebrate mitochondrial bc ₁ complexes available in theprotein databases and structures from yeast and bacterial sources are expected soon. Thisreview summarizes the new information with emphasis on the avian cytochrome bc ₁ complex(PDB entries 1BCC and 3BCC). The Rieske iron–sulfur protein is mobile and this has beenproposed to be important for catalysis. The binding sites for quinone have been located basedon structures containing inhibitors and, in the case of the quinone reduction site Qi, thequinone itself.     

Novel Mechanism of Inhibition of Dendritic Cells Maturation by Mesenchymal Stem Cells via Interleukin-10 and the JAK1/STAT3 Signaling Pathway

Mesenchymal stem cells (MSCs) can suppress dendritic cells (DCs) maturation and function, mediated by soluble factors, such as indoleamine 2,3-dioxygenase (IDO), prostaglandin E₂ (PGE₂), and nitric oxide (NO). Interleukin-10 (IL-10) is a common immunosuppressive cytokine, and the downstream signaling of the JAK-STAT pathway has been shown to be involved with DCs differentiation and maturation in the context of cancer. Whether IL-10 and/or the JAK-STAT pathway play a role in the inhibitory effect of MSCs on DCs maturation remains controversial. In our study, we cultured MSCs and DCs derived from rat bone marrow under different culturing conditions. Using Transwell plates, we detected by ELISA that the level of IL-10 significantly increased in the supernatants of MSC-DC co-cultures at 48 hours. The cell immunofluorescence assay suggested that the MSCs secreted more IL-10 than the DCs in the co-cultures. Adding exogenous IL-10 to the DCs monoculture or MSC-DC co-cultures stimulated IL-10 and led to a decrease in IL-12, and lower expression of the DCs surface markers CD80, CD86, OX62, MHC-II and CD11b/c. Supplementing the culture with an IL-10 neutralizing antibody (IL-10NA) showed precisely the opposite effect of adding IL-10. Moreover, we demonstrated that the JAK-STAT signaling pathway is involved in inhibiting DCs maturation. Both JAK1 and STAT3 expression and IL-10 secretion decreased markedly after adding a JAK inhibitor (AG490) to the co-culture plate. We propose that there is an IL-10 positive feedback loop, which may explain our observations of elevated IL-10 and enhanced JAK1 and STAT3 expression. Overall, we demonstrated that MSCs inhibit the maturation of DCs through the stimulation of IL-10 secretion, and by activating the JAK1 and STAT3 signaling pathway.     

SERR Spectroelectrochemical Study of Cytochrome cd 1 Nitrite Reductase Co-Immobilized with Physiological Redox Partner Cytochrome c 552 on Biocompatible Metal Electrodes

Cytochrome cd1 nitrite reductases (cd ₁NiRs) catalyze the one-electron reduction of nitrite to nitric oxide. Due to their catalytic reaction, cd ₁NiRs are regarded as promising components for biosensing, bioremediation and biotechnological applications. Motivated by earlier findings that catalytic activity of cd ₁NiR from Marinobacter hydrocarbonoclasticus (Mhcd ₁) depends on the presence of its physiological redox partner, cytochrome c ₅₅₂ (cyt c ₅₅₂), we show here a detailed surface enhanced resonance Raman characterization of Mhcd ₁ and cyt c ₅₅₂ attached to biocompatible electrodes in conditions which allow direct electron transfer between the conducting support and immobilized proteins. Mhcd ₁ and cyt c552 are co-immobilized on silver electrodes coated with self-assembled monolayers (SAMs) and the electrocatalytic activity of Ag // SAM // Mhcd ₁ // cyt c ₅₅₂ and Ag // SAM // cyt c ₅₅₂ // Mhcd ₁ constructs is tested in the presence of nitrite. Simultaneous evaluation of structural and thermodynamic properties of the immobilized proteins reveals that cyt c ₅₅₂ retains its native properties, while the redox potential of apparently intact Mhcd ₁ undergoes a ~150 mV negative shift upon adsorption. Neither of the immobilization strategies results in an active Mhcd ₁, reinforcing the idea that subtle and very specific interactions between Mhcd ₁ and cyt c ₅₅₂ govern efficient intermolecular electron transfer and catalytic activity of Mhcd ₁.     

Brownie,a Gene Involved in Building Complex Respiratory Devices in Insect Eggshells

Background

Insect eggshells must combine protection for the yolk and embryo with provisions for respiration and for the entry of sperm, which are ensured by aeropyles and micropyles, respectively. Insects which oviposit the eggs in an egg-case have a double problem of respiration as gas exchange then involves two barriers. An example of this situation is found in the cockroach Blattella germanica, where the aeropyle and the micropyle are combined in a complex structure called the sponge-like body. The sponge-like body has been well described morphologically, but nothing is known about how it is built up.

Methodology/Principal Findings

In a library designed to find genes expressed during late chorion formation in B. germanica, we isolated the novel sequence Bg30009 (now called Brownie), which was outstanding due to its high copy number. In the present work, we show that Brownie is expressed in the follicle cells localized in the anterior pole of the oocyte in late choriogenesis. RNA interference (RNAi) of Brownie impaired correct formation of the sponge-like body and, as a result, the egg-case was also ill-formed and the eggs were not viable.

Conclusions/Significance

Results indicate that the novel gene Brownie plays a pivotal role in building up the sponge-like body. Brownie is the first reported gene involved in the construction of complex eggshell respiratory structures.