In the presented studies, chitin synthase containing nanoparticles (chitosomes) from the yeast Saccharomyces cerevisiae lacking the chs3 gene were investigated. Two step centrifugations using sucrose gradients led to considerable purity of the chitosomal complexes. Chitin synthase I activity was determined via a previously described ELISA based WGA assay and a novel assay using the Streptomyces chitin binding protein CHB1, which provided good tools to follow the purification procedure. In collaboration, it could be shown that the complexes produce fibers in the presence of the substrates uridine-diphosphate-N-acetylglucosamine (UDP-GlcNAc) and N-acetylglucosamine (GlcNAc) and this reaction was inhibited by addition of chitin synthase inhibitor nikkomycin Z. These results demonstrate for the first time that CSI containing chitosomes can be gained. Investigation of the purified nanocomplexes with CSI activity led to the additional conclusion that proteins of the glycolytic pathway such as glyceraldehyde-3-phosphate (GAPDH isoform Tdh3), enolase (Eno1), pyruvate decarboxylase (Pdc1) and pyruvate kinase (Pyk1) are also concentrated around the peak of CSI activity. The presence of these proteins in the pure chitosomes was further verified via testing for their individual enzymatic activities and by antibody studies. The relative levels of GAPDH, Pdc1 and Pyk1 were found to be higher in comparison to enolase; however GAPDH and Pdc1 proteins had a broad distribution across the purification gradient and were also found in neighboring fractions of peak of CSI activity. In addition to these, two high molecular weight proteins showing similarity to glucan synthase and fatty acid synthase were also found in such fractions as analyzed via MALDI-MS. In future it will be worthwhile to ascertain the active functional relationships among the different proteins found in chitosomal preparations using immuno fluorescence co-localization studies.

No description available.

Doctor of Philosophy / Department of Entomology / Kun Yan Zhu / Chitin metabolism represents an attractive target site for combating insect pests as insect growth and development are strictly dependent on precisely toned chitin synthesis and degradation and this process is absent in humans and other vertebrates. However, current understanding on this process and the involved enzymes is rather limited in insects. In this study, two chitin synthase genes (AgCHS1 and AgCHS2 or AgCHSA and AgCHSB), and 20 chitinase and chitinase-like genes (groups I-VIII) presumably encoding the enzymes for chitin biosynthesis and degradation, respectively, were identified and characterized in African malaria mosquito, Anopheles gambiae. Immunohistochemistry analysis and developmental stage- and tissue-dependent transcript profiling by using reverse transcription PCR, real-time quantitative PCR, and in situ hybridization revealed new information on these genes. Current understanding on chitin synthases is extended by the expression profiles such as the localization of AgCHS1 and AgCHS2 transcripts in eggs, AgCHS2 transcripts in the posterior larval midgut, AgCHS1 and AgCHS2 proteins in the compound eyes, and AgCHS2 enzyme in pupal inter-segments. Chitinase and chitinase-like genes are highly diverse in their gene structure, domain organization, and stage- and tissue-specific expression patterns. Most of these genes were expressed in several stages. However, some genes are stage- and tissue-specific such as AgCht8 mainly in pupal and adult stages, AgCht2 and AgCht12 specifically in foregut, AgCht13 exclusively in midgut. Functional analysis of each chitin synthase gene was conducted by using the chitosan/dsRNA nanoparticle-based RNA interference (RNAi) through larval feeding. The repression of the AgCHS1 transcripts which are predominantly expressed in carcass initiated from the mosquito larval feeding of dsRNA suggests the systemic nature of RNAi in mosquito larvae. In addition, silencing of AgCHS1 increased larval susceptibilities to diflubenzuron, whereas silencing of AgCHS2 enhanced the peritrophic matrix disruption and thus increased larval susceptibilities to calcofluor white or dithiothreitol. Furthermore, a non-radioactive method was adapted and optimized to examine the chitin synthase activity in mosquitoes. By using this method, diflubenzuron and nikkomycin Z show limited in vitro inhibition on chitin synthase at high concentration in cell free system, whereas no in vivo inhibition was observed.

This thesis concerns work on structure and membrane interactions of enzymes involved in lipid synthesis, biomembrane and cell wall regulation and cell defense processes. These proteins, known as glycosyltransferases (GTs), are involved in the transfer of sugar moieties from nucleotide sugars to lipids or chitin polymers. Glycosyltransferases from three types of organisms have been investigated; one is responsible for vital lipid synthesis in Arabidopsis thaliana (atDGD2) and adjusts the lipid content in biomembranes if the plant experiences stressful growth conditions. This enzyme shares many structural features with another GT found in gram-negative bacteria (WaaG). WaaG is however continuously active and involved in synthesis of the protective lipopolysaccharide layer in the cell walls of Escherichia coli. The third type of enzymes investigated here are chitin synthases (ChS) coupled to filamentous growth in the oomycete Saprolegnia monoica. I have investigated two ChS-derived MIT domains that may be involved in membrane interactions within the endosomal pathway. From analysis of the three-dimensional structure and the amino-acid sequence, some important regions of these very large proteins were selected for in vitro studies. By the use of an array of biophysical methods (e.g. Nuclear Magnetic Resonance, Fluorescence and Circular Dichroism spectroscopy) and directed sequence analyses it was possible to shed light on some important details regarding the structure and membrane-interacting properties of the GTs. The importance of basic amino-acid residues and hydrophobic anchoring segments, both generally and for the abovementioned proteins specifically, is discussed. Also, the topology and amino-acid sequence of GT-B enzymes of the GT4 family are analyzed with emphasis on their biomembrane association modes. The results presented herein regarding the structural and lipid-interacting properties of GTs aid in the general understanding of glycosyltransferase activity. Since GTs are involved in a high number of biochemical processes in vivo it is of outmost importance to understand the underlying processes responsible for their activity, structure and interaction events. The results are likely to be useful for many applications and future experimental design within life sciences and biomedicine. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript.</p>

A maior parte dos insetos possui uma estrutura anatômica em forma de filme (membrana peritrófica, MP) composta de quitina e proteínas (peritrofinas), que separa o alimento do epitélio do intestino médio. A MP protege o epitélio de microorganismos e da abrasão, e possui outras funções baseadas no fato de que a MP promove a compartimentalização de enzimas, que incluem: aumento da eficiência digestiva através da diminuição da taxa de excreção das enzimas e de outros mecanismos postulados que são testados nesta tese. A síntese das peritrofinas é mais conhecida do que a da quitina componente da MP, tornando desejável um esforço no detalhamento dessa última. Foram realizadas a caracterização e expressão de genes de S. frugiperda que codificam uma peritrofina e enzimas responsáveis pela síntese e degradação de quitina (quitina sintases 1 (SfCHS1) e 2 (SfCHS2), e quitinase (SfCHI), respectivamente). As sequências dos cDNAs correspondentes foram determinadas através da amplificação de fragmentos de PCR que se sobrepõem. Os padrões de expressão dos genes envolvidos no metabolismo da quitina da MP foram analisados durante o desenvolvimento do inseto por RT-PCR. SfCHS2 é expresso no intestino médio durante os estágios de alimentação da larva, enquanto que SfCHI é expresso durante as fases de pós-alimentação, pré-pupa, e pupa. Ambos os genes são predominantemente expressos na região anterior no intestino médio com um gradiente decrescente de expressão ao longo do tubo digestivo. A citolocalização da quitina revelou que o polissacarídeo está presente somente quando SfCHS2 é expresso e não há quitina no intestino médio quando SfCHI é expresso. Esses resultados levaram a formulação da hipótese de que SfCHS2 é responsável pela síntese da quitina da MP durante o estágio larval e SfCHI degrada a quitina da MP durante a muda larva-pupa, sugerindo padrões inversos de expressão desses genes. Em Spodoptera frugiperda, Tenebrio molitor e Musca domestica é possível prever o sítio de secreção das enzimas digestivas (ventrículo anterior, médio ou posterior) a partir da distribuição antero-posterior das enzimas no espaço endoperitrófico. Também foi possível mostrar, usando vários modelos experimentais, que a separação de compartimentos luminais pela MP: a) impede a inibição de despolimerazes por remover oligômeros do espaço endoperitrófico; b) evita a inibição de oligômero hidrolases restringindo-as ao espaço ectoperitrófico e impedindo que entrem em contato com o alimento e c) anula a inibição de enzimas envolvidas na digestão terminal presentes na superfície do epitélio, impedindo que o alimento entre em contato com elas. / Most insects have a film-like anatomical structure (peritrophic membrane, PM) composed of chitin and proteins (peritrophins), which separates food from midgut tissue. It protects the epithelium against food abrasion and microrganisms and has other functions based on compartmentalization of enzymes, which include: increasing digestive efficiency by decreasing enzyme excretion and by other mechanisms that were tested in this thesis. The peritrophin synthesis is less known than PM chitin synthesis, which needs to be better understood. The characterization and expression of S. frugiperda genes encoding a peritrophin and enzymes responsible for the synthesis and degradation of chitin, chitin synthases 1(SfCHS1) and 2 (SfCHS2), and chitinase (SfCHI), respectively, were analysed. Sequences of corresponding cDNAs were determined by amplification of overlapping PCR fragments and the expression patterns of chitin metabolism genes were analyzed during insect development by RT-PCR. SfCHS2 is expressed in the midgut during the feeding stages, whereas SfCHI is expressed during the wandering and pupal stages. Both genes are predominantly expressed in the anterior portion of the midgut with a decreasing gradient of transcript levels in the medial and posterior portions. Chitin staining revealed that the polysaccharide is present in the PM only when SfCHS2 is expressed. There is little or no chitin in the midgut when SfCHI is expressed. These results support the hypothesis that SfCHS2 is responsible for PM chitin synthesis during the larval stage and SfCHI for PM chitin degradation during larval-pupal molting, suggesting inverse patterns of expression of these genes. The secretion site (anterior, middle or posterior midgut) of digestive enzymes can be predicted in Spodoptera frugiperda, Tenebrio molitor and Musca domestica based on enzyme activity distribution along the endoperitrophic space. We also have shown, using several experimental models, that the luminal compartment separation by PM: a) avoid the polimer hidrolases inhibition by removing oligomer from endoperitrophic space; b) decrease the oligomer hidrolases inhibition by restricting them to the ectoperitrophic space (by avoiding their contact with food); and c) block the inhibition of enzymes located at the cell surface involved in terminal digestion by avoiding their contact with food.

A maior parte dos insetos possui uma estrutura anatômica em forma de filme (membrana peritrófica, MP) composta de quitina e proteínas (peritrofinas), que separa o alimento do epitélio do intestino médio. A MP protege o epitélio de microorganismos e da abrasão, e possui outras funções baseadas no fato de que a MP promove a compartimentalização de enzimas, que incluem: aumento da eficiência digestiva através da diminuição da taxa de excreção das enzimas e de outros mecanismos postulados que são testados nesta tese. A síntese das peritrofinas é mais conhecida do que a da quitina componente da MP, tornando desejável um esforço no detalhamento dessa última. Foram realizadas a caracterização e expressão de genes de S. frugiperda que codificam uma peritrofina e enzimas responsáveis pela síntese e degradação de quitina (quitina sintases 1 (SfCHS1) e 2 (SfCHS2), e quitinase (SfCHI), respectivamente). As sequências dos cDNAs correspondentes foram determinadas através da amplificação de fragmentos de PCR que se sobrepõem. Os padrões de expressão dos genes envolvidos no metabolismo da quitina da MP foram analisados durante o desenvolvimento do inseto por RT-PCR. SfCHS2 é expresso no intestino médio durante os estágios de alimentação da larva, enquanto que SfCHI é expresso durante as fases de pós-alimentação, pré-pupa, e pupa. Ambos os genes são predominantemente expressos na região anterior no intestino médio com um gradiente decrescente de expressão ao longo do tubo digestivo. A citolocalização da quitina revelou que o polissacarídeo está presente somente quando SfCHS2 é expresso e não há quitina no intestino médio quando SfCHI é expresso. Esses resultados levaram a formulação da hipótese de que SfCHS2 é responsável pela síntese da quitina da MP durante o estágio larval e SfCHI degrada a quitina da MP durante a muda larva-pupa, sugerindo padrões inversos de expressão desses genes. Em Spodoptera frugiperda, Tenebrio molitor e Musca domestica é possível prever o sítio de secreção das enzimas digestivas (ventrículo anterior, médio ou posterior) a partir da distribuição antero-posterior das enzimas no espaço endoperitrófico. Também foi possível mostrar, usando vários modelos experimentais, que a separação de compartimentos luminais pela MP: a) impede a inibição de despolimerazes por remover oligômeros do espaço endoperitrófico; b) evita a inibição de oligômero hidrolases restringindo-as ao espaço ectoperitrófico e impedindo que entrem em contato com o alimento e c) anula a inibição de enzimas envolvidas na digestão terminal presentes na superfície do epitélio, impedindo que o alimento entre em contato com elas. / Most insects have a film-like anatomical structure (peritrophic membrane, PM) composed of chitin and proteins (peritrophins), which separates food from midgut tissue. It protects the epithelium against food abrasion and microrganisms and has other functions based on compartmentalization of enzymes, which include: increasing digestive efficiency by decreasing enzyme excretion and by other mechanisms that were tested in this thesis. The peritrophin synthesis is less known than PM chitin synthesis, which needs to be better understood. The characterization and expression of S. frugiperda genes encoding a peritrophin and enzymes responsible for the synthesis and degradation of chitin, chitin synthases 1(SfCHS1) and 2 (SfCHS2), and chitinase (SfCHI), respectively, were analysed. Sequences of corresponding cDNAs were determined by amplification of overlapping PCR fragments and the expression patterns of chitin metabolism genes were analyzed during insect development by RT-PCR. SfCHS2 is expressed in the midgut during the feeding stages, whereas SfCHI is expressed during the wandering and pupal stages. Both genes are predominantly expressed in the anterior portion of the midgut with a decreasing gradient of transcript levels in the medial and posterior portions. Chitin staining revealed that the polysaccharide is present in the PM only when SfCHS2 is expressed. There is little or no chitin in the midgut when SfCHI is expressed. These results support the hypothesis that SfCHS2 is responsible for PM chitin synthesis during the larval stage and SfCHI for PM chitin degradation during larval-pupal molting, suggesting inverse patterns of expression of these genes. The secretion site (anterior, middle or posterior midgut) of digestive enzymes can be predicted in Spodoptera frugiperda, Tenebrio molitor and Musca domestica based on enzyme activity distribution along the endoperitrophic space. We also have shown, using several experimental models, that the luminal compartment separation by PM: a) avoid the polimer hidrolases inhibition by removing oligomer from endoperitrophic space; b) decrease the oligomer hidrolases inhibition by restricting them to the ectoperitrophic space (by avoiding their contact with food); and c) block the inhibition of enzymes located at the cell surface involved in terminal digestion by avoiding their contact with food.

Septins are a highly conserved family of eukaryotic proteins having significant homology within and among species. In the budding yeast, Saccharomyces cerevisiae, a septin-based hierarchy of proteins is required to localize chitin in the bud neck prior to septum formation. However, this process has not been clarified in a filamentous, conidiogenous fungus capable of yeast growth, such as Wangiella dermatitidis, a polymorphic agent of human phaeohyphomycosis. Prior studies of this melanized mold showed that some chitin synthase mutants (wdchsΔ) have defects in yeast septum formation, suggesting that the septins of W. dermatitidis might functionally associate with some of its chitin synthases (WdChsp). To test this hypothesis, four vegetative septin homologs of S. cerevisiae were cloned from W. dermatitidis and designated WdCDC3, WdCDC10, WdCDC11, and WdCDC12. Of the four, only WdCDC3 functionally complemented completely a strain of S. cerevisiae with a ts mutation in the corresponding gene, although WdCDC12 did so partially. Functional characterizations by mutagenesis of the four W. dermatitidis septin genes revealed that resulting mutants (wdcdc[delta]) each had unique defects in yeast growth and morphology, indicating that each septin carried out a distinct function. Furthermore, when a wdcdc10[delta] mutation was introduced into five different wdchs[delta] strains, weak genetic interactions were detected between WdCDC10 and WdCHS3 and WdCHS4, and a strong interaction between and WdCHS5. Cytological studies showed that WdChs5p was mislocalized in some septin mutants, including wdcdc10[delta]. These results confirmed that in W. dermatitidis septins are important for proper cellular morphogenesis, cytokinesis, and especially septum formation through associations with some chitin synthases. / text

Doctor of Philosophy / Department of Biochemistry / Subbaratnam Muthukrishnan / In order to grow and develop, insects must undergo a process of molting, wherein the old cuticle is replaced with a new one. A thin envelope layer has been predicted to act as a physical barrier between molting fluid chitinases and the site of new chitin synthesis ensuring selective protection of newly synthesized chitin. The factors that help the new exoskeleton withstand the deleterious effects of chitinolytic enzymes remain poorly understood. In the current study a mechanistic role for two proteins, Knickkopf (Knk) and Retroactive (Rtv), was explored in organization and protection of the newly synthesized procuticular chitin. Our study demonstrated colocalization of molting fluid chitinases (chitinase-5) with chitin in T. castaneum pharate adult elytral cuticle. Presence of chitinases in the new cuticle, disproved the old theory of the envelope being a protective barrier against chitinases. Confocal and transmission electron microscopic imaging of T. castaneum pharate adult elytral cuticle suggested that Knk protein selectively colocalizes with chitin in the new procuticle, organizes chitin into laminae and protects it from the activity of molting fluid chitinases. Down-regulation of Knk expression resulted in reduction of procuticular chitin, disruption of the laminar architecture of the procuticle and severe molting defects that are ultimately lethal at all stages of insect growth. The presence and activity of Rtv protein ensures the trafficking of Knk into the procuticle. Down regulation of Rtv transcripts showed molting defects and a significant decrease in chitin content similar to those following Knk dsRNA treatment. Confocal microscopic analysis revealed an essential role for Rtv in proper trafficking of Knk from epithelial cells to within the newly synthesized procuticule. Once released into the procuticle, Knk organizes and protects chitin from chitinases. The conservation of Knk and Rtv in all insect species suggests a critical role for these proteins in maintenance and protection of chitin in the insect exoskeleton.

Some species of Oomycetes are well studied pathogens that cause considerable economical losses in the agriculture and aquaculture industries. Currently, there are no chemicals available that are environmentally friendly and at the same time efficient Oomycete inhibitors. The cell wall of Oomycetes consists of b-(1à3) and b-(1à6)-glucans, cellulose and in some species minute amounts of chitin. The biosynthesis of cellulose and chitin in Oomycetes is poorly understood. However, cell wall synthesis represents a potential target for new Oomycete inhibitors. In this work, cellulose and chitin synthase genes and gene products were analyzed in the plant pathogen Phytophthora infestans and in the fish pathogen Saprolegnia monoica.   A new Oomycete CesA gene family was identified, containing four subclasses of genes designated as CesA1 to 4. The gene products of CesA1, 2 and 4 contain pleckstrin homology (PH) domains located at the N-terminus, which is unique to the Oomycete CesAs. Our results show that the SmCesA2 PH domain binds to phosphoinositides, F-actin and microtubules in vitro and can co-localize with F-actin in vivo. Functional characterization of the CesA genes by gene silencing in P. infestans led to decreased cellulose content in the cell wall. The cellulose synthase inhibitors DCB and Congo Red inhibited the growth of the mycelium of S. monoica and had an up-regulating effect on SmCesA gene expression. Zoospores from P. infestans treated with DCB were unable to infect potato leaves. In addition, two full-length chitin synthase genes (Chs) were analyzed from S. monoica.  Expression of SmChs2 in yeast yielded an active recombinant protein. The biochemical characterization of the in vitro product of SmChs2 confirmed that the protein is responsible for chitin formation. The chitin synthase inhibitor nikkomycin Z inhibited the SmChs2 both in vivo and in vitro.   Altogether these results show that at least some of the CesA1-4 genes are involved in cellulose biosynthesis and that synthesis of cellulose is crucial for infection of potato by P. infestans. The PH domain is involved in the interaction of CesA with the cytoskeleton. In addition, we firmly demonstrate that the SmChs2 gene encodes a catalytically active chitin synthase. / QC 20110531

Doctor of Philosophy / Department of Biochemistry / Karl J. Kramer / Subbarat Muthukrishnan / Chitin, a linear homopolymer of beta-1,4-linked N-acetylglucosamine, is the second most abundant biopolymer next to cellulose. It is the major structural polysaccharide in the insect’s exoskeleton and gut lining. An extensive study of two of the major genes encoding enzymes involved in chitin metabolism, chitin synthases (CHSs) and beta-N-acetylglucosaminidases (NAGs), was undertaken. CHS genes from the tobacco hornworm, Manduca sexta, and NAG genes from the red flour beetle, Tribolium castaneum, were identified and characterized. In general, chitin deposition occurs in two major extracellular structures of insects, the cuticle that overlays the epidermis, and the peritrophic membrane (PM) that lines the midgut. Only two CHS genes were identified in M. sexta using Southern blot analysis. Extensive expression studies of both M. sexta CHS genes, MsCHS1 and MsCHS2, suggest a strict functional specialization of these two genes for the synthesis of epidermal and PM-associated chitin, respectively. Furthermore, two alternatively spliced transcripts of MsCHS1, MsCHS1a and MsCHS1b, were identified. Analysis of the levels of these transcripts in different tissues and stages of development indicated that the MsCHS1a transcript predominates in the integument during the feeding and pupal stages, whereas the MsCHS1b transcript is more abundantly present in the tracheae, foregut, and hindgut during all developmental stages tested. Four genes encoding putative NAGs (TcNAG1, TcNAG2, TcNAG3, and TcNAG4) were identified by searching the Tribolium genomic database. The full-length cDNAs for all four NAGs were cloned and sequenced, and the exon-intron organizations were determined. Studies on developmental expression patterns of each gene indicated that they are expressed during most developmental stages with TcNAG1 being the predominant one. The function of each NAG was assessed by down regulating the level of each transcript at various developmental stages using RNA interference. Selective knock down of each transcript, without significant reduction in the expression levels of the other NAG transcripts, was verified and the resulting phenotypes were documented. Knockdown of TcNAG1 interrupted larval-larval, larval-pupal, and pupal-adult molting, and the insects were unable to completely shed their old cuticles.