Stomatal immunity against fungal invasion comprises not only chitin-induced stomatal closure but also chitosan-induced guard cell death.

Many pathogenic fungi exploit stomata as invasion routes, causing destructive diseases of major cereal crops. Intensive interaction is expected to occur between guard cells and fungi. In the present study, we took advantage of well-conserved molecules derived from the fungal cell wall, chitin oligosaccharide (CTOS), and chitosan oligosaccharide (CSOS) to study how guard cells respond to fungal invasion. In Arabidopsis, CTOS induced stomatal closure through a signaling mediated by its receptor CERK1, Ca2+, and a major S-type anion channel, SLAC1. CSOS, which is converted from CTOS by chitin deacetylases from invading fungi, did not induce stomatal closure, suggesting that this conversion is a fungal strategy to evade stomatal closure. At higher concentrations, CSOS but not CTOS induced guard cell death in a manner dependent on Ca2+ but not CERK1. These results suggest that stomatal immunity against fungal invasion comprises not only CTOS-induced stomatal closure but also CSOS-induced guard cell death.

Chitin and chitosan remodeling defines vegetative development and Trichoderma biocontrol.

Fungal parasitism depends on the ability to invade host organisms and mandates adaptive cell wall remodeling to avoid detection and defense reactions by the host. All plant and human pathogens share invasive strategies, which aid to escape the chitin-triggered and chitin-targeted host immune system. Here we describe the full spectrum of the chitin/chitosan-modifying enzymes in the mycoparasite Trichoderma atroviride with a central role in cell wall remodeling. Rapid adaption to a variety of growth conditions, environmental stresses and host defense mechanisms such as oxidative stress depend on the concerted interplay of these enzymes and, ultimately, are necessary for the success of the mycoparasitic attack. To our knowledge, we provide the first in class description of chitin and associated glycopolymer synthesis in a mycoparasite and demonstrate that they are essential for biocontrol. Eight chitin synthases, six chitin deacetylases, additional chitinolytic enzymes, including six chitosanases, transglycosylases as well as accessory proteins are involved in this intricately regulated process. Systematic and biochemical classification, phenotypic characterization and mycoparasitic confrontation assays emphasize the importance of chitin and chitosan assembly in vegetative development and biocontrol in T. atroviride. Our findings critically contribute to understanding the molecular mechanism of chitin synthesis in filamentous fungi and mycoparasites with the overarching goal to selectively exploit the discovered biocontrol strategies.

Vascular bundle sheath and mesophyll cells modulate leaf water balance in response to chitin.

Plants can detect pathogen invasion by sensing microbe-associated molecular patterns (MAMPs). This sensing process leads to the induction of defense responses. Numerous MAMP mechanisms of action have been described in and outside the guard cells. Here, we describe the effects of chitin, a MAMP found in fungal cell walls and insects, on the cellular osmotic water permeability (Pf ) of the leaf vascular bundle-sheath (BS) and mesophyll cells (MCs), and its subsequent effect on leaf hydraulic conductance (Kleaf ). BS is a parenchymatic tissue that tightly encases the vascular system. BS cells (BSCs) have been shown to influence Kleaf through changes in their Pf , for example, after sensing the abiotic stress response-regulating hormone abscisic acid. It was recently reported that, in Arabidopsis, the chitin receptors-like kinases, chitin elicitor receptor kinase 1 (CERK1) and LYSINE MOTIF RECEPTOR KINASE 5 (LYK5) are highly expressed in the BS as well as the neighboring mesophyll. Therefore, we studied the possible impact of chitin on these cells. Our results revealed that BSCs and MCs exhibit a sharp decrease in Pf in response to chitin treatment. In addition, xylem-fed chitin decreased Kleaf and led to stomatal closure. However, Atlyk5 mutant showed none of these responses. Complementing AtLYK5 in the BSCs (using the SCARECROW promoter) resulted in the response to chitin that was similar to that observed in the wild-type. These results suggest that BS play a role in the perception of apoplastic chitin and in initiating chitin-triggered immunity.

Damage, repair and regeneration in insect cuticle: The story so far, and possibilities for the future.

The exoskeleton of an insect can contain countless specializations across an individual, across developmental stages, and across the class Insecta. Hence, the exoskeleton's building material cuticle must perform a vast variety of functions. Cuticle displays a wide range of material properties which are determined by several known factors: the amount and orientation of the chitin fibres, the constituents and degree of cross-linking and hydration of the protein matrix, the relative amounts of exo- and endocuticle, and the shape of the structures themselves. In comparison to other natural materials such as wood and mammal bone, relatively few investigations into the mechanical properties of insect cuticle have been carried out. Of these, very few have focussed on the need for repair and its effectiveness at restoring mechanical stability to the cuticle. Insect body parts are often subject to prolonged repeated cyclic loads when running and flying, as well as more extreme "emergency" behaviours necessary for survival such as jumping, wedging (squeezing through small holes) and righting (when overturned). What effects have these actions on the cuticle itself? How close to the limits of failure does an insect push its body parts? Can an insect recover from minor or major damage to its exoskeleton "bones"? No current research has answered these questions conclusively.

Cuticular body hairs mediate clumping of small Camponotus floridanus larvae.

The body surface of insects usually carries cuticular hairs. Commonly, important functions of these structures are to prevent drowning and to defend against predators. Here, we report on our studies on hairs at the surface of larvae of the ant species Camponotus floridanus and Camponotus sericeiventris. First, we present data supporting the hypothesis that anti-drowning properties of the surface might rely on cuticular hairs. Second, we show that especially in young larvae body hairs serve as attachment and interlocking devices mediating clumping of larvae facilitating transport by workers. Based on our observations, we speculate that clumping also enhances larval perceptibility. Taken together, larval cuticular hairs seem to have at least two important functions augmenting chances of larval survival. Obviously, despite their immobility, young Camponotus larvae support childcare in the ant colony providing an arsenal of cuticular hairs on their body surface.

'Strengthening the fungal cell wall through chitin-glucan cross-links: effects on morphogenesis and cell integrity'.

The cross-linking of polysaccharides to assemble new cell wall in fungi requires transglycosylation mechanisms by which preexisting glycosidic linkages are broken and new linkages are created between the polysaccharides. The molecular mechanisms for these processes, which are essential for fungal cell biology, are only now beginning to be elucidated. Recent development of in vivo and in vitro biochemical approaches has allowed characterization of important aspects about the formation of chitin-glucan covalent cell wall cross-links by cell wall transglycosylases of the CRH family and their biological function. Covalent linkages between chitin and glucan mediated by Crh proteins control morphogenesis and also play important roles in the remodeling of the fungal cell wall as part of the compensatory responses necessary to counterbalance cell wall stress. These enzymes are encoded by multigene families of redundant proteins very well conserved in fungal genomes but absent in mammalian cells. Understanding the molecular basis of fungal adaptation to cell wall stress through these and other cell wall remodeling enzymatic activities offers an opportunity to explore novel antifungal treatments and to identify potential fungal virulence factors.

Biosynthesis, Turnover, and Functions of Chitin in Insects.

Chitin is a major component of the exoskeleton and the peritrophic matrix of insects. It forms complex structures in association with different assortments of cuticle and peritrophic matrix proteins to yield biocomposites with a wide range of physicochemical and mechanical properties. The growth and development of insects are intimately coupled with the biosynthesis, turnover, and modification of chitin. The genes encoding numerous enzymes of chitin metabolism and proteins that associate with and organize chitin have been uncovered by bioinformatics analyses. Many of these proteins are encoded by sets of large gene families. There is specialization among members within each family, which function in particular tissues or developmental stages. Chitin-containing matrices are dynamically modified at every developmental stage and are under developmental and/or physiological control. A thorough understanding of the diverse processes associated with the assembly and turnover of these chitinous matrices offers many strategies to achieve selective pest control.

SEM characterization of anatomical variation in chitin organization in insect and arthropod cuticles.

The cuticles of insects and arthropods have some of the most diverse material properties observed in nature, so much so that it is difficult to imagine that all cutciles are primarily composed of the same two materials: a fibrous chitin network and a matrix composed of cuticle proteins. Various factors contribute to the mechanical and optical properties of an insect or arthropod cuticle including the thickness and composition. In this paper, we also identified another factor that may contribute to the optical, surface, and mechanical properties of a cuticle, i.e. the organization of chitin nanofibers and chitin fiber bundles. Self-assembled chitin nanofibers serve as the foundation for all higher order chitin structures in the cuticles of insects and other arthropods via interactions with structural cuticle proteins. Using a technique that enables the characterization of chitin organization in the cuticle of intact insects and arthropod exoskeletons, we demonstrate a structure/function correlation of chitin organization with larger scale anatomical structures. The chitin scaffolds in cuticles display an extraordinarily diverse set of morphologies that may reflect specific mechanical or physical properties. After removal of the proteinaceous and mineral matrix of a cuticle, we observe using SEM diverse nanoscale and micro scale organization of in-situ chitin in the wing, head, eye, leg, and dorsal and ventral thoracic regions of the periodical cicada Magicicada septendecim and in other insects and arthropods. The organization of chitin also appears to have a significant role in the organization of nanoscale surface structures. While microscale bristles and hairs have long been known to be chitin based materials formed as cellular extensions, we have found a nanostructured layer of chitin in the cuticle of the wing of the dog day annual cicada Tibicen tibicens, which may be the scaffold for the nanocone arrays found on the wing. We also use this process to examine the chitin organizations in the fruit fly, Drosophila melanogaster, and the Atlantic brown shrimp, Farfantepenaeus aztecus. Interestingly many of the homologous anatomical structures from diverse arthropods exhibit similar patterns of chitin organization suggesting that a common set of parameters, govern chitin organization.

Quorum sensing-regulated chitin metabolism provides grazing resistance to Vibrio cholerae biofilms.

Association of Vibrio cholerae with chitinous surfaces of zooplankton is important for its persistence in marine environments, as it provides accessibility to nutrients and resistance to stresses. Predation by heterotrophic protists has a major impact on the survival of V. cholerae. V. cholerae forms biofilms as its main defensive strategy, and quorum sensing (QS) additionally regulates the production of antiprotozoal factors. The role of chitin and QS regulation in V. cholerae grazing resistance was investigated by exposing V. cholerae wild-type (WT) and QS mutant biofilms grown on chitin flakes to the bacteriotrophic, surface-feeding flagellate Rhynchomonas nasuta. V. cholerae formed more biofilm biomass on chitin flakes compared with nonchitinous surfaces. The growth of R. nasuta was inhibited by WT biofilms grown on chitin flakes, whereas the inhibition was attenuated in QS mutant biofilms. The chitin-dependent toxicity was also observed when the V. cholerae biofilms were developed under continuous flow or grown on a natural chitin source, the exoskeleton of Artemia. In addition, the antiprotozoal activity and ammonium concentration of V. cholerae biofilm supernatants were quantified. The ammonium levels (3.5 mM) detected in the supernatants of V. cholerae WT biofilms grown on chitin flakes were estimated to reduce the number of R. nasuta by >80% in add-back experiments, and the supernatant of QS mutant biofilms was less toxic owing to a decrease in ammonium production. Transcriptomic analysis revealed that the majority of genes involved in chitin metabolism and chemotaxis were significantly downregulated in QS mutant biofilms when grown on chitin compared with the WT biofilms.

A chemically functionalized magnetic nanoplatform for rapid and specific biomolecular recognition and separation.

We have developed a target-molecule-functionalized magnetic nanoparticle (MNP)-based method to facilitate the study of biomolecular recognition and separation. The superparamagnetic property of MNPs allows the corresponding biomolecules to be rapidly separated from crude biofluids with a significant improvement in recovery yield and specificity. Various MNPs functionalized with tag molecules (chitin, heparin, and amylose) were synthesized for recombinant protein purification, and several probe-functionalized MNPs, such as nitrilotriacetic acid (NTA)@MNP and P(k)@MNP, exhibited excellent extraction efficiency for proteins. In a cell recognition study, mannose-functionalized MNPs allowed specific purification of Escherichia coli with FimH adhesin on the surface. In an immunoprecipitation assay, the antibody-conjugated MNPs reduced the incubation time from 12 to 1 h while maintaining a comparable efficiency. The functionalized MNPs were also used in a membrane proteomic study that utilized the interaction between streptavidin-functionalized MNPs and biotinylated cell membrane proteins. Overall, the functionalized MNPs were demonstrated to be promising probes for the specific separation of targets from proteins to cells and proteomics.

Chitin elicits CCL2 from airway epithelial cells and induces CCR2-dependent innate allergic inflammation in the lung.

Chitin exposure in the lung induces eosinophilia and alternative activation of macrophages and is correlated with allergic airway disease. However, the mechanism underlying chitin-induced polarization of macrophages is poorly understood. In this paper, we show that chitin induces alternative activation of macrophages in vivo but does not do so directly in vitro. We further show that airway epithelial cells bind chitin in vitro and produce CCL2 in response to chitin both in vitro and in vivo. Supernatants of chitin-exposed epithelial cells promoted alternative activation of macrophages in vitro, whereas Ab neutralization of CCL2 in the supernate abolished the alternative activation of macrophages. CCL2 acted redundantly in vivo, but mice lacking the CCL2 receptor, CCR2, showed impaired alternative activation of macrophages in response to chitin, as measured by arginase I, CCL17, and CCL22 expression. Furthermore, CCR2 knockout mice exposed to chitin had diminished reactive oxygen species products in the lung, blunted eosinophil and monocyte recruitment, and impaired eosinophil functions as measured by expression of CCL5, IL-13, and CCL11. Thus, airway epithelial cells secrete CCL2 in response to chitin and CCR2 signaling mediates chitin-induced alternative activation of macrophages and allergic inflammation in vivo.

The role of peritrophic membrane in the resistance of Anticarsia gemmatalis larvae (Lepidoptera: Noctuidae) during the infection by its nucleopolyhedrovirus (AgMNPV).

The aim of this study was to analyze morphologically the peritrophic membrane (PM) of Anticarsia gemmatalis larvae resistant (RL) and non-resistant (susceptible) (SL) to the A. gemmatalis multicapsid nucleopolyhedrovirus (AgMNPV), in the presence of viral infection. Also, in this investigation the results between SL and RL were compared to improve the understanding of the resistance mechanisms to the virus. The PM of SL of A. gemmatalis was less efficient as a barrier against the viral infection since it was found to be more fragile than the PM of RL. The lower chitin content as seen from weaker fluorescent staining in SL as well as the abundance of non-solubilized vesicular materials in the ectoperitrophic space, would cause the malformation of this membrane, facilitating the passage of the virus toward the epithelium of the midgut. On the other hand, in RL, the intensity of WGA (wheat germ agglutinin)-conjugated FITC (fluorescein) reaction of the PM was greater than in SL, making this insect more resistant to infection. We can conclude that the effectiveness of the PM in protecting against pathogens is dependent on the integrity of the epithelial cells of the midgut and of the structural preservation of the PM, being directly implicated in the resistance of A. gemmatalis larvae to AgMNPV.

Chitin modulates innate immune responses of keratinocytes.

BACKGROUND: Chitin, after cellulose the second most abundant polysaccharide in nature, is an essential component of exoskeletons of crabs, shrimps and insects and protects these organisms from harsh conditions in their environment. Unexpectedly, chitin has been found to activate innate immune cells and to elicit murine airway inflammation. The skin represents the outer barrier of the human host defense and is in frequent contact with chitin-bearing organisms, such as house-dust mites or flies. The effects of chitin on keratinocytes, however, are poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: We hypothesized that chitin stimulates keratinocytes and thereby modulates the innate immune response of the skin. Here we show that chitin is bioactive on primary and immortalized keratinocytes by triggering production of pro-inflammatory cytokines and chemokines. Chitin stimulation further induced the expression of the Toll-like receptor (TLR) TLR4 on keratinocytes at mRNA and protein level. Chitin-induced effects were mainly abrogated when TLR2 was blocked, suggesting that TLR2 senses chitin on keratinocytes. CONCLUSIONS/SIGNIFICANCE: We speculate that chitin-bearing organisms modulate the innate immune response towards pathogens by upregulating secretion of cytokines and chemokines and expression of MyD88-associated TLRs, two major components of innate immunity. The clinical relevance of this mechanism remains to be defined.

An epidermis-specific chitin synthase CDNA in Choristoneura fumiferana: cloning, characterization, developmental and hormonal-regulated expression.

Chitin synthase catalyzes chitin synthesis in the exoskeleton, tracheal system and gut during insect development. A chitin synthase 1 (CfCHS1) cDNA was identified and cloned from the spruce budworm, Choristoneura fumiferana. The CfCHS1 cDNA is 5,300 bp in length and codes a 1,564-amino acid protein with a molecular mass of 178 kDa. The deduced protein contains 16 transmembrane helixes in its domains A and C. The single copy CfCHS1 gene expressed during each of the larval molts from the 3rd to the 6th instar. The gene expressed highly and periodically in the epidermis during each of molts, whereas no transcripts were detected in the midgut and fat body. 20-hydroxyecdysone and the ecdysone agonist RH5992 suppressed CfCHS1 expression, whereas the juvenile hormone analog methoprene induced CfCHS1 expression. These results implicate that CfCHS1 is involved in the chitin synthase and new chitin formation during molting in the insect.

Eggshell chitin and chitin-interacting proteins prevent polyspermy in C. elegans.

Development requires fertilization by a single sperm. In Caenorhabditis elegans, fertilization occurs in a sperm-filled spermatheca, implying the barrier to polyspermy is generated in this compartment. Eggshell chitin synthesis is initiated at fertilization, and chitin is deposited before the zygote exits the spermatheca. Whereas polyspermy is very rare in wild-type, here we report an incidence of 14%-51% in zygotes made chitin deficient by loss of chitin synthase-1 (CHS-1), the CHS-1 substrate UDP-N-acetylglucosamine, the CHS-1-interacting protein EGG-3, or the sperm-provided protein SPE-11. The spe-11(hc90) mutant deposits chitin at the male end but fails to complete a continuous layer. The polyspermy barrier is also compromised by loss of the chitin-binding protein CBD-1 or the GLD-1-regulated LDL receptor-like EGG-1, together with its homolog, EGG-2. Loss of CBD-1 or EGG-1/2 disrupts oocyte cortical distribution of CHS-1, as well as MBK-2 and EGG-3. In CBD-1 or EGG-1/2 deficiency, chitin is synthesized but the eggshell is fractured, suggesting aberrantly clustered CHS-1/MBK-2/EGG-3 may fail to support construction of a continuous eggshell. Together, our results show that eggshell chitin is required to prevent polyspermy in C. elegans, in addition to its previously reported requirement in polar body extrusion and polarization of the zygote.

A rice fungal MAMP-responsive MAPK cascade regulates metabolic flow to antimicrobial metabolite synthesis.

Plants recognize potential microbial pathogens through microbial-associated molecular patterns (MAMPs) and activate a series of defense responses, including cell death and the production of reactive oxygen species (ROS) and diverse anti-microbial secondary metabolites. Mitogen-activated protein kinase (MAPK) cascades are known to play a pivotal role in mediating MAMP signals; however, the signaling pathway from a MAPK cascade to the activation of defense responses is poorly understood. Here, we found in rice that the chitin elicitor, a fungal MAMP, activates two rice MAPKs (OsMPK3 and OsMPK6) and one MAPK kinase (OsMKK4). OsMPK6 was essential for the chitin elicitor-induced biosynthesis of diterpenoid phytoalexins. Conditional expression of the active form of OsMKK4 (OsMKK4(DD) ) induced extensive alterations in gene expression, which implied dynamic changes of metabolic flow from glycolysis to secondary metabolite biosynthesis while suppressing basic cellular activities such as translation and cell division. OsMKK4(DD) also induced various defense responses, such as cell death, biosynthesis of diterpenoid phytoalexins and lignin but not generation of extracellular ROS. OsMKK4(DD) -induced cell death and expression of diterpenoid phytoalexin pathway genes, but not that of phenylpropanoid pathway genes, were dependent on OsMPK6. Collectively, the OsMKK4-OsMPK6 cascade plays a crucial role in reprogramming plant metabolism during MAMP-triggered defense responses.

Exoskeletal chitin scales isometrically with body size in terrestrial insects.

The skeletal system of animals provides the support for a variety of activities and functions. For animals such as mammals, which have endoskeletons, research has shown that skeletal investment (mass) scales with body mass to the 1.1 power. In this study, we ask how exoskeletal investment in insects scales with body mass. We measured the body mass and mass of exoskeletal chitin of 551 adult terrestrial insects of 245 species, with dry masses ranging from 0.0001 to 2.41 g (0.0002-6.13 g wet mass) to assess the allometry of exoskeletal investment. Our results showed that exoskeletal chitin mass scales isometrically with dry body mass across the Insecta as M(chitin) = a M(dry) (b), where b = 1.03 +/- 0.04, indicating that both large and small terrestrial insects allocate a similar fraction of their body mass to chitin. This isometric chitin-scaling relationship was also evident at the taxonomic level of order, for all insect orders except Coleoptera. We additionally found that the relative exoskeletal chitin investment, indexed by the coefficient, a, varies with insect life history and phylogeny. Exoskeletal chitin mass tends to be proportionally less and to increase at a lower rate with mass in flying than in nonflying insects (M(flying insect chitin) = -0.56 x M(dry) (0.97); M(nonflying insect chitin) = -0.55 x M(dry) (1.03)), and to vary with insect order. Isometric scaling (b = 1) of insect exoskeletal chitin suggests that the exoskeleton in insects scales differently than support structures of most other organisms, which have a positive allometry (b > 1) (e.g., vertebrate endoskeleton, tree secondary tissue). The isometric pattern that we document here additionally suggests that exoskeletal investment may not be the primary limit on insect body size.

Peritrophic matrix of Phlebotomus duboscqi and its kinetics during Leishmania major development.

Light microscopy of native preparations, histology, and electron microscopy have revealed that Phlebotomus duboscqi belongs to a class of sand fly species with prompt development of the peritrophic matrix (PM). Secretion of electron-lucent fibrils, presumably chitin, starts immediately after the ingestion of a blood meal and, about 6 h later, is followed by secretion of amorphous electron-dense components, presumably proteins and glycoproteins. The PM matures in less than 12 h and consists of a thin laminar outer layer and a thick amorphous inner layer. No differences have been found in the timing of the disintegration of the PM in females infected with Leishmania major. In both groups of females (infected and uninfected), the disintegration of the PM is initiated at the posterior end. Although parasites are present at high densities in the anterior part of the blood meal bolus, they escape from the PM at the posterior end only. These results suggest that L. major chitinase does not have an important role in parasite escape from the PM. Promastigotes remain in the intraperitrophic space until the PM is broken down by sand-fly-derived chitinases and only then migrate anteriorly. Disintegration of the PM occurs simultaneously with the morphological transformation of parasites from procyclic forms to long nectomonads. A novel role is ascribed to the anterior plug, a component of the PM secreted by the thoracic midgut; this plug functions as a temporary barrier to stop the forward migration of nectomonads to the thoracic midgut.

Chitin is a size-dependent regulator of macrophage TNF and IL-10 production.

Chitin is a ubiquitous polysaccharide in fungi, insects, and parasites. We hypothesized that chitin is a size-dependent regulator of innate immunity. To test this hypothesis, we characterized the effects of chitins of different sizes on murine bronchoalveolar or peritoneal macrophages. In these studies, large chitin fragments were inert, while both intermediate-sized chitin (40-70 microm) and small chitin (SC; <40 microm, largely 2-10 microm) stimulated TNF elaboration. In contrast, only SC induced IL-10 elaboration. The effects of intermediate-sized chitin were mediated by pathways that involve TLR2, dectin-1, and NF-kappaB. In contrast, the effects of SC were mediated by TLR2-dependent and -independent, dectin-1-dependent pathways that involved the mannose receptor and spleen tyrosine kinase. Chitin contains size-dependent pathogen-associated molecular patterns that stimulate TLR2, dectin-1, and the mannose receptor, differentially activate NF-kappaB and spleen tyrosine kinase, and stimulate the production of pro- and anti-inflammatory cytokines.