Functional analysis of the fungal/plant class chitinase family in Aspergillus fumigatus

A quintuple mutant was constructed to delete the entire family of the fungal/plant (class III) chitinases of Aspergillus fumigatus. Only a limited reduction in the total chitinolytic activity was seen for the different chitinase mutants including the quintuple mutant. In spite of this reduction in chitinolytic activity, no growth or germination defects were observed in these chitinase mutants. This result demonstrated that the fungal/plant chitinases do not have an essential role in the morphogenesis of A. fumigatus. A slight diminution of the growth during autolysis was seen for the quintuple mutant suggesting that class III chitinases may play only a nutritional role during this phase of the cycle, retarding fungal death.     

Septins from the phytopathogenic fungus Ustilago maydis are required for proper morphogenesis but dispensable for virulence

Background

Septins are a highly conserved family of GTP-binding proteins involved in multiple cellular functions, including cell division and morphogenesis. Studies of septins in fungal cells underpin a clear correlation between septin-based structures and fungal morphology, providing clues to understand the molecular frame behind the varied morphologies found in fungal world.

Methodology/Principal Findings

Ustilago maydis genome has the ability to encode four septins. Here, using loss-of-function as well as GFP-tagged alleles of these septin genes, we investigated the roles of septins in the morphogenesis of this basidiomycete fungus. We described that septins in U. maydis could assemble into at least three different structures coexisting in the same cell: bud neck collars, band-like structures at the growing tip, and long septin fibers that run from pole to pole near the cell cortex. We also found that in the absence of septins, U. maydis cells lost their elongated shape, became wider at the central region and ended up losing their polarity, pointing to an important role of septins in the morphogenesis of this fungus. These morphological defects were alleviated in the presence of an osmotic stabilizer suggesting that absence of septins affected the proper formation of the cell wall, which was coherent with a higher sensitivity of septin defective cells to drugs that affect cell wall construction as well as exocytosis. As U. maydis is a phytopathogen, we analyzed the role of septins in virulence and found that in spite of the described morphological defects, septin mutants were virulent in corn plants.

Conclusions/Significance

Our results indicated a major role of septins in morphogenesis in U. maydis. However, in contrast to studies in other fungal pathogens, in which septins were reported to be necessary during the infection process, we found a minor role of septins during corn infection by U. maydis.     

Chitinases of filamentous fungi: a large group of diverse proteins with multiple physiological functions

Chitin is the second most abundant natural biopolymer and the main structural component of invertebrate exoskeletons and cell walls of filamentous fungi. Fungal chitinases have multiple physiological functions including the degradation of exogenous chitin and cell wall remodelling during hyphal growth, but the regulation of the chitinolytic systems of filamentous fungi is not well understood. Fungi have on average between 10 and 25 different chitinases, but only the increasing number of fungal genome sequencing projects in the last few years has enabled us to assess the whole range and diversity of fungal chitinases. In this review the variety, domain architecture and subgroups of chitinases of filamentous fungi are shown, and how these data integrate with that from molecular biological studies on chitinases are discussed.     

Killer toxin-like chitinases in filamentous fungi: Structure,regulation and potential roles in fungal biology

Fungal chitinases are hydrolytic enzymes responsible for degradation of chitin. Chitinases are involved in several aspects of fungal biology, including cell wall remodelling during hyphal growth, conidial germination, autolysis, mycoparasitism and nutrient acquisition. They are divided into three distinct phylogenetic groups; A, B and C. Chitinases from the C group show structural similarities with the killer toxin zymocin produced by the yeast Kluyveromyces lactis and it is speculated that they have a similar function in filamentous ascomycetes, by facilitating penetration of toxins into cells of competing individuals. Genome analyses show that certain fungal species with a mycoparasitic lifestyle contain high numbers of killer toxin-like chitinases, compared with specialized saprotrophs and plant pathogens. Recent developments within this research field have revealed considerable variation in the modular structure and regulation of killer toxin-like chitinases, suggesting more diverse roles than merely fungal-fungal interactions. In this review, we summarize the current knowledge about this intriguing class of chitinases, including their modular structure, evolution, gene regulation, and functional analyses in mycoparasitic as well as in saprotrophic species. We also propose important questions for future research.     

Retroactive Maintains Cuticle Integrity by Promoting the Trafficking of Knickkopf into the Procuticle of Tribolium castaneum

Molting, or the replacement of the old exoskeleton with a new cuticle, is a complex developmental process that all insects must undergo to allow unhindered growth and development. Prior to each molt, the developing new cuticle must resist the actions of potent chitinolytic enzymes that degrade the overlying old cuticle. We recently disproved the classical dogma that a physical barrier prevents chitinases from accessing the new cuticle and showed that the chitin-binding protein Knickkopf (Knk) protects the new cuticle from degradation. Here we demonstrate that, in Tribolium castaneum, the protein Retroactive (TcRtv) is an essential mediator of this protective effect of Knk. TcRtv localizes within epidermal cells and specifically confers protection to the new cuticle against chitinases by facilitating the trafficking of TcKnk into the procuticle. Down-regulation of TcRtv resulted in entrapment of TcKnk within the epidermal cells and caused molting defects and lethality in all stages of insect growth, consistent with the loss of TcKnk function. Given the ubiquity of Rtv and Knk orthologs in arthropods, we propose that this mechanism of new cuticle protection is conserved throughout the phylum.     

Dimorphism inBenjaminiella poitrasii: Involvement of intracellular endochitinase andN-acetylglucosaminidase activities in the yeast-mycelium transition

The chitinase and N-acetylglucosaminidase activities in cell-wall-bound and free fractions in the dimorphic fungus Benjaminiella poitrasii were studied as a function of morphological (unicellular yeast-mycelium) transition. The specific activities of chitinases of cell-wall-free, particularly in the membrane fraction, were significantly different in the yeast and mycelial forms. During the yeast-mycelium transition, the N-acetylglucosaminidase activity isolated in a membrane preparation increased steadily. The activity of the yeast cells (0.83 +/- 0.17 nkat/mg protein) increased 17-fold to 14.2 +/- 1.7 nkat/mg protein in 1-d-old mycelial cells. The endochitinase activity increased 12-fold between 6 and 12 h and thereafter practically remained unchanged up to 24 h. A reverse trend in the chitinolytic activities was observed during the mycelium-yeast transition. Isoelectrofocussing (pH range 3.5-10) of mixed membrane fraction free of particulate fraction of parent and morphological (Y-5, yeast-form) mutant cells separated endochitinase and N-acetylglucosaminidase activity into two pH ranges, viz. 4.3-5.7 and 6.1-7.7, respectively. The predominant N-acetylglucosaminidase activity observed at pH 6.9 and 7.1 for the parent strain membrane fraction was undetected in the mutant preparation. The results suggested that the membrane-bound (either tightly or loosely) chitinolytic enzymes, particularly, N-acetylglucosaminidase, significantly contributed to the morphological changes in B. poitrasii.     

Evidence Supporting a Role for Mammalian Chitinases in Efficacy of Caspofungin against Experimental Aspergillosis in Immunocompromised Rats

Objectives

Caspofungin, currently used as salvage therapy for invasive pulmonary aspergillosis (IPA), strangely only causes morphological changes in fungal growth in vitro but does not inhibit the growth. In vivo it has good efficacy. Therefore the question arises how this in vivo activity is reached. Caspofungin is known to increase the amount of chitin in the fungal cell wall. Mammals produce two chitinases, chitotriosidase and AMCase, which can hydrolyse chitin. We hypothesized that the mammalian chitinases play a role in the in vivo efficacy of caspofungin.

Methods

In order to determine the role of chitotriosidase and AMCase in IPA, both chitinases were measured in rats which did or did not receive caspofungin treatment. In order to understand the role of each chitinase in the breakdown of the caspofungin-exposed cells, we also exposed caspofungin treated fungi to recombinant enzymes in vitro.

Results

IPA in immunocompromised rats caused a dramatic increase in chitinase activity. This increase in chitinase activity was still noted when rats were treated with caspofungin. In vitro, it was demonstrated that the action of both chitinases were needed to lyse the fungal cell wall upon caspofungin exposure.

Conclusion

Caspofungin seemed to alter the cell wall in such a way that the two chitinases, when combined, could lyse the fungal cell wall and assisted in clearing the fungal pathogen. We also found that both chitinases combined had a direct effect on the fungus in vitro.     

Synthesis of Extracellular Chitinase by Wild-Type B-10 and Mutant M-1 Strains of Serratia marcescens

Molecular weights of extracellular chitinases from wild-type B-10 (62, 54, 43, 38, and 21 kDa) and mutant M-1 strains of Serratia marcescens (62, 52, 43, 38, and 21 kDa) were estimated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. In the absence of chitin inductors, chitinolytic enzymes were not found in the culture liquid of B-10, whereas M-1 cells produced the chitinase complex (to 470 pU/cell). Crystalline chitin insignificantly stimulated the synthesis of chitinases with molecular weights of 62, 54, and 21 kDa by B-10 (up to 20 pU/cell), but caused oversynthesis of all chitinases by the mutant strain (up to 2600 pU/cell). Colloidal chitin induced the production of chitinases by cells of both strains. Two peaks of chitinolytic activity were observed during cultivation of strains B-10 (350 and 450 pU/cell) and M-1 (2200 and 2400 pU/cell). The first peak of cell productivity was associated with biosynthesis of the chitinase complex. The second peak was related to the synthesis of enzymes with molecular weights of 54, 43, 38, and 21 kDa (B-10) or 43, 38, and 21 kDa (M-1).     

Multiple components and induction mechanism of the chitinolytic system of the hyperthermophilic archaeon <Emphasis Type="Italic">Thermococcus chitonophagus</Emphasis>

Thermococcus chitonophagus produces several, cellular and extracellular chitinolytic enzymes following induction with various types of chitin and chitin oligomers, as well as cellulose. Factors affecting the anaerobic culture of this archaeon, such as optimal temperature, agitation speed and type of chitin, were investigated. A series of chitinases, co-isolated with the major, cell membrane-associated endochitinase (Chi70), and a periplasmic chitobiase (Chi90) were subsequently isolated. In addition, a distinct chitinolytic activity was detected in the culture supernatant and partially purified. This enzyme exhibited an apparent molecular mass of 50 kDa (Chi50) and was optimally active at 80°C and pH 6.0. Chi50 was classified as an exochitinase based on its ability to release chitobiose as the exclusive hydrolysis product of colloidal chitin. A multi-component enzymatic apparatus, consisting of an extracellular exochitinase (Chi50), a periplasmic chitobiase (Chi90) and at least one cell-membrane-anchored endochitinase (Chi70), seems to be sufficient for effective synergistic in vivo degradation of chitin. Induction with chitin stimulates the coordinated expression of a combination of chitinolytic enzymes exhibiting different specificities for polymeric chitin and its degradation products. Among all investigated potential inducers and nutrient substrates, colloidal chitin was the strongest inducer of chitinase synthesis, whereas the highest growth rate was obtained following the addition of yeast extract and/or peptone to the minimal, mineralic culture medium in the absence of chitin. In rich medium, chitin monomer acted as a repressor of total chitinolytic activity, indicating the presence of a negative feedback regulatory mechanism. Despite the undisputable fact that the multi-component chitinolytic system of this archaeon is strongly induced by chitin, it is clear that, even in the absence of any chitinous substrates, there is low-level, basal, constitutive production of chitinolytic enzymes, which can be attributed to the presence of traces of chito-oligosaccharides and other structurally related molecules (in the undefined, rich, non-inducing medium) that act as potential inducers of chitinolytic activity. The low, basal and constitutive levels of chitinase gene expression may be sufficient to initiate chitin degradation and to release soluble oligomers, which, in turn, induce chitinase synthesis.     

Cross Talk between NDR Kinase Pathways Coordinates Cytokinesis with Cell Separation in Schizosaccharomyces pombe

NDR (nuclear Dbf-2-related) kinases constitute key regulatory nodes in signaling networks that control multiple biological processes such as growth, proliferation, mitotic exit, morphogenesis, and apoptosis. Two NDR pathways called the septation initiation network (SIN) and the morphogenesis Orb6 network (MOR) exist in the fission yeast Schizosaccharomyces pombe. The SIN promotes cytokinesis, and the MOR drives cell separation at the end of cytokinesis and polarized growth during interphase. We showed previously that cross talk exists between these two pathways, with the SIN inhibiting the MOR during cytokinesis through phosphorylation of the MOR component Nak1 by the SIN Sid2 kinase. The reason for this inhibition remained uncertain. We show here that failure to inhibit MOR signaling during cytokinesis results in cell lysis at the site of septum formation. Time-lapse analysis revealed that MOR signaling during cytokinesis causes cells to prematurely initiate septum degradation/cell separation. The cell lysis phenotype is due to premature initiation of cell separation because it can be rescued by mutations in genes required for cell separation/septum degradation. We also shed further light on how the SIN inhibits the MOR. Sid2 phosphorylation of the MOR proteins Sog2 and Nak1 is required to prevent cell lysis during cytokinesis. Together, these results show that SIN inhibition of the MOR enforces proper temporal ordering of cytokinetic events.     

Comparative studies of chitinases A,B and C from Serratia marcescens

Serratia marcescens produces three chitinases, ChiA, ChiB and ChiC which together enable the bacterium to efficiently degrade the insoluble chitin polymer. We present an overview of the structural properties of these enzymes, as well as an analysis of their activities towards artificial chromogenic chito-oligosaccharide-based substrates, chito-oligosaccharides, chitin and chitosan. We also present comparative inhibition data for the pseudotrisaccharide allosamidin (an analogue of the reaction intermediate) and the cyclic pentapeptide argadin. The results show that the enzymes differ in terms of their subsite architecture and their efficiency towards chitinous substrates. The idea that the three chitinases play different roles during chitin degradation was confirmed by the synergistic effects that were observed for certain combinations of the enzymes. Studies of the degradation of the soluble heteropolymer chitosan provided insight into processivity. Taken together, the available data for Serratia chitinases show that the chitinolytic machinery of this bacterium consists of two processive exo-enzymes that degrade the chitin chains in opposite directions (ChiA and ChiB) and a non-processive endo-enzyme, ChiC.     

Genomic comparison of chitinolytic enzyme systems from terrestrial and aquatic bacteria

Chitin degradation ability is known for many aquatic and terrestrial bacterial species. However, differences in the composition of chitin resources between aquatic (mainly exoskeletons of crustaceans) and terrestrial (mainly fungal cell walls) habitats may have resulted in adaptation of chitinolytic enzyme systems to the prevalent resources. We screened publicly available terrestrial and aquatic chitinase‐containing bacterial genomes for possible differences in the composition of their chitinolytic enzyme systems. The results show significant differences between terrestrial and aquatic bacterial genomes in the modular composition of chitinases (i.e. presence of different types of carbohydrate binding modules). Terrestrial Actinobacteria appear to be best adapted to use a wide variety of chitin resources as they have the highest number of chitinase genes, the highest diversity of associated carbohydrate‐binding modules and the highest number of CBM33‐type lytic polysaccharide monooxygenases. A ctinobacteria do also have the highest fraction of genomes containing β‐1, 3‐glucanases, enzymes that may reinforce the potential for degrading fungal cell walls. The fraction of bacterial chitinase‐containing genomes encoding polyketide synthases was much higher for terrestrial bacteria than for aquatic ones supporting the idea that the combined production of antibiotics and cell‐wall degrading chitinases can be an important strategy in antagonistic interactions with fungi.     

A novel strain of Brevibacillus laterosporus produces chitinases that contribute to its biocontrol potential

A novel strain exhibiting entomopathogenic and chitinolytic activity was isolated from mangrove marsh soil in India. The isolate was identified as Brevibacillus laterosporus by phenotypic characterization and 16S rRNA sequencing and designated Lak1210. When grown in the presence of colloidal chitin as the sole carbon source, the isolate produced extracellular chitinases. Chitinase activity was inhibited by allosamidin indicating that the enzymes belong to the family 18 chitinases. The chitinases were purified by ammonium sulfate precipitation followed by chitin affinity chromatography yielding chitinases and chitinase fragments with 90, 75, 70, 55, 45, and 25 kDa masses. Mass spectrometric analyses of tryptic fragments showed that these fragments belong to two distinct chitinases that are almost identical to two putative chitinases, a 89.6-kDa four-domain chitodextrinase and a 69.4-kDa two-domain enzyme called ChiA1, that are encoded on the recently sequenced genome of B. laterosporus LMG15441. The chitinase mixture showed two pH optima, at 6.0 and 8.0, and an optimum temperature of 70 °C. The enzymes exhibited antifungal activity against the phytopathogenic fungus Fusarium equiseti. Insect toxicity bioassays with larvae of diamondback moths (Plutella xylostella), showed that addition of chitinases reduced the time to reach 50 % mortality upon infection with non-induced B. laterosporus from 3.3 to 2.1 days. This study provides evidence for the presence of inducible, extracellular chitinolytic enzymes in B. laterosporus that contribute to the strain’s antifungal activity and insecticidal activity.     

Extracellular chitinase production by wild-type B-10 and mutant M-1 strains of Serratia marcescens

Molecular weights of extracellular chitinases from wild-type B-10 (62, 54, 43, 38, and 21 kDa) and mutant M-1 strains of Serratia marcescens (62, 52, 43, 38, and 21 kDa) were estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the absence of chitin inductors, chitinolytic enzymes were not found in the culture liquid of B-10, while M-10 cells produced the chitinase complex (to 470 pU/cell). Crystalline chitin insignificantly stimulated the synthesis of chitinases with molecular weights of 62, 54, and 21 kDa by B-10 (up to 20 pU/cell), but caused overproduction of all chitinases by the mutant strain (up to 2600 pU/cell). Colloidal chitin induced the production of chitinases by cells of both strains. Two peaks of chitinolytic activity were observed during cultivation of strains B-10 (350 and 450 pU/cell) and M-1 (2200 and 2400 pU/cell). The first peak of cell productivity was associated with biosynthesis of the chitinase complex. The second peak was related to the production of enzymes with molecular weights of 54, 43, 38, and 21 kDa (B-10) or 43, 38, and 21 kDa (M-1).     

Inhibitory activity of 1-farnesylpyridinium on the spatial control over the assembly of cell wall polysaccharides in Schizosaccharomyces pombe

The modes of actions of 1-farnesylpyridinium (FPy) on yeast cell growth were investigated on the basis of its effects on cell cycle progression, morphogenesis and the related events for construction of cell wall architecture in Schizosacchromyces pombe. FPy predominantly inhibited the growth of the yeast cells after various cycles of cell division so that cells were arrested at the phase of separation into daughter cells accompanying morphological changes to swollen spherical cells at 24 h of incubation. FPy-treated cells were osmotically stable but were susceptible to the lytic action of (1, 3) beta-D-glucanases, and characterized by serious damages to the cell wall architecture as represented by a rough and irregular surface outlook. The isolated cell wall fraction gave a similar hexose composition with or without FPy treatment, suggesting that FPy did not inhibit the synthesis of each cell wall polysaccharide. FPy was permissive for the extracellular accumulation of amorphous cell wall materials and septum development in protoplasts, but absolutely interfered with the following morphogenetic process for construction of the rod-shaped cell wall architecture. Our results suggest the inhibitory activity of FPy on the spatial control over the assembly of cell wall polysaccharides.     

Comparative studies of chitinases A, B and C from Serratia marcescens

Serratia marcescens produces three chitinases, ChiA, ChiB and ChiC which together enable the bacterium to efficiently degrade the insoluble chitin polymer. We present an overview of the structural properties of these enzymes, as well as an analysis of their activities towards artificial chromogenic chito-oligosaccharide-based substrates, chito-oligosaccharides, chitin and chitosan. We also present comparative inhibition data for the pseudotrisaccharide allosamidin (an analogue of the reaction intermediate) and the cyclic pentapeptide argadin. The results show that the enzymes differ in terms of their subsite architecture and their efficiency towards chitinous substrates. The idea that the three chitinases play different roles during chitin degradation was confirmed by the synergistic effects that were observed for certain combinations of the enzymes. Studies of the degradation of the soluble heteropolymer chitosan provided insight into processivity. Taken together, the available data for Serratia chitinases show that the chitinolytic machinery of this bacterium consists of two processive exo-enzymes that degrade the chitin chains in opposite directions (ChiA and ChiB) and a non-processive endo-enzyme, ChiC.     

Monitoring of in planta gene expression for xylan degradation and assimilation in the maize pathogen Bipolaris maydis

Swift and efficient onset of feeding on host tissue by phytopathogenic fungi is a requisite event for their successful infection and propagation. Necrotrophic fungi colonizing host cell walls appear to obtain carbon and energy sources from plant wall degradants, but what they actually utilize for nutrition after host invasion remains unclear. Here we focus on plant wall xylan, the major hemicellulosic polysaccharide in cereal plants, and study its participation in post-invasion nutrition of the maize necrotrophic pathogen Bipolaris maydis (syn: Cochliobolus heterostrophus). Using a fluorescence reporter assay, we demonstrated that a B. maydis β-xylosidase gene, BmXyp1, is strongly upregulated at the beginning of infection, specifically within invading hyphae. Additionally, our time-course measurements of mRNA expression during maize infection revealed that xylan degradation and assimilation are concomitantly induced during an early infection stage. These findings suggest that this fungus can access xylan degradants as an early in planta nutrient source after host penetration; however, mutant strains deficient in xylan-assimilation ability still retained virulence, although the lesion size was decreased as compared with the wild-type strain. Overall, we conclude that xylan degradation and assimilation by B. maydis are initial post-invasion events but do not play an essential role in fungal nutrient acquisition.     

Polar localizing class V myosin chitin synthases are essential during early plant infection in the plant pathogenic fungus Ustilago maydis

Fungal chitin synthases (CHSs) form fibers of the cell wall and are crucial for substrate invasion and pathogenicity. Filamentous fungi contain up to 10 CHSs, which might reflect redundant functions or the complex biology of these fungi. Here, we investigate the complete repertoire of eight CHSs in the dimorphic plant pathogen Ustilago maydis. We demonstrate that all CHSs are expressed in yeast cells and hyphae. Green fluorescent protein (GFP) fusions to all CHSs localize to septa, whereas Chs5-GFP, Chs6-GFP, Chs7-yellow fluorescent protein (YFP), and Myosin chitin synthase1 (Mcs1)-YFP were found at growth regions of yeast-like cells and hyphae, indicating that they participate in tip growth. However, only the class IV CHS genes chs7 and chs5 are crucial for shaping yeast cells and hyphae ex planta. Although most CHS mutants were attenuated in plant pathogenicity, Deltachs6, Deltachs7, and Deltamcs1 mutants were drastically reduced in virulence. Deltamcs1 showed no morphological defects in hyphae, but Mcs1 became essential during invasion of the plant epidermis. Deltamcs1 hyphae entered the plant but immediately lost growth polarity and formed large aggregates of spherical cells. Our data show that the polar class IV CHSs are essential for morphogenesis ex planta, whereas the class V myosin-CHS is essential during plant infection.