Cytochemistry, ultrastructure and x-ray microanalysis methods applied to cell wall characterization of Mucoralean fungi strains

G. M. Campos-Takaki1, S.M.C. Dietrich2,† and G. W. Beakes3 1 NPCIAMB-Nucleus of Research in Environmental Sciences and Biotechnology, Catholic University of Pernambuco-

UNICAP, 50050-590 Recife, Pernambuco, Brazil 2 Botanical Institute of São Paulo, 04301-902 São Paulo, Brazil (†) 3 School of Biology, Newcastle University, Newcastle upon Tyne, NE1 7RU, United Kingdom

Studies on mucoralean fungi encompass reporting on their biochemistry composition, identifying ultrastructural characteristics, and conducting X-ray micro-analysis on cell-wall (CW) microelements. Mechanically isolated, cytoplasm-free CWs of Absidia blaskeleana, Gongronella butleri, Rhizopus arhizus, Mucor javanicus, Cunninghamella elegans, and Syncephalastrum racemosum were analyzed quantitatively and qualitatively by biochemically determining proteins, carbohydrates, lipids, and polyphosphate. Calcium and phosphorus were associated with intact (PTH) and treated with chitinase and pronase CWs, and an X-ray microprobe analysis was applied. The fatty acids and amino acids from lipid and protein compositions in the CWs were analyzed. The association of biochemical studies with ultrastructural data and elemental analysis made it possible to propose an organization model for the cell-wall structure of hyphal Mucoralean fungi.

Keywords: Zygomycetes; Mucoralean fungi; cell-walls; organization model

1. Introduction

The kingdom of fungi is divided into four subgroups, phyla, which are chytridiomycota, zygomycota, ascomycota, and basidiomycota [1]. Among these phyla, zygomycota is the most varied and least studied one and appears to be polyphyletic. It consists of two fungal subphyla, trichomycetes and zygomycetes[2]. Zygomycetes mostly live in soil and dung, and are known as fast growers which are able to form sporangiospores (or conidia) and under appropriate circumstances, include 10 orders. Zygomycetes fungi have been used for several hundred years to produce fermented food, mostly in south Asia, tempe and tofu. Tempe is a soya bean cake covered with filamentous zygomycetes growth, with a high protein content (40-50%) and containing different essential amino acids, while tofu is produced by fermenting soya milk with some strains of zygomycetes fungi [1,3]. The Mucorales order is ubiquitous, consists predominantly of saprobic soil organisms characterized by a usually abundant, rapidly growing mycelium as well as anamorphic structures usually formed in large quantities, and is known to include pathogenic species. However, some species of Mucorales have a doubtful pathogenicity (including an annotated list of species not accepted as pathogenic). The mycelium is typically unseptate or irregularly septate. Anamorphic sporangiospores are produced in multi-spored sporangia, few-spored sporangiola or merosporangia. Chlamydospores, arthrospores and yeast cells are, in the most species, rarely formed. Sporangia are characterized by the inclusion of a variously shaped columela [3,4,5]. The fungal cell wall is a shield that protects the cells against changes in the extracellular environment, and these protective attributes are associated with the composition and strength of the biochemical cell wall, but at the same time, the wall has to be plastic and dynamic to allow cell growth and communication with the external environment [6,7]. The fungal cell wall (CW) is an essential organelle and represents a considerable metabolic investment. In addition, Mucorales fungi also are used for diverse biotechnological applications, including biological transformations, as well as the biotechnological production of bioactive molecules such as enzymes, chitin and chitosan. Their macromolecular composition, molecular organization and thickness can vary greatly depending on environmental conditions. The main components of the Mucoralean cell wall are carbohydrates, proteins, lipids, and polyphosphate [8,9,10]. In this review we report our current results on understanding the structural organization of fungal cell walls which focus on the biochemical composition of the cell-walls of Absidia blaskeleana, Gongronella bulteri, Cunninghamella elegans, Rhizopus arrhizus, Mucor javanicus and Syncephalastrum racemosum with regard to the characteristics of the microfibillar fractions of chitin and the localization of polyphosphate by cytochemistry and X-ray micro-analysis methods.

2. Chemical composition of the cell-walls of Mucoralen fungi

The rapid development of Mucorales fungi cultivation and the biological industry using mycelial fungi as producers of biologically compounds have intensified scientific interest in fungal CWs. Fundamental studies have been undertaken so as to obtain data on the biochemical composition of the cell wall (CW) of mycelial mucoralean fungi grown on modified Hesseltine & Anderson medium [11]. The mechanical isolation of CW was obtained by disintegrating cells

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ultrasonically at 5,000 g, using the method described by Letorneau et al.[7] at 50C, followed by centrifugation for 15 min. The residue was resuspended in cold deionized water (10g/125mL), followed by intermittent shaking by ultrasonic probe (20 KHz) for 60 min and centrifugation under the same conditions. The CW was washed with cold deionized water five times, and then with cold NaCl (0.85%w/v), followed by dialysis against deionized water at 50C [12]. Cytoplasmic-free calcium levels were obtained by optical microscopy examination according to Dietrich [13]. It should be emphasized that isolating the pure CW fraction is a very important procedure, the outcome of which determines the results obtained subsequently with regard to analyzing its chemical composition. The chemical composition of the CWs of mucoralean fungi of Absidia blaskeleana, Gongronella bulteri, Cunninghamella elegans, Rhizopus arrhizus, Mucor javanicus, and Syncephalastrum racemosum were analyzed quantitatively and qualitatively by the biochemical determination of proteins, carbohydrates, polyphosphate, and lipids (Table 1). The biochemical analysis of CWs revealed that polysaccharides make up their highest content (around 40-50%). The structure of the polysaccharides chitin and chitosan, and neutral sugars and uronic acid were observed in all species. The results obtained suggest the presence of D-mucoroic acid, as well as protein, and total lipids in all species. However, lower results were obtained for polyphosphate contents in Mucor javanicus and Syncephalastrum racemosum and these are corroborated in the literature [4,7,14].

Table 1 Biochemical composition of the isolated cell-walls of Mucoralean strains

Mucoralean Fungi

Biochemical composition of Cell-walls (%) Chitin

Chitosan Neutral

sugars, uronic acid

Protein Lipids Poly phosphate

Absidia blaskeleana 15.5 27.5 20.4 4.1 5.0 23.7 Gongronella butleri 13.4 28.0 16.5 4.2 14.1 18.7 Rhizopus arrhizus 15.0 28.0 13.9 10.2 8.6 18.6 Mucor javanicus 10.0 28.0 14.8 13.1 18.8 12.6 Cunninghamella elegans

10.8 28.2 22.0 6.9 7.6 22.7

Syncephalastrum racemosum

11.5 26.0 22.6 10.1 6.4 8.6

Analysis of the fatty acid composition of the Mucorales fungi CWs showed that those with the highest amounts were: palmitic (C16:0), oleic (C18:0), linoleic (C18:1), linolenic (C18:2) and arachidonic (C18:3) (Table 2). Polyunsaturated fatty acids (PUFA) are a family of lipids including some subgroups identified by the position of the last double bond in their structure. PUFA can be used as chemotaxonomic and phylogenetic markers in Zygomycetes [15]. The total suppression of colony forming and cell proliferation occurred at high levels of polyunsaturated fatty acid supplementation, in addition to which this contributes to normalizing triglyceride (TG) levels in patients with combined hyperlipidemia [16]. Additionally, considerable interest has been shown in the potential anti-inflammatory effects of PUFAs in multiple sclerosis (MS) and other autoimmune inflammatory disorders [17]. The results reported in this study were found to be promising for developing an economical process in the pharmaceutical industry as to producing PUFA by Absidia blaskeleana, Cunninghamella elegans, Rhizopus arrhizus and Mucor javanicus. Palmitic acid has many functions in cosmetics, from detergent cleansing agent to emollient. Thus, n−3 PUFAs are potentially potent antiinflammatory agents. As such, they may be of therapeutic use in a variety of acute and chronic inflammatory settings.

Table 2 Fatty acid composition of the cell-walls of Mucoralean strains

Mucoralean Fungi

Fatty acid composition (%) C 12:0 C14:0 C15:0 C16:0 C16:1 C17:0 C18:0 C18:1 C18:2 C18:3

Absidia blaskeleana 1.3 0.6 0.1 11.8 0.6 0.0 23.4 50.7 8.7 2.8 Gongronella butleri 0.3 1.8 0.2 31.9 1.6 0.0 6.1 48.4 5.8 3.9 Rhizopus arrhizus 0.0 3.8 8.6 8.4 4.4 9.9 31.1 15.9 16.0 22.9 Mucor javanicus 0.0 4.4 2.0 19.3 2.8 0.0 7.3 37.3 13.2 13.7 Cunninghamella elegans

0.0 0.8 0.7 19.6 3.4 - 6.3 59.4 6.4 3.4

Syncephalastrum racemosum

1.4 1.3 0.3 25.6 2.0 0.0 4.2 55.1 5.9 4.2

In eukaryotes, cell walls are a diverse set of organelles, but in general they consist of a fibrillar component (protein or polysaccharide) and a cross-linked amorphous matrix (usually glycoprotein). The fungal CW is an essential organelle and represents a considerable metabolic investment. Its macromolecular composition, molecular organization and thickness can vary greatly depending on environmental conditions. Most of the CW proteins are glycoproteins modified

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by a glycolipid and/or oligosaccharides covalently attached to asparagine (N-linked glycosylation) or serine/threonine residues (O-linked glycosylation). These wall proteins play important roles in the integrity and structure of the cell wall, as they sense changes in the extracellular environment, and some of them have adhesive properties and hydrolytic activities [18]. Table 3 shows the amino acid composition of the protein extracted from the CW of all strains of Mucoralean fungi. Similar content was observed for Lysine, Aspartic acid, Glutamic acid, Alanine, and Lucine. However, a lower Glycine content was observed for A. blaskeleana and M. javanicus. All strains showed the presence of essential amino acids [14]. The composition of these amino acids in the CW play a significant role in removal environmental pollutants, and their potential cellular mechanisms may be involved in the process for detoxifying heavy metals and thus in tolerance to metal stress [19]. Table 3 The composition of amino acids of the protein from cell-walls of Mucoralean strains

Composition of

Amino Acids (%)

Mucoralean Fungi A.

blaskeleana

G. butleri

R. arrhizus M.

javanicus C.

elegans S.

racemosum Lysine 8.44 6.66 8.00 8.54 8.00 7.30 Histidine 3.80 4.54 3.49 4.14 5.00 4.20 Arginine 4.90 4.42 5.00 6.36 4.30 4.00 Aspartic acid 10.97 10.30 10.07 11.00 11.20 9.12 Threonine 5.90 7.20 5.85 5.91 4.20 5.11 Serine 6.85 7.86 6.77 5.62 6.80 6.13 Glutamic acid 11.90 9.71 11.30 13.42 10.80 9.44 Proline 5.02 7.20 4.46 3.99 3.00 4.07 Glycine 4.11 9.36 8.03 5.29 8.60 8.70 Alanine 9.20 10.20 8.53 6.73 7.40 7.90 Valine 6.53 6.80 6.05 6.83 6.80 5.28 Methionine 1.26 1.60 1.26 2.49 ND 10.10 Isoleucin 5.43 4.75 5.58 5.88 6.40 4.65 Leucine 8.35 9.40 7.93 9.00 9.00 8.09 Tyrosine 3.27 ND 3.47 ND 4.20 2.65 Phenylalanine 4.07 ND 4.21 4.80 4.30 3.26

ND= Not detected

3. Ultrastructural aspects of polyphosphate (poly-P)

The inorganic polyphosphate (polyP) has a significant role in increasing cell resistance to unfavorable environmental conditions and in regulating different biochemical processes. The chain of polyP consists of tens or many hundreds of phosphate (Pi) residues linked by high-energy phosphoanhydride bonds[20]. The literature suggests that polyphosphate performs numerous and varied biological functions depending on the need and its location in a specie, cell or subcellular compartment, such as being an energy reserve, a chelator of metals, a buffer against alkali, involved in cell envelope formation and function, gene activity control, disposal of pollulant phosphate, and as a substrate for glucose phosphorylation [21,21,22,23]. In addition, the most valuable biological effect of polyphosphate is in its physiological adjustment to growth, development, stress response and nutrient deprivation and its minimizing of the toxic effects of heavy metals, nucleic acid and phospholipid metabolism [24]. Ultrastructural cytochemistry was successfully first used to identify the localization and distribution of polyphosphate in the mycelium of Mucoralean fungi [10,14]. In 2002, Sharia’a et al. [25] using the ultrastructural cytochemistry method described by Campos-Takaki et al. [10], found the localization and distribution of polyphosphate for Absidia cylindrospora, Gongronella butleri and Mucor javanicus. The authors showed that the Zygomycetes studied exhibited a cytochemical polyphosphate labeling in different structures as well as different amounts of this depending on the specie. Werner et al. [25] described a novel method for the specific quantification and visualization of poly P in fungal cell walls. A selective extraction in a high salt buffer revealed large poly P stores in cell walls of Mucorales and lower amounts in most other fungi tested. They suggest that the presence of an extracellular phosphate pool in the form of a strongly negatively charged polymer has important functions as a phosphate source in mycorrhizal interactions, as an antimicrobial compound or as protection against the toxicity of heavy metals. Recently, Lima et al. [27] observed cadmium induced vacuolization, the presence of electron dense deposits in vacuoles, cytoplasm and cell membranes, as well as the distinct behavior of polyphosphate fractions. The authors suggested that precipitation, vacuolization and polyphosphate fractions by C. elegans were associated with cadmium tolerance, and that this species demonstrated a higher potential for bioremediation of heavy metals. Alternatively, it is possible these mechanisms are related to the accumulation/degradation of polyphosphate as a detoxification process.

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Fig. 1 Cytochemical localization of polyphosphate in the cell walls of Absidia blaskeleana; Gongronella bulteri; Mucor javanicus; Rhizopus arrhizus G- Cunninghamella elegans and Syncephalastrum racemosum using the method described by Campos-Takaki et al. [10,14].

4. Characterization of microfibrillar chitin fraction from Mucoralean fungi

In recent years, there has been an increasing interest in biopolymers from fungi, specially chitin and chitosan, due to the fact they are easy to obtain, their wide applicability and their promising features such as their absence of toxicity, biodegradability, biocompatibility and environmentally friendly nature and their wide range of potential industrial applications [28]. Chitin is a structural polysaccharide widely found in nature. It occurs as highly ordered microfibrils in many species such as yeast, fungi, insects, and marine invertebrates. Chitin is a homopolymer of 1-4 linked 2-acetamido-2-deoxy-β-D-glucopyranose, although some of the glucopyranose residues are deacetylated and occur as 2-amino-2-deoxy-β-D-

glucopyranose [29,30]. Chitosan is naturally found in the cell wall of fungi, mainly in Zygomycetes. Chitosan is formed by the deacetylation of chitin, and the N-acetyl group can undergo several degrees of deacetylation, and consequently chitosan is widely used in the pharmaceutical, food, agriculture, cosmetics and textile industries [31,32]. Studies on Mucoralean fungi encompass identifying the ultrastructural characteristics of isolated microfibrilles of chitin from CW of Absidia blaskeleana, Gongronella bulteri, M. javanicus, R. arrhizus, Cunninghamella elegans and Syncephalastrum racemosum fungi (Figure 2). The microfibrillar fraction chitin from Mucoralean fungi was obtained using the method described by Hawes [28], and consisted of mixing glacial acetic acid and hydrogen peroxide (1:1 v/v) at 1000C, for 5 hours. The structure of the hyphal CWs microfibrils was investigated by electron microscopy of shadowed replicas with a mixture of gold and paladium (60:40) in a coating unit, and observed in a scanning electron microscope. The ultrastructural data with an isolate microfibrillar fraction mainly consisted of chitin and was organized into two distinct layers; an outer, thicker layer of randomly oriented microfibrils, and an inner, thin layer of parallel microfibrils in all fungi studied. Chitin-rich fungal cell wall material was used as both a source of microbial N and C; N was predominantly assimilated and C was predominantly metabolized. It is possible that fungal necromass may contribute to N stabilization in these soils. Fragments from chitin and chitosan are known to have eliciting activities leading to a variety of defense responses in host plants in response to microbial infections, including the accumulation of phytoalexins, pathogen-related (PR) proteins and proteinase inhibitors, lignin synthesis, and callose formation. Both chitin and chitosan have demonstrated antiviral, antibacterial, and antifungal properties, and have been explored for many agricultural uses [34]. Recent developments in fungal effectors have raised several questions regarding the interaction that chitin may have with certain secreted proteins and effectors. Many described fungal effectors are cysteine-rich proteins that are often secreted and play a role in virulence. These two proteins are inhibitors of plant cysteine proteases and help protect chitin and the integrity of fungal cell walls against plant chitinases [35,36]. In addition the co-polymers the successful extraction of chitosan and chitin from their CW of Mucoralean fungi it has remain the polymer of first choice for various in their value added medical applications in fields like pharmacodynamic, pharmaceutical & bio-pharmaceutical [37,38].

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Fig. 2 Distribution of microfibrilles of chitin isolated from CWs of Absidia blaskeleana (A); Gongronella bulteri (B); Mucor javanicus (C); Rhizopus arrhizus (D) Cunninghamella elegans (E) and Syncephalastrum racemosum (F).

5. X-Ray analysis of microelements in CWs of Mucoralean fungi

X-Ray microanalyses were made by transmission electron microprobe and the microfibrillar chitin fractions were analyzed in a 1. Linear scan profile and multilinear scan images of X-ray emissions for microelements of magnesium, calcium, phosphorus, chlorine, potassium, and sulphur were compared using the equation described by Appleton [33]. A microprobe analysis using mechanically isolated, cytoplasm-free CW of Absidia blaskeleana, Gongronella bulteri, Cunninghamella elegans, M. javanicus, R. arrhizus, and Syncephalastrum racemosum

was conducted by transmission electron microscopy and by using the X-ray microprobe analysis and diffraction described by Campos-Takaki et al. [10]. Self-organization in CW Mucoralean fungi was found the first time by X-ray microanalysis in microfibrillar fraction chitin associated with microelements of Mg, P, S, Cl, K, and Ca. All CWs showed the presence of phosphorus, followed by magnesium and calcium, except R. arrhizus. Sulfur showed a lower presence and was absent in M. javanicus, while the presence of chlorine and potassium was rarely observed (Table 4). Table 4 Composition of microelements present in the isolated CWs of Mucoralean fungi by X-ray microanalysis

Mucoralean Fungi

Elements in the cell-walls Mg P S Cl K Ca

Absidia blaskeleana + + ± - ± + Gongronella butleri + + ± - - + Mucor javanicus + + - - + + Rhizopus arrhizus - + + - - - Cunninghamella elegans + + ± - - + Syncephalastrum racemosum

+ + + + + +

± = presence in 3 to 6 samples in 10 samples analyzed + = present in7samples in 10 samples analyzed - = absent

Phosphorus was the microelement detected in all CWs of the Mucoralean strains by X-ray microanalysis. This information was used as support for establishing the ratio of calcium/phosphorus in the intact CWs and after pronase and chitinase digestion treatments in accordance with Campos-Takaki [14]. Table 5 shows the percentage of polyphosphate in the intact CW and three groups are formed: a) high phosphorus levels (A. blaskeleana and C. elegans); b) a medium percentage of phosphorus (G. butleri, M. javanicus and R. arrhizus), and c) the lowest phosphorus content was observed in S. racemosum. An effective digestion of chitinase and pronase in the CWs of C. elegans and S. racemosum was observed with an expressive reduction in P/Ca. Treating A. blaskeleana with chitinase did not influence the P/Ca ratio. However, chitinase acted effectively on the CWs of A. blaskeleana (Figure 3). The microfibrillar fraction of chitin showed an absence of phosphorus except for Gongronella butleri. The results described here suggest it is possible calcium is linked to the microfibrils of chitin in Mucoralean fungi.

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Table 5 Polyphosphate content and the P/Ca ratio in the intact and treated (with pronase and chitinase) CWs of Mucoralean fungi

Mucoralean Fungi

Ratio of Phosphorus/Calcium Poly

phosphate %

Intact cell-

wall

Chitinase Treated cell

wall

Pronase Treated cell

wall

Microfibrillar fraction

Absidia blaskeleana 23.7 5.65±0.70 5.88±0.85 2.38±0.55 -P Gongronella butleri 16.7 12.89±2.83 4.69±9.41 5.25±0.68 0.22±0.04 Mucor javanicus 12.6 23.09±4.56 9.37±1.88 0.82±0.15 -P Rhizopus arrhizus 18.6 -Ca 5.65±0.83 1.95±0.27 -P Cunninghamella elegans

22.7 23.23±4.37 3.24±0.39 2.37±0.40 -P

Syncephalastrum racemosum

8.6 19.35±3.10 3.70±0.46 1.15±0.13 -P

-P = total absence of Phosphorus -Ca= total absence of Calcium

Fig. 3 Apical region of hyphae of cell-walls of Absidia blaskeleana. (A) Intact cell-wall; (B) Cell wall treated with chitinase

6. Conclusions on the composition of Mucoralean fungi cell walls

Therefore, the evidence supports the idea that Mucoralean fungal cell walls consist of rigid structures which in the inner layer wall contain chitin linked to calcium and perhaps cross-linked through N-glycosylated glycoproteins containing sulfur in their structure, and chitosan, neutral sugars and uronic acid in the amorphous region. Nevertheless, there is an unusual structural and functional diversity particularly in the enriched proteins of the cell wall. The number of amino acids from different strains varies greatly. The fatty acids form a special group from which important poly unsaturated fatty acids (PUFA) are produced. Inorganic polyphosphate is a rich fraction which when the environment is varied facilitates exploring for new ones. The relative abundance of fungi was highest in CW from Mucoralean fungi and found in fungal PUFAs, while the fatty acids are used as microbial taxonomic groups markers, include palmitic acid (PA), gamma linolenic acid (GLA) and linoleic acid (LA). Chitin and chitosan are versatile and promising biomaterial. The deacetylated chitin derivative, chitosan is more useful and interesting

bioactive biopolymer. In this view, an attempt has been made to increase the understanding of the importance and characteristics of the CW composition in Mucoralean fungi distributing various aspects including the chemical and biological properties, and applications. In view of this, this study will attract the attention of entrepreneurs, industrialists, academicians, and environmentalists for the biotechnological potential of the Mucoralean fungi.

Acknowledgements This work was financially supported by the National Council for Scientific and Technological Development (CNPq), the Coordination unit for the Improvement of Higher Level Education (CAPES), and the Foundation for the Support of Science and Technology of the State of Pernambuco (FACEPE). We are also grateful to NPCIAMB-Catholic University of Pernambuco, Brazil for the use of their facilities.

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