There are four main catalytic classes of proteolytic enzymes: aspartic-, cysteine- (thiol-), serine-, and metalloproteinases [45]. It has been inferred that aspartic proteinases are mostly active under acidic pH conditions, that metallo- and serine proteinases usually work optimally under alkaline conditions, and that cysteine proteinase activity is high over a broader range (pH 4 - 7) [46]. We compared both the total and the class-specific proteolytic activity of attine ant symbionts and their free-living relatives across a gradient of different pH conditions.
Sample material, fungal tissue extract preparation and buffering
Colonies of fungus-growing ants Apterostigma collare (nest number Apcol1), Myrmicocrypta ednaella (Myred1, Myred2), Mycocepurus smithii (Mycsmi9, Mycsmi15, Mycsmi32), Cyphomyrmex costatus (Cycos6, Cycos9, Cycos16), Cyphomyrmex longiscapus (Cylon5, Cylon12, Cylon24), Sericomyrmex amabilis (Serama7, Serama8, Serama12), Trachymyrmex cornetzi (Trcor1, Trcor3, Trcor4, Trcor10), Trachymyrmex sp. 3 (Trsp3-3, Trsp3-6), Trachymyrmex cf. zeteki (Trzet2, Trzet3, Trzet6), Acromyrmex echinator (Acech322), Acromyrmex octospinosus (Acoct367), Atta colombica (Atcol27), Atta sexdens (Atsex1), and Atta cephalotes (Atcep16) were collected in Gamboa, Panama and maintained under standard laboratory conditions at ca. 25°C and 60 - 70% RH. The ants were supplied with oatmeal (Apterostigma, Mycocepurus and Cyphomyrmex), oatmeal and fragmented bramble leaves (Myrmicocrypta, Sericomyrmex and Trachymyrmex) or entire bramble leaves, dry rice and pieces of apple (Atta and Acromyrmex).
Strains of non-symbiotic fungi Agaricus bisporus, Pleurotus ostreatus, P. pulmonarous and Lentinula edodes, which belong to the same fungal order as the leaf-cutting ant symbiont (Agaricales), were obtained from the Department of Mycology and Algology, Moscow State University, Russia. Pure cultures of Leucocoprinus gongylophorus were obtained by inoculating mycelium collected from fungus gardens on potato dextrose agar plates and subsequent incubation at 25°C. Fungal cultures were maintained on wort-agar medium and Czapek medium enriched by tryptone (10 g/L) and peptone (10g/L).
Fungi are known to modify environmental pH by producing pH regulating compounds. To detect whether the acidity of fungus garden extracts was due to instantaneous acid production or active buffering, we examined the buffering properties of the extracts. First buffering abilities of the fungal extracts were determined by mixing one μl of fungus garden water extract (1 g in 1 ml) with an equal volume of 0.04 M acid solution (containing phosphoric, boric and acetic acids) or an alkaline solution (0.02 M NaOH), and the resulting pH levels were measured as color changes on pH test paper. The resulting pH change was compared to the pH change obtained using a control acid solution diluted with an equal volume of distilled water, or an alkaline solution two times diluted with distilled water. Next we determined the buffering capacity of the extracts, and compared it to the buffering capacity of extracts made from related non-symbiotic basidiomycete fungi. To measure the approximate buffering capacity of the different fungal species, one gram of fungal biomass or one gram of fungus garden material was homogenized with a pestle in 1 ml of distilled water. Samples were centrifuged (5 min, 5200g) and the supernatant was used for buffer capacity measurements, i.e. the quantity of 1M NaOH that needed to be added to 1 ml the fungus extract in order to change the pH of the suspension by one unit.
Proteolytic activity assays
Proteolytic activity was measured spectrophotometrically using azocasein (Sigma-Aldrich Co) and the chromogenic p-nitroanilide substrates: Glp-Ala-Ala-Leu-pNa, N-benzoyl-Arg-pNa, and Suc-Ala-Ala-Pro-Phe-pNa (prepared by The State Research Institute of Genetics and Selection of Industrial Microorganisms, Russia). Total and class-specific proteinase activity towards azocasein was tested by determining the rate of hydrolysis after homogenizing pieces of fungus garden material with a pestle in an Eppendorf tube using 2.5 volumes (w/v) of distilled water (in order to keep the natural pH of the sample). Samples were centrifuged at 8000g for 15 minutes and the supernatant transferred to a clean tube. Ten μl of extract was mixed with 15 μl of 2% (w/v) azocasein solution and incubated for 1 hour at 26°C. The reaction was terminated with the addition of 120 μl of 10% TCA after which the suspension was centrifuged for 5 minutes at 14000g and 140 μl of supernatant was added to an equal volume of freshly prepared NaOH (1M). Absorbance was measured at 440 nm using a VERSAmax microplate reader. Reactions in control samples were terminated immediately after adding azocasein. The difference between treatment and control absorbance (A440, at t°C 26°C, 1 hour) was used as a relative measure of enzyme activity. All measurements were performed four times producing means that are presented ± SE.
In order to measure class-specific proteinase activity, the assays were performed in the presence of a protease inhibitor that specifically targets proteases of a certain class. The decrease in activity caused by the inhibitor was used as the class-specific activity value. The inhibition assays were performed using azocasein as described above. 10 μl of sample was preincubated for 3 hours at room temperature with 1 μl of inhibitor resulting in the following final concentrations of the inhibitors (all purchased from Sigma Chemicals Co): For serine proteinase inhibition we used phenylmethane-sulphonul-fluoride (PMSF, 0.57 mM), tosyl lysil chlormethyl ketone (TLCK, 10 μM) and tosyl phenilalanine chlormethyl ketone (TPCK, 10 μM). For cysteine proteinase inhibition we used L-trans-epoxysuccinyl-leucyl-amide-4-guanidino-butane (E64, 5 μM). Activity was also measured after the addition of thyol protecting agent DTT (10mM), which may increase the activity of cysteine proteinases. For metalloproteinase inhibition we used ethylendiaminetetraacetic acid (EDTA, 8 mM) and for aspartyl proteinase inhibition we used pepstatin (2 μM).
Serine proteinases can be further divided into sub-classes based on which substrates they are able to degrade. To determine which sub-classes of serine proteinases were active in fungal gardens, we measured activity towards p-nitroanilides after mixing 5 μl of fungal garden extract, 5 μl of substrate (10mg/ml) and 200 μl of potassium phosphate buffer (0.1M) of pH 5.0 or 7.0 and incubating the reaction mixture at 26°C. The change in absorbance was analyzed using a VERSAmax microplate reader spectrophotometer at 410 nm. The linear part of the obtained kinetic curve (the dependence of absorbance on time) was used to calculate the enzyme activity.
The effect of pH on total and class-specific proteolytic enzyme activity was measured across a pH range of 3 to 8 (actual measurements at 3.0, 4.0, 5.0, 5.2, 6.0, 7.0, 7.5, 8.0) using 0.2 M Britton - Robinson buffers (A mixture of 0.4 M phosphoric-, 0.4 M acetic-, and 0.2 M boric acid was mixed with different quantities of 0.2 M NaOH to give buffer solutions with the required pH values). The relatively high molarity of the buffers was used to make the natural buffering capacity of the extracts negligible compared to the experimentally induced ones.
To measure the pH dependent proteolytic activity of non-symbiotic fungi, culture fluid of A. bisporus was used. Modified Czapek medium (0.7 g KH2PO4, 0.3 g K2HPO4·3H2O, 0.5 g MgSO4·7H2O, 0.01 g FeSO4·7H2O, 23.3 g casein in 1 L H2O) was inoculated with mycelium from seven days old plated fungus culture and incubated for six days on a rotary shaker (130 rpm, 24°C). Culture liquid was centrifuged (14000g, 20 min) and filtered through filter paper. After adding sodium azide (8% water solution, 2.5 μl to 1 ml of culture liquid) to prevent contamination, fifty μl of culture liquid was mixed with 100 μl of Britton - Robinson buffer (0.1M, pH range from 3 to 8; actual measurements at 3, 4, 5, 5.2, 6, 7, 7.5, 8) and 150 μl of 0.5% water azocasein solution. Reactions were kept overnight (37°C) because of relatively low enzyme activity and then terminated by adding 300 μl of 10% TCA. The reactions were placed at 4°C for 30 min and then centrifuged for 20 min (5200g). 400 μl of suspension was mixed with an equal volume of freshly prepared NaOH (0.5 M) and absorbance at 440 nm was measured using a spectrophotometer (Genesys 10 - UV). The reactions of the control samples were terminated with TCA immediately after adding azocasein. The difference between the absorbance of the treatment and control samples was used as a relative measure of enzyme activity. All measurements were performed three times and presented as means ± SE. Class-specific proteinase activity pH optima were measured in the presence of a protease inhibitors PMSF and EDTA as described above.
Proteolytic activities were finally compared across the different stages of advancement of the symbiosis (lower attine ants, higher attine ants, leaf-cutting ants).
Phylogenetic analysis
Sequencing of the LSU rRNA gene and the Elongation Factor 1-alpha gene of the fungal symbionts was done by extracting DNA by the Chelex method [47]. Small amounts of fungal tissue were ground in 200 μl of 10% Chelex-100 and heated for 15 min at 95°C. The samples were centrifuged for 3 min at 10,000g after which 1 μl of supernatant was used for PCR. The primer pair LR0R 5'-ACC CGC TGA ACT TAA GC-3' and LR5 5'-TCC TGA GGG AAA CTT CG-3' was used to amplify a fragment of the LSU rRNA gene of about 920 bps, using the following PCR scheme: one cycle of 95°C for 5 min, then 35 cycles of 95°C for 20 sec, 56°C for 30 sec, and 72°C for 1.5 min, ending with one cycle of 72°C for 7 min. The primer pair EF1a-F 5'-GTT GCT GTC AAC AAG ATG GAC ACT AC-3'. [48] and EF1a-R5 5'-CAG GCA ATG TGG GCT GTG TGA CAA TC-3' was used to amplify a fragment of the Elongation factor 1-alpha gene of about 820 bps, using a PCR scheme similar to the one above, although for some of the samples the annealing temperature had to be decreased to 50°C in order to obtain a PCR product. PCR products were sequenced by Eurofins MWG Operon. Nucleotide sequence data are deposited in GenBank with Accession Numbers HQ191224-HQ191277.
The gene sequences were aligned with Clustal W [49], and after deletion of regions that could not be unambiguously aligned, a phylogeny was constructed by maximum-likelihood PhyML-aLRT [50]. The nucleotide substitution model was GTR [51] and the transition/transversion ratios, the proportion of invariable sites and the Gamma distribution parameter were estimated by maximizing the likelihood of the phylogeny. The substitution rate category was set to four, and the input tree to be refined by the maximum-likelihood algorithm was set to BIONJ. The aLRT statistics were performed using the non-parametric Shimodaira-Hasegawa-like procedure. Two of the fungal colonies (Trsp3-6 Trzet6) died during the experiment, so that only the LSU gene could be used for these two samples when constructing the phylogenetic tree.