Biological activity of Cryptococcus neoformans and Cryptococcus gattii from clinical and environmental isolates Atividade biológica de Cryptococcus neoformans e Cryptococcus gattii provenientes de isolados clínicos e ambientais

First submission on 23/05/12; last submission on 31/12/12; accepted for publication on 14/03/13; published on 20/06/13 1. Doctor im Microbiology by Universidade Federal de Minas Gerais (UFMG); assistant professor at Universidade Federal de Sergipe (UFSE). 2. Resident in Neurology at Universidade Federal de São Paulo (UNIFESP). 3. Resident in Cardiology at Instituto Dante Pazzanese. 4. Doctor in Biotechnology by Rede Nordeste de Biotecnologia (RENORBIO); researcher at UFSE Applied Microbiology Laboratory. 5. Doctor in Microbiology by UFMG; associate professor and researcher at UFSE Applied Microbiology Laboratory. 6. Post-doctorate in Microbiology at UFMG; professor and researcher at UFMG Mycology Laboratory. ABStrACt

mainly among healthy hosts and it is classically described as a tropical disease.Nevertheless, there was an outbreak of C. gattii on Vancouver Island, a temperate climate region, which suggests that C. gattii is not exclusively restricted to tropical and subtropical areas (13,20) .Cryptococcus gattii is endemic in the north and northeast regions of Brazil.It affects mostly children and young adults (25) .Despite the fact that cryptococcosis is a systemic infection that affects mostly HIV patients, there are few studies on molecular epidemiology and clinical aspects, mainly in the northeast region of Brazil (8) .Cryptococcal meningitis arises in 8% to 30% of AIDS patients and the mortality is high in 6% to 29% of the cases (22) .
C. neoformans has been isolated from bird excrements, plant debris and contaminated soil (5) .It has also been found in plants such as Eucalyptus (9) , Brazilian native plants (15) and armadillo burrows in the northeast of Brazil (10,15,16) .C. gattii has been continuously isolated from Eucaliptus spp. (1,9) It has also been isolated from Amazonian native plants (1,10) .Moreover, it has been found in animals from temperate regions such as dolphins (Tursiops truncatus) in the Atlantic ocean (Metropolitan region of Vancouver, Canada) (23) .In this specific case, there was an epidemic affecting both immunocompetent and immunodeficient patients as well as animals (dogs and dolphins) (20,23) . Additinally, strains of this pathogenic yeast were found in plant species from Canada (23) .This species was also found in wasps, bats, koalas, and camels (9,15,16) .Both species are commonly isolated in hen excrements as well as deteriorating wood planks and trunks from living trees (15,16) .
Several factors are involved in the adherence process, destruction and invasion of host tissues.According to the literature, protease production, which ascertains a potential adaptability to adverse environments, is among them.In general, proteinases degrade several human proteins such as albumin, hemoglobin, keratin and immunoglobulin A (IgA), which are present in different sites.This proteolytic activity has been associated with tissue invasion and it is considered a virulence factor (14) .These fungi have several virulence factors that allow invasion of the host and their multiplication (24) .The present study evaluated protease production by Cryptococcus neoformans and Cryptococcus gattii and possible differences in enzymatic activity between clinical and environmental samples.Furthermore, it verified possible differences in thermotolerance and urease activity in clinical and environmental samples in order to confirm, albeit indirectly, these features as virulence factors.

oBJECtivE
To observe the capacity of extracellular enzyme production (urease and proteinase) and biological activity in several temperatures (thermotolerance) in clinical and environmental isolates of Cryptococcus neoformans and Cryptococcus gattii from Sergipe, Brazil.

MAtEriAL AnD MEthoDS
Preparation of microbial strains 70 strains were selected from the collection of the Applied Microbiology Laboratory -Universidade Federal de Sergipe (AML-UFS), including 35 environmental isolates, 35 clinical isolates and two standard strains (ATCC 32608 and ATCC 56990).In the strain description, we analyzed 9 strains of Cryptococcus gattii and 26 strains of Cryptococcus neoformans from cerebrospinal samples collected from patients with meningoencephalitis and assisted at Sergipe Emergency Hospital.
The surveyed pathogenic yeasts were isolated from several environmental samples from Sergipe, including 34 strains of Cryptococcus neoformans and 1 strain of Cryptococcus gattii.The environmental substrates were from bird and bat excrements as well as native and exotic tree trunks from Sergipe.The strains were inoculated in Sabouraud Dextrose and incubated at 25ºC for five days.

Ability to grow in thermotolerance
Five tubes/sample were employed for thermotolerance analysis.We inoculated 5 µl of suspension with 1.2 × 10 9 microorganisms/ml in each tube, which contained 3 ml of Sabouraud broth.Each tube was incubated at different temperatures (25ºC, 30ºC, 37ºC, 40ºC and 42ºC).Turbidity readings were carried out through MacFarland standard and spectrophotometer within 24 hours, 48 hours and seven-day incubation periods (4) .

Enzymatic activity: urease
For the assessment of urease activity, we employed Christensen agar (with Ph adjusted to 5.0) added to a 20% urea solution, distributed into slanted tubes (3 ml/tube).After inoculation, the samples were incubated at 25ºC and the readings were performed after 24 hours, 48 hours and seven days.The results were interpreted according to the following criteria: negative (medium remained yellow), moderate activity (half of the medium became pink) and intense activity (the medium became entirely pink) (2) .

Enzymatic activity: protease
We applied plates with protease agar containing fraction V bovine albumin and Protovit, in which 5 µl of suspension with 1.2 × 10 9 microorganisms/ml were inoculated in duplicate.The inoculated plates were incubated at 37ºC and the reading was conducted after seven days.Light areas around the colonies were interpreted as albumin degradation.The colonies (a) and the colonies plus light halo (b) were assessed.Protease activity was estimated through Pz calculation (a/b) so that the lower Pz, the higher the enzymatic activity.As positive controls, we used six endocervical samples of Candida spp.from AML-UFS strain database (3) .

Statistical analysis of yielded data
All strains were statiscally analyzed through SPSS v. 11 and BioEstat v.5 software.They were also submitted to mean test and comparison between variables through student's t -distribution (thermotolerance and protease) and ANOVA variance analysis (urease).Turkey test was performed after significant p < 0.01 for statistical data differentiation.

rESuLtS
Table 1 shows the microbial concentration of Cryptococcus neoformans special complex in isolates from Sergipe, northeast of Brazil, when tested at different temperatures in order to assess thermotolerance.
Table 2 shows the optical density of clinical isolates of both surveyed yeasts (C.neoformans and C. gattii) for the analysis of growth under thermotolerance biological activity.Table 5 shows statistical studies on optical density and comparison of surveyed temperatures in C. neoformans isolates from both clinical and environmental sites.
Table 5 corroborates data shown in Table 4.
Table 6 presents the profile of urease production in 70 surveyed pathogenic yeasts by comparing both environmental and clinical isolated sites.Table 11 shows data on protease production in C. gattii strains from both environmental and clinical isolates after 24 hours, 48 hours and seven days.

DiSCuSSion
There was statistical difference between environmental and clinical isolates when p < 0.01, regardless of the yeast species tested at 30ºC and 42ºC.There was statistical uniformity in optical density for other temperatures.It is particularly worth mentioning    that in terms of fungal growth there was no statistical difference between environmental and clinical isolates and the strains (70) when surveyed at body temperature(37ºC).
The most significant increase in the descriptive data on both clinical and environmental isolates occurred at the following temperatures: 42ºC, 40ºC, 37ºC, 30ºC and 25ºC.It is important to highlight that the typical average temperature of the studied region is 30ºC.
It was observed that there was statistical difference in both species at 30ºC and 42ºC.There was no statistical difference in the other temperatures.There was growth in C. neoformans and C. gattii clinical isolates at 30ºC and 37ºC, as shown in Table 1.The worst thermotolerance and optical density relation was 40ºC temperature.
As it was mentioned in the previous tables, there was statistical difference at 30ºC and 42ºC in comparison with other temperatures.In these pathogenic yeasts isolated from environmental sites in the state of Sergipe, the highest optical density for C. neoformans was at the following temperatures: 37ºC and 30ºC.On the other hand, C. gattii showed higher optical density for clinical isolates at 30ºC and 37ºC and for environmental isolates at 25ºC, 30ºC and 37ºC.
There was statistical difference in the optical density of this yeast when we compared the following temperatures: 25ºC and 37ºC with 42ºC.There was no statistical difference for the other temperatures.It may be inferred that it is difficult to obtain reference optical density for thermotolerance test at 42ºC, that is to say that there is fungal growth inhibition, mainly when this temperature is required in order to obtain bioproducts and/or bioprocesses relevant to the health area.
By confronting data on C. gattii, though p values were more significant, there was no homogeneity in the statistical analysis of optical density and thermotolerance.
As to thermotolerance, when mean microorganism concentrations from clinical and environmental samples were compared at different temperatures and after seven days, there was significant statistical difference at 30ºC (p < 0.05) and 42ºC (p < 0.05).There was no difference at other temperatures (Tables 2  and 3).By using t test, it was observed that there was no significant statistical difference in the comparison of mean microorganism concentrations from clinical samples at different temperatures, contrasting with environmental samples, in which there was significant statistical difference in the following comparisons: 25ºC vs. 42ºC (p < 0.001) and 37ºC vs. 42ºC (p < 0.001).
The use of microbial growth in several temperatures, mainly in the occurrence of circulating strains, specially in the northeast of Brazil and in plant and animal samples, provides information on the epidemiological aspects of these yeasts (virulence factors) as well as their ecology (biological activity).However, there was no significant result as to microbial growth neither at the tested temperatures (25ºC, 37ºC and 42ºC) nor at the contrasted ones.In high temperatures (30ºC and 42ºC), the clinical samples presented a higher growth with statistical significance in comparison with environmental ones.Moreover, the clinical samples grew regardless of the incubation temperature, which did not occur with environmental ones.

Casadevall et al. claim that thermotolerance (at 37ºC
) is a singular phenomenon in the triggering of systemic mycoses.Therefore, the yeast with this featured virulence factor maximizes the triggering of this disease in humans.Zuyderduyn et al. state that, as far as thermotolerance is concerned, there are several genes involved in the pathogenesis of these fungi.By use of serial analysis of gene expression, the following genes were present: translation elongation factor 1, cyclophilin A, thioredoxin peroxidase, histone H4, ribosomal proteins 40S RPS12 60S RPL21A.The studied strain was C. neoformans H99 (26) .
In the analysis of urease production, there was statistical difference in the adopted temperatures for the analysis of enzymatic production (24 hours, 48 hours and seven days) in both clinical and environmental isolates.In the clinical isolates, there was statistical prevalence of moderate production within 24 and 48 hours.All isolates produced it (100%).After the seventh day of enzymatic analysis, only the clinical strain remained with moderate production whereas the other 34 strains produced urease intensely.There were statistical differences among these data.
As to environmental samples, there was prevalence of moderate urease production in the following readings: 24 and 48 hours.32 from the total of 35 surveyed strains produced it moderately, one did not produce it and two produced it intensely.There were statistical differences in all cases.In the seventh day of reading, 32 from the total 35 strains showed intense production, two revealed moderate production and one did not produce urease.It is worth highlighting that there were statistical differences within yielded data.
As to clinical samples, it was possible to notice that both 24 and 48-hours periods yielded a moderate production of urease (100% of strains).In the seventh day of reading, eight from nine strains produced the enzyme intensely whereas one isolate revealed moderate production.In these cases, there were statistical differences when ANOVA test was adopted (p < 0.01) followed by Turkey test.The only strain of C. gattii isolated from environmental substrate in the state of Sergipe produced the enzyme moderately within both periods (24 and 48 hours).In the seventh day, there was an intense production of this enzyme, corroborating other yielded data.
Ato urease production, there were statistical differences when we compared data from 24 and 48-hours reading periods with the seventh day of data analysis.The moderate enzymatic production prevailed in C. neoformans environmental isolates within both 24 and 48-hours periods.In the seventh day, these strains progressed to intense production of urease.Analyzing the data from environmental isolates, there was also uniformity in the enzymatic production within 24 and 48-hours periods.31 from 34 surveyed strains produced the enzyme moderately, one strain did not produce it and two produced it intensely.In the seventh day of reading, there was intense enzymatic production in 31 from the total of 34 surveyed strains and one of them did not produce any enzyme.There were statistical differences in these data.Therefore, it may be inferred that C. neoformans yields better results for urease in the seventh day of stimulus for enzymatic production.
Urease is an enzyme from the microbial metabolism that plays a major role as a virulence factor of several pathogenic agents such as Cryptococcus neoformans and Cryptococcus gattii (3,4,24) .Urease hydrolyses urea in ammonia and carbamate, resulting in local pH increase, which may be applied in microbial identification as a result from colorimetric changes.Most C. neoformans clinical isolates are positive for urease (2) .Perfect et al. (21) verified that C. neoformans strains that were negative for urease through the inactivation of the gene responsible for the enzymatic production had lower pathogenicity in comparison with the wild mutant reconstituted type, which was demonstrated by the survival of the sample after inoculation.By use of ANOVA test, it was possible to notice that there was a significant statistical difference between clinical and environmental samples.As to urease activity, all types of samples hydrolyzed urea fast, regardless of its type (clinical or environmental).
Torres-Rodríguez et al. (12) report that there are several variables influencing this virulence factor, namely microbial biomass used in the experiment and test accuracy.In the same investigation, they demonstrated that Cryptococcus neoformans had a higher enzymatic activity or urease production in comparison with Cryptococcus gattii, which was not corroborated in the present study.Hsueh et al. (11) reported the occurrence of moderate urease activity (37.5%) in C. neoformans special complex isolates from Taiwan after 48 hours and low activity in urease production (62.5%).These data differ from our present findings.Thus, the strains from this investigation presented potential urease production, which causes higher pathogenicity.
Other virulence factors involved in Cryptococcus neoformans and Cryptococcus gattii pathogenicity are the following enzymes: lipases, proteinases and proteases.They are involved in the destruction of cellular structures in order to offer nutrients to the pathogenic fungi and facilitate their systemic propagation (7) .
Casadevall et al. (3) showed that proteinase is able to degrade proteins from the host, namely collagen, elastin, fibrinogen, immunoglobulins and proteins from the complement system.Furthermore, the authors highlighted that this host protein degradation may protect C. neoformans from the immune system by blocking phagocytosis.
In the data analysis based on proteinase production by encapsulated pathogenic yeasts, the reading time for the visualization of this enzyme in both sites (clinical and environmental) was 48 hours.For the clinical strains, 34 strains did not produce proteinase and one did within 24 hours whereas 12 strains did not produce proteinase and 23 did within 48 hours.In the seventh day of enzymatic reading, only one strain remained without producing the enzyme, whereas 34 strains did.It is particularly worth mentioning that there were statistical differences with result selection (p < 0.01) in all data and studied periods.
As to environmental samples, 100% of them showed no enzymatic production within 24 hours.31 from 35 strains showed enzymatic production and only four strains showed none within 48 hours.In the seventh day of analysis, two from the total of four isolates that did not produce any reading within 48 hours remained without synthesizing the enzyme.In the last period, 33 strains produced proteinase.Standard Cryptococcus neoformans (ATCC 32608) and Cryptococcus gattii (ATCC56990) strains were used for test standardization.Standard ATCC32608 showed enzyme production in all surveyed periods whereas ATCC 56990 strains did not synthesize the enzyme during the experiment.100% of the strains did not produce the enzyme in neither of the sites within 24 hours.18 clinical samples produced proteinase and eight did not synthesize it within 48 hours.As to environmental samples, 30 from 34 surveyed samples produced the enzyme.In the seventh day of biological reading, only one clinical sample and two environmental samples did not produce it.During this period, there was enzyme production in 25 clinical and 32 environmental samples.In the analyzed periods, there was statistical difference with data selection at p < 0.01.
In the clinical C. gattii samples, eight from nine strains did not produce proteinase and one did within 24 hours.There was no statistical difference when we analyzed the production or absence of proteinase within 48 hours.Four from nine strains remained without producing whereas five did.In the studies on C. gattii virulence factors, the number of isolates was lower, which also influenced statistical analysis of experimental data.In the seventh day, 100% of clinical C. gattii synthesized proteinase.The only environmental C. gatti sample that produced proteinase was within 48 hours.
As to proteinase production, when Pz values from clinical and environmental samples were compared through ANOVA test, there was Biological activity of Cryptococcus neoformans and Cryptococcus gattii from clinical and environmental isolates significant statistical difference (p = 0.66).Souza et al. (24) report that isolates from Amazonas showed a high protease production (Pz < 0.64) with mean Pz = 0.3 for both Cryptococcus species, supporting the information found herein and implying the great potential in the production of this virulence factor in the studied pathogenic yeasts.

ACknowLEDgMEntS
To the National Council for Scientific and Technological Development (CNPq), the Ministry of Science and Technology (MST), the Foundation for Supporting Research and Technology Innovation of the state of Sergipe (FAPITEC-SE), the State Secretariat for Economic Development, Science, technology and Tourism and the Government of the State of Sergipe for funding this scientific article.To the Foundation and Institute Parreiras Horta (FIPH) and the Public Health Laboratory of the State of Sergipe (LACEN-SE) for kindly providing the clinical isolates collected over a ten-year collection from the AML-UFS.

Table 4 ,
when we analyze thermotolerance in environmental isolates, it is observable the diminishment in optical density in comparison with clinical isolates, though with no statistical difference when p < 0.01.activity of Cryptococcus neoformans and Cryptococcus gattii from clinical and environmental isolates

tABLE 1 -
Mean * There was statistical difference when t Test was conducted (p < 0.01).tABLE 2 -Mean microorganism concentrations in the seventh day of thermotolerance reading with only clinical samples* There was statistical difference when t Test was conducted (p < 0.01).

tABLE 3 -
Mean microorganism concentrations in the seventh day of thermotolerance reading with only environmental samples * There was statistical difference when t Test was conducted (p < 0.01).

Table 7 indicates
C. neoformans urease production in both environmental and clinical isolates after 24 hours, 48 hours and seven days.

tABLE 4 -
Statistical correlation between mean concentration of Cryptococcus neoformans isolates under thermotolerance and differences in test temperatures * There was statistical difference when t Test was conducted (p < 0.01).

tABLE 5 -
Statistical correlation between mean concentration of Cryptococcus gattii isolates under thermotolerance and the differences in test temperatures * There was statistical difference when t Test was conducted (p < 0.01).tABLE 6 -Urease production activity in clinical and environmental Cryptococcus neoformans and Cryptococcus gattii samples isolated in Sergipe, Brazil : ANOVA test (p < 0.01) followed by Turkey test for statistical data differentiation in all surveyed sites.ANOVA: variance analysis.

tABLE 7 -
Urease production activity in clinical and environmental Cryptococcus neoformans samples isolated in Sergipe, Brazil a, b, c: ANOVA test (p < 0.01) followed by Turkey test for statistical data differentiation in all surveyed sites.ANOVA: variance analysis.

Table 8
shows urease enzymatic production in C. gattii strains in both environmental and clinical isolates.Table 9 demonstrates protease production in Cryptococcus neoformans and Cryptococcus gattii in both environmental and clinical isolates from the collection of UFS/AML.

tABLE 8 -
Urease production activity in clinical and environmental Cryptococcus gattii samples isolated in Sergipe, Brazil

tABLE 9 -
Protease production activity in clinical and environmental Cryptococcus neoformans and Cryptococcus gattii samples isolated in Sergipe, Brazil : ANOVA test (p < 0.01) followed by Turkey test for statistical data differentiation in all surveyed sites.ANOVA: variance analysis.

tABLE 10 -
Protease production activity in clinical and environmental Cryptococcus neoformans samples isolated in Sergipe, Brazil : ANOVA test (p < 0.01) followed by Turkey test for statistical data differentiation in all surveyed sites.ANOVA: variance analysis.

tABLE 11 -
Protease production activity in clinical and environmental Cryptococcus gattii samples isolated in Sergipe, Brazil