Cryptococcus neoformans Virulence Attributes can be Modulated by Sound Stress

Research Article

J Bacteriol Mycol. 2021; 8(4): 1179.

Cryptococcus neoformans Virulence Attributes can be Modulated by Sound Stress

Kellysson GB Mendes1, Fabiana Brandao AS2*, Raul Alberto Laumann3 and Simoni Campos Dias1,4

1Catholic University of Brasilia, Proteomic and Biochemical Analysis Center, Brasilia-DF, Brazil

2University of Brasilia, Department of Pharmacy, Laboratory of Clinical Analysis, Darcy Ribeiro Campus, Asa Norte, Brasilia-DF, 70910-900, Brazil

3Embrapa Genetic Resources and Biotechnology, PqEBAv. W3 Norte (Final) S/N-Asa Norte, Brasilia-DF, 70770-90170770-917, Brazil

4University of Brasilia, Post-Graduation in Animal Biology, Darcy Ribeiro Campus, Asa Norte, Brasilia-DF, 70910-900, Brazil

*Corresponding author: Fabiana Brandao AS, University of Brasilia, Department of Pharmacy, Laboratory of Clinical Analysis, Darcy Ribeiro Campus, Asa Norte, Brasilia-DF, 70910-900, Brazil

Received: May 17, 2021; Accepted: July 01, 2021; Published: July 08, 2021


Sound waves are a prime component making up the environment, and they are present in almost all niches on the planet. In times of increasing noise pollution, the effect of sound stress on humans, animals, and microorganisms is well known. However, the possibility of this kind of pressure in the environment, affecting pathogenic fungi, which live in the background as saprophytes, has not been explored. Fungi can develop attributes and become virulent due to adaptation to selective pressure or stress. In this context, our group has become interested in evaluating the impact of sound stress on the fungus Cryptococcus neoformans, a pathogen that has high phenotypic plasticity. C. neoformans strain H99 was chosen for all assays. The yeasts were cultivated at 30°C, exposed or not to the frequency of 8 kHz. We observed morphological changes in these cells, such as the expression of phenotype virulence attributes: capsule expansion and melanin production. We also analyzed the number of viable cells after exposure, and we observed the yeast’s susceptibility to antifungals. After the treatment with 8 kHz, the cells showed a significant increase in the capsule expansion, an acceleration of the melanin production, and a slight reduction in the number of viable cells. Finally, tests performed with the antifungals showed a decrease in inhibition halo on the plate test. Our results are innovative and suggest that stress caused by sound could incite increased virulence in this fungus.

Keywords: Cryptococcus neoformans, Sound frequency, Virulence Attributes, Stressor environment, Phenotypic Plasticity.


Sound is mechanical energy that disperses in the form of waves, an intrinsic component of the environment [1,2]. From a human perspective, the sound frequencies can be divided into roughly three groups: infrasound (< 20 Hz), audible sound (20 to 20,000 Hz), and ultrasound > 20,000 Hz) [3].

Essentially, all life on the planet interacts with sound waves [4,5]. These interactions can be classified into two groups: 1- Interactions with sound self-produced by the organisms; these intentional interactions are usually involved in organism communication (5), and 2- Interactions that are non-intentional, when the organisms are exposed to environmental noise. The second group can have a major impact on living organisms, which vary according to the frequency range utilized and the organism that is exposed. It has been observed that plants exposed to the frequencies of 1 kHz significantly increased cell division and cell wall fluidity [6-8].In animals, the principal impact is induced by the sound in the audible sound frequencies, causing disorientation that disturbs the ability to communicate and hunt, and spatial orientation [9-12].

Hypotheses about the effect of global warming-related changes [13], radiation[14-16]and the use of pesticides [17] performing as selective pressure on pathogenic fungi in the environment have been raised. Besides, since the industrial revolution, the amount of noise emitted into the environment has significantly increased, considered today to be a problem only exceeded by pollution of air and water, especially in densely populated areas with intense industrial activity [12,18,19].

There is some evidence that sound, as environmental noise, could interfere in microorganism physiology,and this knowledge has been explored for decades in medicine [20-22]. Microbes growing exposed to different sound frequencies demonstrated significant changes such as increased cell permeability, change in cell surface charge, the release of nitric acid, hydrogen, peroxidase, and free radicals [22].

The effect of sound waves on the frequency of 8 kHz was investigated in the E. coliK-12 bacterial model. First, the researchers noticed an acceleration in RNA and protein synthesis, suggesting a periodic oscillation of the bacterial intracellular liquid induced by sound stress [3,23]. In a second experiment, it was shown that sound could induce mechanical stress, causing an influx of small molecules like H2O, Na+, K+, and Ca2+ [23]. A frequency-dependent fungicidal activity was observed in Aspergillus sp. [24-26].

Fungi are ubiquitous microorganisms in nature and have essential functions for maintaining life on Earth. However, some species are highly pathogenic, which is a result of a well-adapted selection of survival and infection in mammalian cells. Further, with the advent of immunosuppression, the number of fungal infections has increased significantly during the last few decades, reaching an alarming threshold of human mortality worldwide, affecting more than a billion people [27,28]. Questions are raised about what would happen in an environment that could promote selection for fungi strains that become increasingly virulent and resistant to the antifungals, and how this adaptation is also emerging at an exponential rate [29-31].

Some pathogenic fungi, such as Cryptococcus neoformans, present a saprophytic life cycle [32-34], and hypotheses discuss the ability of these pathogens to face environmental stresses, and how these events could modulate virulence factors [35-38]. These in turn could result in a more beneficial adaptation of these microorganisms when in the host’s infectious processes [38].

C. neoformans are encapsulated, polysaccharide-coated yeasts frequently found in the environment in association with decaying vegetation and are able to cause disease in humans [34,39,40]. This fungus can invade the Central Nervous System (CNS), causing fungal meningoencephalitis, which is the most common cause of meningitis in adults living with HIV in sub-Saharan Africa [41-43]. The global incidence of cryptococcal meningitis was recently estimated at approximately 220,000 per year [42]. The mortality for those receiving care was estimated at 60% in low-income countries [42,44]. People with compromised immune systems, especially those with AIDS and organ transplants are more susceptible to Cryptococcus infections [45-47]. In addition to its clinical importance, this fungal pathogen displays remarkable phenotypic plasticity in response to host and environment [36,48].

C. neoformans infects a wide range of organisms, from amoebas to insects (Lepidoptera) and plants such as Arabidopsis thaliana[49]. Hypotheses about the effect of global warming-related changes [13], radiation [14-16] and use of pesticides [17] could play a role as a form of selective pressure upon pathogenic fungi in the environment. In a broad view of the types of environmental stresses that would act as selective pressure, it is worth noting that since the industrial revolution, the amount of noise emitted into the environment has significantly increased. It is considered today as a problem on the scale of pollution of air and water, especially in densely populated areas with intense industrial activity [12,18,19].

In the face of this new concern about the role of sound/vibration in the environment, Biotremologyhas arisen as a new science. Biotremology is an emergent discipline that studies the production, transmission, reception, and biological effects of vibrations in a living organism [50,51]. The findings of this new science support studies in several domains, from animal communication to use in growth and pest control [52,53]. In this innovative vision, we investigate the effect of background noise as a source of substrate vibrations on the virulence attributes of the pathogen C. neoformans. The sound frequency of 2 and 8 kHz were evaluated on yeast growth, expansion of the polysaccharide capsule, melanin production, and susceptibility to fluconazole. Our data imply that sound can exert selective pressure on the environment, stimulating micro-organism virulence phenotypes.

Materials and Methods

Yeast strains and growth conditions

In our assays, we used the species Cryptococcus neoformansvargrubii, a well-established pathogenic strain, H99, a widely known virulent strain, with its entire genome sequenced [54]. Fungal strains were stored in 15% glycerol at -80°C until use. The cells were grown in YPD broth (yeast extract [2%], peptone [1%], dextrose [2%]) at 30°C and isolated in the log-phase of microbial growth for further testing.

Background Sound playback

A loudspeaker (of low-frequency response, 8O impedance, membrane diameter 10 cm, Radioshack, Taiwan)was usedfor the emission of the sound waves. Over the speaker was placed a 15 cm acrylic plate, on which the Petri dishes with fungal strains were arranged, produced as described in the previous section, were placed. With this setup, the sound was transmitted as a substrate vibration of the acrylic plate to the Petri dishes.

For the experiments, the complete setup was introduced in a cultivation oven that maintained the temperature at 30°C and alsoacted as an isolation sound chamber.

Two simulation programs were built, using the function synthesis of the software Sound Forge 6.0 software (Sonic Foundry Inc., Madison, WI, U.S.A.). The programs consisted of digital pure tone continuous sequences of 2 or 8 kHz frequencies built in monophonic mode at 24-bit, 96-kHz, 80-dB signal-to-noise ratio. The stimulation program was played back without interruptions (looping reproduction mode of the Sound Forge software) during all duration periods of the experiments, using a computer connected to a sound card (UA-25EX, Edirol-Roland 24bits-96kHz; RolandCorp., Japan) which the loudspeaker was plugged into.

The measure of air sound intensity was assessed by a digital decibelimeter (minipa Model MSL-1355b, SPS, Brazil) connected to a computer and placed in the middle of the cultivation oven. Vibrations transmitted to the acrylic plate were recorded by a laser vibrometer (PDV-100, Polytec, Waldbronn, Germany). The beam of the vibrometer was directed perpendicularly to different points of the acrylic plate and the equipment was placed at a distance of ~30 cm from the vibration surface. To get a better reflection, a small piece of reflective tape was glued on the recording points. Registered signals were amplified and digitized (monophonic mode, 24-bit, 96-kHz, 100-dB signal-to-noise ratio),via the audio capture sound card described above, and computer-stored using Cool Edit Pro 2.0 software (Adobe Systems Inc., San Jose, CA, U.S.A.).

Airborne sound intensity inside the cultivation oven was 57.6 dB. The air pressure generated by the loudspeaker membrane was transmitted efficiently to the acrylic plate and to the Petri dish, Vibration generated by the air pressure varied between -1 and -21 dB, in relation to the intensity measured in the loudspeaker membrane (-50 dB) (Supplementary Figure 1).