Streptomyces griseus Versus Trichoderma viride Chitinase as an Anti-inflammatory and Antifungal Agent Against Human Pathogenic Fungi

Abstract

Fungal pathogens cause over a billion human infections annually, leading to more than 1.6 million deaths each year. The scarcity of available antifungal drugs intensifies the public health threat posed by human pathogenic fungal infections. Therefore there is a critical demand for novel, safe, and effective antifungal agents. Although chitinases are established as effective antifungal agents against phytopathogenic fungi, research on their activity against human pathogenic fungi is limited. The present study seeks to investigate the anti-inflammatory and antifungal activity of bacterial and fungal chitinase against human pathogenic fungi. The antifungal efficacy of bacterial chitinase from Streptomyces griseus, fungal chitinase from Trichoderma viride, and a combination of both was determined by calculating the inhibition percentage in fungal growth, indicated by the reduction in the dry mass of the fungi. Additionally, the anti-inflammatory activity of these chitinases was assessed by measuring the inhibition of albumin denaturation. Results revealed that chitinases exhibited greater antifungal activity compared to the standard. Notably, bacterial chitinase demonstrated higher effectiveness than fungal chitinase against Aspergillus fumigatus, while the bacterial and fungal chitinase had similar effects against different Cryptococcus neoformans and Candida species. The combination of bacterial and fungal chitinase demonstrated the highest antifungal activity against all tested fungi. Furthermore, the anti-inflammatory activity indicated that chitinases prevented 98% of albumin denaturation, marking the first study reporting the anti-inflammatory role of chitinases in preventing albumin denaturation. Additional in-vivo studies are necessary to explore the antifungal activity of chitinases against human pathogenic fungi and investigate the anti-inflammatory mechanisms of chitinase.

Introduction

Fungi pathogens lead to over a billion human infections annually, causing over 1.6 million deaths each year [1]. In recent decades, the rise of pathogenic fungi has increasingly become a public health threat, particularly due to the limited availability of antifungal drugs for treating invasive infections and antibiotic resistance [2, 3]. The World Health Organization (WHO) highlighted specific pathogens of concern in 2022, categorizing them into critical, high, and medium-priority levels. Critical priority pathogens include Cryptococcus neoformans, Candida auris, Aspergillus fumigatus, and Candida albicans. High-priority pathogens encompass Nakaseomyces glabrata (Candida glabrata), Histoplasma spp., Mucorales, Fusarium spp., Candida tropicalis, and Candida parapsilosis. Medium-priority pathogens consist of Scedosporium spp., Lomentospora prolificans, Coccidioides spp., Pichia kudriavzeveii (Candida krusei), Cryptococcus gattii, Talaromyces marneffei, Pneumocystis jirovecii, and Paracoccidioides spp. Thus, finding new safe effective antifungal agents became an urgent need [2, 4].

Chitinase is an enzyme that hydrolyzes chitin, a major component of the cell walls of fungi and some other microorganisms. Research has shown that chitinases have potential antifungal activity against human pathogenic fungi. Chitinase can be produced by various organisms, including bacteria, fungi, plants, and animals [5, 6]. The antifungal activity of chitinase can be through two mechanisms. First, chitinase breaks down chitin, which is a crucial component of fungal cell walls. By degrading the cell wall, chitinase can compromise the structural integrity of the fungi, potentially leading to cell death. Second, chitinase interferes with the growth and development of fungi by inhibiting the synthesis of chitin. This disruption in fungal cell wall formation can impede the progression of the fungal infection [7–9]. Chitinase plays a role in modulating the immune response to fungal infections. Chitinase-generated chitin fragments may contribute to the activation of adaptive immune responses. This involves the stimulation of B cells to produce antibodies and the activation of T cells to mount a more specific and targeted immune response against the fungal pathogen. It can stimulate the host's immune system, leading to an enhanced antifungal defense [10]. The regulation of the immune cells through chitinase gives chitinase a central role in the inflammatory response. Chitinase activity could modulate the phenotype and function of these cells, promoting an anti-inflammatory state. Chitinase may play a role in the resolution phase of inflammation, promoting the clearance of inflammatory mediators and damaged tissues. This can contribute to the restoration of tissue homeostasis and the dampening of the inflammatory response. Also, chitinase activity interferes with signaling pathways involved in inflammation. By disrupting these pathways, chitinase could inhibit the expression of pro-inflammatory genes and reduce the overall inflammatory response [11].

The role of chitinase against human pathogenic fungi needs more investigation. Research in this area is ongoing, and the use of chitinase as a therapeutic agent against human pathogenic fungi is still in the experimental stages. It's important to note that the effectiveness of chitinase can vary depending on the specific fungal species and the chitinase source [6]. From this point of view, the present research tries to investigate the anti-inflammatory and antifungal activity of bacterial and fungal chitinase against critical and high-priority human pathogenic fungi.

Material and Methods

Bacterial and Fungal Chitinase

Bacterial chitinase from Streptomyces griseus (C6137) and fungal chitinase from Trichoderma viride (C8241) was purchased from Sigma Aldrich, Merck KGaA, Darmstadt, Germany.

Antifungal Experiments

Pathogenic Fungi

We studied the antifungal activity of the bacterial and fungal chitinase on human pathogenic fungi. Critical priority pathogens were Cryptococcus neoformans (ATCC 208821), Candida auris (ATCC MYA-5001), Aspergillus fumigatus (ATCC 1022), Candida albicans (ATCC 14053). High-priority pathogens were Candida glabrata (ATCC 90030), Candida tropicalis (ATCC 750), and Candida parapsilosis (ATCC 90018). The studied strains were purchased from the American Type Culture Collection (ATCC). The fungal strains were acquired in the form of frozen ampoules, which were delivered in dry ice. To thaw a frozen ampoule, we immersed it in a water bath set at a temperature between 25 and 30 °C until it was just thawed. Following thawing, we aseptically transferred the contents onto a plate containing Sarbouraud's broth (ATCC medium 28) and incubated at 27 °C till the fungal growth appeared (3 days). Upon fungi growth, we proceeded to subculture the inoculum on the same media at 27 °C for 5 days.

Assay for Antifungal Activity

The antifungal efficacy of chitinase was assessed using a growth inhibition assay against pathogenic fungi. Flasks (250 mL) containing 1/2 Emmons' modification of Sarbouraud's broth (ATCC medium 28) were supplemented with varying concentrations of chitinases (bacterial chitinase, or fungal chitinase, or a mixture of fungal and bacterial chitinase (in 1:1 ratio) (10–50 mg/mL we dissolved the chitinase powder in dist. water with required concentration) and 10 µL of a fungal spore suspension (approximately 10⁶ spores/mL). Positive control cultures were prepared with the addition of different concentrations of fluconazole as a common antifungal agent (10–50 mg/mL). A negative control culture was prepared without additional chitinases. The culture underwent incubation at 26 °C for 5 days to assess fungal growth. Harvesting of fungal growth was accomplished by centrifugation at 4000 rpm for 5 min, followed by rinsing with distilled water. Subsequently, the harvested biomass was dried at 65 °C until a constant weight was achieved to determine the dry biomass. The percentage inhibition of growth was calculated using the formula:

$$\%inhibition=\frac{Controldryweight-Treatmentdrweight}{controldryweight}\times 100$$

Anti-inflammatory Activity

The anti-inflammatory activity assessment involved determining the percentage of inhibition of albumin denaturation, following the methodology outlined by Sakat et al. [12]. The reaction mixture included test extracts (varying concentrations of bacterial or fungal chitinase or fungal and bacterial chitinase mixture (in 1:1 ratio) (10–50 mg/mL we dissolved the chitinase powder in dist. water with required concentration) or Aspirin as positive control) and a 1% aqueous solution of bovine albumin fraction. Subsequently, the tubes underwent incubation at 37 °C for 20 min, followed by heating to 51 °C for an additional 20 min. After cooling, the absorbance was measured spectrophotometrically at 660 nm. The percentage inhibition of protein denaturation was then calculated using the formula:

$$\%inhibition=\frac{Absorbanceofcontrol-AbsorbanceofSample}{Absorbanceofcontrol}\times 100$$

Statistical Analysis

All results were analyzed by SPSS software (version 14). Data was expressed as mean ± SD. A comparison of mean values was done using two-way ANOVA.

Results and Discussion

Antifungal Activity

Chitinase is an enzyme that breaks down chitin, a major component of the cell walls of fungi. The antifungal activity of chitinase is primarily attributed to its ability to degrade chitin, which is an essential structural component for many fungi. Also, chitinase interferes with the growth and development of fungi by inhibiting the synthesis of chitin. This disruption in fungal cell wall formation can impede the progression of the fungal infection [7–9]. Results in Figs. 1 and 2 clarified that Streptomyces griseus (bacterial) and Trichoderma viride (fungal) chitinase have a significantly higher antifungal activity than the standard (Fluconazole). The antifungal activity of chitinases is dose-dependent as the chitinase concentration increases the fungal growth inhibition increases. Bacterial, fungal chitinase, the mixture of them with a concentration of 50U/mL caused more than 90% fungal growth inhibition. The dose-dependent nature of chitinase's antifungal activity is a result of the fundamental principles governing enzyme–substrate interactions. Increasing the concentration of chitinase allows for a more effective and efficient breakdown of chitin in fungal cell walls, leading to a stronger antifungal effect [13].

Fig. 1.

Antifungal activity of different concentrations (10–50 mg/mL) of bacterial chitinase, fungal chitinase, bacterial and fungal chitinase mixture, and fluconazole against human pathogenic fungi with critical priority. Columns are means of three replicates with error bars for standard deviation. Columns followed by different letters are significantly different from each other according to the ANOVA test

Fig. 2.

Antifungal activity of different concentrations (10–50 mg/mL) of bacterial chitinase, fungal chitinase, bacterial and fungal chitinase mixture, and fluconazole against human pathogenic fungi with high priority. Columns are means of three replicates with error bars for standard deviation. Columns followed by different letters are significantly different from each other according to the ANOVA test

Interestingly, the bacterial chitinase exhibited greater antifungal activity against Aspergillus fumigatus compared to the fungal chitinase. Conversely, when tested against other pathogenic fungi such as Cryptococcus neoformans and Candida species, both bacterial and fungal chitinase demonstrated similar antifungal activity. This distinction in antifungal efficacy against Aspergillus fumigatus arises from the higher activity of bacterial chitinase, enabling it to hydrolyze chitin more effectively than its fungal counterpart.

Enzyme fundamentals dictate that high substrate concentration requires a higher enzyme concentration and activity. Given the variability in fungal structure, composition of the cell wall, chitin content, and genetic diversity, bacterial chitinase, with its heightened activity, exhibits greater antifungal activity against fungi characterized by high chitin content in their cell walls [14, 15].

Notably, Aspergillus fumigatus has a higher chitin content compared to other pathogenic fungi. While the chitin content constitutes 1–2% of the dry weight of yeast cell walls (Cryptococcus neoformans and Candida species), filamentous fungi like Aspergillus fumigatus have chitin content ranging from 10 to 20% [16–18]. Therefore, the heightened antifungal activity of bacterial chitinase, particularly against Aspergillus fumigatus, is attributed to its ability to degrade the cell wall, with antifungal efficacy hinging on the composition of the fungal cell wall [14, 15].

Anti-inflammatory Activity

In response to harmful stimuli such as pathogens, tissue injury, or irritants, the body initiates a complex biological response known as inflammation. Fungal infections are among the various triggers capable of inducing this inflammatory response [19]. Inflammatory conditions lead to the denaturation of protein cells and compounds that can prevent this denaturation exhibit anti-inflammatory effects. The inhibition of albumin denaturation is a widely used assay for evaluating the anti-inflammatory activity of compounds [20, 21]. Table 1 presents results indicating that bacterial, and fungal chitinase and their mixture demonstrate significantly higher anti-inflammatory activity compared to aspirin, a standard anti-inflammatory material. Chitinases, in particular, were found to prevent 98% albumin denaturation, marking the first study to highlight their role in protecting albumin from denaturation. Other protein compounds have been reported to protect albumin from denaturation by preserving its three-dimensional structure under stress conditions [22]. Studies suggest that chitinase may be linked to a reduction in the production of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α) and interleukins. Additionally, chitinases play a role in the resolution phase of inflammation by facilitating the clearance of inflammatory mediators and damaged tissues, contributing to an overall anti-inflammatory effect [11].

Table 1.

Anti-inflammatory effect of different concentrations on bacterial and fungal chitinase as albumin denaturation inhibition %

Concentration (U/mL) Sample	10	20	30	40	50
Bacterial-Chitinase	68.395 ± 1.583b	78.204 ± 1.482c	87.158 ± 1.220d	95.178 ± 1.394e	97.593 ± 1.578e
Fungal-Chitinase	56.304 ± 1.782a	67.771 ± 1.382b	82.917 ± 2.001c	96.019 ± 1.321e	97.2049 ± 1.029e
Bacterial–Fungal Chitinase Mixture	78.592 ± 0.893c	88.103 ± 1.291d	96.394 ± 1.003e	97.819 ± 1.010e	97.921 ± 1.192e
Aspirin (standard)	34.207 ± 0.964a	50.692 ± 0.620b	55.496 ± 0.495c	58.102 ± 0.859c	63.291 ± 0.810cd

Values are represented as the mean of three replicates ± standard deviation. Values in the same column followed by different letters are significantly different from each other according to the ANOVA test

It's crucial to note that the relationship between chitinase and inflammation can be complex and context-dependent. Therefore, further studies are needed to explore the therapeutic role of chitinases [6].

Conclusion

Bacterial and fungal chitinase showed high antifungal activity against critical and high-priority human pathogenic fungi. Interestingly, chitinases showed anti-inflammatory activity by inhibiting the albumin denaturation. More studies are needed to explore the antifungal activity of chitinases against human pathogenic fungi and investigate the anti-inflammatory mechanism of chitinase.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical Approval

This study does not contain any studies on humans or animals.

Consent to Participate

Not applicable.

Consent for Publication

Not applicable.