1. Introductions

1. 简介

Cryptococcus. neoformanss is a human pathogenic fungus belonging to the phylum Basidiomycota ^[1][2]. It has a well-defined bipolar mating system, consisting of two different mating types, MATa and MATα ^[3][4], but is also capable of self-fertilization within cells of the same mating type ^[5][6].

C. neoformans is widely distributed in the environment, particularly in bird droppings, soil, and trees ^[7]. It poses a particular danger to immunocompromised hosts, such as HIV/AIDS patients and organ transplant recipients, as it can easily cause meningitis ^{[8][9][10][11][12][13]}. For instance,

C. neoformans is estimated to cause approximately 223,100 new cases and 181,100 deaths annually among HIV/AIDS patients ^[14][15]. According to the classification by the World Health Organization (WHO), these species are classified as one of the four priority fungal pathogens affecting humans. Cryptococcus neoformans infection typically occurs through inhalation of fungal spores or desiccated yeast cells in the environment ^[16][17], but can also occur through skin wounds ^[18]. These species are able to overcome host defenses and enter the bloodstream, subsequently invading the central nervous system and causing meningitis ^[16][19]. The diagnosis of cryptococcal meningitis typically involves a comprehensive evaluation of clinical symptoms and signs, combined with various diagnostic methods ^{[20][21][22][23]}. These methods include assessing the patient's medical history and performing physical examinations to identify symptoms associated with cryptococcal meningitis. Lumbar puncture is a routine procedure used to obtain and analyze cerebrospinal fluid (CSF) to determine the presence of

Cryptococcus species. Laboratory tests include India ink staining of the CSF, culture of Cryptococcus species, or antigen testing to confirm the presence of Cryptococcus infection. Neuroimaging techniques such as computed tomography (CT) and magnetic resonance imaging (MRI) can be used to examine the brain for signs of meningitis or other abnormalities. The combined application of these methods is crucial for accurate diagnosis and exclusion of other potential causes. It has been suggested that the composition of the gut microbiota may play a potential role in the diagnosis of cryptococcal meningitis. Studies have shown that alterations in the gut microbiota may be associated with fungal infections, including Cryptococcus species ^[24]. C. neoformans is able to impair the accurate recognition of fungal antigens by the host and evade host immune responses, including the coordinated action of macrophages, dendritic cells, T lymphocytes, B lymphocytes, innate lymphoid cells, and cytokines ^[25][26]. Furthermore,

C. neoformans utilizes melanin production to evade host immunity and enhance its infectivity in the host. Melanin in the cell wall of

C. neoformans is a major virulence factor with multiple functions, such as resistance to oxidative stress, reduction of antifungal drug efficacy, and modulation of interactions with phagocytic cells ^[16][27][28]. Researchers believe that melanin may be a potential target for the development of drugs against

Cryptococcus infections ^[29][30]. A notable feature of C. neoformans is its inability to produce melanin pigments from endogenously synthesized compounds, such as tyrosine, but instead utilizes catecholamines, such as dopamine, norepinephrine, and epinephrine, to oxidize and generate melanin in the brain tissue through the action of laccases (Lac1 and Lac2) ^[31][32][33]. The neurotropism of

C. neoformans is closely related to the presence of catecholamines in the brain and low glucose concentrations in the central nervous system, as these conditions favor laccase production ^[32][34][35].

2黑色

Infection and Dysbiosis of the Gut Microbiota  

 The latest advances in our understanding and analysis of the gut microbiota have revealed significant impacts of alterations on human health. The gut microbiota is a complex and diverse microbial ecosystem that plays crucial roles in various aspects of host physiology. It actively participates in host immune responses, influences metabolism, promotes biosynthesis, and defends against pathogenic yeast infections ^{[36][37][38][39]}. In this regard, it has been found that the composition of the gut bacterial microbiota can influence the production of pulmonary IL-17 responses during opportunistic human fungal pathogen Aspergillus fumigatus infection in mice. Mice with specific gut bacterial compositions exhibited stronger IL-17 responses, which are critical for combating fungal infections ^[40]. Recently, Li and colleagues reported that

C. neoformanss infection induces changes in the human gut microbiota ^[24]. The authors performed alpha and beta diversity analyses, comparing the gut microbiota of cryptococcal meningitis patients with healthy controls. The results showed a significant reduction in alpha diversity in cryptococcal meningitis patients compared to the healthy control group, indicating gut dysbiosis. They identified a total of 72 differentially abundant bacterial species and 8 differentially abundant fungal species between these two groups (Figure 1).

Figure 1. Changes in the diversity of gut bacterial and fungal microbiota in patients with cryptococcal meningitis. There are notable differences in the composition of gut bacterial and fungal microbiota between patients with cryptococcal meningitis and healthy controls. A total of 72 bacterial species and 8 fungal species were found to have differential abundance between these two groups ^[24].

2. Melanization phenomenon of

Cryptococcus. neoformmans in human brain tissue

The deposition of melanin on the surface of Cryptococcus neoformans cells is one of its important features, and this deposition contributes to its pathogenicity ^[41][42]. The deposition of melanin depends on the composition and flexibility of the cell wall of

C. neoformans ^[43]. The deposition of melanin within the cell wall provides several advantages for the pathogen ^[44]. Firstly, melanin acts as a protective barrier and can resist host immune reactions, including phagocytosis by immune cells. Studies have shown that melanin can inhibit the production of reactive oxygen species and reduce the activity of antifungal drugs, thereby enhancing

C. neoforman resistance to host defenses. In addition, melanin is associated with the dissemination of

C. neoformans within the host. Melanized fungal cells can be detected in various organs, including the brain and lungs, indicating the important role of melanization in the invasion and establishment process in different infected tissues ^[45]. The main sign of melanization during

C. neoformans infection is the presence of acid-resistant melanin ghost particles. These particles can be isolated from infected animal and human tissues, as well as from cells cultured on agar plates ^[46][47]. The mammalian nervous system is a rich source of catecholamines, which are a class of nitrogen-containing diphenolic compounds, including dopamine, adrenaline, and noradrenaline, among others ^[48][49].

C. neoformans synthesizes melanin by using the oxidation process of exogenous catecholamine substrates catalyzed by laccase ^[50][51][52]. Melanized Cryptococcus neoformans cells can be detected in brain tissue samples from patients with cryptococcal meningitis [60]. The melanin synthesized by Cryptococcus neoformans in brain tissue may vary in different anatomical regions, as it can bind to multiple catecholamines simultaneously (see Figure 2). This is because the relative proportions of these neurotransmitters may vary greatly in different brain regions ^[48][49]. The chemical structure determines the variability of the types of melanin synthesized when added to the culture medium. It is worth noting that Baker et al. made important observations on the ability of Cryptococcus neoformans to produce different forms of melanin using a mixture of catecholamines in the human brain (0.6 mM dopamine, 0.33 mM adrenaline, and 0.07 mM noradrenaline). This melanin can provide protection against UV radiation and oxidants.

Figure 2. Melanization of Cs

. neoformans in different brain tissues. The composition of melanin produced during the infection process may vary depending on the composition of catecholamines in the brain tissue. It is likely that melanin synthesis in vivo is generated through the polymerization of various precursor compounds ^[27]. Distribution of catecholamines in the brain: the red region represents dopamine enrichment, the green region represents norepinephrine enrichment, and the yellow region represents the overlap of dopamine and norepinephrine. In the cell wall of

C. neoformans, the melanin produced in different brain regions can be visualized as red, green, and yellow.

4. Potential impact of the gut microbiota on melanization of

Cryptococcus. neoformans

4.1. The gut microbiota may influence the levels of melanin substrates

The Gut-brain axis is a bidirectional communication system connecting the gut microbiota and the central nervous system ^[53][54]. It is generally believed that the gut microbiota has the ability to influence various aspects of brain function, such as emotions, behavior, and cognition ^[55][56]. This influence may occur through different pathways, such as affecting the production of neurotransmitters and other signaling molecules that can impact the function of the central nervous system [70, 72]. While there is no direct evidence linking the gut-brain axis to the synthesis of melanin in C新福曼斯

. neoformans, there is evidence suggesting that the gut microbiota may influence the levels of catecholamines (such as dopamine and norepinephrine) (see Figure 3), which are precursors for melanin synthesis in

neoformans.

Figure 3. Potential model of the interplay between

s SC利用人脑儿茶酚胺混合物（0.6 mM多巴胺，0.33 mM去甲肾上腺素和0.07 mM肾上腺素）产生多型黑色素的能力进行了重要观察。这种黑色素可防止紫外线和氧化剂。

图2.

C. neoformans infection, gut microbiota, and host molecules. It has been observed that there is an association between

C. neoformans infection and alterations in the gut microbiota ^[24]. The microbiota-gut-brain axis may regulate the levels of melanin sunstrate catecholamines (such as dopamine or norepinephrine) ^[57][58][59], as well as modulate the nutritional signals (such as glucose or amino acids) that influence Cryptococcus neoformans melanin production in the brain ^[60].

4.2. The gut microbiota may influence the generation of

s SC的细胞壁中，不同大脑区域产生的不同类型的黑色素可以可视化为红色，绿色和黄色。

4. 肠道微生物组对新甲虫脑黑色化的可能影响

4.1. 肠道微生物组影响黑色素底物的水平

肠脑轴是连接肠道微生物组和中枢神经系统的双向通信系统[68，69]。人们普遍认为，肠道微生物组有能力影响大脑功能的各个方面，如情绪、行为和认知[70，71]。这被认为是通过不同的途径发生的，例如神经递质和其他信号分子的产生，这些信号分子可以影响中枢神经系统的功能[70，72]。虽然没有直接证据表明肠脑轴与新福曼梭菌黑色素的合成有关，但有证据表明肠道微生物组有可能影响儿茶酚胺的水平（图3），如多巴胺和去甲肾上腺素，它们是脑组织中新福曼梭菌SC菌株的黑色素底物。

图3.

Cryptococcus. neoformans melanin by modulating nutritional signals

The ability to rapidly adapt to fluctuating external conditions is crucial for the survival and proliferation of microorganisms. This is especially important for pathogenic microorganisms like

C. neoformans, as they need to adapt to the transition from the environment to the host and initiate appropriate responses to establish infection. The host environment presents various challenging conditions, such as changes in available nutrients, oxygen levels, pH, and temperature, as well as potential threats from the host immune response ^[61][62]. It is noteworthy that the mechanisms for adapting to nutrient availability not only contribute to microbial proliferation but also play a role in regulating their virulence ^[63].