To infect the brain, the fungus learns to make itself smaller in guano!

Fungal meningitis is usually caused by the lungs inhaling fungal spores in the environment, and the ability of the fungus to infect is determined by the smooth transfer from the lungs to the brain
.
Cryptococcus neoformans is a common pathogen that causes fungal meningitis and is prevalent in soil, decaying wood and guano in
the wild.
By the age of five, human exposure to the fungus is as high as 70 percent, making prevention a daunting task
for people at high risk of CD4+ T cell deficiency, an important immune cell.

A new study published in Cell Host & Microbe by Jessica C.
S.
Brown's team from the University of Utah titled "A dissemination-prone morphotype enhances extrapulmonary organ entry by Cryptococcus neoformans" shows (Figure 1) [1] that once Cryptococcus neoformans enters the body, It only takes a few days to shrink in size and better infect the brain
.
The development of drugs that weaken or even curb this ability may provide new means
of treating fungal meningitis.

Figure 1 Research results (Source: [1])

Different responses by hosts to invading microorganisms or cancer cells will create a variety of environmental stressors, such as cells around the lesion being more vulnerable to attack by immune cells, while cells in the core region may face oxygen and nutrient deficiencies
.
Phenotypic switching and heterogeneity are clever ways for many microbes and cancer cells to survive in a fluctuating host environment, such as bacteria and parasites that avoid adaptive immune responses
through antigen switching.
Significant epigenetic changes that affect pathogenesis, such as yeast/hyphae switching in type II fungi, have also occurred, however, this morphological difference is evident
.
This study aims to show that subtle morphological changes can also have a profound impact on the infectivity of fungi, and the phenotypic heterogeneity of Cryptococcus neoformans in terms of capsule and somal size plays an important role
in its transmission in the host.

01 Deep behind enemy lines: small cells enter organs outside the lungs at a higher rate

In the lungs, Cryptococcus has a wide variety of cells of various sizes and appearances, while in the brain, the types are surprisingly homogeneous, suggesting that there is a reason why these cells can penetrate further into the body
.
To do this, the researchers measured the size of fungal somas and capsulas in the lungs, blood, spleen and brain at 3 and 17 days after vaccination with Cryptococcus in mice, and found that cells were limited in size in the spleen and brain, and brain fungi rarely exceeded 10 millimeters
in diameter.
Further by intravenously injecting mice with fungal ex vivo cells of different sizes to mimic the lung infection process, it was found that after 3 h of inoculation, the number of small ex vivo cells reaching the liver, spleen and brain was higher than that of medium and large ex vivo cells
.
Intracranial inoculation showed that all ex vivo cells grew well in the brain, suggesting that small cells have the advantage of multiplying after transmission rather than entering extrapulmonary organs (Figure 2).

Figure 2 Small ex vivo cells spread to organs outside the lungs at a higher rate (Source: [1])

02 Trojan horse meter: Mannose is the key for small cells to carry macrophages into organs outside the lungs

The researchers found that large cells bind more easily to complement protein C3 (p<0.
05) than medium-sized and small cells, while surfactant protein D (SPD) preferentially binds to small and medium-sized cells (p<0.
01).

This shows that the changes that occur in Cryptococcus are not only morphological size, but also accompanied by changes in immune recognition and cell surface structure
.
The researchers also used two lectins to measure exposure to microbial signatures at different depths of the cell wall and found that exposure to mannose was significantly increased in small ex vivo cells, while there was no difference
in exposure to chitin.
Given the pathogen clearance function of liver macrophages and platelets, the researchers measured changes in the number of fungi in the liver after macrophages and platelets were depleted, and found that the presence of macrophages did not play a role in removing fungi, but led to a large increase
in the number of cryptococci in the liver.
Exogenous mannose will reduce the binding rate of macrophages to small cells, suggesting that mannose exposure in small cells is key to mediating macrophage recognition (Figure 3).

Figure 3 Macrophage recognition of mannose helps small ex vivo cells to be uptaken by organs (Source: [1])

03 One side of soil and water culture and one side of "bacteria": the pigeon fecal environment enhances the organ entry ability of Cryptococcus neoformans

The results of the venous transmission model showed that cell size was not the only factor affecting the rate of diffusion, and that the culture conditions of the fungus also greatly influenced the distribution in the organ
.
The researchers further performed RNA sequencing on cells with different in vitro culture conditions and ex vivo cells of different sizes, and found that among all ex vivo cells, small cells were the most unique in transcription, with a large number of differentially expressed genes
.

The term gene ontology (GO) enrichment analysis showed that in vitro-cultured cells under specific conditions where diffusion performance was closest to small ex vivo cells, the expression of genes involved in phosphate acquisition and storage was not the highest, but higher than in the control group (Figure 4).

To identify whether phosphate is the component that induces small cell formation, the researchers conducted in vitro culture and found that only phosphate was sufficient to stimulate the transition of cells to smaller sizes, and that Cryptococcus neoformans shifted
to smaller forms when extracellular concentrations were higher or phosphate utilization increased due to lower pH.
This is also confirmed by experiments that limit phosphate in the body
.

In addition, the infection process itself can increase the extracellular phosphate content, which in turn forms a positive feedback loop
.
Phosphate-rich pigeon droppings are another important source of phosphate, with in vitro culture cells with the greatest
variation in cell diameter induced by pigeon droppings compared to other conditions.
Brown believes this could demonstrate how the pathogenicity of the fungus arose in the first place: "Evolutionary pressures from environmental niches, such as pigeon droppings, somehow give Cryptococcus neoformans the ability to
infect mammals.
" ”

Figure 4 Expression of phosphate acquisition genes correlates with changes in cell and capsule size (Source: [1])

Because of its key role in the invasion and reproduction of organs outside the lungs, the researchers suggest that these small, transcriptionally distinct cells from other populations are called "seed cells," rather than simply being seen as part
of a continuous change in cell size.
In the future, drugs that block this seed cell may help prevent or treat this fungal meningitis
.