Journal of Analytical & Bioanalytical Techniques
Open Access

Fungal infections; Plants; Host cells; Morphology

Results

The ability of filamentous C. albicans cells to enter and develop invasively in substrates of varied stiffness has been investigated in the current work using PDMS micro-fabrication. We demonstrate that invasive growth within a stiff substrate is characterised by severe filament buckling and a stiffness-dependent drop in extension rate, and that there is a penetration barrier that corresponds to a stiffness of less than 200 kPa. We noticed a startling change in cell morphology during invasive growth, which is reduced cell compartment length and increased diameter. This change is not brought on by the depolarization of active Cdc42, but rather by mechanical compression occurring far away from the site of development.

Conclusions

Our findings show that active Rho1 depolarizes, as seen in membrane protrusions, whereas active Cdc42 levels rise at the apex in response to this compression. Our findings indicate that during invasive growth, cell growth and morphology are altered, suggesting that stiffness controls which host cells C. albicans can enter.

Background

Walled cells like fungus and plants can explore their environment for food and mates using polar tip development, which restricts extension to the apical surface, while still preserving their surface to volume ratio. Using turgor pressure, cell wall elastic properties, and secretion rate, Campas and Mahadevan have derived straightforward scaling equations for cell geometry and discovered a single dimensionless parameter that is adequate to explain variance in the form of tip developing cells. However, little is understood about how mechanical stress affects tip-growing cells. There are five main categories of mechanical stress: bending, torsion, torsion, compression, and shear. Fungal pathogens in humans and plants are capable of penetrating the host tissue, and it is expected that this penetration will result in compressive stress and invasive growth. Infection and successful dissemination of such fungal diseases depend on growth within a spatially constrained context in addition to tissue penetration. Tip development is a recurrent element in the various techniques fungal diseases use to interact with their surroundings. Both human and plant fungal diseases depend on penetration of the host tissue, which necessitates not only the production of enough force but also adhesion to the host cells to balance this force. Turgor pressures for fungi are in the MPa range, and for human fungi, this turgor pressure is greater than the host cell’s resistance to penetration. Although the critical stress for material rupture is decisive, such host cells have elastic moduli that are in the 1-100 kPa range. Candida albicans and Aspergillus fumigatus, two human fungal infections, may both actively infiltrate host tissue, which is a crucial step in the infection process. Previous research has shown that C. albicans can enter cells actively or by host-induced endocytosis. While both endocytosis and active penetration are crucial for C. albicans invasion of the oral epithelia, active penetration virtually exclusively causes invasion of the intestinal epithelia (small intestine enterocytes). Active penetration is the main method of tissue invasion, even with oral epithelia, in the early stages of infection. Therefore, a deeper comprehension of active penetration ought to provide light on the first stage of tissue harm for mucosal infections. The fungus peptide toxin candidalysin promotes C. albicans translocation through intestinal epithelial layers. Previous research has demonstrated that C. albicans hyphal ends have an asymmetrical posture when growing on a stiff surface, or a “nose down” morphology, and that perpendicular development and contact to a stiff topographical ridge causes the ridge to be indented. We used micro-fabrication and time-lapse imaging to examine the link between substrate stiffness and C. albicans penetration and invasive growth. We demonstrate that invasive growth within a stiff substrate is characterised by severe filament buckling and a stiffness-dependent drop in extension rate, and that there is a penetration barrier that corresponds to a stiffness of less than 200 kPa. However, only a tiny proportion of cells can enter 200 kPa PDMS, indicating that these cells may be crucial to infection, much like persister cells in biofilms. Additionally, during invasive growth, we noticed a startling change in cell shape that wasn’t brought on by the depolarization of active Cdc42 but rather by mechanical forces acting at a great distance from the site of development. Our findings demonstrate that, similarly to PDGF-induced fibroblast membrane protrusions, C. albicans increases active Cdc42 at the apex whereas active Rho1 is depolarized in response to mechanical stresses.

Results

Observing the development of Candida albicans filaments in microfabricated chambers

We made use of micro-fabrication techniques with the elastomer polydimethylsiloxane (PDMS), which has been specifically mentioned as a single-cell force sensor for fission yeast cells, to study C. albicans hyphal development. We created PDMS arrays with roughly 105 microchambers that had a cylindrical shape, a diameter of 10 mm, a depth of 5 mm, and an interchamber distance of 15 mm. With the aid of inverted microscopes, C. albicans cells were observed in microfabricated PDMS chamber arrays; imaging was done through an upright array of 150–200 m thick PDMS. An XZ confocal reflectance image through a PDMS microarray is shown in text, with the chambers and media at the top (highest position) and the supportslip below. Fetal calf serum was combined with C. albicans cells, introduced to the PDMS array, and then the development of filamentous growth was monitored over time. With low-stiffness PDMS (which has a high polymer to cross-linker ratio of 40:1), we saw two main types of filamentous development: invasive growth inside of PDMS and noninvasive growth on the surface. Additionally, we noticed that when a PDMS filament was inserted, the blastospore (round cell) portion of the filamentous cells that were growing in the microchambers pushed back against the chamber wall and the filament frequently buckled within the elastomer, most likely as a result of the resistive force created by the cells’ growth within the elastomer. These findings show that PDMS is compatible with C. albicans filamentous development in addition to possessing perfect optical characteristics.

Introduction

The stiffness of the substrate affects growth modes

By adjusting the ratio of polymer to cross-linker, we were able to monitor C. albicans filamentous growth in PDMS of various stiffnesses, or the degree to which an object resists [1-6] deformation in response to an applied force. In chambers with varying PDMS stiffness, we saw two distinct cell growth patterns: invasive growth, which was more common in chambers with softer PDMS, and dramatic bending, which was more common in chambers with stiffer PDMS (30:1).

Access into and exit from PDMS

We looked more closely at this process in PDMS since active penetration is essential for C. albicans epithelial invasion. Depicts a filamentous cell that enters PDMS at 4 minutes (II; take note that I is before the filament contacts the chamber wall); grows invasively therein (III); deforms the adjacent chamber (IV), causing a dramatic invagination; and then leaves PDMS at 2 minutes and 4 seconds (V), penetrating the opposing chamber at 2 minutes and 8 seconds (VI), and continuing to grow therein (2:12; VII). The portion of the filament inside PDMS that buckled during this time (1:22-2:02), resulting in an S-shaped filament, suggests that the resistive force revealed by the filament’s buckling and the initial chamber’s deformation during invasive growth (III), likely increases upon deformation and subsequent piercing into the adjacent well (IV). The filament’s tension was relieved when it left PDMS and entered the nearby well (V), as seen by the filament’s tip appearing to advance (2:04). The filament (part in the well) buckled as a result of the resistive [7-10] force from the last development stage (VII), forming a M shape (2:42–3:00). In some ways, this PDMS escape is comparable to macrophage filaments protruding from the cell. Here, the filament pushes into a circle, causing a deformation that requires local invagination of the chamber rather than surface area expansion, which is easier to see.

Materials and Methods

We monitored cells in which GFP was targeted to the plasma membrane, by confocal spinning disc imaging acquisition throughout a range of z-positions, to more clearly see the invasive development within PDMS over these several processes. A typical time-lapse acquisition is shown in text, where inspection of the cell outline did not demonstrate a significant change in the form of the filament tip [7] during invasive expansion and bursting into the next well. There were no modifications after the cell broke out of the PDMS, and the radius of the tip’s curvature matched that of surface-growing cells.

Resistance has an impact on the shape and expansion of hyphae

When the filaments buckled and the PDMS wells deformed during invasive development, it was clear that the filaments were responding to the resistive force, the strength of which we had calculated in the physical model. Young’s modulus affects the proportion of cells that enter PDMS, and investigations of the percentage of PDMS invasion at two stiffness values show that the invasion threshold is between 120 and 200 kPa.

Results and Discussion

Dependence of cell morphology on substrate stiffness

Our findings show that PDMS resistive forces cause changes in morphology since effects on cell morphology are only seen in filaments inside the material. Surprisingly, even when the compartment was more than 10 m away from the hyphal tip, we saw a steady increase in compartment diameter during growth in the stiffest PDMS. The cell wall in this proximal compartment may have undergone additional change, which might have resulted in a less rigid cell wall, an increase in turgor pressure, or mechanical deformation of the filament. As the cell compartment volume grows and around 60% of invasively growing hyphae buckle in this PDMS stiffness (150 kPa), we favour the latter two options even though we cannot rule out the possibility that the cell wall in this proximal compartment is less rigid during invasive growth. As the proximal compartment rose 25% more than the tip diameter during invasive growth, the significant change in filament shape was not entirely caused by a broadened tip. These morphological alterations are probably the result of increased turgor pressure and mechanical pressures from developing against a resistant substrate.

Conclusions

According to our findings, the sort of host cells that C. albicans can penetrate depends on how stiff they are. We noticed a small number of cells that were able to penetrate the substrate even with stiffer PDMS ( 200 kPa), which is intriguing and suggests these cells have unique qualities that may be helpful during epithelial invasion.

Author Contributions

The diagnosis and treatment of this cat were handled exclusively by Jennifer Weng and Harry Cridge. This report was written by Jennifer Weng, and Harry Cridge gave it a critical appraisal. The final draught of the manuscript has received the approval of both Jennifer Weng and Harry Cridge.

Conflict of Interest

According to the authors, there are no conflicts of interest that might be thought to compromise the objectivity of the research presented.

Ethics Statement

The case described in this report was handled as part of the regular clinical caseload at the university teaching hospital; an IACUC or other ethical approval was not necessary. All facets of this patient’s care had the owner’s consent.

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