Cryptococcus neoformans (CN) is a microscopic fungus much like the very common Baker’s Yeast (Saccharomyces cerevisiae); however, it possesses some special characteristics, which make it unique and bestow upon it the ability to cause disease (pathogenic potential).
What are these characteristics, otherwise known as Virulence Factors? The very first thing one notices about CN is the presence of an external covering, called the Capsule, outside the cell membrane. The capsule is a polysaccharide, a molecule composed of several types of sugars (‘carbohydrates’), that are laid out in strands from a backbone. Structurally, there are two different polysaccharides that make up the capsule in varying quantities. Why is this important? Because both polysaccharides are important for the disease caused by this fungus – known as cryptococcosis.
One polysaccharide, found in copious quantities (90-95%) in the capsular material, is a large molecule called GXM or GlucuroXyloMannan – composed of repeating units, made of strands of Glucuronic acid (a sugar acid) and Xylose (a small sugar) attached to a backbone of chains of mannose (another small sugar). The other polysaccharide, present in smaller quantities (5-8%), is a smaller molecule called GalXM or GalactoXyloMannan – composed of repeating units of mannose and xylose strands attached to a Galactose (a small sugar, very similar to glucose) backbone. There is a minute proportion of a protein containing mannose subunits (a.k.a. mannoprotein), but we don’t know much about it or its function in the cryptococcal capsule.
Can we see the capsule? Well, yes, but… The capsule, being highly negatively charged, retains a lot of water, and is difficult to see under the microscope – unless special techniques are used. For example, India Ink (colloidal carbon particles suspended in water) or Nigrosin (an aniline dye which is excluded by living cells), a drop of which makes the medium surrounding the microbe darker. The carbon particles or the dye cannot enter the area covered by the capsule, making the microbe visible by contrast (‘negative staining’). Or, chemical substances such as the PAS (Periodic Acid Schiff) stain (which stains polysaccharides purple-magenta), the Mucicarmine stain (which stains the cryptococcal capsule pink-red), as well as certain other stains, which make CN visible in tissue sections (‘positive staining’). Or, antibody-based techniques, in which antibodies that recognize structural determinants on the capsule are attached to fluorescent molecules (such as Fluorescein Isothiocyanate, FITC), and allowed to bind to the capsule, making the capsule brilliantly visible under the microscope.
Left Panel: Cryptococcal capsule made visible by Nigrosin stain; Middle Panel: Hematoxylin-Eosin stain showing CN in brain section; Right Panel: Cryptococcal capsule made visible by a FITC-conjugated capsule-specific monoclonal antibody 18B7 (right panel image courtesy Dr. Magdia De Jesus, acknowledged with gratitude)
One of the unique features of the polysaccharide capsule of CN is that when the organism grows and divides (in tissues during disease, or in artificial medium during in vitro cultures), the capsular material is shed in copious amounts, so that it is detectable by antibody-based techniques in culture supernatants (i.e. the cell-free spent culture medium), as well as in various body fluids (depending upon the location of infection) – such as serum (the straw-colored fluid part of blood), expectorated sputum, bronchial washings (technically known as ‘broncho-alveolar lavage or BAL fluid), CSF (‘cerebrospinal fluid’ contained within the brain and the spinal chord), or even urine. In fact, the detection of the cryptococcal antigen in the body fluids is an adjunct diagnostic sign that indicates current or immediate past infection, and may be used to monitor the progress of the disease and therapy.
Chemistry-based techniques have been used to purify the capsular polysaccharide from culture supernatants, and the purified material has so long formed the basis of many, many studies that have elucidated the chemical structure, properties, and functions of the cryptococcal polysaccharide, although it was unclear whether the material simply sloughs off, or whether the microbe actively releases the material in its environment. However, a fascinating 2008 study by Susana Frases-Carvajal and others at the Albert Einstein College of Medicine has indicated that the polysaccharide on the cryptococcal capsule, and the exopolysaccharide that is shed have markedly different physico-chemical, as well as antigenic, properties.
Be that as it may, how do we know about the importance of this capsule in causing disease? Through studies, of course. Mouse studies done in early 80s and 90s demonstrated that natural and/or laboratory-created acapsular (i.e. lacking a capsule) mutants of CN had much reduced virulence, or ability to cause disease; in contrast, the parent strain of the lab-generated mutant, as well as a reconstituted/complemented mutant (in which the ability to make the capsule was returned by genetic manipulation), retained or regained the virulence.
So, why is the capsule a virulence factor? This I am going to discuss in the next post in this series. Don’t run away!
P.S. If interested, check out these excellent images of tissue sections stained to demonstrate fungal infections, including cryptococcosis.