Cryptococcus: the types

In the previous post in this series, I promised to talk about some different types of the cryptococcal organism, including their clinical significance. But before I point out the types, let’s recapitulate quickly the conventions of scientific naming of biological organisms, a.k.a. binomial nomenclature, a system in which living organisms (bacteria, plants, animals, and so forth) are identified with a set of two names, a generic name (indicating its Genus) and a specific name (indicating the species). Simply put, Genus (always written with an initial capital) refers to a particular group with a family of organisms, and species (always written in lowercase) refers to specific members within that group, who share genetic similarity to the point of being capable of interbreeding amongst themselves and producing fertile offsprings. This definition of species is, however, slightly foggy because under different circumstances, both natural and artificial, members of different species within the same genus have been able to mate. Be that as it may, in this discussion, we shall use the names of different cryptococcal species to indicate different organisms under the genus Cryptococcus. It is possible to identify (and discriminate between) these organisms in the laboratory using a variety of techniques, biochemical and molecular biological.

Currently the cryptococcal organisms that carry serious clinical significance are Cryptococcus neoformans variety (abbreviated as ‘var.’) grubii, C. neoformans var. neoformans, and C. gattii. In the older literature, C. gattii used to be known as C. bacillisporus and C. neoformans var. gattii; however, in late 2002, a group of taxonomists proposed the name C. gattii as more scientifically accurate¹ and it has since been accepted in the mycology community.

This is not to say that there are no other members of the genus Cryptococcus. Indeed, there are several non-neoformans Cryptococci in the environment, with their distinct biochemical and physiological profiles, as well as geographical habitats. However, they are mostly incapable of causing disease in animal hosts. A few (most notably, C. albidus and C. laurentii) have been implicated² in human diseases, albeit with very low incidence and prevalence, mostly in situations where the host was already severely immune-suppressed. We shall focus our current discussion on Cn var. grubii, Cn var. neoformans, and C. gattii, which are the major pathogens in clinical situations.

One major means of discriminating between these species is (or rather, used to be) immunological. In 1982, a Japanese scientist, Ikeda Reiko, developed a set of four rabbit antibodies³ which could produce a specific pattern of binding on the cryptococcal capsule; based on the binding pattern, four subtypes, named A, B, C and D, could be distinguished amongst the cryptococcal isolates. These types were called ‘serotypes’, because the determining tool was antibody-based (‘serological’). We now know that Cn var. grubii and Cn var. neoformans belong, respectively, to serotype A and D, whereas C. gattii comprises serotypes B and C. (Note: Ambiguous serotypes, or inter-varietal hybrids, such as AD and BC, as well as untypable strains – not reacting with any antibody – are occasionally observed, but their clinical significance is unknown.)

Another easy way of separating varieties of Cn from C. gattii is a biochemical test of growth on artificial medium containing specific chemical substances. The medium contains (a) some substance (such as L-canavanine or Cycloheximide) which – at a specific concentration – prevents the growth of Cn, but not C. gattii; (b) an amino acid called glycine; and (c) a pH indicator dye (such as phenol red or bromothymol blue). (a) and (b) serve as screens; C. gattii can, but Cn mostly cannot, subsist on glycine as the sole source of carbon and nitrogen (the basic elements or building blocks of life). Again, those Cn that manage to survive on glycine, cannot grow in presence of the specific concentration of, say, L-canavanine. Therefore, in this medium, only C. gattii can grow. When they grow, they release carbond di-oxide, making the medium more acidic. This is observed by the change in color of the pH indicator; for example, Bromophenol Blue changes to a deep ‘Cobalt blue’ color. Therefore, if C. gattii or Cn isolates are put on this medium to grow, only C. gattii will grow and change the medium to a deep blue color. The CGB (‘L-canavanine/ Glycine/ Bromothymol Blue’)-containing medium is still in use in microbiology laboratories to differentiate C. gattii from Cn.

Image Credit: My own work

However, scientists have to guard against the possibility that there may be other organisms which may grow on this medium as well. Therefore, usually, they would make sure that the isolates they are going to test on CGB are indeed Cryptococcus. This they do – as shown in the above figure – by utilizing two biochemical principles, two enzymes that Cryptococcus makes: (a) urease, an enzyme capable of breaking down urea, and (b) laccase (a.k.a. phenoloxidase), another enzyme which allow the fungus to break down certain organic substances to make the black pigment, melanin (yes, similar to the melanin we have in our skin and hair). In urea-containing medium, the breakdown of urea causes a change in pH, which shows up as a change of color of the medium (which contains, as before, an indicator). And in special medium containing phenolic substances, growth of Cryptococcus is accompanied by the production of a deep brown pigment. These tests, when positive, indicate the presence of cryptococcal strains. (Note: Rarely, cryptococcal strains that don’t make these enzymes are found; however, they are generally found to be incapable of causing disease, indicating that both these enzymes may independently contribute to the organism’s pathogenic potential.)

Hope you have enjoyed the series this far. In the next installment, I would talk about the geography and clinical significance of these cryptococcal species. Ta!

Technical reading, if interested:

1. Kwon-Chung et al. (2002) Taxon 51: 804-6
2. Khawcharoenporn et al. (2007) Infection 35: 51-8
3. Ikeda et al. (1982) Journal of Clinical Microbiology 16: 22–29