Tag: cryptococcosis

Cryptococcus gattii Gone Wild on World Tour


By now you know, dear readers, that Cryptococcus gattii (CG), the deadly fungal pathogen and a native of tropical and subtropical regions of the world, has stealthily charted itself a course of world domination, starting with the Pacific Northwest of North America. I have also alerted you to the possibilities about its transmission – (a) that CG may have spread as a result of human activity, human and avian migration, and other natural means of dispersal; and (b) that slow, but sustained, elevation of global temperatures and corresponding changes in the long-term climate of different regions may have created micro-climates suitable for CG growth within zones with a temperate climate ordinarily considered inhospitable to the fungus.

Well, it is emerging that CG‘s plans are rather grandiose, total world domination – if possible.

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Stealthy emergence of Cryptococcus gattii in North America


What can the members of multiple animal species (cat, dog, bird, ferret, llama, alpaca, elk, goat, sheep, horse, porpoise) have in common with humans? Deeper philosophical questions aside, all of them have fallen prey to a deadly fungus spreading gradually, but steadily, in western North America (southwest Canada; US states of the Pacific Northwest, PNW) for over a decade.1

Well, what-ho, what-ho Cryptococcus gattii (CG), I believe we have been introduced.

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Cryptococcal Virulence Factor: Capsule 2

As I asked in the previous part: Why is the capsule a virulence factor? What special properties make it a key player in the process by which Cryptococcus neoformans (CN) causes disease?

Well, while acapsular mutants (lack external capsule, but contain GalXM on the cell wall), avirulent in normal hosts, may still cause disease in severely immunocompromised hosts, the burden of virulence associated with CN has traditionally fallen on the capsule, because of the extra-ordinary effects that it has on the immune system (quick reminder: these studies were largely done with what we now know as the ‘exopolysaccharide’, i.e. the shed capsular material).

Interestingly, CN appears to have the ability to modify the size and structure of the capsule in response to various stimuli, including the micro-environment of the target organ, stage of infection, stage of fungal cellular growth, and so forth. In mouse infection models, the capsule size increases rapidly (within hours) following infection; strains isolated from severely immunocompromised individuals (such as HIV/AIDS patients) often show large capsular diameter. The capsule composition (including ratio of various sugars, and arrangement of repeating units) may change at various stages during the infection. Since the host immune system presumably encounters the capsule first, these changes likely modulate how the host immune components react against CN.

various capsule sizes of Cryptococcus neoformans
Cryptococcal cells of various capsule sizes (all images under same magnification)

Let me try to summarize some of the major deleterious effects which the capsular polysaccharides (including the exopolysaccharide) have on the host immune system. Needless to say, these effects are crucial in helping CN establish the disease in the host. [Note: The host immunity plays an unwitting role in the establishment of the cryptococcal disease, but we shall discuss those factors later.]

  • Capsule interferes with normal functioning and immune functions of epithelial cells: Two types of epithelial cells (lining the surface of cavities) encounter inhaled CN first, the bronchial epithelium (inner lining of the branches of trachea, the air-tube) and the alveolar epithelium (inner lining of the air sacs – pockets of air – where exchange of gases takes place). Both epithelial cell types are capable of responding to the capsule, initiate a signal via release of messenger proteins called cytokines, and call for backup in form of an immune cell called neutrophil, which can kill microbes. However, the capsule suppresses cytokine release from bronchial epithelial cells; in alveolar epithelium, however, the capsule causes CN to be internalized in the epithelial cell, which kills the host cells. The resulting host cell damage likely allows CN to cross the epithelial barrier to reach inside the lung tissue.
  • Capsule helps CN evade capture by largely inhibiting phagocytosis: Phagocytic cells (cells which engulf and kill foreign substances, including invading microbes), such as the Macrophages in the alveolar pockets of the lung tissue, are among the first line of defence against invading microbial pathogens. The engulfment is initiated either by direct interaction between macrophages and the microbe, or via components of the humoral (i.e. non-cellular) immune system (antibodies and/or complements) of the host. Apart from killing the microbe, this process also presents bits and pieces of microbial material (‘antigen presentation’) to another immune cell, the helper T-lymphocytes (‘T-cells’), which ultimately allow the antibody-producing B-lymphocytes (‘B-cells’) to produce specific antibodies recognizing the microbe, as well as create immunologic memory. The cryptococcal capsule helps the microbe evade capture (thereby, also interfering with antigen presentation) by largely inhibiting the phagocytic process
  • Capsule also interferes with additional immune mechanisms that enable phagocytosis: Even in presence of capsule, antibodies and/or complements (mentioned above) can bind to the capsule and/or cell wall, and enable phagocytosis. However, a large capsule may effectively mask the binding sites for these immune proteins.
  • Capsule allows intracellular parasitism of phagocytosed CN: Even when the first-line immune mechanisms can successfully help Macrophages engulf the cryptococcal cell, the capsule actively interferes with their antimicrobial properties, so that internalized CN is not killed, but thrives, with the ability to spread to other cells, tissues, and organs.
  • Capsule aids dissemination of CN: Along with other mechanisms (including various cryptococcal enzymes), the capsule allows the spread of CN from lungs to the rest of the body. The microbe is able to change the composition of the capsule, presumably in order to adapt to different micro-environments. The structure and composition of the capsule are also important for the ability of CN to cross various physiological barriers to reach the target tissue.
  • Capsule is able to induce immunological unresponsiveness to CN (a.k.a. immune paralysis): The polysaccharide is known to be able to inhibit antibody production by B-cells, and growth and proliferation of T-cells; this is achieved via inhibition of antigen presentation by phagocytes (mentioned above), modulation of the production and release of certain cytokines, as well as induction of a certain subset of T-cells whose secreted products make other T-cells unresponsive. In addition, the direct binding of the polysaccharide to certain receptors (known as FCRγII) in various phagocytic cells leads to a profound immune suppression. The capsule also interferes with the functions of various other immune effector cells, such as neutrophils. Most of these effects have been described for the GXM component (see previous part) of the capsule, but GalXM and the mannoprotein components have also been implicated in similar effects. GXM and GalXM both can induce antigen-presenting phagocytic cells and T-cells to commit suicide (via a process called ‘apoptosis’).
Cryptococcus colony morphologies  on SDA
Click to embiggen: Various types of cryptococcal colony morphologies on artificial medium; it has been hypothesized that changes in colony morphology, controlled genetically, may be associated with changes in structure and composition of the capsule.

CN is abundantly present in the environment (especially in endemic areas, in association with certain trees and birds); therefore, one enduring mystery is the reason why it would need to evolve to make a capsule with such high virulence potential in mammalian hosts (primates, quadrupeds, as well as aquatic mammals). Extensive work done in the laboratory of Arturo Casadevall at the Albert Einstein College of Medicine in New York, as well as elsewhere, have shown that, in the environment, CN has to interact with various hosts, including certain amoebas, slime mould, nematode worms and insects – many of which can kill the microbe; the capsule affords protection against those marauders in a way that is remarkably similar to its interaction with the mammalian immune cells. Pathogenicity and virulence of CN in mammalian hosts may, therefore, be a consequence of that process.

In the next installment, I shall describe some different types of the cryptococcal organism, including their clinical significance.

Further reading:

Review of the Cryptococcal Capsule, by Oscar Zaragoza et al. Advances in Applied Microbiology, 2009, 68:133-216.

Cryptococcal Virulence Factor: Capsule 1

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.

cryptococcus capsule stains
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.

Cryptococcus, the Silent Menace: A Primer

Sir Arthur Conan Doyle (1859-1930), the celebrated author of Sherlock Holmes and the Sci-Fi Professor Challenger series, was also a physician and prone to poetry. In his second poetry collection, called Songs of the Road (published in 1911), he included several of his narrative verses and songs, as well as philosophical verses – some of which stand testaments to his abiding interest in the field of Medical Mycology, the study of clinically-relevant fungi. One particularly poignant poem, titled Mind and Matter, details his awe about spore-bearing pathogenic fungi and related systemic fungal infections, which were – in the early 20th century – probably almost always fatal. (Broad spectrum antifungals of today didn’t appear until the 60s.)

Mind and Matter

Great was his soul and high his aim,
He viewed the world, and he could trace
A lofty plan to leave his name
Immortal ‘mid the human race.
But as he planned, and as he worked,
The fungus spore within him lurked.

Though dark the present and the past,
The future seemed a sunlit thing.
Still ever deeper and more vast,
The changes that he hoped to bring.
His was the will to dare and do;
But still the stealthy fungus grew.

Alas the plans that came to nought!
Alas the soul that thrilled in vain!
The sunlit future that he sought
Was but a mirage of the brain.
Where now the wit? Where now the will?
The fungus is the master still.

Powerful imagery, right? To me, no fungus brings home this sense of foreboding and inevitability more, than does the yeast-like pathogen, Cryptococcus neoformans (let’s call it CN). (I admit I have a bias towards this microbe, having worked on it for the longest time in my academic career.)

Why Cryptococcus, you may well ask. Surely there are other sporulating (spore-forming) fungal pathogens that fit the bill? There are, of course. But I think, it was the mention of the brain in the third last line, that settled CN firmly in my mind. You see, CN is known to have a peculiar predilection for the brain and the central nervous system, even though the microbe – in form of spores (technical term: infectious propagules) – comes into the body from the environment most likely via the nose and the respiratory pathway, depositing itself into the lungs.

In healthy people, the body can usually fight off this invasion, with the aid of various components of the immune armamentarium – such as alveolar Macrophages, residents of the lungs and the first line of defence – cells that engulf the microbe and kill it internally; helper T-lymphocytes (a.k.a. CD4+ T-cells) which recognize fragments of the microbe and help B-lymphocytes to produce more and specific antibodies; immune effector cells, cytotoxic T-lymphocytes (a.k.a. CTL, or CD8+ T-cells) and Natural Killer (NK) cells; and so forth.

However, sometimes, and especially those who have a suppressed or compromised immunity for any reason, the microbe can overwhelm this defence and escape the lungs. One of the major systems it primarily attacks is the central nervous system (CNS), the brain and the spinal cord. Immune-compromised patients lie within a wide spectrum; suppression of immune defences may be secondary to HIV infection and AIDS, aggressive chemotherapy for cancer, therapeutic immune suppression for the purpose of organ or tissue transplants, or even metabolic immune-suppression that may result from chronic disorders such as diabetes. In these patients, the microbe CN is able to evade the sub-optimal immune defences and reach the CNS to cause a serious inflammatory condition called meningitis or meningoencephalitis. This is why CN is primarily considered to be an ‘Opportunistic Pathogen’, one that causes disease only in the context of suppressed immunity. This distinction is no longer strictly correct, because there are well-documented instances of cryptococcal infection of apparently-healthy individuals, too. However, a majority of the cases of cryptococcal meningitis occur in immunocompromised patients; in fact, cryptococcal disease of any organ other than the lung (a.k.a. extra-pulmonary cryptococcosis) is considered an AIDS-defining illness in an HIV infected individual, according to the Centers for Disease Control and Prevention’s (CDC) guidelines.

In the next few posts, I would describe more of the importance of this micro-organism, especially in the context of the United States. Stay tuned for part deux!