The influence of bacterial interaction on the virulence of Cryptococcus neoformans

Abstract

Microbes exist in complex communities in the environment. The interaction between fungi, such as the opportunistic pathogen Cryptococcus neoformans, and antagonistic environmental bacteria, such as Acinetobacter spp., may influence fungal evolution through the production of fungal defence mechanisms that inadvertently enhance fungal pathogenicity. Such changes include alteration of biofilm formation and increased capsule production. The molecular mechanisms responsible for such changes, both from a bacterial and fungal point of view, are of great interest to understanding the evolution of pathogenicity. Additionally, further elucidation of the stability of the induced changes in C. neoformans, and the impacts of these change on the disease-causing potential of this fungus, is of great interest.

Keywords:

• Acinetobacter baumannii
• C. neoformans
• evolution
• polymicrobial interaction
• virulence

This article is referred to by:

Fungal serotype-specific differences in bacterial-yeast interactions

Microbiologists favor a reductionist approach to the study of microorganisms, but this belies the fact that microbes exist in complex communities rife with antagonistic interactions that can contribute to pathogen evolution. Fungal and bacterial interactions are widespread in soil and water, as well as within plant and animal hosts. Fungal and bacterial species that co-inhabit a niche show diversified phenotypic properties not seen in their individual counterparts. These changes can bring about adverse effects to the host such as the case of Candida albicans, an opportunistic pathogen affecting immunocompromised individuals, whose ability to cause invasive candidiasis can be enhanced by the presence of oral streptocoocci.¹ In addition to a contribution to virulence, altered phenotypes can enhance resistance toward antimicrobial drugs, therefore it is extremely important to understand the mechanism of fungal and bacterial interactions.

Cryptococcus neoformans is a commensal fungus found in the pigeon gastrointestinal tract and in soil contaminated with pigeon excreta. C. neoformans is an opportunistic facultative intracellular pathogen that may cause skin infections (serotype D) or lung infections with a risk of progression to meningoencephalitis (serotype A), especially in immunocompromised individuals. C. neoformans owes its pathogenesis to the presence of a polysaccharide capsule that protects it from phagocytosis and helps it survive inside the macrophage, while the formation of biofilms may favor environmental survival and potentiate disease.² In this issue, Abdulkareem et al.³ analyzed the outcome of interactions between C. neoformans serotype A and serotype D and a model soil bacterium, Acinetobacter baumannii. They found that in laboratory co-culture the fungus had reduced viability (to around 30% – 60%), with serotype A isolates having higher survival rates than serotype D. Both serotypes of C. neoformans produced more robust biofilms, a thicker capsule and released more capsular polysaccharide upon exposure to A. baumannii; these phenotypes were more pronounced for serotype A. The authors speculate that these changes initiated through interaction with soil microbes in the environment may place selective pressure on C. neoformans, enhancing the disease causing potential of this pathogen. They also suggest that the more prominent response for serotype A strains may influence the geographical distribution of this serotype and contribute to the ability of this serotype to cause a different spectrum of disease.

This study has some limitations. The authors use A. baumannii as a model soil organism; while other Acinetobacter species are ubiquitous in soil and water, this is not the case for A. baumannii, which is only occasionally isolated from environmental sources.^4,5 The analysis of the A. baumannii influence on C. neoformans biofilm architecture was limited to only one isolate for each serotype, precluding conclusions on the role that biofilm structure may play in different disease outcomes or geographical distribution for each serotype.

An obvious question arises from this study; what is the molecular mechanism that causes these changes in C. neoformans? Is change due to direct contact with cell surface-exposed factors, effectors directly injected into the yeast cells, or secreted compounds in the extracellular milieu? The A. baumannii outer membrane porin OmpA has previously been shown to play a direct antagonistic role against the yeast Candida albicans.⁶ An intact type VI secretion system allows Acinetobacter strain M2 to outcompete E. coli in co-culture in a contact-dependent manner,⁷ suggesting that direct injection of effectors is used by A. baumannii to kill competitors in an ecological niche. Secreted factors are likely to be responsible for changes in biofilm formation of the fungus because in the assay used, a membrane physically separates the protagonists. An examination of A. baumannii mutants in some of these factors may reveal the basis of the bacterial side of this interplay.

This study also shows that the bacterial-yeast antagonism is bidirectional, with only 40% of A. baumannii surviving incubation with yeast. Other studies have found that yeast can inhibit A. baumannii growth. For example, Candida albicans secretes a quorum sensing molecule, farnesol, which inhibits the growth of A. baumannii.⁸ Further examination of these factors is also of interest.

Polymicrobial interactions with C. neoformans have previously been investigated with the bacterium Staphylococcus aureus.⁹ Viability of C. neoformans was decreased when co-cultured with S. aureus, while S. aureus was unaffected. Notably, the C. neoformans capsule was protective and direct attachment of S. aureus cells was a prerequisite to exert its lethal effect on C. neoformans. Abdulkareem et al., ³ report a similar observation for interaction between A. baumannii and C. neoformans; the higher survival of C. neoformans serotype A strains compared to serotype D strains correlates with thicker capsule and more capsular polysaccharide (glucuronoxylomannan) production.

This study opens up numerous avenues for further investigation. As mentioned above, the mechanism of interaction between A. baumannii and C. neoformans is an important aspect to examine. The role of capsular polysaccharide, specifically glucuronoxylomannan in virulence and protection of C. neoformans against A. baumannii should be elucidated. It would also be interesting to analyze the regulation of known pathways of biofilm formation and capsule production to see if they are induced by interaction with A. baumannii.

Besides polysaccharide capsule, the phospholipase activity of C. neoformans is another important virulence factor ¹⁰ but what is its role in bacterial interaction? Could stable genetic variants be selected through prolonged co-incubation of C. neoformans and A. baumannii? These may have constitutively increased capsule production, or altered biofilm formation leading to an increase in resistance to killing by A. baumannii. Finally, to ascertain the role of these changes in disease causation, could A. baumannii-exposed fungus be introduced into an animal host to see if it has enhanced virulence?

This study makes an interesting contribution to the concept of how microbial evolution may be driven by environmental factors such as polymicrobial interactions. The interaction with other microbes may enhance the production of factors that contribute to the disease-causing potential of C. neoformans. Further work into the mechanisms driving these changes, and how these changes may influence infection, will be enlightening.