Role of laccase in the virulence of Talaromyces marneffei: A common link between AIDS-related fungal pathogens?

KEYWORDS:

• AIDS
• Cryptococcus neoformans
• fungus
• Histoplasma capsulatum
• immunoregulation
• pathogen
• Talaromyces marneffei
• virulence

This article refers to:

Talaromyces marneffei laccase modifies THP-1 macrophage responses

Talaromyces (Penicillium) marneffei is an important AIDS-associated opportunist that causes disseminated infections involving the reticuloendothelial system, often causing lymphadenopathy, hepatomegaly and splenomegaly.^1,2 Up to 10% of patients in AIDS patients in Hong Kong have been reported infected with T. marneffei, making this an important AIDS-related fungal pathogen. Other immune deficiencies also associated with T-cell activation deficits such as autoantibody to IFN-γ and hematological malignancies such as Hodgkins lymphoma have been reported to be predisposing conditions.^3,4. Disseminated infections result in a chronic wasting syndrome characterized by fever, fatigue and anorexia as well as cough, dyspnea and chest pain. Skin manifestations are quite unique and suggest the diagnosis, described as papule-or mulluscum-contagiosum-like.⁵ Most infections occur in Southeast Asia where the organism has been found in soil as well as in the bamboo rat whose infected feces may contaminate surrounding surfaces.⁶ The fungus grows as a granular colony of septated hyaline hyphae at 25°C on Sabaouraud's dextrose agar and as yeast-like colonies at elevated host temperatures. The yeast form predominates in the host and can be found either in peripheral blood monocytes⁷ or within tissue biopsies such as those of lymph nodes where yeasts with a characteristic transverse septum can be obseerved using silver methenamine stains of cytology specimens from a fine needle aspiration.⁸

One of the distinguishing features of both yeast and mold forms of T. marneffei is the production of a red soluble pigment. Along with other putative virulence factors such as cell adhesion,^9,10 dimorphism ¹¹ and an ability to utilize alternative sugars through the glyoxylate cycle,¹² laccase-like activities detected in protein extracts of the fungus have been proposed to have a role in virulence.¹³ Laccases are a class of copper metaloenzymes first described in plants and are named because of their role in lacquer polymerization.¹⁴ Fairly promiscuous in their enzymatic reactions, laccases have an ability to oxidize a variety of diphenolic compounds to the corresponding highly reactive o-quinone, which can form adducts with a variety of nucleophile-containing proteins,¹⁵ as well as self-polymerize into molecules, including melanin-like pigments.^16,17 Other activities of laccases include oxidation of transition metals such as Fe II¹⁸ which block macrophage fungicidal activities, and the synthesis of immunosuppressing oxylipid molecules such as prostaglandin E2.¹⁹ The presence of multiple-enzymatic activities have often made it difficult to determine which one contributes to laccase-dependent virulence under a given condition. With regards to pigment production, some fungi such as the AIDS-related fungus Cryptococcus neoformans are wholly dependent on exogenous substrates to produce melanin-like pigments, making it difficult to determine whether sufficient tissue catecholamine substrate is available and whether sufficient pigments are produced to affect virulence. However, T. marneffei self-generates these pigments independent of exogenous materials, utilizing internally produced progenitors, ensuring their presence as putative laccase virulence factors. However, another problem has made it difficult to establish laccase as a virulence factor of T. marneffei. Previously, molecular analyses had demonstrated a requirement for a pbrB laccase gene for full pigment formation in conidia of T. marneffei;²⁰ however, additional laccases also contribute to the overall synthesis of fungal pigments in this organism. Thus a challenge in implicating laccases of T. marneffei in virulence has been the presence of these multiple, perhaps redundant laccase activities in the fungus. Under such circumstances, single genetic deletions may not have significantly altered phenotypes to demonstrate roles in virulence, although such redundancy in function often points to the overall importance to the organism during evolution within diverse environments.

Thus, in the present study, Sapmak and collegues ²¹ undertook the formidable task of constructing a quadruple mutant of the fungus, to test the role of laccases in the virulence of the organism. Despite the technical challenges of such transformations in T. marneffei genetic backgrounds, for which limited experience (and transformation markers) existed for the fungus, deletion strains were obtained after multiple transformations which showed robust wild-type growth rates. As predicted, targeted single, double and triple gene deletions of LacA, lacB and LacC showed no altered phenotype, confirming a redundancy of function. Only after a fourth gene, pbrB was deleted were phenotypic changes exhibited. These included delayed conidiation as well as increased sensitivity to the oxidant hydrogen peroxide and detergents such as sodium dodecyl sulfate and antifungal agents. Conidia of the quadruple mutant also showed increased phagocytosis and killing by the monocyte cell line THP-1, suggesting protection against these important immune cells by products of these laccases. Laccases were also found to protect against immune recognition as the mutants were able to induce higher levels of pro-inflammatory cytokines TNF-a, IL-1B and IL-6 from THP-1 cells infected with the quadrupole mutant. Importantly, these phenotypes were reversed after complementation of the quadruple mutant with a wild-type copy of pbrB, suggesting that defective virulence attributes were not due to defects introduced during the transformation procedure, an important control which strengths the overall results.

Such findings are fascinating and important and also present interesting questions for further study. For example, an influence of melanin-like pigments on cytokine production may suggest that pigment production or other laccase products reduce immune recognition of the fungus, potentially interfering with monocytic signaling pathways. In vitro-melanized C. neoformans strains have also been shown to modulate inflammatory responses during lung infections, suggesting the effect could be due directly to production of melanin-like pigments.²² Other fungi causing disseminated infections in HIV/AIDS include Histoplasma capsulatum which has also been found to contain a laccase-like activity and to form pigments in the presence of exogenous catecholamines, suggesting a common link among AIDS-related fungal infections.²³ Importantly, the pigment itself may be more significant for T. marneffei infections than other fungi such as C. neoformans since this dimorphic fungus produces robust pigmentation at 37°C independent of exogenous diphenolic compounds that may or may not be present in sufficient quantities during infections.¹⁶ Furthermore, in a fungus with multiple laccases such as T. marneffei, different enzymatic activities or expression patterns could result in a unique immune response at different phases of infection or within specific tissues. Other alternative enzymatic properties common to laccases may also be present in T. marneffei, contributing to a complex pattern of immune repression.¹⁹ Interestingly, the laccase-expressing fungi represent important AIDS-related pathogens and a shared virulence factor such as laccase suggests commonality in immune-mediating effects that could synergize with HIV-induced immune suppression to make these fungi particularly virulent in AIDS. For example, AIDS-related responses to C. neoformans have a minimal, but retained macrophage signaling component that contributes significantly to pathology during anti-retroviral-associated immune reconstitution syndromes.^24,25 Degradation of these residual macrophage responses by fungal laccases as demonstrated by Sapmak et al.,²¹ may thus provide a key common factor in determining the prevalence of laccase-expressing fungal pathogens in AIDS-related infections. Thus, further study of laccases in T. marneffei may provide molecular and immunological clues as to why saprophytic organisms have become such potent AIDS-related pathogens.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed

Funding

This work was funded, in part, by the Intramural Research Program of the NIH, NIAID, NICHD, grant numbers AI001123, AI109657, and AI001124.