Effects of silencing 14-3-3 protein in Paracoccidiodes brasiliensis infection

Keywords:

• Adhesins
• P. brasiliensis
• 14-3-3 protein
• RNA silencing
• virulence

This article refers to:

Decreased expression of 14-3-3 in Paracoccidioides brasiliensis confirms its involvement in fungal pathogenesis

Paracoccidiomycosis is a fungal infection caused by 2 different species, Paracoccidiodes brasiliensis and P. lutzii that are endemic in Latin America. The Paracoccidiodes spp. are thermally dimorphic fungi that switch from mycelial or conidia forms found in the environment (at 26°C) to a yeast form during mammalian infection (at 37°C). The lungs are the primary infection site since inhalation of the conidia or mycelial propagules is the most common manner of transmission. There are 2 common forms of subsequent disease: acute/subacute and chronic. The acute/subacute form predominantly occurs in children and young adults, and is typically manifested as a highly lethal disseminated disease of the reticuloendothelial system. In contrast, the chronic form is predominantly pulmonary and/or mucocutaneous and represents a reactivation of latent pulmonary or lymph node foci.^1,2 Therefore, the development of the infection is a complex event that depends on several factors such as the expression of fungal virulence traits and the fitness of the host immune system.

Paracoccidioides spp have several mechanisms that facilitate adherence and invasion of host tissues. The adhesion process has been extensively studied since it contributes to the initial colonization by the fungus, which then leads to the subsequent dissemination and evasion of the host immune system.³ In particular, the fungus can interact with molecules present on host cell surfaces and with proteins of the extracellular matrix (ECM), such as type I and IV collagens, fibronectin and laminin.⁴ Several adhesins and their roles during this interaction have been described in Paracoccidiodes spp.: Gp43,^4-6 14-3-3 protein,^7,8 enolase,^9-11 triosephosphate isomerase (TPI),¹² glyceraldehyde-3-phosphate dehydrogenase (GAPDH) ¹³ and PbHAD32, a 32 kDa hydrolase.^14,15

Given the difficulties in the genetic manipulation of Paracoccidiodes spp most of the information available on the roles of adhesins in this fungus comes from studies using recombinant proteins produced by Saccharomyces cerevisiae, or by interfering with fungal-host interactions using specific blocking antibodies. However, despite the usefulness of those methods, such results are indirect. Antisense RNA technology and Agrobacterium tumefaciens mediated-transformation are excellent tools to study the roles of these proteins in the biology and virulence of Paracoccidioides spp. Indeed, these techniques have already been successfully used to study Gp43 and PbHAD32 proteins.^15,16

In this issue of Virulence, the authors of the article entitled “Decreased expression of 14-3-3 in Paracoccidiodes brasiliensis confirms its involvement in fungal pathogenesis” utilized antisense RNA to generate a 14-3-3 silenced strain of P. brasiliensis and studied the importance of this protein for the pathogenesis of the fungus.¹⁷ The 14-3-3 protein was first identified as an overexpressed 30 KDa protein after challenging mice with P. brasiliensis.⁸ Later, this protein was localized in the cell wall of the fungus and described as a laminin ligand that purportedly played a role in the adhesion to epithelial cells in the lungs.¹⁸ However, its role in the biology of the fungus remained not clear.

Marcos et al. successfully “knocked-down” Pb14-3-3 (~55% silenced) using siRNA. Pb14-3-3 silenced cells were unable to undergo either yeast-to-mycelium or mycelium-to-yeast transitions, which is particularly significant as the parasitic form (yeast form) is more resistant to killing by host effector cells>.¹⁷ Additionally, the 14-3-3 silenced Paracoccidoides cells were smaller and produced lower numbers of buds compared to untreated yeast cells. Importantly, the virulence of 14-3-3 siRNA treated Paracoccidioides yeast cells was significantly attenuated in a Galleria mellonella model of infection.¹⁷ Together these results indicate that Pb14-3-3 plays a major role not only in Paracoccidioides virulence, but also in the morphological program of this fungus. Morphological changes during interaction with the host are critical for the pathogenicity of different fungi, such as Candida albicans (yeast to hyphal growth),¹⁹ H. capsulatum (mycelium to yeast) ²⁰ and Cryptococcus neoformans (capsule enlargement and titan cell formation).²¹ Despite knowing some of the key actors in these processes in Paracoccidioides, the mechanisms involved in morphogenesis are largely unknown. However, it seems clear, that the fungus uses these morphological changes to evade killing by the host immune system and to promote yeast cell dissemination.

Furthermore, the authors confirmed the role of Pb14-3-3 in the adhesion process, a critical early factor in the development and subsequent progression of invasive infection. Their studies found that Pb14-3-3 was important for binding to laminin and fibronectin, which was especially significant at the initial stages of interaction with pneumocytes. Recently, de Oliveria et al carried an in depth study to elucidate the importance of different adhesins in the virulence of Paracoccidiodes ³ and they found that 14-3-3, along with enolase, were the most highly expressed adhesins during host-pathogen interactions, which supports the results presented in this issue of Virulence.

Since paracoccidioidomycosis is initiated through inhalation of conidia followed by adhesion to pneumocytes, improving our knowledge on this process is essential to develop strategies against the acquisition of and establishment of disease by this fungus. Additionally, this work further validates the use of RNA silencing method as an extremely appropriate approach since it allows the study of the whole fungus during host cell interactions, which can effectively augment our understanding of both the biology of the target protein within the microorganism and the downstream effects of the protein during pathogenesis.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.