Changes in the Pseudogymnoascus destructans transcriptome during White-nose Syndrome reveal possible mechanisms for both virulence and host resistance

KEYWORDS:

• enzymes
• epidermis
• hibernation
• lipids
• Pseudogymnoascus destructans

This article refers to:

Pseudogymnoascus destructans transcriptome changes during white-nose syndrome infections

White-nose Syndrome (WNS) was first observed in 6 New York State sites during the winter of 2007–08, and has since spread to > 190 sites in 31 U.S. states and 5 Canadian provinces. WNS produces over-winter mortality rates of 75 – 98% for 4 of the 6 bat species that over-winter in the northeast, including the little brown bat (Myotis lucifugus). The fungus that causes this disease is Pseudogymnoascus destructans which grows on the muzzle, wings, and ears of bats during torpor.¹ The hyphae penetrate the epidermis and dermis, consuming hair follicles, sebaceous and sweat glands.² The optimal temperature for the growth of P. destructans is 4.0 −15.8°C,³ and it was introduced to North America.⁴ Bats with extensive P. destructans infections arouse more frequently from torpor during hibernation, and this leads to a premature depletion of body fat reserves, which causes death.^5,6 Some North American bat species are more resistant to P. destructans. Big brown bats (Eptesicus fuscus) over-wintering where P. destructans occurs do not develop cutaneous infections and hibernate normally.⁷ The Eastern small-footed bat (Myotis leibii) also appears to be more resistant to cutaneous infection with P. destructans.⁸ The factors that enable some bat species to resist P. destructans infections are poorly understood. Understanding the mechanism by which P. destructans breaches the epidermis during WNS is fundamental to determining this species-specific resistance.

The mammalian epidermis is composed chiefly of epithelial cells called keratinocytes arranged in 4 strata; they are produced in the deepest stratum (the stratum basale), and migrate to the top stratum (the stratum corneum). Epidermal lipids are mixtures secreted by keratinocytes into the intracellular matrix, and sebum secreted by the sebaceous glands. The lipid mixture secreted by keratinocytes contains free sphingosine bases, ceramides, cholesterol, and free fatty acids (FFAs), whereas the sebum is composed of triacylglycerols, diacylglycerols, FFAs, wax esters, squalene, cholesterol, and cholesterol esters.^9,10 The epidermal lipids of bats also contain cerebrosides and monoacylglycerols.^11,12 Sebum accounts for almost all of the lipids found on the skin surface of mammals¹³, and the epidermis of M. lucifugus has a total lipid content of 47% dry matter mass.¹⁴ The hyphae of P. destructans breach this outer lipid barrier during WNS. Lipolytic enzymes have long been implicated in the virulence of other fungal pathogens.¹⁵

In this issue of Virulence, Reeder et al.¹⁶ compare the transcriptome of P. destructans infecting free-ranging M. lucifugus during hibernation, to that of P. destructans grown on Sabouraud dextrose agar (SDA) plates in the laboratory under similar conditions. They found that a total of 94 genes were more highly expressed during the cutaneous infection (WNS) of M. lucifugus, and 117 were more highly expressed in P. destructans during growth on SDA. They identified 39 of these genes as having functions that could contribute to virulence. The expression of lipase 1 and squalene monooxygenase by P. destructans both increased substantially during WNS. Lipase 1 hydrolyses triacylglycerols, whereas squalene monooxygenase catalyzes the first step of a pathway that converts squalene to sterols. These enzymes may be important to the virulence of P. destructans because sebum is on average 45% triacylglycerol and 12% squalene.¹³ These two enzyme together thus can degrade half of the sebum lipids found on the epidermis.

The authors also examined the expression of genes for 6 different proteases, known as subtilases, which can degrade the extracellular matrix of host tissues. The expression of 5 of these genes by P. destructans was lower during WNS, indicating that the increased production of subtilases is probably not associated with virulence. Interpreting these findings together reveals that one of the factors which enables P. destructans to invade the epidermis of some bats is the facultative production of enzymes capable of degrading the sebum.

Some epidermal lipids are known to have anti-microbial effects.¹⁷ The wing epidermis of E. fuscus contains almost twice the mean levels of the fatty acids called myristic, palmitoleic, and oleic acids as wing epidermis of M. lucifugus. Experiments revealed that palmitoleic, oleic, and linoleic acids all inhibit the growth of P. destructans.¹⁴ The mechanism by which certain lipid classes inhibit fungal growth is unknown.¹⁷ The findings of Reeder et al.¹⁶ suggest that P. destructans can easily degrade the triacylglycerols and squalene found in/on the epidermis. It is therefore possible that the epidermal lipids of bat species susceptible to P. destructans contain relatively more of the specific lipid classes that can be degraded by this fungus, such as triacylglycerols and squalene, than the epidermal lipids of bats that are more resistant.

The findings of Reeder et al.¹⁶ demonstrate that both lipolytic enzymes and cutaneous lipids may play a significant role in the virulence of P. destructans. This study also provides a model of gene expression changes in P. destructans associated with the transition from saprophytic to parasitic growth that involves genes for heat shock proteins, the importation of several metal ions, cell wall production, as well as proteases and lipases. Further examination of this differential gene expression, along with that of the corresponding changes in protein expression, will provide key insights into why some bat species are more susceptible to P. destructans, and thus WNS as well.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Funding

This study was supported by NSF grant IOS-0818222 awarded to C.L.F.