Caenorhabditis elegans and Tenebrio molitor-New tools to investigate Malassezia species

Malassezia species are part of human commensal microbiota and is also related to diseases. Little is known about the interaction of these microorganisms with their host. Here we established two standard culture conditions for Malassezia sp. to perform infection assays using C. elegans and T. molitor. Invertebrate hosts infected by Malassezia sp. cultured in M9M resulted in higher death rate on survival assays when compared to yeasts cultured in the standard Dixon medium, indicating that M9M cultured Malassezia species have increased virulence. The culture and infection conditions established in this work, using invertebrate models, are valuable tools to understand Malassezia-host interaction. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 1 October 2018 doi:10.20944/preprints201810.0001.v1


Introduction
Malassezia genus comprises the more abundant lipophilic and/or lipodependent yeasts in the human resident microbiota, establishing a commensal relationship with their hosts [1; 2; 3].However, when immune system homeostasis is impaired Malassezia can act as pathogens.In this context, some Malassezia cellular characteristics takes place at pathological processes acting as virulence factors: cell wall structure, lipases, morphological transition and indole compounds [3; 4].Despite many years of Malassezia genus studies, the knowledge about species interaction with human host is not completely understood [3; 5; 6; 7].
Invertebrate host models to study microorganism-host interactions is becoming very popular to understand many aspects of microbial virulence [8; 9].The nematode Caenorhabditis elegans, an invertebrate model used since the 1960s in several areas of scientific research, and more recently the coleopteran Tenebrio molitor, represent excellent tools to study fungal diseases [8; 10; 11; 12; 13; 14; 15; 16].
We evaluated these alternative hosts as models to study infection and diseases associated with Malassezia species using two distinct and standardized culture conditions and we showed that nutrients starved Malassezia sp.significantly reduces C. elegans end T. molitor survival, and thus promotes increased virulence of these pathogenic yeasts.

Malassezia sp. culture
Malassezia species used in this study are clinical samples from the yeast library of the Biotechnology and Ecology of Yeasts Laboratory (Biological Sciences Institute -Federal University of Minas Gerais).Three clinical isolates, previously identified by PCR and sequencing (unpublished data), were selected: M. furfur (MIE), M. sympodialis (A88) and M. yamatoensis (MZ2).

Cultivation and adaptation to alternative medium M9M
The M9 buffer, commonly used for C. elegans manipulation [18], supplemented or not with agar [2% (w/v)], was established as an alternative culture medium for Malassezia species and named in this work as M9M (M9 Medium).When necessary selected species where grown in liquid Dixon medium and inoculated in M9M for at least 3 days at 30°C (in a rotatory shaker for liquid cultures).Malassezia species were adapted to M9M through at least three successive passages before infection assays.
Caenorhabditis elegans culture and synchronization C. elegans N2 Bristol (wild-type strain kindly provided by CGC) were maintained in Petri dishes containing NGM media and a layer of Escherichia coli OP50 [18].
Plates were maintained at 16°C until the experimental procedures were performed.In order to obtain worms at the same larval stage C. elegans where subjected to synchronization as described by Porta de la Riva et al [19].

Caenorhabditis elegans survival assays
For the survival tests, one isolated colony of Malassezia sp., picked from Dixon or M9M agar, was inoculated into a sterile Falcon tube containing 8 mL of the selected culture medium (Dixon or M9M) and maintained at 30°C for 3 days.On the experiment day these cultures were centrifuged (4000 rpm during 10 minutes in conical tubes), washed with M9 buffer and cell density was measured (OD660).Cell suspensions were diluted in order to obtain a final concentration of 1.25 x 10 6 UFC/mL [20] and 10 μL were placed at the top M9M agar supplement with streptomycin (50 μg/mL -Sigma) and 5-fluoro-2'deoxyuridine (FUDR -80 µg/mL-Sigma) in a 24-well plate.To survival determination, 10-15 young adults (L4) C. elegans were added to the pathogenic yeast lawn and incubated at 25°C.Worms viability was examined at 24h intervals and those not responding to mechanical stimulus with a platinum wire were considered dead [15].

Tenebrio molitor survival assays
T. molitor beetle larvae were commercially obtained and maintained at our laboratory.To perform survival analyses, larvae presenting 0.1 -0.2g without signs of contamination, spots and exoskeleton changes, were selected.Each larva, previously cleaned with 70% ethanol, were infected directly into the hemocele using a Hamilton syringe (701N, caliber 26, 10μL capacity).T. molitor was injected with 5μL of yeasts suspension (2.65 x 10 7 cells), cultured in Dixon or M9M.Experimental group were composed of 10 larvae each, belonging to three experimental groups: 1-Dixon-cultured Malassezia, 2-M9M-cultured Malassezia and 3-negative control.The control group was inoculated with 5 μl of Phosphate Buffered Saline (PBS).Infected larvae and negative control were incubated in Petri dishes at 37ºC and viability was examined at 24h intervals for ten days, being considered dead those larvae not responding to mechanical stimulus.

Statistical analyzes
C. elegans and T. molitor survival curves were plotted, and estimation of differences (log-rank and Wilcoxon tests) in survival analyzed with the Kaplan-Meier method performed using GraphPad Prism software 5.01 (GraphPad Software).A p-value of 0.05 was considered significant.In this work we cultured and adapted Malassezia species in M9M, as previously described, to prevent an observed overgrowth of these yeasts in the standard C elegans culture medium NGM.Also, we tried to perform C. elegans assays using the Malassezia standard medium Dixon, that showed to be toxic to nematodes.When Malassezia sp. were subjected to successive passages through liquid M9M cells remained viable, as determined by MTT assays (data not shown) and plating in Dixon agar, but presented reduced growth when compared to those cultivated in Dixon (data showed for M. furfur -Figure 1).In addition, we observed that yeasts are subjected to morphological changes in M9M, presenting filamentous growth (Figure 2).

Discussion
Malassezia species are described in literature as lipophilic or lipid-dependent yeasts according to its colonization site and availability of lipidic components [5; 21].At the laboratory these yeasts only grow in media containing external lipid sources.In this work we showed that M. furfur, M. sympodialis, M. yamatoensis can grow in M9 (named here as M9M), a C. elegans physiological buffer, as well as are viable in this culture condition.External lipid source is absent in M9M and the observed growth of Malassezia sp. can be explained by stable storage of lipdis.Malassezia spp.can store lipids in cytoplasmic droplets as occur with Saccharomyces cerevisae, and these sources could be used when yeasts are growing in M9M as other conditions when lipids are scarce [22].
Microbial cells have extremely adaptable metabolism in order to survive to diverse conditions, for example, when they are in a hostile environment as in the case of infection of host tissues, and even during commensalism.Nutritional stress is a very common example of metabolism dynamics, in which microbial cells became more vulnerable and hence can adapt, sometimes influencing its morphology and virulence [23,24,25].Stress inducing morphogenesis and virulence changes can be clearly observed when microorganisms are submitted to culture conditions that limit nutrients and were demonstrated by several works.[25].Rodríguez-Gomes et al. [26] observed Beauveria bassiana virulence changes using different culture conditions and found that Sabouraud-dextrose agar increased virulence for T.
molitor when compared to fungal cells that were grown in other culture media.
As occur with other yeasts, here we show that Malassezia sp. have optimal growth in Dixon medium, but when adapted and cultured in M9M can form hyphae (Figure 2), and thus can be potentially more invasive and virulent.
Corroborating our growth data, Malassezia species adapted to M9M (liquid or agar) showed a significant increase in virulence to invertebrate hosts, evidenced by the rapid death of C. elegans and T. molitor when compared to Dixon cultured cells (Figures 3 and 4).
There are few studies about the mechanisms involved in altering Malassezia spp.commensal state to pathogenic life [3; 27].Existing studies are mostly restricted to in vitro tests using cell cultures or in vivo using murine models [3].
Brilhante et al. evaluated the virulence of M. pachydermatis using C. elegans as invertebrate model and describe it as a valuable model to investigate this yeast [28].In this work we could define a standard culture condition that increase invertebrate models to expand the knowledge in pathogen-host interaction research field [8; 10; 11; 12; 13; 14].

Conclusion
Here we established two alternative invertebrate model hosts to investigate Malassezia sp.infections, adding one more tool to understand its virulence and pathogenesis.Moreover, this is the first work demonstrating Malassezia sp.
culture in the absence of external lipid sources that potentiates its virulence to C. elegans and T. molitor.

Figure 4 -
Figure 4 -Malassezia furfur adapted to M9M decreases T. molitor larvae Malassezia sp.virulence by simply removing nutrients and lipid exogenous sources.In addition, we established C. elagans and T. molitor as new models to study this pathogenic genus thus contributing to future works to investigate malasseziosis pathogenesis and treatment, reinforcing the excellence of Preprints (www.preprints.org)| NOT PEER-REVIEWED | Posted: 1 October 2018 doi:10.20944/preprints201810.0001.v1

Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 1 October 2018 doi:10.20944/preprints201810.0001.v1
Weerasekera et al. evaluated how culture conditions influence growth, adhesion and biofilm formation in C. albicans and C. tropicalis [24].They found that maximal growth of C. albicans and C. tropicalis was favored in Sabouraud Dextrose medium, but C. tropicalis presented optimal growth in the Yeast Nitrogen Base medium.In paralell, they observed a facilitated adherence and biofilm formation in RPMI 1640 medium in both species.Differently, O'Connor and cols.showed that C. dubliniensis cultivation in nutrient limited conditions can promote yeast transition to filamentous growth