Hortaea werneckii

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Classification

Higher order taxa

Eukaryote; Ascomycota; Pezizomycotina; Dothideomycetes; Mycosphaerellales; Teratosphaeria; Hortaea

Species

NCBI: [1]

Hortaea werneckii

Description and significance

H. werneckii is a halophilic fungus common in hypersaline environments, as well as adjacent sea-water habitats ranging from temperate to tropical and subtropical [2]. H. werneckii causes a rare skin infection known as tinea nigra and has long been classified as a BioSafety Level 2 organism due to its pathogenic status [3]. Taxonomic analysis has revealed that the genotypes of H. werneckii strains found in the environment and among humans are intermingled [2]. Current research focuses on whole genome duplication (WGD) that H. werneckii is thought to have undergone in recent years, which is similar to what has been observed in the yeast S. cerevisiae [4]. H. werneckii also switches between yeast-like budding and filamentous growth, a process that is not well-understood [2]. Because H. werneckii is the only fungus known to grow from 0-30% NaCl, it is a useful model organism for studying halotolerance in Eukarya [2].

Genome structure

The genome of H. werneckii has been sequenced using PacBio sequencing technology. The genome of strain EXF-2000 contains 49.9 Mb, or almost 50 million base pairs, including 38,282 exons and 15, 974 genes [4]. The genome has 53.5% Guanine/Cytosine (GC) content and the average coding exon length is 342 base pairs. H. werneckii has 148 tRNAs [4].

The size of the H. werneckii genome is larger than that of similar species, such as Baudoinia compniacensis and Zymoseptoria tritici. This large genome size is caused by an unusually high number of protein-coding genes, as well as longer gene, protein, and exon lengths compared to its close relatives, with 69.9% of the genome coding for proteins [4]. The H. werneckii genome encodes many proteins to achieve halotolerance, including alkali metal cation transporters (Trk1, Trk2, Tok1, Nha1, Ena proteins, Pho89, Kha1, Mrs7, Vnx1, and Nhx1), which allow the fungus to maintain low internal amounts of Na+ across environmental NaCl concentrations [5]. Another key protein group is the high-osmolarity glycerol (HOG) pathway, which regulates the expression of many halotolerance genes in the presence of high NaCl concentrations [6]. 36 of the 95 H. werneckii genes that are expressed only in high saline concentrations are associated with HwHog1. Other pathways for adapting to changing salinities have not yet been studied [6].

95% of the genes in the genome are repeated, suggesting that H. werneckii has undergone a whole genome duplication. The gene duplicates have an average of only 5% protein sequence difference between them, suggesting that the whole genome duplication event happened relatively recently [4].

Cell structure

H. werneckii is polymorphic, meaning its cell morphology depends in part on the medium it is grown in. When H. werneckii is exposed to high levels of salt, it can form thick cell walls with more melanin than under low salt levels [2]. H. werneckii can grow in both yeast-like and filamentous forms, with most strains growing some type of fungal threads called mycelia [2]. The mycelial form has a generally tubular architectural model with branches. Scars from the budding of new yeast cells appear in various shapes, including round and scale-like. Yeast-like cells have similar bud scars, but with a higher proportion of round ones [7].

Metabolic processes

H. werneckii is halotolerant, meaning that it can withstand extremely high levels of salt, and it is able to do so with the High-osmolarity glycerol (HOG) pathway [8]. In H. werneckii, this involves two key kinases (HwHog1A and HwHog1B) that help act as sensors to indicate to the microbe that it is in a high saline environment. These kinases are only activated in environments with a salinity of greater than 3.0 M NaCl, which then moves into the nucleus to help express genes to help protect the microbe from the extreme environment [6]. Using a chemical called BPTIP as an inhibitor, researchers found that with no or low levels of HwHog1A/B, the H. werneckii’s growth was stunted. Another molecule that possibly contributes to H. werneckii’s growth and cell wall maintenance is melanin as a lack of melanin led to a change of the fungus’s size and shape [9].

H. werneckii mainly secretes enzymes that recognize and break down plant cells rather than animal cells ([10]. H. werneckii produces amylase, lipase, esterase, pectinase and/or cellulase, which are enzymes that recognize plant cell components but not animal cells. In addition, H. werneckii did not produce albuminase, keratinase, phospholipase and DNAse, which recognize animal cell components only [10]. This is consistent with the fact that H. werneckii is an obligate aerobe and requires oxygen to grow [11].

A recent study suggests that H. werneckii uses both fission and budding for its cell division and growth [12]. The first division cycle of H. werneckii tends to be a fission-like process, followed by budding. This pattern continues in an alternating fashion and differs from what is normally seen in “classic model” yeasts that usually only utilize one division method. At the moment, there are no studies that clearly explain the advantages of this alternation. However, researchers hypothesize that it is because a certain type of division method could be more advantageous depending on the environment [12].

Ecology

H. werneckii is typically found in tropical and subtropical regions of the world. Historically, the fungus is found in Central America, South America, the Caribbean, Africa, and Asia. Within these regions, it typically grows on tropical plants [10]. However, current research indicates that the organism can be found in the Mediterranean Sea [13]. Because of its ability to produce amylase, lipase, esterase, pectinase and/or cellulase, but not albuminase, keratinase, phospholipase or DNAse[10], H. werneckii is believed to primarily live on plants rather than animals [10].

H. werneckii can survive in environments with high levels of salt, making the organism a halotolerant. The fungus can live and grow in solutions of 30% NaCl [10]. The organism’s ability to survive in high-salinity environments is largely due to the High-osmolarity glycerol pathway [6]. Two kinases, HwHog1A and HwHog1B assist by acting as sensors to inform the organism whether or not it is in a high salt level environment. Once the organism is in a high saline environment, these kinases reach the nucleus where they express genes designed to protect H. werneckii from the extreme environment [6].

Melanin plays a role in the growth and development of H. werneckii when in environments of high-salinity. When exposed to the melanin inhibitor 2,3-dihydrobenzofuran and a high-salinity environment, the fungus changed color, size and shape depending on what inhibitor was being used. The researchers predict that a lack of melanin can interfere with cell wall integrity [9].

Pathology

H. werneckii is associated with the superficial skin infection known as tinea nigra. Tinea nigra is quite uncommon and primarily occurs in tropical locations [3]. It is typically characterized by painless dark brown patches on the soles of the feet and the palms that appear 2-7 weeks after exposure to H. werneckii. The fungus invades cuts or openings in the skin, but does not penetrate into deeper tissue layers, and does not pose any notable risks [3]. It is diagnosed by KOH analysis, in which a potassium hydroxide solution is added to skin scrapings for microscopic examination [14]. The infection is quite easily treated with salicylic acid and topical antifungals [3].

While tinea nigra is a harmless skin condition, several cases of H. werneckii operating as an opportunistic systemic pathogen have been reported. In 2005, two Malaysian patients with acute myelomonocytic leukemia were found to have H. werneckii in the blood, and in a splenic abscess [15]. The isolated strains were resistant to the antifungals, amphotericin B and flucytosine, but are typically susceptible to most antifungals [10]. While the mode of infection was not determined, H. werneckii may be considered a potential opportunistic pathogen in the future [15].

Current Research

In 2020, the antibiotic activity of three strains of H. werneckii from the coast of the Red Sea in Saudi Arabia was analyzed. The isolated strains generated significant responses against and inhibited the growth of S. aureus (MRSA), C. jejuni, and S. typhimurium. It was determined that several compounds within the H. werneckii strains possessed antimicrobial activity, as well as the production of melanin. While preliminary, this study was the first to indicate how H. werneckii may be used against pathogens that have incurred resistance to common antibiotics [16].

While H. werneckii is typically found in tropical regions of the world, one study in 2019 found the fungus in the Mediterranean Sea. While conducting research at off-shore locations stretching from western Italy to Greece, an abundance of H. Werneckii was found at depths up to 2,500 meters below sea level. In addition, they found the fungus in areas that are typically considered to have low species diversity. The study suggested that H. werneckii might be found in more regions than previously thought. It has been difficult to identify how long H. werneckii has been in the Mediterranean Sea and if it could be found anywhere else [13].

References

[1] Schoch CL, et al. (2020). NCBI Taxonomy: a comprehensive update on curation, resources and tools. Database (Oxford).

[2]Zalar, P., Zupančič, J., Gostinčar, C., Zajc, J., de Hoog, G. S., De Leo, F., Azua-Bustos, A., and Gunde-Cimerman, N. 2019. The extremely halotolerant black yeast Hortaea werneckii—A model for intraspecific hybridization in clonal fungi. IMA Fungus 10:10. https://doi.org/10.1186/s43008-019-0007-5

[3]Bonifaz, A., Badali, H., de Hoog, G. S., Cruz, M., Araiza, J., Cruz, M. A., Fierro, L., and Ponce, R. M. 2008. Tinea nigra by Hortaea werneckii, a report of 22 cases from Mexico. Studies in Mycology 61:77–82. https://doi.org/10.3114/sim.2008.61.07

[4]Sinha, S., Flibotte, S., Neira, M., Formby, S., Plemenitaš, A., Cimerman, N. G., Lenassi, M., Gostinčar, C., Stajich, J. E., and Nislow, C. 2017. Insight into the Recent Genome Duplication of the Halophilic Yeast Hortaea werneckii: Combining an Improved Genome with Gene Expression and Chromatin Structure. G3 7:2015–2022. https://doi.org/10.1534/g3.117.040691

[5]Lenassi, M. , Gostinčar, C., Jackman, S., Turk, M., Sadowski, I., Nislow, C., Jones, S., Birol, I., Cimerman, N. G., & Plemenitaš, A. 2013. Whole genome duplication and enrichment of metal cation transporters revealed by de novo genome sequencing of extremely halotolerant black yeast Hortaea werneckii. PLoS One 8:e71328. https://doi.org/10.1371/journal.pone.0071328

[6]Kejžar, A., Grötli M., Tamás M. J., Plemenitaš A., and Lenassi, M. 2015. HwHog1 kinase activity is crucial for survival of Hortaea werneckii in extremely hyperosmolar environments. Fungal Genetics and Biology 74:45–58. https://doi.org/10.1016/j.fgb.2014.11.004

[7]Nishimura, K., & Miyaji, M. 1984. Hortaea, a new genus to accommodate Cladosporium werneckii. Japanese Journal of Medical Mycology 25(2):139–146. https://doi.org/10.3314/jjmm1960.25.139

[8]Saito, H., & Posas F. 2012. Response to Hyperosmotic Stress. Genetics 192(2):289–318. https://doi.org/10.1534/genetics.112.140863

[9]Kejžar, A., Gobec, S., Plemenitaš, A., and Lenassi, M. 2013. Melanin is crucial for growth of the black yeast Hortaea werneckii in its natural hypersaline environment. Fungal Biology 117(5):368–379. https://doi.org/10.1016/j.funbio.2013.03.006

[10]Formoso, A., Heidrich, D., Felix, C. R., Tenório, A. C., Leite, B. R., Pagani, D. M., Ortiz-Monsalve, S., Ramírez-Castrillón, M., Landell, M. F., Scroferneker, M. L., and Valente, P. 2015. Enzymatic activity and susceptibility to antifungal agents of brazilian environmental isolates of Hortaea werneckii. Mycopathologia 180(5–6):345–352. https://doi.org/10.1007/s11046-015-9920-3

[11][Petrovic, U., Gunde-Cimerman, N., & Plemenitas, A. 2002. Cellular responses to environmental salinity in the halophilic black yeast Hortaea werneckii. Molecular Microbiology 45(3):665–672. https://doi.org/10.1046/j.1365-2958.2002.03021.x]

[12]Mitchison-Field, L. M. Y., Vargas-Muñiz, J. M., Stormo, B. M., Vogt, E. J. D., Van Dierdonck, S., Pelletier, J. F., Ehrlich, C., Lew, D. J., Field, C. M., and Gladfelter, A. S. 2019. Unconventional cell division cycles from marine-derived yeasts. Current Biology 29(20):3439-3456.e5. https://doi.org/10.1016/j.cub.2019.08.050

[13]De Leo, F., Lo Giudice, A., Alaimo, C., De Carlo, G., Rappazzo, A. C., Graziano, M., De Domenico, E., and Urzì, C. 2019. Occurrence of the black yeast Hortaea werneckii in the Mediterranean Sea. Extremophiles: Life Under Extreme Conditions 23(1):9–17. https://doi.org/10.1007/s00792-018-1056-1

[14]Ponka, D., Baddar, F. 2014. Microscopic potassium hydroxide preparation. Can Fam Physician 60(1):57.

[15]Ng, K. P., Soo-Hoo, T. S., Na, S. L., Tay, S. T., Hamimah, H., Lim, P. C., Chong, P. P., Seow, H. F., Chavez, A. J., and Messer, S. A. 2005. The mycological and molecular study of Hortaea werneckii isolated from blood and splenic abscess. Mycopathologia 159(4):495–500. https://doi.org/10.1007/s11046-005-1154-3

[16]Hodhod, M. S. E.-D., Gaafar, A.-R. Z., Alshameri, A., Qahtan, A. A., Noor, A., and Abdel-Wahab, M. 2020. Molecular characterization and bioactive potential of newly identified strains of the extremophilic black yeast Hortaea werneckii isolated from Red Sea mangrove. Biotechnology, Biotechnological Equipment 34(1):1288–1298. https://doi.org/10.1080/13102818.2020.1835535

Edited by Julia Hermann, Andrew Kwon, Devon McAuley, Caroline Pane, and Bridget Yates, students of of Jennifer Bhatnagar for BI 311 General Microbiology, 2021, Boston University.