Sloth Hair As a Novel Source of Fungi with Potent Anti- Parasitic, Anti-Cancer and Anti-Bacterial Bioactivity
Total Page：16
File Type：pdf, Size：1020Kb
Sloth Hair as a Novel Source of Fungi with Potent Anti- Parasitic, Anti-Cancer and Anti-Bacterial Bioactivity Sarah Higginbotham1*, Weng Ruh Wong2, Roger G. Linington2, Carmenza Spadafora3, Liliana Iturrado1, A. Elizabeth Arnold4 1 Smithsonian Tropical Research Institute, Panama, Republic of Panama, 2 Department of Chemistry and Biochemistry, University of California Santa Cruz, Santa Cruz, California, United States of America, 3 Instituto de Investigaciones Cientı´ficas y Servicios de Alta Tecnologı´a, Panama, Republic of Panama, 4 School of Plant Sciences, University of Arizona, Tucson, Arizona, United States of America Abstract The extraordinary biological diversity of tropical forests harbors a rich chemical diversity with enormous potential as a source of novel bioactive compounds. Of particular interest are new environments for microbial discovery. Sloths – arboreal mammals commonly found in the lowland forests of Panama – carry a wide variety of micro- and macro-organisms on their coarse outer hair. Here we report for the first time the isolation of diverse and bioactive strains of fungi from sloth hair, and their taxonomic placement. Eighty-four isolates of fungi were obtained in culture from the surface of hair that was collected from living three-toed sloths (Bradypus variegatus, Bradypodidae) in Soberanı´a National Park, Republic of Panama. Phylogenetic analyses revealed a diverse group of Ascomycota belonging to 28 distinct operational taxonomic units (OTUs), several of which are divergent from previously known taxa. Seventy-four isolates were cultivated in liquid broth and crude extracts were tested for bioactivity in vitro. We found a broad range of activities against strains of the parasites that cause malaria (Plasmodium falciparum) and Chagas disease (Trypanosoma cruzi), and against the human breast cancer cell line MCF-7. Fifty fungal extracts were tested for antibacterial activity in a new antibiotic profile screen called BioMAP; of these, 20 were active against at least one bacterial strain, and one had an unusual pattern of bioactivity against Gram-negative bacteria that suggests a potentially new mode of action. Together our results reveal the importance of exploring novel environments for bioactive fungi, and demonstrate for the first time the taxonomic composition and bioactivity of fungi from sloth hair. Citation: Higginbotham S, Wong WR, Linington RG, Spadafora C, Iturrado L, et al. (2014) Sloth Hair as a Novel Source of Fungi with Potent Anti-Parasitic, Anti- Cancer and Anti-Bacterial Bioactivity. PLoS ONE 9(1): e84549. doi:10.1371/journal.pone.0084549 Editor: Joy Sturtevant, Louisiana State University, United States of America Received July 31, 2013; Accepted November 15, 2013; Published January 15, 2014 Copyright: ß 2014 Higginbotham et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the International Cooperative Biodiversity Groups program (ICBG-Panama http://www.icbg.org/, grant number 2 U01 TW006634-06) and the College of Agriculture and Life Sciences at the University of Arizona. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] Introduction b-lactam antibiotics, griseofulvin, cyclosporine, fusidic acid, and lovastatin [7]. However, in recent years the hit-rate for bioactivity Despite vast increases in spending on international healthcare from fungi has slowed, possibly pointing to the impending over the last 20 years, communicable diseases continue to exhaustion of ‘low hanging fruit’ – the easily and frequently represent an enormous burden to global health [1]. Chronic accessed fungi such as soil microfungi – as sources of new bioactive infectious illnesses such as malaria and diverse neglected tropical metabolites. diseases (NTDs) affect millions of people each year, mainly women Conservative estimates suggest that the total number of fungal and children in developing countries [2]. The rapid spread of species in existence exceeds 5 million, yet fewer than 100,000 antibiotic resistance has decreased the arsenal available to treat fungal species have been described [8][9]. This suggests that many infectious diseases, with recent emergence of pandrug exploring new environments that may be home to previously resistance yielding illnesses that are recalcitrant to treatment with undescribed fungi could be extremely productive. For example, any known drug [3]. Concurrently non-communicable diseases recent examination of tropical fungal endophytes has described are rising in incidence compared to communicable diseases: in tropical leaves as a ‘biodiversity hotspot’ [10][11][12] that is 2008 cancer was responsible for about 7.8 million deaths (about proving fruitful as a source of new bioactive compounds (see [13] 13% of all deaths worldwide; [4]). for a review). Natural products represent one of the most significant sources of Gloer [14] proposed that production of secondary metabolites new drugs today. Approximately 50% of all medicines introduced by fungi may be influenced by the selective pressures imparted by between 1981 and 2006 were of natural product origin [5], with other organisms. This suggests that environments where many an additional 34 natural product-based drugs launched between different species exist in close proximity may have particular 1997 and 2007 [6]. Since the discovery of penicillin over 80 years potential as sources of bioactive metabolites. One such unexplored ago, fungi have contributed enormously to natural product drug environment is the fungal microbiome associated with the coarse, discovery, providing a host of invaluable medicines including the PLOS ONE | www.plosone.org 1 January 2014 | Volume 9 | Issue 1 | e84549 Exploring the Sloth Microbiome sponge-like hair of the three-toed sloth (Bradypus variegatus), an adjacent portion of the nuclear ribosomal large subunit arboreal mammal commonly found in the lowland tropical forests (LSUrDNA) were amplified as a single fragment using primers of Central America. ITS1F or ITS5 and LR3 following Hoffman et al. [18]. PCR The coat of three-toed sloths consists of two distinct layers: an products were visualized using SYBR green following electropho- inner layer of fine, soft hair close to the skin, and an outer layer of resis on a 1% agarose gel and submitted to the University of coarse hairs that are oval in cross section and are approximately Arizona Genetics Core for cleanup, normalization, and bidirec- 0.4 mm wide [15]. These outer hairs carry transverse cracks that tional Sanger sequencing. Sequences were assembled automati- are home to an apparently ubiquitous green alga, most commonly cally and bases called using phred and phrap [19][20] with of the genus Trichophilus [16]. The alga is popularly thought to orchestration by Mesquite [21], followed by manual editing in provide camouflage for sloths against the mottled background of Sequencher 4.5 (GeneCodes Corp.). Sequences were compared their habitat, but this has not been confirmed with data. Suutari against the NCBI non-redundant database using BLASTn [22] to et al. [16] suggested that exopolymeric substances produced by the estimate taxonomic placement and were submitted to GenBank alga might encourage the growth of beneficial bacteria. Sloth hair under accession nos. KF746076-KF746159 (Table S1). is commonly home to cyanobacteria and diatoms, as well as a Because identification based only on BLAST matches must be variety of macro-organisms (e.g., cockroaches, roundworms, and treated with caution (e.g., [23]) we used phylogenetic analyses to moth larvae; [16]). However, little is known about communities of provide stronger inference regarding taxonomic affiliation. fungi on sloth hair. Sequence data for 84 fungal isolates were loaded as one group In an effort to uncover new sources of drugs for treating vector- into Mesquite [21] and aligned by MUSCLE [24]. The resulting borne diseases, cancer, and bacterial infections, we used a culture- alignment, which identified clusters of easily alignable sequences, based approach to examine fungal communities associated with was inappropriate for a single phylogenetic analysis due to the the coarse, outer hair of Bradypus variegatus in Panama. Here we prevalence of ambiguous and unalignable regions. Sequences that report the isolation of fungi with bioactivity against Trypanosoma aligned coherently to one another were partitioned into groups of cruzi, the causal agent of Chagas disease; Plasmodium falciparum, the apparently closely related strains. Resulting groups were then causal agent of malaria; the human breast cancer cell line MCF-7; analyzed as separate data sets as follows: All sequences in each and a range of Gram-negative and Gram-positive human group were compared against the NCBI database using BLASTn. pathogenic bacteria. Phylogenetic analyses were used to identify The top 50 hits for each sequence were downloaded and filtered to these fungi at a finer and more robust level than is possible using remove (1) redundant sequences, and (2) sequences from typical database-matching methods alone. Our results suggest that potentially mis-identified strains and unvouchered specimens (see sloth hair is