Omics analyses and biochemical study of Phlebiopsis gigantea elucidate its degradation strategy of wood extractives

Wood extractives, solvent-soluble fractions of woody biomass, are considered to be a factor impeding or excluding fungal colonization on the freshly harvested conifers. Among wood decay fungi, the basidiomycete Phlebiopsis gigantea has evolved a unique enzyme system to efficiently transform or degrade conifer extractives but little is known about the mechanism(s). In this study, to clarify the mechanism(s) of softwood degradation, we examined the transcriptome, proteome, and metabolome of P. gigantea when grown on defined media containing microcrystalline cellulose and pine sapwood extractives. Beyond the conventional enzymes often associated with cellulose, hemicellulose and lignin degradation, an array of enzymes implicated in the metabolism of softwood lipophilic extractives such as fatty and resin acids, steroids and glycerides was significantly up-regulated. Among these, a highly expressed and inducible lipase is likely responsible for lipophilic extractive degradation, based on its extracellular location and our characterization of the recombinant enzyme. Our results provide insight into physiological roles of extractives in the interaction between wood and fungi.

Identification of suitable Botryococcus braunii strains for non-destructive in situ hydrocarbon extraction

The process of milking microalgae is a promising approach to reduce the downstream costs for the production of valuable substances from microalgae by avoiding the steps of harvest, dewatering, and cell disruption of the common process chain (cultivation, harvesting, dewatering, cell disruption, extraction, purification). The green microalga Botryococcus braunii is particularly suited for this process due to its ability to produce large amounts of long-chain hydrocarbons accumulating in an extracellular matrix. The extracellular location of hydrocarbons is an enormous advantage in comparison with other microalgae that accumulate lipids in intracellular lipid bodies. At present, only a few B. braunii strains (UTEX 572, CCAP 807/2, SAG 807/1, FACHB 357, Bot22, and SCCAP 1761) have been examined for the process of long-term repetitive milking. In order to identify promising candidates for the milking process, twelve different B. braunii strains (SAG 30.81, SAG 807/1, UTEX 572, UTEX 2441, CCAP 807/2, ACOI 58, ACOI 1257, SCCAP K-1489, var. Showa, Bot22, SCCAP K-1761, and CCALA 779) were investigated in terms of growth, lipid accumulation, nutrient uptake, solvent compatibility, and extracellular hydrocarbon extractability. Based on these results, a ranking was defined in view of eligibility for non-destructive hydrocarbon extraction. Results indicate a particular potential for hydrocarbon milking for two of those twelve B. braunii strains. The strain Showa (71 out of 75 ranking points), which has not yet been examined for long-term repetitive milking, and the strain Bot22 (64 out of 75 ranking points) seem to be the most suitable strains for the milking process. They both possess good extractant compatibility including hydrocarbon extractability as well as high biomass and lipid productivity.

Moonlighting Proteins at the Candidal Cell Surface

The cell wall in Candida albicans is not only a tight protective envelope but also a point of contact with the human host that provides a dynamic response to the constantly changing environment in infection niches. Particularly important roles are attributed to proteins exposed at the fungal cell surface. These include proteins that are stably and covalently bound to the cell wall or cell membrane and those that are more loosely attached. Interestingly in this regard, numerous loosely attached proteins belong to the class of “moonlighting proteins” that are originally intracellular and that perform essentially different functions in addition to their primary housekeeping roles. These proteins also demonstrate unpredicted interactions with non-canonical partners at an a priori unexpected extracellular location, achieved via non-classical secretion routes. Acting both individually and collectively, the moonlighting proteins contribute to candidal virulence and pathogenicity through their involvement in mechanisms critical for successful host colonization and infection, such as the adhesion to host cells, interactions with plasma homeostatic proteolytic cascades, responses to stress conditions and molecular mimicry. The documented knowledge of the roles of these proteins in C. albicans pathogenicity has utility for assisting the design of new therapeutic, diagnostic and preventive strategies against candidiasis.

Serpins: Purification and characterization of potent protease inhibitors from Clostridium thermocellum

Clostridium thermocellum produces an extracellular cellulosome (a multiprotein complex produced by firmicutes bacteria), which, owing to its extracellular location, is open to protease attack. Serine protease inhibitors (serpins) protect bacteria against protease attack. However, their structure and function are poorly characterized. This study identified and amplified the serpin 1270 gene from the C. thermocellum genome. Purified serpins were cloned into the pTXB1 vector using the one-step sequence and ligation-independent cloning reaction and transformed into Escherichia coli BL21 DE3 cells. Enzyme overexpression and purification and enzyme inhibitory assays were performed. The results showed that serpin 1270 has 89% inhibition against Bacillus subtilisin and 64% inhibition against trypsin, chymotrypsin, and papain.

Evidence for an Intramacrophage Growth Phase of Mycobacterium ulcerans

ABSTRACT Mycobacterium ulcerans is the etiologic agent of Buruli ulcer (BU), an emerging tropical skin disease. Virulent M. ulcerans secretes mycolactone, a cytotoxic exotoxin with a key pathogenic role. M. ulcerans in biopsy specimens has been described as an extracellular bacillus. In vitro assays have suggested a mycolactone-induced inhibition of M. ulcerans uptake by macrophages in which its proliferation has not been demonstrated. Therefore, and uniquely for a mycobacterium, M. ulcerans has been classified as an extracellular pathogen. In specimens from patients and in mouse footpad lesions, extracellular bacilli were concentrated in central necrotic acellular areas; however, we found bacilli within macrophages in surrounding inflammatory infiltrates. We demonstrated that mycolactone-producing M. ulcerans isolates are efficiently phagocytosed by murine macrophages, indicating that the extracellular location of M. ulcerans is not a result of inhibition of phagocytosis. Additionally, we found that M. ulcerans multiplies inside cultured mouse macrophages when low multiplicities of infection are used to prevent early mycolactone-associated cytotoxicity. Following the proliferation phase within macrophages, M. ulcerans induces the lysis of the infected host cells, becoming extracellular. Our data show that M. ulcerans, like M. tuberculosis, is an intracellular parasite with phases of intramacrophage and extracellular multiplication. The occurrence of an intramacrophage phase is in accordance with the development of cell-mediated and delayed-type hypersensitivity responses in BU patients.

VII. EnteropathogenicEscherichia coli: physiological alterations from an extracellular position

Enteropathogenic Escherichia coli (EPEC) is primarily associated with infantile diarrhea in developing countries. This intriguing pathogen exerts numerous physiological effects on its host target tissue, the intestinal epithelium, all from an extracellular location. Expression of a type III secretory apparatus allows this organism to transfer bacterial effector molecules directly into host cells. As a result of EPEC attachment to and/or translocation of proteins into intestinal epithelial cells, many signaling cascades are activated. Ultimately, host functions are perturbed, including alteration of ion transport, disruption of the tight junction barrier, and activation of the inflammatory response.