The Antimicrobial Peptide Histatin-5 Causes a Spatially Restricted Disruption on the Candida albicans Surface, Allowing Rapid Entry of the Peptide into the Cytoplasm

Antimicrobial peptides play an important role in host defense against microbial pathogens. Their high cationic charge and strong amphipathic structure allow them to bind to the anionic microbial cell membrane and disrupt the membrane bilayer by forming pores or channels. In contrast to the classical pore-forming peptides, studies on histatin-5 (Hst-5) have suggested that the peptide is transported into the cytoplasm of Candida albicans in a non-lytic manner, and cytoplasmic Hst-5 exerts its candicidal activities on various intracellular targets, consistent with its weak amphipathic structure. To understand how Hst-5 is internalized, we investigated the localization of FITC-conjugated Hst-5. We find that Hst-5 is internalized into the vacuole through receptor-mediated endocytosis at low extracellular Hst-5 concentrations, whereas under higher physiological concentrations, Hst-5 is translocated into the cytoplasm through a mechanism that requires a high cationic charge on Hst-5. At intermediate concentrations, two cell populations with distinct Hst-5 localizations were observed. By cell sorting, we show that cells with vacuolar localization of Hst-5 survived, while none of the cells with cytoplasmic Hst-5 formed colonies. Surprisingly, extracellular Hst-5, upon cell surface binding, induces a perturbation on the cell surface, as visualized by an immediate and rapid internalization of Hst-5 and propidium iodide or rhodamine B into the cytoplasm from the site using time-lapse microscopy, and a concurrent rapid expansion of the vacuole. Thus, the formation of a spatially restricted site in the plasma membrane causes the initial injury to C. albicans and offers a mechanism for its internalization into the cytoplasm. Our study suggests that, unlike classical channel-forming antimicrobial peptides, action of Hst-5 requires an energized membrane and causes localized disruptions on the plasma membrane of the yeast. This mechanism of cell membrane disruption may provide species-specific killing with minimal damage to microflora and the host and may be used by many other antimicrobial peptides

The Expression of a Xylanase Targeted to ER-Protein Bodies Provides a Simple Strategy to Produce Active Insoluble Enzyme Polymers in Tobacco Plants

Background Xylanases deserve particular attention due to their potential application in the feed, pulp bleaching and paper industries. We have developed here an efficient system for the production of an active xylanase in tobacco plants fused to a proline-rich domain (Zera) of the maize storage protein γ-zein. Zera is a self-assembling domain able to form protein aggregates in vivo packed in newly formed endoplasmic reticulum-derived organelles known as protein bodies (PBs). Methodology/Principal Findings Tobacco leaves were transiently transformed with a binary vector containing the Zera-xylanase coding region, which was optimized for plant expression, under the control of the 35S CaMV promoter. The fusion protein was efficiently expressed and stored in dense PBs, resulting in yields of up to 9% of total protein. Zera-xylanase was post-translationally modified with high-mannose-type glycans. Xylanase fused to Zera was biologically active not only when solubilized from PBs but also in its insoluble form. The resistance of insoluble Zera-xylanase to trypsin digestion demonstrated that the correct folding of xylanase in PBs was not impaired by Zera oligomerization. The activity of insoluble Zera-xylanase was enhanced when substrate accessibility was facilitated by physical treatments such as ultrasound. Moreover, we found that the thermostability of the enzyme was improved when Zera was fused to the C-terminus of xylanase. Conclusion/Significance In the present work we have successfully produced an active insoluble aggregate of xylanase fused to Zera in plants. Zera-xylanase chimeric protein accumulates within ER-derived protein bodies as active aggregates that can easily be recovered by a simple density-based downstream process. The production of insoluble active Zera-xylanase protein in tobacco outlines the potential of Zera as a fusion partner for producing enzymes of biotechnological relevance. Zera-PBs could thus become efficient and low-cost bioreactors for industrial purposes.This work was mainly supported by ERA Biotech (www.erabiotech.com). Additional support was supplied by grant SGR 2009/703 funded by the Generalitat de Catalunya (www10.gencat.net) and grants CDS2007/00036 of Consolider Ingenio program and TRA 2009/0124 of TRACE program funded by Ministerio de Ciencia e Inovación (MICINN, www.micinn.es). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewe

Antimicrobial resistance (AMR) nanomachines: mechanisms for fluoroquinolone and glycopeptide recognition, efflux and/or deactivation

In this review, we discuss mechanisms of resistance identified in bacterial agents Staphylococcus aureus and the enterococci towards two priority classes of antibiotics—the fluoroquinolones and the glycopeptides. Members of both classes interact with a number of components in the cells of these bacteria, so the cellular targets are also considered. Fluoroquinolone resistance mechanisms include efflux pumps (MepA, NorA, NorB, NorC, MdeA, LmrS or SdrM in S. aureus and EfmA or EfrAB in the enterococci) for removal of fluoroquinolone from the intracellular environment of bacterial cells and/or protection of the gyrase and topoisomerase IV target sites in Enterococcus faecalis by Qnr-like proteins. Expression of efflux systems is regulated by GntR-like (S. aureus NorG), MarR-like (MgrA, MepR) regulators or a two-component signal transduction system (TCS) (S. aureus ArlSR). Resistance to the glycopeptide antibiotic teicoplanin occurs via efflux regulated by the TcaR regulator in S. aureus. Resistance to vancomycin occurs through modification of the D-Ala-D-Ala target in the cell wall peptidoglycan and removal of high affinity precursors, or by target protection via cell wall thickening. Of the six Van resistance types (VanA-E, VanG), the VanA resistance type is considered in this review, including its regulation by the VanSR TCS. We describe the recent application of biophysical approaches such as the hydrodynamic technique of analytical ultracentrifugation and circular dichroism spectroscopy to identify the possible molecular effector of the VanS receptor that activates expression of the Van resistance genes; both approaches demonstrated that vancomycin interacts with VanS, suggesting that vancomycin itself (or vancomycin with an accessory factor) may be an effector of vancomycin resistance. With 16 and 19 proteins or protein complexes involved in fluoroquinolone and glycopeptide resistances, respectively, and the complexities of bacterial sensing mechanisms that trigger and regulate a wide variety of possible resistance mechanisms, we propose that these antimicrobial resistance mechanisms might be considered complex ‘nanomachines’ that drive survival of bacterial cells in antibiotic environments

Transformation of Saccharomyces cerevisiae and other fungi: Methods and possible underlying mechanism

Transformation (i.e., genetic modification of a cell by the incorporation of exogenous DNA) is indispensable for manipulating fungi. Here, we review the transformation methods for Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida albicans, Pichia pastoris and Aspergillus species and discuss some common modifications to improve transformation efficiency. We also present a model of the mechanism underlying S. cerevisiae transformation, based on recent reports and the mechanism of transfection in mammalian systems. This model predicts that DNA attaches to the cell wall and enters the cell via endocytotic membrane invagination, although how DNA reaches the nucleus is unknown. Polyethylene glycol is indispensable for successful transformation of intact cells and the attachment of DNA and also possibly acts on the membrane to increase the transformation efficiency. Both lithium acetate and heat shock, which enhance the transformation efficiency of intact cells but not that of spheroplasts, probably help DNA to pass through the cell wall

Calmodulin dissociation regulates Myo5 recruitment and function at endocytic sites

The type I myosin Myo5 promotes actin-dependent membrane remodelling. Calmodulin binds to Myo5 and maintains it in a closed and inactive conformation. Calmodulin dissociation from Myo5 leads to local activation of actin polymerization

Protein uptake by bacteria: An endocytosis-like process in the planctomycete Gemmata obscuriglobus

Endocytosis is a fundamental process of membrane-trafficking in eukaryotes, but has not been known to occur in bacteria or archaea. The origin of endocytosis is central to the understanding of evolution of the first eukaryotes and their endomembrane systems. In a recent study we have established that an endocytosis-like process for uptake of proteins into cells occurs in a bacterium, Gemmata obscuriglobus, a member of the distinctive phylum Planctomycetes of peptidoglycan-less budding bacteria. Members of this phylum characteristically possess cells divided into compartments separated by internal membranes and in the case of G. obscuriglobus these compartments include one where a double membrane envelope surrounds its nucleoid DNA, as well as an outer ribosome- free region of cytoplasm. Proteins can be internalized by cells from the external milieu and collected into this ribosome-free compartment, and this process is energy-dependent and appears to be receptor-mediated. As in eukaryote endocytosis, internalized proteins are associated with vesicles, and can be subjected to proteolytic degradation. The discovery of this process in a bacterium has significant implications for our understanding of the origins of endocytosis in eukaryotes

Recombinant cellulase accumulation in the leaves of mature, vegetatively propagated transgenic sugarcane

The cost of enzymes that hydrolyse lignocellulosic substrates to fermentable sugars needs to be reduced to make cellulosic ethanol a cost-competitive liquid transport fuel. Sugarcane is a perennial crop and the successful integration of cellulase transgenes into the sugarcane production system requires that transgene expression is stable in the ratoon. Herein, we compared the accumulation of recombinant fungal cellobiohydrolase I (CBH I), fungal cellobiohydrolase II (CBH II), and bacterial endoglucanase (EG) in the leaves of mature, initial transgenic sugarcane plants and their mature ratoon. Mature ratoon events containing equivalent or elevated levels of active CBH I, CBH II, and EG in the leaves were identified. Further, we have demonstrated that recombinant fungal CBH I and CBH II can resist proteolysis during sugarcane leaf senescence, while bacterial EG cannot. These results demonstrate the stability of cellulase enzyme transgene expression in transgenic sugarcane and the utility of sugarcane as a biofactory crop for production of cellulases