Antibacterial activity of vB_AbaM_PhT2 phage hydrophobic amino acid fusion endolysin, combined with colistin against Acinetobacter baumannii.

Phage lytic enzymes are promising antimicrobial agents. In this study, an endolysin derived from vB_AbaM_PhT2 (vPhT2), was identified. This endolysin represented the conserved lysozyme domain. Recombinant endolysin (lysAB- vT2) and hydrophobic fusion endolysin (lysAB-vT2-fusion) were expressed and purified. Both endolysins showed lytic activity against bacterial crude cell wall of Gram-negative bacteria. The MIC of lysAB-vT2-fusion was 2 mg/ml corresponding to 100 µM, while the MIC of lysAB-vT2 was more than 10 mg/ml (400 µM). Combination of lysAB-vT2-fusion with colistin, polymyxin B or copper was synergistic against A. baumannii (FICI value as 0.25). Antibacterial activity of lysAB-vT2-fusion plus colistin at the fractional inhibitory concentrations (FICs) revealed that it can inhibit Escherichia coli, Klebsiella pneumoniae and various strains of extremely drug-resistant A. baumannii (XDRAB) and phage resistant A. baumannii. The lysAB- vT2-fusion still retained its antibacterial activity after incubating the enzyme at 4, 20, 40 and 60 °C for 30 min. The lysAB-vT2-fusion could inhibit the mature biofilm, and incubation of lysAB-vT2-fusion with T24 human cells infected with A. baumannii led to a partial reduction of LDH release from T24 cells. In summary, our study highlights the antimicrobial ability of engineered lysAB-vT2-fusion endolysin, which can be applied for the control of A. baumannii infection.

Atg8 family proteins, LIR/AIM motifs and other interaction modes.

The Atg8 family of ubiquitin-like proteins play pivotal roles in autophagy and other processes involving vesicle fusion and transport where the lysosome/vacuole is the end station. Nuclear roles of Atg8 proteins are also emerging. Here, we review the structural and functional features of Atg8 family proteins and their protein-protein interaction modes in model organisms such as yeast, Arabidopsis, C. elegans and Drosophila to humans. Although varying in number of homologs, from one in yeast to seven in humans, and more than ten in some plants, there is a strong evolutionary conservation of structural features and interaction modes. The most prominent interaction mode is between the LC3 interacting region (LIR), also called Atg8 interacting motif (AIM), binding to the LIR docking site (LDS) in Atg8 homologs. There are variants of these motifs like "half-LIRs" and helical LIRs. We discuss details of the binding modes and how selectivity is achieved as well as the role of multivalent LIR-LDS interactions in selective autophagy. A number of LIR-LDS interactions are known to be regulated by phosphorylation. New methods to predict LIR motifs in proteins have emerged that will aid in discovery and analyses. There are also other interaction surfaces than the LDS becoming known where we presently lack detailed structural information, like the N-terminal arm region and the UIM-docking site (UDS). More interaction modes are likely to be discovered in future studies.

A yeast two-hybrid screening identifies novel Atg8a interactors in Drosophila.

Macroautophagy/autophagy-related protein Atg8/LC3 is important for autophagosome biogenesis and required for selective degradation of various substrates. In our recent study, we performed a yeast two-hybrid screening to identify proteins that interact with Atg8a, the Drosophila homolog of Atg8/LC3. The screening identified several Atg8a-interacting proteins. These proteins include: i) proteins which have already been experimentally verified to bind Atg8a, such as Atg1, DOR, ref(2)P and key (Kenny); ii) proteins for which their mammalian homologs interact with Atg8-family members, like Ank2, Atg4, and Nedd4; and iii) several novel Atg8a-interacting proteins, such as trc/STK38 and Tak1. We showed that Tak1, as well as its co-activator, Tab2, both interact with Atg8a and are substrates for selective autophagic clearance. We also determined that SH3PX1 interacts with Tab2 and is necessary for the effective regulation of the immune-deficiency (IMD) pathway. Our findings suggest a mechanism for the regulatory interactions between Tak1-Tab2-SH3PX1 and Atg8a, which contribute to the fine-tuning of the IMD pathway.

Selective autophagy controls innate immune response through a TAK1/TAB2/SH3PX1 axis.

Selective autophagy is a catabolic route that turns over specific cellular material for degradation by lysosomes, and whose role in the regulation of innate immunity is largely unexplored. Here, we show that the apical kinase of the Drosophila immune deficiency (IMD) pathway Tak1, as well as its co-activator Tab2, are both selective autophagy substrates that interact with the autophagy protein Atg8a. We also present a role for the Atg8a-interacting protein Sh3px1 in the downregulation of the IMD pathway, by facilitating targeting of the Tak1/Tab2 complex to the autophagy platform through its interaction with Tab2. Our findings show the Tak1/Tab2/Sh3px1 interactions with Atg8a mediate the removal of the Tak1/Tab2 signaling complex by selective autophagy. This in turn prevents constitutive activation of the IMD pathway in Drosophila. This study provides mechanistic insight on the regulation of innate immune responses by selective autophagy.

Assays to Monitor Mitophagy in Drosophila.

Autophagy is a central pathway utilized by many eukaryotic cells in order to recycle intracellular constituents, particularly under periods of nutrient scarcity or cellular damage. The process is evolutionarily conserved from yeast to mammals and can be highly selective with regard to the contents that are targeted for degradation. The availability of Drosophila transgenic lines and fluorophore-labeled autophagic markers allows nowadays for the more effortless visualization of the process within cells. Herein, we provide two protocols to prepare Drosophila samples for confocal and transmission electron microscopy for in vivo monitoring of mitophagy, a specific type of autophagy for the clearance of damaged or superfluous mitochondria from cells.

Caspase involvement in autophagy.

Caspases are a family of cysteine proteases widely known as the principal mediators of the apoptotic cell death response, but considerably less so as the contributors to the regulation of pathways outside cellular demise. In regards to autophagy, the modulatory roles of caspases have only recently begun to be adequately described. In contrast to apoptosis, autophagy promotes cell survival by providing energy and nutrients through the lysosomal degradation of cytoplasmic constituents. Under basal conditions autophagy and apoptosis cross-regulate each other through an elaborate network of interconnections which also includes the interplay between autophagy-related proteins (ATGs) and caspases. In this review we focus on the effects of this crosstalk at the cellular level, as we aim to concentrate the main observations from research conducted so far on the fine-tuning of autophagy by caspases. Several members of this protease-family have been found to directly interact with key ATGs involved in different tiers across the autophagic cascade. Therefore, we firstly outline the core mechanism of macroautophagy in brief. In an effort to emphasize the importance of the intricate cross-regulation of ATGs and caspases, we also present examples of autophagy's contribution to apoptotic cell death during development.

Immuno-Gold Labeling of Drosophila Follicles for Transmission Electron Microscopy.

Detecting the localization of cellular components using gold nanoparticles has come to offer tremendous advantages in cell biology, allowing for the high resolution imaging of the cellular organization at the subcellular level. This is further aided by the breakthroughs in the cryopreparation of samples, which focus at the retention of antigenicity in efforts to mirror the native state of the tissues and cells as closely as possible. Herein, we describe the methodology for immuno-gold labeling of Drosophila follicles, following preparation of the samples using the Tokuyasu method for ultracryosectioning.