SpHyastatin was first identified as a new cationic antimicrobial peptide in hemocytes of the mud crab Scylla paramamosain. Based on the amino acid sequences deduced, it was predicted that this peptide was composed of two different functional domains, a proline-rich domain (PRD) and a cysteine-rich domain (CRD). The recombinant product of SpHyastatin displayed potent antimicrobial activities against the human pathogen Staphylococcus aureus and the aquatic animal pathogens Aeromonas hydrophila and Pseudomonas fluorescens. Compared with the CRD of SpHyastatin, the PRD presented better antimicrobial and chitin binding activities, but both regions were essential for allowing SpHyastatin complete antimicrobial activity. The binding properties of SpHyastatin to different microbial surface molecules suggested that this might be an initial and crucial step for performing its antimicrobial activities. Evaluated using propidium iodide uptake assays and scanning electron microscopy images, the antimicrobial mechanism of SpHyastatin was found to be prone to disrupt cell membrane integrity. Interestingly, SpHyastatin exerted its role specifically on the surface of S. aureus and Pichia pastoris whereas it directly killed P. fluorescens through simultaneous targeting the membrane and the cytoplasm, indicating that SpHyastatin could use different antimicrobial mechanisms to kill different species of microbes. As expected, the recombinant SpHyastatin increased the survival rate of crabs challenged with Vibrio parahaemolyticus. In addition, SpHyastatin could modulate some V. parahaemolyticus-responsive genes in S. paramamosain.

Natural antimicrobial peptides (AMPs) are potential antibiotic alternatives. Marine crustaceans are thought to generate more powerful and various AMPs to protect themselves from infections caused by pathogenic microorganisms in their complex aquatic habitat, thus becoming one of the most promising sources of AMPs or other bioactive substances. In the study, a novel protein was identified as an interacting partner of male-specific AMP SCY2 in Scylla paramamosain and named scyreprocin. The recombinant product of scyreprocin (rScyreprocin) was successfully expressed in Escherichia coli. rScyreprocin exerted potent, broad-spectrum antifungal, antibacterial, and anti-biofilm activity (minimum inhibitory concentrations from 0.5 to 32 μM) through differential modes of action, including disruption of cell membrane integrity and induction of cell apoptosis, and has rapid bactericidal (in 0.5-2 h) and fungicidal (in 8-10 h) kinetics. In addition to its fungicidal activity against planktonic fungi, rScyreprocin also prevented the adhesion of fungal cells, inhibited biofilm formation, and eradicated the mature biofilms. Moreover, rScyreprocin showed a profound inhibitory effect on spore germination of Aspergillus spp. (minimum inhibitory concentrations from 4 to 8 μM). This peptide was not cytotoxic to murine and mammalian cells and could increase the survival rate of Oryzias melastigma under the challenge of Vibrio harveyi. Taken together, the novel AMP scyreprocin would be a promising alternative to antibiotics used in aquaculture and medicine.

Hydrogen sulfide (H2S) is thought to signal through protein S-sulfuration (persulfidation; S-sulfhydration) in both mammalian systems and bacteria. We previously profiled proteome S-sulfuration in Staphylococcus aureus (S. aureus) and identified two thioredoxin-like proteins, designated TrxP and TrxQ, that were capable of reducing protein persulfides as a potential regulatory mechanism. In this study, we further characterize TrxP, TrxQ and the canonical thioredoxin, TrxA, by identifying candidate protein substrates in S. aureus cells using a mechanism-based profiling assay where we trap mixed disulfides that exist between the attacking cysteine of a FLAG-tagged Trx and a persulfidated cysteine on the candidate substrate protein in cells. Largely non-overlapping sets of four, 32 and three candidate cellular substrates were detected for TrxA, TrxP, and TrxQ, respectively, many of which were previously identified as global proteome S-sulfuration targets including for example, pyruvate kinase, PykA. Both TrxA (k cat = 0.13 s-1) and TrxP (k cat = 0.088 s-1) are capable of reducing protein persulfides on PykA, a model substrate detected as a candidate substrate of TrxP; in contrast, TrxQ shows lower activity (k cat = 0.015 s-1). This work reveals that protein S-sulfuration, central to H2S and reactive sulfur species (RSS) signaling, may impact cellular activities and appears to be regulated in S. aureus largely by TrxP under conditions of sulfide stress.

Enterovirus 71 (EV71) is one of the most important etiological agents for hand-foot-mouth disease. Compared with coxsackievirus A16 infection, EV71 infection is often associated with severe central nervous system complications, such as encephalitis, encephalomyelitis, and acute flaccid paralysis in infants and young children. In this study, we constructed a recombinant baculovirus with T7 ribonucleic acid polymerase under the control of a cytomegalovirus promoter and simultaneously engineered the T7 promoter upstream of a full-length EV71 complementary deoxyribonucleic acid. After transduction into mammalian cells, typical cytopathic effects (CPEs) and VP1 signals were detected in cells transfected with recombinant baculovirus. Additionally, viral particles located in the cytoplasm of human rhabdomyosarcoma cells (Rd) and Vero cells were observed by electron microscope, indicating that EV71 was recovered using a Bac-to-Bac expression system in vitro. After four passages, the rescued virus had a growth curve and plaque morphology similar to those of the parental virus. Furthermore, the Vp1 gene and the protein from the mouse brain were detected by reverse transcription polymerase chain reaction and immunohistochemistry after intracerebral injection of purified recombinant baculovirus. Typical CPEs were observed after inoculation of the supernatant from mouse brain to Rd cells, revealing a reconstruction of EV71 in vivo. Thus, we established a new approach to rescue EV71 based on a baculovirus expression system in vitro and in vivo, which may provide a safe and convenient platform for fundamental research and a strategy to rescue viruses that currently lack suitable cell culture and animal models.