A g-Type Lysozyme from Deep-Sea Hydrothermal Vent Shrimp Kills Selectively Gram-Negative Bacteria

Abstract

Lysozyme is a key effector molecule of the innate immune system in both vertebrate and invertebrate. It is classified into six types, one of which is the goose-type (g-type). To date, no study on g-type lysozyme in crustacean has been documented. Here, we report the identification and characterization of a g-type lysozyme (named LysG1) from the shrimp inhabiting a deep-sea hydrothermal vent in Manus Basin. LysG1 possesses conserved structural features of g-type lysozymes. The recombinant LysG1 (rLysG1) exhibited no muramidase activity and killed selectively Gram-negative bacteria in a manner that depended on temperature, pH, and metal ions. rLysG1 bound target bacteria via interaction with bacterial cell wall components, notably lipopolysaccharide (LPS), and induced cellular membrane permeabilization, which eventually caused cell lysis. The endotoxin-binding capacity enabled rLysG1 to alleviate the inflammatory response induced by LPS. Mutation analysis showed that the bacterial binding and killing activities of rLysG1 required the integrity of the conserved α3 and 4 helixes of the protein. Together, these results provide the first insight into the activity and working mechanism of g-type lysozyme in crustacean and deep-sea organisms.

A Type Ib Crustin from Deep-Sea Shrimp Possesses Antimicrobial and Immunomodulatory Activity

“…The bacterial growth was shifted to 16 °C and continued for 12 h. The cells were then collected by centrifugation and disrupted by sonication. Recombinant proteins were purified with Ni-NTA affinity columns (QIAGEN, Germantown, TN, USA) as reported previously [ 21 , 22 ]. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was applied to examine the proteins.…”

“…Protein binding to the bacteria and bacterial components was determined by ELISA as described previously [ 21 ]. Briefly, bacteria (10 8 CFU/mL) or bacterial cell wall components (PGN, LTA, or LPS, 200 μg/mL) were each added to a 96-well microtiter plate.…”

“…Deep-sea hydrothermal vents are extreme environments that nurture thriving ecosystems constituted mainly by microorganisms and macro–invertebrates such as shrimp [ 18 ]. In particular, the Alvinocarididae family is the dominant shrimp in deep-sea vents, and has been found to encode a variety of antibacterial molecules [ 19 , 20 , 21 ]. In a previous study, we studied the antibacterial activity of a Type Ia crustin, Crus1, from Alvinocarididae in the deep sea of Manus Basin [ 22 ].…”

Heterogeneity of Lipopolysaccharide as Source of Variability in Bioassays and LPS-Binding Proteins as Remedy

“…However, not every (G-) bacterial strain was affected equally by this binder. Some bacteria-e.g., Escherichia coli and Pseudoalteromonas hodoensis-were lysed already at low concentrations of rLysG1, whereas others-such as different Vibrio strains-were not affected at all [193]. The changes that LPS binders have on the morphology of bacteria can be observed using electron microscopy.…”

Functional characterization and expression analysis of c-type and g-like-type lysozymes in yellowtail clownfish (Amphiprion clarkii)

“…The reason might be that g-type lysozyme has a different binding ability to bacteria or bacteria cell wall components. Due to these different binding abilities, g-type lysozymes selectively kill gram-negative bacteria in a non-enzymatic mode by damaging bacterial membranes (Luo et al, 2021). Collectively both lysozymes can be proposed as potential players in the host antibacterial defense in A. clarkii.…”