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

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.


Introduction
The innate immune system is an important mechanism of the host to defend against pathogens, especially for invertebrate, which lack adaptive immune systems. Lysozyme (muramidase, EC 3.2.1.17) is one of the key molecules of the innate immune system. It catalyzes the cleavage of the beta-1,4-glycosidic bond between the C-1 of N-acetylmuramic acid (MurNAc) and the C-4 of N-acetylglucosamine (GlcNAc) in peptidoglycan [1], an essential component of the bacterial cell wall. As a result, lysozyme is widely employed by animals as a weapon against bacterial infection. Since, compared to Gram-negative bacteria, Gram-positive bacteria have a peptidoglycan cell wall that is much thicker and exposed to the extracellular milieu, lysozyme is generally more efficient at killing Grampositive bacteria. However, some lysozymes are known to kill effectively Gram-negative bacteria [2][3][4][5]. In recent years, lysozyme has been reported to have functions other than muramidase, such as isopeptidase, chitinase, and non-enzyme antibacterial activities [5]. In some cases, the mechanism of the non-enzymatic antibacterial effect was attributed to the extreme cationic nature of the lysozymes [6,7]. In addition to its antibacterial activity, lysozyme has an influence on the inflammatory response [8,9]. For example, hen egg lysozyme (HEL) can alleviate the immune response to inflammatory bowel disease [10], and human lysozyme can affect inflammation by regulating serum complement activation [11].

LysG1 Possesses the Conserved Structure Features of g-Type Lysozyme
LysG1 was identified in the shrimp Rimicaris sp. from a hydrothermal vent in Desmos, Manus basin. LysG1 protein is composed of 188 amino acid residues, with a calculated molecular weight of 20.0 kDa and a predicted pI of 9.06. It contains the conserved residues of the sugar binding site (residues E72, 94QID96, F124, 150YN151, and G153) and catalytic site (E72 and D96) of g-type lysozyme. LysG1 was predicted to form 8 α-helixes and 2 β-pleated sheets ( Figure 1A,B). LysG1 is homologous to the g-type lysozymes of crustacean, teleost, and mammal, and shares the highest sequence identity (63.8%) with the g-type lysozyme of Lates calcarifer ( Figure 1A). To date, no study on crustacean gtype lysozyme has been reported, however, two g-type lysozyme sequences from Caligus rogercresseyi and Macrobrachium nipponense (Accession no. ACO11114.1 and AKC42440.1, respectively) have been deposited in GenBank, which are 52.98% and 53.76%, respectively, identical to LysG1 ( Figure 1A). Phylogenetic analysis of the crustacean lysozymes showed that LysG1 was grouped into the clade of g-type lysozyme and clustered together with the g-type lysozyme of M. nipponense ( Figure 1C).

Recombinant LysG1 (rLysG1) Kills Selectively Gram-Negative Bacteria in a Regulated Manner
To facilitate study, rLysG1 was prepared ( Figure S1). rLysG1 was not able to digest peptidoglycan (PGN) ( Figure S2). However, rLysG1 exhibited apparent killing effect on a

rLysG1 Binds Target Bacteria via Interaction with LPS and Attenuates LPS-Induced
Inflammatory Response rLysG1 exhibited relatively strong binding to Gram-negative bacteria, including those, such as the Vibrios, that were immune to the killing of rLysG1, whereas the binding activity of rLysG1 to Gram-positive bacteria was comparatively much lower or barely detectable ( Figure 3A). Microscopy confirmed localization of rLysG1 on the cell surface following incubation of rLysG1 with the bacteria ( Figure 3B). In contrast, no binding of rTrx, a control protein, to bacteria was detected ( Figure 3B). Consistent with these observations, rLysG1 bound efficiently to the cell wall components of Gram-negative bacteria (LPS and lipid A), and much weakly to that of Gram-positive bacteria (PGN) ( Figure 3C). The presence of Cu 2+ , however, impaired the binding of rLysG1 to bacteria, LPS and lipid A ( Figure S3). Interaction with exogenous LPS significantly inhibited the ability of rLysG1 to bind and kill bacteria in a manner that depended on the concentration of LPS ( Figure 3D). To examine whether the LPS-binding capacity of rLysG1 would have any effect on LPSinduced inflammatory response, RAW264.7 cells were treated with LPS and examined for the expression of IL-1β, IL-6, and TNF-α. All these cytokines were significantly suppressed by rLysG1 in a dose-dependent manner ( Figure 4).

rLysG1 Kills Bacteria by Disrupting Bacterial Membrane
When grown in the presence of rLysG1, E. coli and P. hodoensiswas formed significantly reduced numbers of colonies compared to growth in the absence of rLysG1 ( Figure 5A), suggesting that most of the parental bacterial cells were killed by rLysG1. Following treatment with rLysG1, the bacterial cells were stained positive by propidium iodide (PI) ( Figure 5B), which passes into the cells only when the cellular membrane is permeabilized or damaged. Scanning electron microscope (SEM) revealed time-dependent change in the morphology of rLysG1-treated bacterial cells. The cellular membrane became rough and formed blebs that increased in number and size with time ( Figure 5C). The cells were eventually completely disrupted in structure.

rLysG1 Kills Bacteria by Disrupting Bacterial Membrane
When grown in the presence of rLysG1, E. coli and P. hodoensiswas formed significantly reduced numbers of colonies compared to growth in the absence of rLysG1 ( Figure 5A), suggesting that most of the parental bacterial cells were killed by rLysG1. Following treatment with rLysG1, the bacterial cells were stained positive by propidium iodide (PI) ( Figure 5B), which passes into the cells only when the cellular membrane is permeabilized or damaged. Scanning electron microscope (SEM) revealed timedependent change in the morphology of rLysG1-treated bacterial cells. The cellular membrane became rough and formed blebs that increased in number and size with time ( Figure 5C). The cells were eventually completely disrupted in structure.

Discussion
In the present study, we identified a g-type lysozyme, LysG1, from the Rimicaris shrimp found in a hydrothermal vent of Manus Basin. LysG1 exhibits the conserved carbohydrate binding site and catalytic site of g-type lysozyme, but apparently lacks a signal peptide. Previous reports showed that almost all g-type lysozymes in birds and mammals contain predicted signal sequences for secretion, whereas most fish g-type lysozymes and some invertebrate g-type lysozymes do not have the characteristic secretion signals, suggesting that they may function within the cells [14,18,36]. In invertebrate, studies on g-type lysozyme are very limited, chiefly in Haliotis discus discus, Chlamys farreri, and Ciona [16,18,20]. In fact, g-type lysozyme was even long considered to be absent in arthropods based on the absence of g-type lysozyme-encoding genes in the available genomes of the most abundant class of arthropods-the insect [37]. Indeed, a search of NCBI database yielded only two deposited g-type lysozyme sequences from crustacean, i.e., C. rogercresseyi and M. nipponense. LysG1 was phylogenetically clustered

Discussion
In the present study, we identified a g-type lysozyme, LysG1, from the Rimicaris shrimp found in a hydrothermal vent of Manus Basin. LysG1 exhibits the conserved carbohydrate binding site and catalytic site of g-type lysozyme, but apparently lacks a signal peptide. Previous reports showed that almost all g-type lysozymes in birds and mammals contain predicted signal sequences for secretion, whereas most fish g-type lysozymes and some invertebrate g-type lysozymes do not have the characteristic secretion signals, suggesting that they may function within the cells [14,18,36]. In invertebrate, studies on g-type lysozyme are very limited, chiefly in Haliotis discus discus, Chlamys farreri, and Ciona [16,18,20]. In fact, g-type lysozyme was even long considered to be absent in arthropods based on the absence of g-type lysozyme-encoding genes in the available genomes of the most abundant class of arthropods-the insect [37]. Indeed, a search of NCBI database yielded only two deposited g-type lysozyme sequences from crustacean, i.e., C. rogercresseyi and M. nipponense. LysG1 was phylogenetically clustered together with the gtype lysozyme of M. nipponense, and differs from the two crustacean lysozymes by 47% and 46.2%, respectively, in sequence. These results indicate that LysG1 has evolved differently and acquired new sequence features that are distinct from that of other crustaceans.
The antimicrobial mechanism of lysozymes can be classified as the enzyme-dependent mode and the non-enzymatic mode [14]. Most vertebrate and invertebrate lysozymes are active muramidases and can act upon a broad spectrum of bacteria [20,[38][39][40][41][42]. A small group of lysozymes exert antibacterial effects in a muramidase-independent manner. For example, a T4 lysozyme and two i-type lysozymes from red swamp crayfish were able to kill bacteria independent of enzyme activity [5,43]. In our study, rLysG1 could not degrade PGN and, consistently, showed no evident effect on the survival and growth of Gram-positive bacteria. In contrast, rLysG1 effectively killed some Gram-negative bacteria. This property implies that rLysG1 may be used as a bactericidal agent against infections caused only by Gram-negative bacterial pathogens, such as E. coli. In agreement with these observations, rLysG1 bound more efficiently to Gram-negative bacteria, as well as their cell wall components, than to Gram-positive bacteria, and pre-binding of free LPS diminished the bacterial killing effect of rLysG1. These results suggest that rLysG1 bound target bacteria via interaction with LPS, which is a prerequisite for the bactericidal activity of rLysG1.
A number of studies have demonstrated that pH, temperature, and metal ions can influence the activity of some lysozymes [38][39][40]44,45]. The structure of lysozyme can change greatly at different pH [46]. Metal ions, such as Ca 2+ and Mn 2+ , are known to affect the activity of lysozymes [44,45]. Similarly, in our study, we found that the bactericidal activity of rLysG1 was optimal only at certain pH and temperature under the specified experimental conditions. Cu 2+ and Zn 2+ had a significant inhibitory effect on rLysG1, which may not be due to the charged status of these ions, since other divalent ions failed to produce any effect. Together, these results indicate that the antibacterial activity of rLysG1 is regulated by multiple factors. These factors, such as metal ions, pH, and temperature, may be used to manipulate the working effect of rLysG1.
A study of hen egg white lysozyme (HEWL) showed that the enzyme rapidly increased the permeability of the outer membrane of E. coli due to large size pore formation [3]. It also observed a direct delayed activity of lysozyme against the inner membrane, but without evidence of perforations [3]. In our study, we found that bacteria treated with rLysG1 were stained positive by PI, suggesting cellular membrane permealization. In support of this observation, scanning electron microscopy revealed time-dependent disruption of the surface structure of rLysG1-treated bacteria. Similar membrane damages by lysozymes or lysozyme-derived antimicrobial peptides have been reported previously [47][48][49]. The membrane lytic effect of some non-enzymatic lysozymes was thought to be related to the extreme cationic charges of the enzymes [6]. In the case of LysG1, it is rich in arginine and lysine residues and exhibits a high pI of 9.06. Mutation of two of the basic amino acid residues, i.e., R43 and K48, located in the α3 and 4 helixes caused significant reductions in the activity of LysG1, implying an essential role of these residues in the interaction with target bacteria.
In addition to its direct bacteriolytic effect, lysozyme also plays a part in limiting inflammation [9]. During the infection of bacterial pathogens, lysozyme can reduce the pathogen-induced inflammation by inhibiting the growth of the bacteria [50]. For the lysozymes with muramidase activity, they can digest bacterial peptidoglycan to reduce the production of anaphylatoxins, hence limiting the inflammatory response [51]. In our study, rLysG1 was found to bind not only LPS but also lipid A, the endotoxin that is essentially responsible for the toxic effect of Gram-negative bacteria. In accordance with this property, rLysG1 effectively reduced the expression of inflammatory genes in LPS-stimulated macrophages. Inflammation is an important immune response against pathogen infection. However, excessive inflammation can cause tissue damage and disease development. Hence, the attenuating effect of LysG1 on LPS-induced inflammatory response may be beneficial to the host.

Sequence and Phylogenetic Analysis
The sequence of LysG1 was obtained from the transcriptome of Alvinocarididae shrimp [28]. The sequence was analyzed with DNAMAN 6.0 (Lynnon Biosoft, San Ramon, CA, USA). Sequence BLAST was performed using the Protein BLAST algorithm of the NCBI (http://www.ncbi.nlm.nih.gov/blast (accessed on 3 December 2020)). Signal peptide search was performed using the SignalP program (http://www.cbs.dtu.dk/services/ SignalP (accessed on 3 December 2020)). Multiple sequence alignment was performed with the ClustalX software version 2.1. The output picture was generated using ESPript 3.0 (https://espript.ibcp.fr/ESPript/ESPript/index.php (accessed on 1 December 2020)). The structure modeling of LysG1 was predicted with the SWISS-MODEL (https://swissmodel. expasy.org/interactive (accessed on 14 December 2020)). The phylogenetic tree was constructed using neighbour-joining method with the MEGA software version 7.0.26, and the reliability of the tree was assessed by bootstrapping (1000 replications).

Plasmid Construction and Protein Purification
To construct the plasmid pET28a-LysG1, which expresses recombinant LysG1 (rLysG1) with a His-tag, the coding sequence of LysG1 was inserted into the expression plasmid pET28a at the NdeI/XhoI sites, resulting in pET28a-LysG1. The pET28a-LysG1 and pET32a (for purification of His-tagged Trx as a control protein) were separately introduced into Escherichia coli BL21 (DE3) by transformation. The transformants were cultured in LB medium at 37 • C to OD 600 of 0.4, and then 0.04 mM Isopropyl-β-D-Thiogalactopyranoside (IPTG) was added. The culture was continued at 16 • C for 15 h with shaking (100 rpm). The cells were then harvested by centrifugation. The proteins with His-tag were purified with nickel-nitrilotriacetic acid (Ni-NTA) columns (QIAGEN, Germantown, MD, USA) as described previously [52]. The recombinant LysG1 and Trx eluates were dialyzed extensively against PBS (pH 7.2) supplemented with 10% glycerol at 4 • C for 24 h. The purified proteins were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). To prepare the mutant LysG1 variants, the plasmids expressing the mutants were constructed by inserting the coding sequences of LysG1 bearing alanine substitution at R43, K45, K48, K50, K139, KK141, K144 and R43K48 into pET28a as above. The mutant proteins were purified as above.

Muramidase Activity
The DNS (3, 5-Dinitrosalicylic acid) method was used to detect the muramidase activity of rLysG1 as reported previously [53]. Briefly, a standard curve of N-acetylglucosamine reaction with DNS was generated as reported previously [53]. To determine the muramidase activity of rLysG1, 16 µM of rLysG1 or lysozyme (Sigma, St Louis, MO, USA) (positive control) was added to PBS buffer containing 10 mg/mL peptidoglycan (PGN). After incubation at 37 • C for 4 h, DNS (Sangon Biotech, Shanghai, China) was added to terminate the reaction, and the Eppendorf tube containing the sample was placed in boiling water for 5 min for color to develop. The solution was centrifuged at 6000 rpm for 10 min, and the optical density of the supernatant was measured at 520 nm.

Bactericidal Activity
To determine the minimal bactericidal concentration (MBC) of rLysG1, bacteria were grown to logarithmic phase and resuspended in PBS to 10 3 CFU/mL with PBS. Then 0.5 µM to 16 µM (2-fold dilution) of rLysG1 was added to 200 µL bacterial suspension. PBS was added to the control bacterial suspension. The samples were incubated at 37 • C for 4 h, and bacterial survival was determined by plate count [34]. To examine the effect of temperature and pH on the bactericidal activity of rLysG1, E. coli was incubated with or without (control) rLysG1 (8 µM) at different temperatures (4, 16, 37 and 42 • C) or pH (5.6, 6.0, 6.4, 7.0, 7.6 and 8.0) for 4 h. To examine the effect of metal ions, E. coli was incubated with or without rLysG1 (8 µM) in PBS containing 100 µM FeCl 2 , CuCl 2 , CaCl 2 , MgCl 2 , MnCl 2 , CoCl 2 , or ZnCl 2 . To determine the effect of LPS on the bactericidal activity of rLysG1, rLysG1 (8 µM) was pre-incubated with LPS (Sigma, St Louis, MO, USA) (0.4, 0.8, or 2 mg/mL) at 37 • C for 1 h. E. coli was then added to the mixture, and the mixture was incubated at 37 • C for 4 h. To examine the bactericidal activity of the rLysG1 mutants, E. coli was incubated with or without each of the rLysG1 mutants (8 µM) at 37 • C for 4 h. In all cases, bacterial survival was determined as above.

Binding of rLysG1 to Bacteria and Bacterial Cell Wall Components
Bacterial cells were placed into a 96-well microtiter plate containing coating buffer (1.59 g Na 2 CO 3 and 2.93 g NaHCO 3 /L, pH 9.6) at the final concentration of 10 8 CFU/mL. The plate was incubated at 4 • C overnight. LPS or PGN (Sigma, St Louis, MO, USA) dissolved in the coating buffer to 200 µg/mL was added to a 96-well microtiter plate, followed by incubation at 4 • C overnight. Lipid A (Sigma, St Louis, MO, USA) (200 µg/mL in methanol) was added to a 96-well microtiter plate, and the plate was left to dry at room temperature for 2 h. All the plates were washed 3 times with PBST (PBS with 0.05% Tween 20) and blocked with 150 µL 5% skim milk powder (Solarbio, Beijing, China) at 37 • C for 1 h. The plates were washed as above, and different concentrations of rLysG1 or PBS was added to the wells. The plates were incubated at 37 • C for 1 h and washed as above. The HRPconjugated mouse anti-His antibody (Abcam, Cambridge, MA, USA) (1/1000 dilution) was added to the plates. The plates were incubated at 37 • C for 1 h and washed as above. Color development was performed using TMB substrate solution (Tiangen, Beijing, China) and terminated by adding ELISA stop solution (Solarbio, Beijing, China). Finally the absorbance at 450 nm was measured. To examine binding of rLysG1 to bacteria by microscopy, E. coli and P. hodoensiswas were incubated with rLysG1 or rTrx for 1 h at 37 • C and washed 5 times with PBS. Then the cells were successively treated with the mouse anti-His antibody (Abcam, Cambridge, MA, USA) (1/1000 dilution) and FITC-labeled goat anti-mouse secondary antibody (Abcam, Cambridge, MA, USA) (1/1000 dilution) for 1 h at 37 • C. After washing 5 times with PBS, the cells were observed with a fluorescence microscope (TiS/L100, Nikon, Tokyo, Japan).

Examination of Bacterial Cell Membrane Integrity
E. coli and P. hodoensiswas were cultured to OD 600 of 1.0 and resuspended in PBS to 5× 10 8 CFU/mL. The cells were incubated with rLysG1 or rTrx for 2 h at 37 • C and washed 5 times with PBS. The cells were stained with propidium iodide (50 µg/mL) for 5 min and washed as above. The cells were then observed with a fluorescence microscope as above. For electron microscopic examination of bacterial structural change caused by rLysG1, E. coli and P. hodoensiswas were incubated with rLysG1 or rTrx and washed with PBS as above. The cells were fixed and processed as reported previously [34]. The samples were then examined with a scanning electron microscope (S-3400N, Hitachi, Tokyo, Japan).

Conclusions
In Summary, we identified and investigated the activity of a g-type lysozyme from deep-sea shrimp. rLysG1 killed selectively Gram-negative bacteria in a non-enzymatic mode by damaging bacterial membrane. The bactericidal activity of rLysG1 was regulated by multiple factors and required the integrity of the α3 and 4 helixes of the protein. The results of our study provide insights into the characteristics and function of g-type lysozyme in crustacean and deep-sea organisms.
Supplementary Materials: The following are available online. Figure S1: SDS-PAGE analysis of rLysG1, Figure S2: Analysis of the potential digestive effect of rLysG1 on peptidoglycan (PGN), Figure S3: The effect of Cu 2+ on the binding of rLysG1 to bacteria and bacterial components, Table S1: Primers used in this study.