Lj-RGD3, an RGD (Arg-Gly-Asp) toxin protein from the salivary gland of
, exhibits antifungal activity against
. Lj-RGD3 has three RGD motifs and shows homology to histidine-rich glycoprotein. We synthesised two mutant derivatives of Lj-RGD3: Lj-26, which lacks all three RGD motifs and contains no His residues; and Lj-112, which lacks only the three RGD motifs. We investigated the effects of the wild-type and mutated toxins on a gram-positive bacterium (
), a gram-negative bacterium (
), and a fungus (
). rLj-RGD3 and its mutants exhibited antifungal but not antibacterial activity, as measured by a radial diffusion assay. The
inhibition zone induced by rLj-112 was larger than that induced by the other proteins, and its inhibitory effect on
was dose-dependent. In viable-count assays, the rLj-112 MIC was 7.7 μM, whereas the MIC of the positive control (ketoconazole) was 15 μM. Time-kill kinetics demonstrated that rLj-112 effectively killed
at 1× and 2× MIC within 12 and 6 h, respectively. Electron microscopy analysis showed that rLj-RGD3 and rLj-112 induced
lysis. Our results demonstrate a novel anticandidal activity for rLj-RGD3 and its mutant derivatives.
play an important role in multiple innate immune systems. Most antibacterial peptides have two common features, in that they are highly cationic (pI= 8.9–12) and amphipathic. The cationicity allows antibacterial peptides to bind to acidic phospholipids, polysaccharides, and LPS on the external surface of the microbial phospholipid bilayer but not the cholesterol-rich neutral plasma cell surface of human hosts. Thus, these peptides are not only specifically antibacterial but also non-toxic to mammalian cells. The amphipathicity allows the antibacterial peptides to insert into the bacterial cell membrane to form a hydrophobic channel. Cationic antimicrobials cluster on the cell membrane surface and alter the lipid bilayer structure, thereby destroying cell membrane function and killing the invading microorganisms. Some cationic antimicrobial peptides also target DNA, RNA, and cellular mitochondrial metabolism. Antimicrobial peptides (AMPs), first isolated from human leukocyte extracts by Zeya and Spitznagel in 1963
, were subsequently discovered in invertebrates
and coldblooded vertebrates
. To date, there are more than 1,200 types of known antimicrobial peptides. Recently, an increasing number of antibacterial peptides have been shown to have multiple functions
, such as chemotaxis (defensins, LL-37)
, apoptosis (lactoferricin, LL-37)
, and angiogenesis (PR-39, LL-37)
. In contrast, some molecules that were not considered to be antibacterial peptides, such as chemokines
, and hormones
, have recently been shown to have antibacterial activity.
RGD3) is a toxin protein with three RGD (Arg-Gly-Asp) domains and is secreted from the buccal glands of
. Like many toxins, Lj-RGD3 inhibits angiogenesis, tumor growth, and platelet aggregation
. The mutant Lj-112, which lacks the three RGDs, is a classic HRG-like protein.
Histidine-rich glycoprotein (HRG) is a single-polypeptide chain α2-plasma glycoprotein of approximately 75 kDa and 507 amino acid residues. HRG was first isolated from human serum by Heimburger in 1972
and was subsequently isolated and characterized in human plasma
. HRG is found in the plasma of other vertebrates, including mice, rabbits, rats, chickens, and cows. Structurally
, the HRG molecule consists of three primary domains: the N-terminal domain, which contains two cystatin (cysteine protease inhibitor)-like regions, and the C-terminal domain and a histidine-rich region (HRR), which are flanked by proline-rich regions (PRRs). Owing to its structure, HRG can interact with multiple different cell surface receptors and bind a wide range of ligands, such as heparin, heparan sulfate, platelet thrombospondin (TSP), fibrinogen, plasminogen, complement proteins, divalent metal ions, and immunoglobulins. In addition, HRG has been implicated in the regulation of numerous biological functions, notably angiogenesis
, immune complex clearance
, and antibacterial activity
. The observation that Lj-RGD3 shares many structural (histidine-rich) and functional (antiangiogenic, antitumor, antithrombotic) features with HRG led us to hypothesize that Lj-RGD3 or its derivative mutants possess antibacterial activity. Here, we demonstrate that Lj-RGD3 and its derivative mutants exert specific anticandidal activity.
Materials and Methods
(AS 1.349) and
(AS 1.72) were acquired from the China General Microbiological Culture Collection Center. A fungal strain
(NCPF 3179) was acquired from the China Center of Industrial Culture Collection.
- Gene Synthesis, Expression, Purification, and Verification of Wild-Type Lj-RGD3 and Mutants Lj-112 and Lj-26
was obtained from Heilong River, China. Total RNA was extracted from the buccal gland of
using Trizol reagent (Invitrogen) and reversely transcribed to cDNA with a RT-PCR kit (TaKaRa Biotechnology Co., Ltd., Dalian, China). The primers annealing to the 5’ and 3’ sequences of the ORF regions of the Lj-RGD3 mRNA are as follows: 5’-primer: 5’-
tcaacgttc atcaacggaacc-3’ (the
I site is underlined); 3’-primer: 5’-
dIII site is underlined). The primers synthesizing and DNA sequencing were performed by TaKaRa Biotechnology Co., Ltd. (Dalian, China). To generate fusion proteins with the C-terminal His-tag, the PCR products were digested with
dIII and ligated into the pET23b vector.
Based on the wild-type L j-RGD3, we synthesised two mutant derivatives of Lj-RGD3: Lj-26, which lacks all three RGD motifs and contains no His residues; and Lj-112, which lacks only the three RGD motifs. We designed complete gene sequences for each mutation. For ligating into the pET23b vector to generate fusion proteins,
dIII were added at the ends of the gene sequences.
The resulting constructs (pET23b-Lj-RGD3, pET23b-Lj-112, and pET23b-Lj-26) were transformed into
BL21 using the CaCl
BL21 containing pET23b-Lj-RGD3, pET23b-Lj-112, or pET23b-Lj-26 was grown at 30℃ for 12 h in LB medium, and IPTG (final concentration 1 mmol/l) was added to induce the expression of the soluble recombinant proteins. The fusion proteins (rLj-xx-His) were purified using His·Bind columns (Novagen) according to the manufacturer’s instructions. The cells were harvested, resuspended in ice-cold binding buffer (5 mmol/l imidazole, 0.5 mol/l NaCl, and 20 mmol/l Tris-HCl at pH 7.9), and sonicated on ice. After centrifugation of the lysate at 14,000 ×
for 20 min, the supernatant was filtered through a 0.45 μm membrane and incubated with Ni-NTA resin. The bound fusion protein was eluted with increasing amounts of imidazole. The protein concentration was determined by the Lowry method
, and the purified protein was resolved on a 16.5% Tricine SDS-PAGE gel
- Radial Diffusion Assay (RDA)
The Oxford cup method was used to determine the antibacterial and antifungal activities of the purified fusion proteins.
were grown to mid-logarithmic phase in Mueller–Hinton (MH) broth. Next, 0.1 ml of the diluted microorganisms (10
CFU/ml) was spread on the surface of the agar plates (90 mm Petri dishes). Sterilized Oxford cups (5 mm diameter) were then placed on the agar medium, and 200 μl or less of the test sample was added to each cup. Equivalent amounts of ketoconazole were used as controls. The plates were placed at 4℃ for 1 h to allow the peptides to diffuse. Antimicrobial activity was determined by measuring the zone of clearance around each well after 16 h of incubation at 37℃ for the bacteria and 24 h at 30℃ for the fungi. The antimicrobial activity was evaluated by measuring the diameter of the zone of clearance around each well. The experiments were repeated three times, and the data were calculated as the mean ± SD.
- Determination of Minimum Inhibitory Concentrations (MICs)
The MICs of the peptides towards the microorganisms were measured with a standard microdilution method using 96-well microtiter plates
was grown to midlogarithmic phase in MH broth. The microorganisms were then diluted to 1 × 10
– 2 × 10
CFU/ml in broth. For dose-response experiments, purified rLj-RGD3 was serially diluted to concentrations of 0.48, 0.96, 1.92, 3.85, 7.7, 15.4, 30.8, and 61.6 μM, and rLj-112 was serially diluted to concentrations of 0.35, 0.7, 1.4, 2.8, 5.6, 11.3, 22.6, and 45.2 μM. Four groups were included: A, 50 μl of protein was incubated with 50 μl of bacteria (10
CFU/ml); B, 50 μl of protein was incubated with 50 μl of MH broth; C, 50 μl of water was incubated with 50 μl of bacteria (10
CFU/ml); and D, 50 μl of water was incubated with 50 μl of MH broth. All of the incubations were performed at 30℃ for 24 h. The antimicrobial activity was evaluated by measuring the optical density at 595 nm (OD
). The survival rate was calculated according to the following formula: Survival Rate (%) = (A − B)/(C − D) × 100%.
The MIC was defined as the lowest concentration of the protein that completely inhibited the growth of each tested bacterial and fungal strain. The MIC was measured in triplicate for each sample.
- Fungicidal Kinetics
The fungicidal activity was assessed using time-kill curves. Briefly, each protein was added to log-phase cultures (5 × 10
at final concentrations of 0.5×, 1×, and 2× the MIC value, and the cultures were then incubated at 30℃ for 0-24 h. The cells were then washed twice with sterile MH broth, and the surviving fungi were diluted 10
-fold and counted using a spiralplating system (Don Whitley Scientific, Shipley, UK). A nopeptide control was included. The fungicidal kinetics was determined by plotting the number of surviving fungi over time.
- Electron Microscopy
was grown in MH broth at 30℃ to mid-logarithmic phase. Suspensions of
CFU/ml) were incubated for 24 h at 30℃ with rLj-RGD3, rLj-112, or rLj-26 at identical concentrations. We included untreated
as a control. The cells were washed three times with 0.15 M phosphate-buffered saline (PBS), pH 7.2, and fixed in 3% (v/v) glutaraldehyde, pH 7.2, for 2 h at 4℃. The fixed cells were subsequently washed and dehydrated in an ethanol gradient (30%, 50%, 70%, 80%, 90%, and 100% (v/v)). Between each step, the cells were centrifuged for 5 min at 8,000 ×
. The samples were then dried, adhered to slides, gold-plated, and examined using a Jeol JEM 1230 electron microscope (Jeol, Tokyo, Japan) operated at 5 kV accelerating voltage.
- Statistical Analysis
All of the experiments were repeated three times, each in triplicate, and the data were calculated as the mean ± SD. Statistical significance of differences in means between groups was determined using analysis of variance.
values less than or equal to 0.05 were considered significant for our analyses.
- Predicted Structure of Lj-RGD3
Sequence analysis demonstrated that the Lj-RGD3 cDNA (GenBank No. FJ416333) is 354 bp long, and the deduced amino acid sequence contains 118 amino acids, including 2 cysteines, 17 histidines, 17 arginines, 20 threonines, and 3 RGD motifs. Its theoretical isoelectric point (pI) is 10.01 and the theoretical molecular mass is 13,633.74 Da. The molecular mass of recombinant rLj-RGD3 is 14,447.85 Da.
The amino acid composition of wild-type Lj-RGD3 is as follows: Ala Arg Asn Asp Cys Glu Gln Gly His Ile Leu Lys Met Phe Pro Ser Thr Trp Tyr Val Gla Hyp Nle Pyr Czs (
The analysis of the secretion characteristics using Signal P and Secretome 2.0P showed that wild-type Lj-RGD3 is a nonclassical secreted protein that lacks a signal peptide and has an NN-score of 0.856 (nonclassical secreted proteins have NN-scores of greater than 0.5).
The predicted Lj-GRD3 secondary structure includes four α-helices that are connected by a random-coil peptide. The three RGD motifs of Lj-RGD3 are distributed throughout the random coils (
The structure of Lj-RGD3. (A) The cDNA sequences and deduced amino acid sequence of rLj-RGD3. The stop codon is indicated with an asterisk. (B) The secondary structure of Lj-RGD3, as deduced using ExPASy Proteomics software. (C) The super-secondary structure of Lj-RGD3. Lj-RGD3 has four α-helices, which are connected by random coils. The three Lj-RGD3 RGD motifs are found within the random coils.
- The rLj-RGD3 Derivative rLj-112 is an HRG-Like Protein
Sequence alignment indicated that Lj-RGD3 not only contains the RGD motif that is characteristic of RGD toxin proteins but also shares 40% identity with residues 21 to 102 of a histidine-rich glycoprotein from
. Lj-RGD3 shares 41% identity with residues 5 to 93 of the T-cell receptor beta chain ANA 11 from
, 30% identity with the human His/Pro-rich domain, and 36% identity with a His/Gly-rich domain (residues 408 to 468) of the human high-molecular-weight kininogen (HK) (
If the three RGD motifs are removed, Lj-RGD3 resembles a classical HRG-like protein. We designed two mutants of wild-type Lj-RGD3. The mutant Lj-112 retains His but lacks the three RGDs, and the theoretical molecular mass is 12,648 Da (
B); the mutant Lj-26 lacks both the RGDs and His, and the theoretical molecular weight is 10,311Da (
C). The cDNAs of Lj-RGD3, Lj-112, and Lj-26 were cloned into the pET23b vector. The recombinant plasmids (pET23b-Lj-RGD3, pET23b-Lj-112, and pET23b-Lj-26) were transformed into
BL21, and the recombinant protein was expressed.
BLAST queries and the Lj-RGD3 mutants. (A) BLAST hits for Lj-RGD3. HRGP: Histidine-rich glycoprotein, Brugia malayi (filarial nematode); TCR β: T-cell receptor beta chain ANA 11, Brugia malayi (filarial nematode). Identical residues are highlighted in grey. (B) The cDNA sequence and deduced amino acid sequence of rLj-112. (C) The cDNA sequence and deduced amino acid sequence of rLj-26.
- rLj-RGD3 Exhibits Antifungal Activity
Recombinant Lj-RGD3 and the two mutants were purified using Ni-NTA columns, and the molecular mass and purity of each protein were confirmed by SDS-PAGE (
A). To determine whether recombinant Lj-RGD3 possesses antibacterial activity, we initially investigated the effects of purified recombinant Lj-RGD3 and the mutants on
(Gram-positive bacterium), and
(Gram-negative bacterium). Lj-RGD3 and the mutants exhibited inhibitory activity towards
(results from the
tests are not shown). As shown in
B, 10 μM of rLj-112 showed similar inhibition in an RDA compared with the identical amount of positive control ketoconazole. However, wild-type rLj-RGD3 and the mutant rLj-26, which lacks His residues and the three RGDs, showed no inhibition at the same concentration. The diameters of the ketoconazole, rLj-RGD3, rLj-112, and rLj-26 inhibition zones were 31, 0, 33, and 0 mm, respectively. Despite the lack of RGD and His, rLj-26 also showed inhibition in an RDA at a concentration of 20 μM (
Anticandidal activity of rLj-RGD3. (A) An identical amount of purified proteins was resolved on a 16.5% Tricine SDS-PAGE gel and stained with Coomassie Blue. M, molecular weight marker; 1, purified rLj-RGD3 protein; 2, purified rLj-26 protein; 3, purified rLj-112 protein. (B) The zones of clearance correspond to the inhibition by each protein after incubation at 30℃ for 18-24 h. Elution buffer was included as a negative control, and the antifungal drug ketoconazole was used as a positive control. a, ketoconazole; b, 10 μM rLj-RGD3; c, 10 μM rLj-112; d, 10 μM rLj-26; e, 20 μM rLj-RGD3; f, 20 μM rLj- 26; g, elution buffer. This image shows only one representative plate from among the replicate experiments. (C) Diameters of the C. albicans inhibition zones shown in Fig. 3B. The values are the means of three independent experiments; each experiment included duplicate trials. Data are expressed as the mean ± SD. *P < 0.05 versus the respective vehicle (in the absence of proteins). (D) The zones of clearance correspond to inhibition by rLj-112 at concentrations ranging from 2.5 to 15 μM after incubation at 30℃ for 18-24 h. a, ketoconazole; b, 2.5 μM rLj-112; c, 5 μM rLj-112; d, 7.5 μM rLj-112; e, 10 μM rLj-112; f, 12.5 μM rLj-112; g, 15 μM rLj-112; h, elution buffer. (E) Diameters of the C. albicans inhibition zones shown in Fig. 3D. The values are the means of three independent experiments performed in duplicate. Data are expressed as the mean ± SD. *P < 0.05 versus the respective vehicle (in the absence of proteins). (F) Minimum inhibitory concentrations (MICs) of rLj-RGD3 and rLj-112 and the survival rate of the representative fungus C. albicans. C. albicans (108 CFU/ml) was incubated with rLj-RGD3 at concentrations ranging from 0.48 to 61.6 μM or with rLj-112 at concentrations ranging from 0.35 to 45.2 μM. (G) MICs of rLj-112 and ketoconazole and the survival rate of C. albicans. C. albicans (108 CFU/ml) was incubated with rLj-112 at concentrations ranging from 0.0875 to 45.2 μM or ketoconazole at concentrations ranging from 0.003 to 37.6 μM. The values shown are the means of three independent experiments performed in duplicate. (H) In vitro killing kinetics of C. albicans treated with rLj-112. Log-phase C. albicans (106 CFU/ml) was treated with rLj-112 (0.5×, 1×, and 2× MIC) for 0-24 h. The surviving fungi were diluted 103-fold and counted using a spiral-plating system. A control with no protein was assayed under the same conditions. Each result represents an average of three independent experiments. (I) Electron microscopy analysis of C. albicans treated with rLj-RGD3 or the two mutant proteins. a, PBS; b, rLj-RGD3; c, rLj-112; d, rLj-26. C. albicans was incubated for 16 h at 30℃ with 15 μM of rLj-RGD3, rLj-112, or rLj-26 and analyzed by electron microscopy.
As described above, rLj-112 induced a larger
inhibition zone than the other proteins. To determine whether the effect of rLj-112 was dose-dependent, as is common for antifungal drugs, we used the Oxford cup method to analyze. As shown in
D, when the concentration of rLj-112 was increased, the inhibition zone became larger. The diameters of the inhibition zones were 2 mm (2.64 μM), 14 mm (5.28 μM), 22 mm (7.92 μM), 34 mm (10.56 μM), 37 mm (13.20 μM), and 40 mm (15.84 μM) (
E). These results indicate that the inhibition of
by mutant rLj-112 was dose-dependent.
The MIC is the minimal peptide concentration required for the total inhibition of microbial growth in liquid medium. We measured the MICs of the antimicrobial peptides using a standard microdilution method in 96-well microtiter plates. The MICs were as follows: rLj-RGD3, 42 μM; rLj-112, 7.7 μM; and ketoconazole, 15 μM. The
survival rate was calculated using the linear regression equation shown in Materials and Methods (
After determining the MIC of rLj-112, we investigated the
fungicidal activity of rLj-112 against
at 0 .5×, 1×, and 2× MIC. The
colony counts decreased gradually during a 24 h period for the three concentrations of rLj-112, and the anticandidal activity increased gradually with time (
H). The higher the concentrations of rLj-112, the greater its inhibition rate of
. We observed >98% inhibition of
by rLj-112 at 1× and 2× MIC within 12 and 6 h, respectively. However, rLj-112 showed 50% inhibition at 0.5× MIC after 24 h.
To examine whether rLj-RGD3 and the mutant proteins interacted with and induced ruptures in plasma membranes,
was incubated with rLj-RGD3, rLj-112, or rLj-26 at identical concentrations (15 μM) and analyzed by electron microscopy. We noted clear differences in the morphology of protein-treated fungi in comparison with the control (
I). rLj-112 caused local perturbations and ruptures throughout the
plasma membranes, and intracellular material was released from the cells. The other proteins, rLj-RGD3 and rLj-26, induced a slight rupturing of the fungal membranes.
The primary finding of our study is the identification of the novel potent anticandidal activity of recombinant Lj-RGD3 and its derivative mutants. Recombinant Lj-112, the mutant lacking all three RGD motifs, has a greater inhibitory effect than the other proteins. These results have implications for our understanding of novel rLj-RGD3 properties and will enable the future development of rLj-112-derived antifungal peptides for therapeutic use.
In this study, we investigated Lj-RGD3 and two derivative mutants: rLj-112, which lacks three RGD motifs; and rLj-26, which lacks three RGD motifs and all His residues. At the same dosage, rLj-112 exhibited stronger anticandidal activity than the wild-type rLj-RGD3 or the mutant rLj-26. Peptides rich in His exert anticandidal functions; however, in the presence of RGDs, this activity is inhibited. Primary and secondary structure analyses showed that the three RGDs are present in the areas between the α-helices. Deletion of the three RGDs may have altered the overall structure of the protein, which could influence the antifungal activity.
HRRs are not the only feature of rLj-112 that is important for anticandidal activity. The mutant rLj-26, which did not contain any His residues, still exhibited anticandidal activity. Compared with a mutant protein that contained only the first or second RGD, the mutant protein that contained the third RGD exhibited stronger anticandidal activity (data not shown) and was most similar to rLj-112. Therefore, we suggest that the antifungal fragment of rLj-112 is present at its N-terminus, and the antifungal activity of rLj-112 is closely related to its amino acid sequence. However, the shortest effective fragment of rLj-RGD3 remains to be determined.
The anticandidal mechanism described here may be similar to that exhibited by human salivary histatins, a family of histidine-rich proteins that are secreted from human and primate parotid, submandibular, and sublingual glands. Pollock
first reported that HRPs inhibit
demonstrated that at least 14 residues and an α-helix at the HRP C-terminus show effective anti-
activity, and the most important functional residues are His15, 18, 19, and 21, Lys11 and 13, and Arg12. In addition, this fragment can kill pathogenic yeasts such as
C. glabrata via
a special mechanism. Histatin has also been shown to kill fungi
. Histatin binds to a specific protein on the fungal cell membrane and is internalized. Inside the cell, it binds to mitochondria and suppresses respiration. This leads to changes in cell morphology and, ultimately, cell death. In addition to killing fungi, histatin also exhibits antibacterial activity, unlike rLj-RGD3, which is specific to fungi. Thus, rLj-RGD3 could be used for targeted treatment.
The growing problem of resistance to conventional antibiotics and the observation that mammals utilize AMPs to counter microbial infections have generated considerable interest in the discovery and development of novel AMPs for therapeutic use. Because AMPs can lyze bacterial as well as mammalian membranes, one of the challenges in designing new peptides is the development of AMPs with a high specificity for bacterial or fungal cells; that is a high therapeutic index (minimal hemolytic concentration/minimal inhibitory concentrations). In this study, we demonstrated that the MIC of rLj-112 is 7.7 μM, which is lower than the MIC of the antifungal drug ketoconazole (15 μM). In addition, rLj-RGD3 displayed no toxicity towards mammalian cells
. This suggests that there is a high degree of dissociation between antifungal and anti-eukaryotic activities. Taken together, our results suggest that rLj-RGD3 has a high therapeutic index and has significant potential for use in clinical treatment.
This project was funded by a grant from the National High Technology Research and Development Program (“863” Program, No. SS2014AA091602), National Nature Science Foundation of China (No. 30770297), Public Science and Technology Research Funds Projects of Ocean (No. 201305016-5), and Major Scientific and Technological Research Projects of Dalian (No. 2013E11SF056).
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