Chinese herb medicine against Sortase A catalyzed transformations, a key role in gram-positive bacterial infection progress

Abstract

Many Gram-positive bacteria can anchor their surface proteins to the cell wall peptidoglycan covalently by a common mechanism with Sortase A (SrtA), thus escaping from the host’s identification of immune cells. SrtA can complete this anchoring process by cleaving LPXTG motif conserved among these surface proteins and thus these proteins anchor on the cell wall. Moreover, those SrtA mutants lose this capability to anchor these relative proteins, with these bacteria no longer infectious. Therefore, SrtA inhibitors can be promising anti-infective agents to cure bacterial infections. Chinese herb medicines (CHMs) (chosen from Science Citation Index) have exhibited inhibition on SrtA of Gram-positive pathogens irreversibly or reversibly. In general, CHMs are likely to have important long-term impact as new antibacterial compounds and sought after by academia and the pharmaceutical industry. This review mainly focuses on SrtA inhibitors from CHMs and the potential inhibiting mechanism related to chemical structures of compounds in CHMs.

Keywords:

• Chinese herb medicines
• Gram-positive bacteria
• inhibitors
• LPXTG-containing protein
• Sortase A

Introduction

Many Gram-positive bacteria are extremely prevalent and responsible for the mortality in the bacteria disease around the world. While for the bacteria themselves, surface proteins and/or pili proteins of Gram-positive pathogenic bacteria particularly play the key role during the infection process. These proteins could either adhere to host’s specific organ tissues or provide some way for bacteria to escape from host’s immune response1. Among these pathogens, Staphylococcus aureus (S. aureus) is particularly concerned. This Gram-positive pathogen is the leading cause of infections in the lower respiratory tract, bloodstream, skin and soft tissue nearly all over the world, causing diverse infections between human and animals2,3. These extracellular proteins are covalently anchored to peptidoglycan by SrtA enzyme on bacterial cell wall after secreted by cells, making pathogens unrecognized during infection4,5. In addition, the LPXTG motif is conserved among these special extracellular proteins with facilitating roles for bacteria to escape immune recognition, considering this we call them LPXTG-containing proteins. This SrtA-dependent anchoring of LPXTG-containing proteins is believed to be a necessary strategy for bacterial survival during infection6,7.

Sortase is a kind of cell surface anchored transpeptidase, and cleaves specific target proteins at its specific sorting signal, for the covalent attachment to the peptidoglycan (Table 1). It is dispensable for microbial growth and survival, but necessary for bacterial infection process8. Sortase homologs have been found in most available Gram-positive bacteria genomes; in most cases, more than one sortase genes have been identified1,9. Recently, four commonly described classes of sortases in gram-positive bacteria were divided10,11. According to the sorting signals recognized and cleaved by the five subfamilies, subfamilies 1, 2 and 3 are, respectively, affiliated with the sortase A, B and C classes, respectively; and subfamilies 4 and 5 are affiliated to the sortase D class (Table 1).

Table 1. Cell wall sorting signals categorized by subfamily type.

sortases	Subfamily	1	2	3	4	5	6
Sortase A class	Family 1	L	P	X	T	G	X
Sortase B class	Family 2	N	P	X	T	N	D
Sortase C class	Family 3	L	P	X	T	G	G
Sortase D class	Family 4	L	P	X	T	A	X
	Family 5	L	A	X	T	G	X

Column 1–6: amino acid sequences of different sorting signals recognized by different sortases.

One of the most important sortases among is the SrtA enzyme, which can recognize, cleave, and anchor specific LPXTG-containing proteins which contribute to bacterial infection process. As discussed above, SrtA inhibitors can interrupt the LPXTG-containing proteins anchoring process, and thus these proteins, which could combine to immunized host, failed in anchoring on the bacteria, making pathogens easily recognized by host during infection, which makes SrtA inhibitor a promising candidate for the development of treatments and/or prevents for major S. aureus’s infection11,12. By contrast, there are significantly less SrtB identified than SrtA13. SrtB anchored its substrates deeper into the cell wall envelope due to the location of enzyme active site deep into cell wall, while proteins anchored by SrtA were displayed on the cell surface14,15. As membrane enzymes, SrtA would be more easily targeted by inhibitors and activity was wreaked, compared to intracellular bacterial targets. SrtC involves in flagella synthesis9,16, and similar to SrtA, SrtC could recognize and anchor the LPXTG-containing proteins17, and covalently tether the pilin subunits in Staphylococcus pneumonia, Enterococcus faecalis and Streptococcus agalactiae18–20, but it also requires help from SrtA to terminate assembly and to tether pili to the cell10. The function of SrtD is not so clear up to now. Recent studies on the high GC % Gram-positive bacteria S. coelicolor suggested that SrtD enzymes may be related to mycelium formation in specific developmental conditions21,22. In comparison, SrtA is well studied, and researches on the structure and catalytic mechanism of SrtA are extensive and detailed. In addition, considering the important role of SrtA paly in bacterial infection, we beleived SrtA inhibitors would be the key factor to cure bacterial infections.

S. aureus has the unique ability to generate suppurative lesions in virtually all organ systems by intruding into the deeper layers of host barriers5. Significant reduction in virulence was observed in the S. aureus SrtA (SaSrtA) mutant (SrtA⁻); lacking of the SrtA activity, bacteria are unable to establish persistent infections, and bacterial cells are easily recognized by the immune system of host, while deletion or inactivation of other sortases does not obviously affect the infection of Gram-positive pathogens23,24, suggesting that the SrtA isoform plays a critical role in the pathogenesis of S. aureus by modulating the ability of the bacteria to adhere virulence-associated proteins to the host tissue7,24–26. Moreover, there are no sortase homologs existing in eucaryon, and therefore selective toxicity is feasible. Above all, researches showed SrtA is not necessary for the growth and viability of S. aureus, and hence, SrtA inhibitors wouldn’t likely induce selection pressure for bacterial resistance27. Considering the important function of SrtA in S. aureus, SrtA inhibitors might consequently be promising novel candidates for the treatment and/or prevention of S. aureus infections without inducing selective pressures.

At prime tense, people tend to use antibiotics to treat bacterial infection disease. Afterwards, due to the abuse of antibiotics, bacterial strains resistant to antibiotics therapy appear, greatly threating the public health. Especially, the hospital strains of S. aureus are usually resistant to a variety of different antibiotics28. Bacterial resistance to antibiotics is threatening the world, and has appeared in many fields of antibiotics application29. Antibiotics treat bacterial infection diseases by preventing the assembly or synthesis of key materials which are essential for bacterial growth and reproduction of needs that are for growth. As a result, the target bacterium gradually formed resistant subpopulations under substantial stress30. Most virulence factors produced by bacterium are not necessary for bacterial growth, and searching inhibitors for virulence factor is of great significance to cure bacterial infections by resistant bacteria, not inducing selective pressures at the same time. Surface adhesion proteins produced by S. aureus were the key virulence factors during infecting mammalian hosts, and SrtA is involved in the catalytic process of anchoring these surface adhesion proteins31. Hence, destruction the anchoring process of these proteins by inhibiting the activity of SrtA could reduce bacterial infection effectively. Moreover, SrtA inhibitors should be less likely to cause drug resistance or induce selective pressures. Currently, there have been some papers in the literature describing inhibitors of sortase running from synthetic products like small molecules to natural products32,33. Mazmanian et al.34 were the first researchers to publish a paper on SrtA inhibitors, where they showed that methanethiosulfonates or organomercurials displayed inhibitory effects on the SrtA reaction. However, people preferred to use CHMs for treatment now, especially in China, and therefore, novel antibacterial drug against SrtA are pressing needs. Plant-derived antimicrobial agents are always a source of novel therapeutics35, and CHMs have been accepted by doctors and patients as superior and a unique valuable property in China for a long time36,37. The long history of CHMs exhibited it has enormous value in medicine field38–40, and from this perspective, identifying antibacterial drugs from natural products like CHMs is urgently needed. Another reason for the renewed focus on medical plant antibacterial in the past 30 years has been own to increasing studies of CHMs treatment for bacterial infection diseases, and with the experiment conditions and technology greatly increased, many important components of CHMs has been clarified or are about to determine, which is of great use for us to determine the active inhibitors in the related CHM. Researches showed there were many plant extracts display strong inhibitory activity on SrtA34,41, and the active substances of these CHMs could effectively inhibit SrtA activity at a concentration far lower than the minimal inhibitory concentration (MIC). That is, these CHMs could inhibit activity of SrtA without affecting the growth of bacteria or forming resistant subpopulations. Compared to traditional antibiotics, CHMs are easy to get42,43, and pharmacological activity of CHMs are based on their diversity of chemical components, and several components can simultaneously involve in the treatment of bacterial infections, while antibiotics nearly can’t be used simultaneously in case of adverse drug reactions or even death. Therefore, medicinal plants have received a significant attention as new sources of anti-microbial agents, and searches for effective SrtA inhibitors have shifted more attention to herbal medicine extracts.

Studies showed extracts from Curcuma longa, Fritillaria verticillata, Coptis chinensis, Radix isatidis, Radix Sophorae flavescentis, Radix Pulsatillae, both well-known traditional CHMs, have been widely used in medical practice in China for thousands of years. Moreover, the extracts of these CHMs exhibit inhibitory effect on SrtA with a relatively high MIC41.

Sortase A

Three-dimensional structure SrtA

SrtA is a cell surface anchored thiol transpeptidase that cleaves LPXTG-containing proteins between threonine and glycine at the LPXTG motif4. In Gram-positive pathogens, LPXTG-containing proteins promote adhesion to specific organ tissues, thus escaping from the identification of immune cells7,44,45. SaSrtA (SrtA of S. aureus, Genebank) is a 206-amino acid polypeptide with a hydrophobic N-terminal segment probably functioning as both a secretion signal peptide and a stop transfer signal for membrane anchoring7. Quaternary structure of SaSrtA (modeled by SWISSMODEL46 http://swissmodel.expasy.org/) contains a unique β-barrel structure including eight β-pleated sheets, and three other shorter β-pleated sheets (Figure 1a and b).

Figure 1. The predicted quaternary structure of SaSrtA. The predicted His₁₂₀, Cys₁₈₄ and Arg₁₉₇ in the active site are highlighted. The α-helix and β-sheet regions of the putative protein are indicated with cylinders and bands, respectively. Multiple sequence alignments revealed many conserved domains. The three amino acid residues thought to be involved in the formation of the catalytic site were conserved in those domains (highlights) of all 25 sequences. His₁₂₀, Cys₁₈₄ residues, participating in protein–protein interaction, are conserved between these bacteria above.

The key amino acids of SrtA

Three β-pleated sheets of the β-barrel structure were curved and folded, forming a notch, the active site of SrtA. The active site of hydrophobicity contains His₁₂₀, Cys₁₈₄, Arg₁₉₇, Ala₁₁₈, Val₁₆₁ Val₁₆₆, Ile₁₈₂ (both of them are amino acids, and the number represents position on the peptide chain)47–49, The catalytic site residue Cys₁₈₄ is in close proximity with His₁₂₀, forming a putative active site cysteine-histidine ion pair50,51. Histidine and Cysteine are both of great importance in maintaining the activity of SrtA50,52, and so is Arg₁₉₇53, making the cleavage of Thr and Gly more easily by ionizing Cys₁₈₄54. The sulfhydryl group of Cys₁₈₄ can also attack the T-G peptide bond of LPXTG-containing proteins, resulting in an enzyme-acyl intermediate formation34,55. While CHMs possess aryl (β-amino) ethyl ketones, vinyl sulfones as well as other compounds of a nucleophilic attack could inhibit SrtA activity by conjugating to the thiol of Cys₁₈₄ of SrtA56, as well as those reacting with Histidine and Arginine, thus irreversibly inhibiting SrtA activity57,58. Besides, hydrophobic compounds could also penetrate into the active site though hydrophobic interaction, and threonine analogs can also inhibit the activity of SrtA59.

Phylogentic analysis of SrtA

The available complete genome sequences enabled us to identify twenty-five SrtAs from nine Gram-positive bacteria species (Clustal X 2.0 program http://clustalx.software.informer.com/2.0/). Multiple sequence alignments revealed some conserved domains among these sequences (Figure 1). As expected, the three amino acid residues (His₁₂₀, Cys₁₈₄, Arg₁₉₇) thought to be involved in the formation of the catalytic site were conserved in those domains between these bacteria studied (highlights in Figure 1) of all twenty-five sequences. Nine groups of these twenty-five SrtA enzymes, were chosen to construct the phylogenetic tree (Figure 2) by using MEGA 6.0.6 software60 (http://www.megasoftware.net/), Bacillus, Brevibacillus, Exiguobacterium, Lactococcus, Listeria, Lysinibacillus, Paenibacillus, Staphylococcus, Streptococcus. Universally, Brevibacillus, Listeria, Lysinibacillus, Staphylococcus, Streptococcus, and most of Exiguobacterium are clustered definitely into their own group, which implied a conserved evolutionary relationship among these species. Nevertheless, the Lactococcus exhibited an ambiguous relationship with a few Exiguobacterium (such as Exiguobacterium antarcticum), Paenibacillus and Bacillus (Bacillus subtilis and cereus). In conclusion, SrtAs exhibited a certain degree of homology among these nine groups, thus SrtA inhibitors of S. aureus can also reduce infection of pathogens among these nine groups other than S .aureus.

Figure 2. Phylogenetic relationship of the SrtA of various species. The SrtA used for phylogenetic tree construction are as the amino acid sequences used for homology alignment above. Numbers below the branches are the neighbor joining bootstrap values. The evolutionary distance is reflected by the numbers above the branches and the branch lengths proportional to the degree of amino acid substitutions. SrtAs exhibited a certain degree of homology among these nine groups.

The catalytic process of SrtA

LPXTG-containing proteins, anchored by SrtA, mostly harbor an N-terminal signal peptide and a C-terminal cell wall sorting signal consisting of the LPXTG motif, hydrophobic domain and a positively charged tail in all pathogenic Gram-positive bacteria (Figure 3)61–63.

Figure 3. The structure of the LPXTG proteins precursor. (The length of rectangle does not stand for the real length of peptide). The LPXTG proteins, anchored by SrtA, mostly harbor an N-terminal signal peptide and a C-terminal cell wall sorting signal consisting of the LPXTG motif, hydrophobic domain and a positively charged tail have been found in all pathogenic Gram-positive bacteria, besides the anchored protein section.

After synthesized in the cytoplasm, LPXTG-containing proteins are released into extracellular space through the secretary pathway with N-terminal leader peptide moved. Then, SrtA catalyzes the breakage of amide bond between threonines and glycine in the LPXTG motif, and captures the C-terminal carboxyl of cleaved surface proteins by forming a thioester bond with its active site sulfhydryl on the cell surface64. Subsequently, the nucleophilic attack of the amino group of the lipid II peptidoglycan precursor propels LPXTG-containing proteins to form an amide bond with the cell wall cross-bridge, regenerating the active site sulfhydryl of SrtA52,64 (Figure 4). During anchoring process, the scissile T-G peptide bond is positioned between the two active site residues, Cys₁₈₄ and Arg₁₉₇, and the active site is stabilized by His₁₂₀65, forming a hydrogen bond with Arg₁₉₇66. Altogether, SrtA completes the transpeptidation sorting reaction based on the surface protein secretion and cell wall synthesis pathways.

Figure 4. Cell wall sorting progress in Gram-positive bacteria. (1,2) Cell wall synthesis begins with the assembly of nucleotide linked wall peptides in the bacterial cytoplasm. Then the precursor, the lipid І, is transferred to bactoprenol pyrophosphate in the cytomembrane, next, N-Acetylglucosamine (G) connect to lipid І (MurNAc-(pentapeptide)-pyrophosphoryl-undecaprenol), forming the lipid II (GlcNAc-β-1,4-MurNAc-(pentapeptide)-pyrophosphoryl-undecaprenol). Finally lipid II molecules are transported across the cytoplasmic membrane for the polymerization reactions in bacteria. (3–6) Surface proteins are synthesized as precursors in the bacterial cytoplasm bearing an N-terminal signal peptide and a C-terminal sorting signal. The sorting signal contains an LPXTG sequence motif, a hydrophobic domain and a tail of positively charged residues. After surface protein synthesized, the N-terminal signal peptide will be removed before secreted out, and then the hydrophobic domain is embedded within the membrane and the charged tail remained in cell, retaining surface proteins in the secretory pathway. (5) The precursor cleaved by SrtA, a membrane-anchored transpeptidase, between threonines and glycine in the LPXTG motif, generating an acyl enzyme intermediate. (6) The thioester bond between SrtA and surface proteins, is resolved by the nucleophilic attack of the amino group of the cross-bridge (Gly₅) within lipid II precursor. Finally, surface proteins linked to lipid II can be incorporated into the cell wall envelope.

LPXTG-containing Proteins anchored by SrtA

SrtA anchors a number of proteins, such as protein A, fibronectin-binding proteins, collagen-binding proteins and pili proteins, covalently attached to the bacterial cell wall by a common mechanism that is ubiquitous amongst Gram-positive organisms.

Surface proteins

S. aureus is a human and animal pathogen, which causes diverse infections67. Staphylococci lack pili and instead rely on surface protein-mediated adhesion to host cells or invasion of tissues as a strategy for escape from immune defenses by surface proteins. These surface proteins68 covalently anchored by SrtA include the fibronectin-binding proteins69,70, collagen adhesion protein, and protein A. Fibronectin-binding proteins consist of FnbpA and FnbpB71–73, which can interact with the fibronectin of mammalian cells74 (Table 2). In addition, Staphylococcal strains causing connective osteomyelitis or tissue infections regularly through the collagen adhesion proteins84,85, and these collagen adhesion proteins can bind to host’s collagens86. Besides, S. aureus protein A (Spa) binds to the Fc termini of mammalian immunoglobulins in a nonimmune fashion, resulting in the uniform coating of Staphylococci with antibodies87 (Table 2).

Table 2. Proteins anchored by SrtA.

Proteins anchored by SrtA	Binding ligands	Function	Recognition sequence (LPXTG)	Reference
Fibronectin binding proteins (FnbpA and FnbpB)	FibronectinFibrinogen	Invasion cells	LPETG	(75–77)
Protein A	Von Willebrand factor Antibody Tumor necrosis factor receptor	Infection process Antiphagocytic	LPFTG	(78)
Clumping factors (clfA and clfB)	Fibrinogen	Bacterial adhesion	clfA: LPDTGclfB: LPETG	(79,80)
Plasmin and fibronectin-binding protein A (PfbA)	PlasminogenFibronectin	Surface adhesion Invasion	LPKTG	(81)
SpaABC of SpaA-type pilus	Unknown	Bacterial adhesion	LPKTG	(82,83)

Flagellin in Gram-positive bacteria

Expression of filamentous structures extending from bacterial surface is the common mechanism of bacteria to infect host tissues during colonization. The pili proteins have been demonstrated to improve the progression of infective diseases that affect periodontal, gastrointestinal, urinary, and respiratory tissue. Besides, flagella have been implicated in a number of biological processes, including biofilm formation, cell aggregation, host cell adhesion and invasion, and as potent inducers of the host’s inflammatory response88,89. Particularly, the protein components of flagella are covalently polymerized by sortase enzymes in pathogenic bacteria90, similar to those surface proteins involved in the covalent attachments to the peptidoglycan cell wall of Gram-positive bacteria88. The adhesion moieties in flagella are distally located from the bacterial surface, which provide a significant advantage in the initial steps for establishing a successful colonization. Flagella are polymeric virulence factors necessary for bacterial colonization and pathogenesis91, during the assembly process of flagella in Streptococci, SrtA is responsible for anchoring the polymerized pilus to the cell wall92,93, catalyzing proteins of LPXTG motif anchoring94, and transferring the entire pilus structure onto lipid II, and ultimately, it becomes incorporated into peptidoglycan, forming the main site of infection90,95. Deletion of SrtA releases a large portion of SpaA polymers into the culture medium96, indicating SrtA does indeed play some role in pilus assembly, specifically, in the anchoring phase20.

Chinese Herb medicines as inhibitors of SrtA

Fritillaria verticillata

Fritillaria verticillata (Chinese name: Bei mu cao, Liliopsida) can be also used as one herbal medicine to inhibit SrtA activity, and it mainly produced in northern Xinjiang province, a member of Liliaceae family97. Bulbs of Fritillaria have been used as one of the most important antitussive expectorant and anti-hypertensive drugs in TCMs. Extracts from Fritillaria species bulbs are used to treat scrofula, coughs, asthma, tumors, etc98. Extracts from the bulbs of Fritillaria verticillata had significant inhibitory effect on S. aureus99. The β-sitosterol-3-O-glucopyranoside (Table 3) isolated from the bulbs of Fritillaria verticillata by bioassay-guided chromatographic fractionation had an antibacterial activity against Bacillus subtilis, Staphylococcus aureus and Micrococcus leuteus100 with MIC values of 50, 200 and 400 mg/mL, respectively. Once using extracts of Fritillaria verticillata bulbs to incubate S. aureus and analyzed the SrtA activity by fluorescence spectrophotometer100, results showed that the isolated β-sitosterol-3-O-glucopyranoside from Fritillaria verticillata extracts exhibited inhibiting SrtA activity with an IC₅₀ of 18.3 μg/ml (Table 3, Figure 5), almost two times more potent than the positive control, pHMB.

Figure 5. Structures of S. aureus SrtA inhibitors.

Table 3. Antibacterial activity of the herbal extracts on SrtA of S. aureus and effective material.

Inhibitor classes	Potent inhibitor	Plant name	SrtA IC50 (μg/mL)	MIC (μg/mL)
Sterolglycoside compoundes	β-Sitosterol-3-O-glucopyranoside	Fritillaria verticillata	18.3	>200
Isoquinoline alkaloids	Berberine chloride	Copis chinensis franch	8.7	50–400
Indole alkaloid	Aaptamines	Radix isatidis	3.7	50
Flavonoids	Quercetin	Radix isatidis	15.91	>900
	Myricetin	Rhus verniciflua	13.99	>900
	Kurarinol	Radix Sophorae flavescentis	48.8	99.54
	Rutin	Ruta graveolens L	57	>1024
Phenylpropanoid	Curcumin	Curcuma longa	13.8 ± 0.7	>200
	Caffeic acid	Radix Pulsatillae	3.363	125
	Rosmarinic acid		7.242	ND
	Chlorogenic acid	Flos Lonicerae	33.86	256

ND: not determined.

The β-sitosterol-3-O-glucopyranoside is one of sterolglycoside compoundes, of great hydrophobicity, and it can destroy S. aureus adhesion to fibronectin through fibronectin-binding protein101. The OH of the alkyl cyclopentane and alkyl cyclohexane in β-sitosterol-3-O-glucopyranoside can form H-bonds with the guanidine group of Arg₁₉₇, and the aromatic rings can form strong lipophilic interactions with the hydrophobic active site of SrtA102, thus inhibiting the activity of SrtA. In addition, the hydroxyl group of the cyclohexane could also form another hydrogen bond with the backbone carbonyl of Gly₁₉₂ and keeps the β7/β8 loop in a closed state. Moreover, the glucopyranoside side chain moiety was found to be the active component of the compound because elimination of this side chain moiety led to a reduction in inhibitory activity103, probably also because OH of the cyclohexane moiety of the glucopyranoside side chain moiety can form hydrogen bonds with Arg₁₉₇104, both of which are important for Cysteine to complete the catalytic process. Hence, we can find out whether other CHMs containing sterol glycoside compounds can inhibit the activity of SrtA, which can provide one approach to find more herbs to inhibit SrtA, in addition figure out whether several kinds of CHMs taken together can strengthen inhibitory activities on SrtA.

Coptis chinensis Franch

Coptis chinensis Franch (Chinese name: Huang lian), a perennial herb, another valid inhibitor of SrtA, is a member of the Ranunculaceae family, grows in environment of low temperature and high air humidity105. Coptis chinensis Franch as one TCM has a long history. As recorded in Compendium of Materia Medica, Coptis chinensis Franch has functions on many diseases. It has been used as antimicrobial agents, anti-inflammatory, hypolipidemic, hypoglycemic106.

Generally, the extracts of Coptis chinensis Franch rhizome showed antibacterial effect especially on S. aureus107. Berberine in Coptis chinensis Franch well inhibited the infection ability of S. aureus108 and many other bacteria109, and S. aureus was most sensitive to berberine. Berberine chloride is one type of isoquinoline alkaloid (Table 3, Figure 5), from the rhizomes of Coptis chinensis Franch appeared a potent inhibitory effect on SrtA, with an IC₅₀ value of 8.7 μg/ml and also had antibacterial activity against Gram-positive bacteria, with a MIC more than 100 μg/ml, made a comparison to available isoquinoline alkaloids berberine sulfate, palmatine chloride, and β-hydrastine, which are all antibacterial substances12.

As previously revealed, similar to curcumin, the aromatic rings of berberine chloride could combine to the hydrophobic side chains (the side chains of from the β6/β7 loop) through hydrophobic effect, embedded into the active site of SrtA, thus inhibiting SrtA activities104. In addition, the nitrile group of Berberine chloride can form a hydrogen bond with SrtA catalytic residue Arg₁₉₇ via hydrogen bonding102. Coptis chinensis Franch as one CHM to treat bacterial infection contains many other compounds except isoquinoline alkaloid. Furthermore, isoquinoline alkaloids have been found mainly in some plant families, such as Papaveraceae, Magnoliaceae, Rutaceae and Ranunculaceae as well as Fumaria capnoides Linn, providing more herb medicines for us to study to find more inhibitors (Table 3).

Radix isatidis

Radix isatidis (Chinese name: Ban lan gen), a member of Cruciferae, Dicotyledoneae, is also known as Indigowoad Root, and this herb is cultivated in various regions of northern China110. The roots are harvested during the autumn and dried, and then get Radix isatidis, which are usually processed into granules called Banlangen granules. This product, commonly dissolved in hot water, is very popular throughout China, and used to remove toxic heat, soothe sore throat and to treat pharyngitis, laryngitis, erysipelas as well as carbuncle, in addition, it possesses an antibacterial, antiviral, improve immunity, antitumor effect111,112. Especially the prevention and cure of SARs, Radix isatidis again became the focus and hotspot of people’s research into medicinal value. In recent years, the study of Radix isatidis, especially the study of its chemical composition has been sharply increasing110,113,114. The chemical composition isolated from this herb mainly contains the following categories: indole alkaloid and isoquinoline type alkaloid, flavonoid, lignin, organic acids, ketone, sterols and amino acids114. The extracts of Radix isatidis displayed inhibitory effect on Gram-positive bacteria as well as Gram-negative bacteria, S. aureus, S. epidermidis, B. subtilis115. The method of systematic solvents was used to identify inhibitory effects of the different organic solvents extracts on S. aureus116, and results showed that the total extract exhibited the higher antibacterial effect than others and that the antibacterial effects of Radix isatidis were not limited in one active portion110.

As previously revealed, indole alkaloid and isoquinoline type alkaloid possess a prominent inhibitory effect on SrtA117, which were rich in nitrile groups and phenyl rings. The nitrile groups could form hydrogen bonds with the catalytic residue Arg₁₉₇, and the aromatic rings formed strong lipophilic interactions with the amino acids in the hydrophobic binding pocket of the SrtA active site. Aaptamines from the tropical sponge, also in Radix isatidis114, is one of indole alkaloid and can inhibit the activity of SrtA118. The extract of Radix isatidis contains lots of indole alkaloids, such as aaptamines, 5-hydroxyoxindole, indigo, indirubin, indole-3-acetonitrile-6-O-β-D-glucopyranoside, 2-[(3′-indole)-cyanomethylene]-3-indolinone, 2,3-dihydro-4-hydroxy-2-oxo-indole-3-acetonitril, 3-(3′,5′-Dimethoxy-4″-hydroxybenzylidene)-2-indolinone. These indole alkaloids possess acetonitrile groups mostly, which could combine to the sulfhydryl of Cys₁₈₄ in SrtA, thus inhibiting the activity of SrtA59. Hence, the inhibitory effect of Radix isatidis can be due to the acetonitrile groups in these indole alkaloids. In addition, trytanthrin, another compound in the extract of Radix isatidis, is an isoquinoline type alkaloid. The aromatic rings in aaptamines, as hydrophobic groups, can combine to the hydrophobic side and occupy the sorting signal recognition region through hydrophobic interactions with the side chains from the β6/β7 loop104 (Figure 5), preventing LPXTG-containing proteins’ combination with SrtA, while the amide carbonyl, NH groups and the oxygen atom in aaptamines, can link to Cys₁₈₄, leading to a failure of the amide bond between SrtA and LPXTG-containing proteins. According to the research about aaptamines, trytanthrin probably can inhibit the activity of SrtA. Moreover, there are still some flavonoids in Radix isatidis, such as quercetin and myricetin. Both myricetin and quercetin were potent SrtA inhibitors, and they inhibited SrtA activity with IC₅₀ values of 44.03 μM (13.99 μg/ml) and 52.70 μM (15.91 μg/ml)119, respectively. Both of the two flavonoids reduced bacterial clumping to fibrinogen in a dose-dependent manner trough fibronectin binding proteins (FnbpA and FnbpB), which anchoring process was catalysis by SrtA (Table 2). All these two flavonols tested in S.S. Kang’s studies showed they exhibited nearly no growth inhibition effect on S. aureus Newman strain with a MIC values greater than 300 μM.

Radix Sophorae flavescentis

Radix Sophorae flavescentis is another effective inhibitor of SrtA, a member of Leguminosae family, throughout Russia, Japan, India, Sorth Korea and China. The dried root of Radix Sophorae flavescentis, is well known to possess anti-inflammatory, anti-arrhythmic, anti-pyretic, anti-asthmatic, as well as anti-ulcerative effects and is used for the treatment of diarrhea, gastrointestinal hemorrhage and eczema120. Radix Sophorae flavescentis is rich in flavonoids, of a wide range of biological activities, including anti-cancer, anti-inflammatory and anti-bacterial properties121. The flavonols kurarinol was the most effective inhibitors of SrtA compared to others isolated from Radix Sophorae flavescentis, with an IC₅₀ of 48.63 μg/mL, and a MIC of 99.54 μg/mL almost with no effect on S. aureus growth activity121 (Table 3), which is better than kurarinone, another compound from Radix Sophorae flavescentis, with one less hydroxyl group compared to kurarinone. The inhibitory activity of flavonols depend mainly on the hydroxyl substitution in flavonols119,122. The mechanism of the inhibitory effect of kurarinol on SrtA may be due to the hydroxyl groups in kurarinol forming hydrogen bonds with both the side CHA amino of Arg₁₉₇ and the backbone of Gly₁₉₂⁵4, resulting in SrtA activity weakened, and thus failing in intermediate product formation (SrtA-T-surface protein). Moreover, the aromatic rings could also interact with the side chains and combine to the hydrophobic active site of SrtA, occupying the sorting signal recognition region of SrtA104.

Kurarinol is one kind of flavonoids, hence, other Chinese herbs containing flavonols flavonoids maybe potentially possess SrtA inhibition. Flavones and flavonols are usually extracted from vegetables, fruit, tea, and red wine, and among natural flavonols, Flavonoids such as rutin, morin123, myricetin, and quercetin are active against SrtA119. Rutin in Ruta graveolens L. could inhibit SrtA activity in vitro experiments, with an IC₅₀ of 57 μg/mL and little bacteria inhibition124. Similar to the above mentioned CHMs, the aromatic rings of rutin can also make a difference in hindering the catalytic process of SrtA. Besides, the oxygen atom can form hydrogen bond with His₁₂₀ and Arg₁₉₇ residues of SrtA. In addition, there are two flavonoids, myricetin and quercetin in the bark of Rhus verniciflua119 also existing in Radix isatidis, exhibited strong SrtA inhibitory activity (see previous section in Radix isatidis). Moreover, although the two flavonoids structure is similar, they exhibited different inhibitory activity. The number of hydroxyl groups at the specific location of the particular aromatic rings gave them different inhibitory capacity on SrtA119. Another Chinese herb rich in flavonols is Rhizoma Scutellariae, and the major components of R. scutellariae include baicalein, baicalin, wogonin, and wogonoside. The root of Rhizoma Scutellariae is a traditional medicine, displaying a better anti-bacteria effect, and the extract of Rhizoma Scutellariae reveals an obvious inhibitory effect on S. aureus in vivo and vitro125,126. Although, we did not know which flavonoids exhibited inhibitory effect on S. aureus, according to the above conclusion that many flavonoids could inhibit SrtA activity due to their hydroxyl groups and aromatic rings, Rhizoma Scutellariae might be a potential candidate inhibitor127 (Figure 5).

Curcuma longa

Curcuma longa (Chinese name: Jiang Huang), an effective inhibitor of SrtA and also known as turmeric a perennial herb. It is a member of the Zingiberaceae (ginger) family, and can be cultivated extensively in India, China, and other countries with a tropical climate116. The rhizome of Curcuma longa appears irregular ovoid, heavy yellow, rough and shrinking grain in the surface, and therefore the rhizome is widely used in foods for its flavor and color. Moreover, the rhizome of Curcuma longa has a long traditional use in the Chinese and Ayurvedic systems of medicine, particularly as an anti-inflammatory drug and also for the treatment of flatulence, jaundice, menstrual difficulties, hematuria, hemorrhage, and colic128. Curcuma longa can also be applied topically in poultices to relieve pain and inflammation129. The use of Curcuma longa extracts for the antimicrobial activity has long been known. Curcuma longa and its extracts have various beneficial effects on human health130. Curcumin derivatives is one kind of important ingredients from Curcuma longa extracts, and curcumin can exhibit antioxidative131, anticarcinogenic132, anti-inflammatory133 and hypoglycemic potency134,135.

Many pharmacological properties of Curcuma longa have been reported to date including its inhibitory effects on SrtA103, but its inhibitory mechanism on the Gram-positive bacteria SrtA has not been figured out. Analysis of the active compounds isolated from Curcuma longa, their inhibitory effects on SrtA and the minimum inhibitory concentrations (MICs) against S. aureus, showed that the curcumin could inhibited the S. aureus SrtA, resulting in failure of S. aureus cell adhesion to fibronectin135. with an IC₅₀ (50% inhibiting concentration) value of 0.7 μg/mL, more effective than pHMB (p-hydroxymercuribenzoic acid, IC₅₀: 1.2 μg/mL)135. In addition, curcumin showed no obvious growth inhibitory activity against S. aureus with MICs greater than 200 μg/mL135, and therefore SrtA inhibitors should disrupt bacterial infections without inducing drug resistance7. Altogether, Curcuma longa is an excellent candidate in the development of a bacterial SrtA inhibitor.

The active site of SrtA was a large hydrophobic binding pocket136, which was composed of lipophilic side chains of the amino acids, such as Val₁₉₃, Trp₁₉₄, Ala₉₂, Ala₁₀₄, Leu₁₆₉, Val₁₆₈, and Ile₁₈₂102 and the location of the hydrophobic binding pocket as shown in Figure 1. These hydrophobic side chains have relatively strong lipophilic interactions with the phenyl rings of curcumin through the hydrophobic effect. In addition, two aromatic ring of curcumin are located in the trans orientation, similar to the structural formula of (Z)-3–(2,5-dimethoxyphenyl)-2–(4-methoxyphenyl) acrylonitrile (DMMA), one proved inhibitor of SrtA102,137. Besides, the aromatic ring could form hydrophobic interactions with the side chains of residues from the β6/β7 loop (Val₁₆₉, Leu₁₆₈) and Arg₁₉₇, occupying the sorting signal recognition region of SrtA104 (Figure 1). In conclusion, curcumin of Curcuma longa, as an inhibitor of SrtA, maybe interdict the catalysis process of SrtA by embedding the two aromatic rings into its active site through hydrophobic interaction111 and forming hydrogen bonds, and as a result, LPXTG-containing proteins can’t combine to cell wall of the bacteria by SrtA. Other curcumin derivatives from extracts of Curcuma longa also exhibited inhibitory effect on SrtA135, which would replenish Curcuma longa could be used for SrtA inhibitor as a potential medicine for bacterial infection. Besides, curcumin belongs to polyphenolic compounds, also known as a dicarbonyl compounds, shows an inhibitory effect on SrtA, thus we can find out whether other CHMs containing dicarbonyl compounds of aromatic rings can inhibit the activity of SrtA (Table 3, Figure 5).

Radix Pulsatillae

Radix Pulsatillae, Chinese Pulsatilla Root (shennong baicaojing) or the Korean pasque flower, one member of Ranunculaceae family, Dicotyledons class. Radix Pulsatillae is a perennial plant growing in China and Korea, and used as a traditional CHM for a long time138,139. The rhizomes of Radix Pulsatillae have been used as a CHM for malaria, leucorrhea, epistaxis, scrofula, amebic dysentery and internal hemorrhoids, and the root is anti-inflammatory and anti-parasitic140. It contains several medically active constituents including anemonin as well as saponins141.

The rosmarinic acid, phenyl propanoids caffeic acid, exhibited a potent inhibitory effect on SrtA of Streptococcus mutants isolated from human oral cavity; what’s more, these two compounds from the extract of Radix Pulsatillae demonstrate similar inhibition degree142. Rosmarinic acid, a kind of natural multifunctional phenylpropanoids compounds, has been used as anti-infective, anti-inflammatory, anti-virus and anti-tumor. Phenyl propanoids caffeic acid, one kind of phenylpropionic acids, possesses similar functions to rosmarinic acid.

The IC₅₀ of rosmarinic acid and phenyl propanoids caffeic acid is 20.1 and 20.2 μM (IC₅₀), respectively, with pMHB of 35.1 μM142. One mechanism of the inhibitory effect of rosmarinic acid and phenyl propanoids caffeic acid on SrtA may be due to the location of hydroxyl group in these compounds. The presence of a hydroxyl group at the position 3 of caffeic acid exhibited greatly important compared with other phenylpropionic acids of Radix Pulsatillae, possessing an obviously higher degree of inhibition, so dose rosmarinic acid. The hydroxyl group at the position 9 could also affect inhibitory activity on SrtA. In addition, the aromatic rings of the compounds owns a similar function to those in the CHMs mentioned above. Besides, the carboxyl of these two compounds can be as strong nucleophilic groups connected the Cys₁₈₄ of SrtA, resulting in the failure of intermediate product formation.

Natural phenyl propanoids caffeic acid mainly exists in feverfew, ranunculaceae, rutaceae plants143, and natural rosmarinic acid is widely distributed in plants, mainly contained in labiatae, boraginaceae, umbelliferae, cucurbitaceae and tiliaceae plants144. Natural chlorogenic acid, one phenylpropanoids compounds similar to rosmarinic acid, is rich in plants of Lonicera, Artemisia, such as Flos Lonicerae. Chlorogenic acid is one main antibacterial, antiviral active ingredient in Flos Lonicerae, and it could cause S. aureus infection failure through SrtA inhibition with an IC₅₀ 33.86 μg/ml, interfering in Fg/Fn binding and attenuate the virulence of S. aureus104. Chlorogenic acid could localize to the “activity” region of SrtA, and the hydroxyl group of the cyclohexane in this phenylpropanoids compound formed hydrogen bonds with both amino acids Arg₁₉₇ and the backbone of Gly₁₉₂. Moreover, Chlorogenic acid could also form Van der Waals interactions with the side chains of Cys₁₈₄ and Ile₁₈₂104. This new finding laid the foundation for us to search the phenylpropanoids-containing Chinese herbs as SrtA inhibitors to cure bacterial infection145 (Figure 5).

Summary and conclusion

Sortase enzymes, anchoring proteins with different sorting signals, allow gram-positive bacteria to complete protein location and functional display within the cell wall envelope, and response to environment or specific host signals during infection. SrtA, one transpeptidase positioned on the outside of bacterial cells, catalyze the process of amide bond exchange, using LPXTG-containing proteins and peptidoglycan as substrates, play an very important role during Gram-positive bacterial infection process. Thus, finding SrtA inhibitors became the key point. However, in view of the abuse of antibiotics, the number of bacterial drug resistance is increasing, so we turn to CHMs, of medicinal value and with long history, to find out inhibitors of SrtA rather than antibiotics in case of more drug resistance.

Here, we list some traditional Chinese herbs as inhibitors of SrtA from S. aureus, Curcuma longa, Fritillaria verticillata, Coptis chinensis Franch, Radix isatidis, Radix Sophorae flavescentis and Radix Pulsatillae, and their effective constituents belong to which kind of chemical compounds. Hence, we can go on searching for other CHMs with similar above mentioned chemical compounds as new inhibitors of SrtA. In addition, these demonstrated inhibitors from other natural sources, like the sponge Spongosorites sp.32 and ascidian Synoicum sp.146 can also put a good foundation for studying herb medicines with similar compounds. Evidences from published data have shown that compounds isolated from these CHMs are effective against SrtA from S. aureus and other pathogenic microorganisms.

However, these research on CHMs are mostly conducted in vitro, thus, the inhibition of these herbs solely on the basis of IC₅₀ values must be carefully considered. A determination of the mechanism about the inhibition on SrtA by these classes is required to completely and clearly understand, as well as their relative effectiveness and specificity; i.e., is the inhibition competitive in the presence of substrate? Do these inhibitors reversibly or irreversibly react with SrtA? What are the affinities and kinetics of inhibition? In light of the fact that many compounds can inhibit enzyme activity based on redox action, promiscuous aggregation, denaturation, pH changes, and so forth, not to mention possible quenching of fluorescence or spurious reaction with peptide substrate during the experiment process when considering the sortase activity assay, thus it seems prudent to carefully investigate the nature of the inhibition147,148. Finally, we should also consider how to use these inhibitors and what form taken by us to get the best medicinal value in vivo. In addition, we should also consider how these inhibitors can aggregate in targeted points. In one word, we need further more experiments for minimum amount and the best curative effect of herb medicines in the future, unlike antibiotics resulting in drug abuse.

Current research and prospect forecast

Recently, there are more and more studies on the research about SrtA and biological function in Gram-positive pathogens, thus we figure out the catalytic mechanism of SrtA and its vital function in those pathogens. Considering that bacterial resistance to antibiotics has become a serious threat to public health, novel drugs against Gram-positive bacterial infection are urgently needed, and plant-derived antimicrobial agents are always a source of novel therapeutics, thus CHMs draw our attention on the treatment for bacterial disease35,149. Moreover, once we make it clear that how the active constituents of these herb medicine interact with SrtA, and make sure their chemical construction and spatial structure, as well as the key functional groups of these compounds interacting with the active site of SrtA, we can design some analog inhibitors similar to those natural being as new inhibitors of SrtA. Moreover, figuring out mechanisms between inhibitors and SrtA, reversible or irreversible, will undoubtedly be the focus of in-depth investigations in order to develop future environmental, biomedical applications.

Recent studies has also shown that SrtA can be used for protein modification, such as SrtA has been involved in site-specific modification, to find out their applications in the development of therapeutic antibodies, which allows the spatial and temporal control of proteins in vivo, as well as single molecule tracking150. For example, Lactic acid bacteria (LAB) are a diverse group of Gram-positive bacteria found in a vast array of environments including dairy products and the human gastrointestinal tract. What’s more, genomic sequencing of LAB and annotation has allowed for the identification of sortase (especially SrtA) and sortase-dependent proteins (LPXTG-anchored proteins). Nowadays, we also use SrtA for protein modification by LPXTG motif; therefore, we can use SrtA for drug design. Specifically, the use of the LPXTG motif was investigated for in vitro vaccine delivery using food grade and probiotic lactobacilli as the presentation vector for the antigen151. Intriguingly, SrtA was used for semienzymatic cyclization of disulfide-rich peptides for drug design152.

Up to now, the sortase-mediated ligation (especially SrtA) has been successfully applied to many fields such as protein lipidation152,153, PEGylation154, protein immobilization for drug-design and protein-modification155.

Declaration of interest

The authors state no conflict of interest. This work was supported by the National Natural Science Foundation of China (20776051) and the Scientific Returned Overseas Chinese Scholars, State Education Ministry (2005546).