Application of Chinese Herbal Medicine in the lnhibition of Salmonella and its virulence

Xinnan Meng, Wangmin Hu, Yulei Yang, Shanshan Jia, Ying Wen, Mei Zhang

1 School of Chinese Materia Medica, Beijing University of Chinese Medicine, Fangshan District, Beijing 102488, China.

Abstract Salmonellosis poses a serious challenge to public health and poultry industries worldwide.With the emergence of multi-drug resistant Salmonella, the burden caused by Salmonella infections has increased steadily.Enhancing the understanding of the Salmonella infection process may aid the development of new treatments and effective preventive strategies against the disease.This review outlined the Salmonella infection process and introduced the pathogenesis of Salmonella and its virulence factors, such as Type III Secretion Systems effectors.The author summarized the recent advances in the application of Chinese herbal medicine (CHM) in the inhibition of drug-resistant Salmonella and its virulence and discussed the possible mechanisms of CHM inhibiting Salmonella growth and affecting its virulence.CHM has been shown to be effective in inhibiting bacterial growth and alter the virulence properties of pathogens.It is shown that CHM is much less likely to produce drug resistance to bacteria comparing to conventional antibiotics.One reason for this is that CHM may enhance host immunity to control the bacterial infection indirectly whereas conventional antibiotics directly kill the bacteria or inhibit their growth.At present, the percentage of antibiotic-resistant Salmonella isolates is increasing globally.CHM shows great potential for the development of new medicines.It may be an alternative solution to preventing and treating Salmonellosis caused by antibiotic-resistant strains.Further studies on the molecular mechanisms of the active ingredients of CHM may help the future development of novel antibacterial drug designs to contain bacterial infections.

Keywords:Salmonellosis, Drug-resistant Salmonella, Chinese herbal medicine, Virulence, Antibacterial

Background

Salmonellosis remains a serious challenge to public health worldwide.Livestock, poultry, meat products, and eggs are the main causative carriers of Salmonellosis.The causative agent,Salmonella, is a Gram-negative bacterium that is usually pathogenic in human and warm-blooded animals [1].Salmonellaspecies have more than 2500 serotypes around the world [2].VariousSalmonellastrains cause a wide variety of illnesses including food poisoning, gastroenteritis, typhoid fever, and paratyphoid fever.Salmonellainfection has high morbidity and mortality worldwide and costs billions of dollars in global economic losses each year [3].According to statistics, food poisoning caused bySalmonellahas repeatedly ranked first, about 70-80% of all foodborne outbreaks in humans.Furthermore,Salmonellaalso poses huge challenges for the food industry, such as the poultry and egg production factories [4].With the emergence and spread of multi-drug resistantSalmonella, the burden caused bySalmonellainfections has increased in recent years [5].Therefore, it is essential to improve our understanding of theSalmonellainfection process and to develop novel preventive measures as well as new drugs that can effectively treat Salmonellosis.Conventional antibiotics currently used to treat salmonellosis have become increasingly less effective due to drug-resistance worldwide.We discuss here that the active ingredients of Chinese herbal medicine (CHM) and natural medicine are important sources for the development of new antibiotics.

CHM has been very popular in many parts of the world [6].The molecular mechanisms of how most CHM functions remain elusive.Some of them have been found to possess antibacterial activities and others may play roles in regulating the host immune system.Here, we outlined theSalmonellainfection process and summarized recent applications of CHM in the inhibition of drug-resistantSalmonellaand its virulence.

Pathogenesis of Salmonella

Salmonellaspecies are highly adapted to the environment which they survive and infect.The bacteria are usually introduced into the host through oral ingestion of contaminated food, such as eggs and raw meat.The bacteria have incredible ability to survive the acidic environment in the stomachs of humans and animals.Once they reach the small intestine, they invade and enter the intestinal epithelial cells and macrophages.Incredibly,Salmonellacan survive inside both the epithelial and macrophages leading to gastroenteritis [7].Once they pass through the intestinal epithelial layers, they may spread to the mesenteric lymph nodes and the spleen, causing severe systematic infections.Salmonellahave evolved complex machinery to enter and colonize host cells [8].These virulence properties ofSalmonellamainly come fromSalmonellaPathogenicity Islands (SPIs), as well as the virulence plasmid pSLT, adhesin, flagella and so on [9].

Salmonella Pathogenicity Islands and Type III Secretion Systems

SPIs are clusters of genes located on theSalmonellachromosome, but non-pathogenicE.coliwhose genome is similar toSalmonelladoes not have SPIs [10].Numerous SPIs have been identified and at least five of them play important roles in virulence [11].In particular, SPI-1 and SPI-2 have been studies in-depth among all the SPIs.SPI-1 is responsible for bacterial invasion, while SPI-2 is required for the survival and replication ofSalmonellainside the host cells.SPI-1 and SPI-2 each encodes one Type III Secretion System (T3SS), T3SS-1 and T3SS-2, that enable the bacteria to transport numerous effector proteins into host cells to facilitate the bacterial invasion and replication, respectively [10].These effectors regulate host cell functions, such as cytoskeleton rearrangement, signal transduction, and cytokine production [12].The combined action of these effectors enablesSalmonellato invade non-phagocytic cells and survive and replicate inside the formed “Salmonella-containing vacuoles (SCVs)” [13].Intracellular SCV provides a unique space for bacteria to replicate and evade host immune responses during infection [14].

To invade a non-phagocytic cell, T3SS-1 assembles a complex pore in the host cell membrane to inject bacterial effectors into the cell [15], including SipA, SipB, SipC, SopA, SopB, SopD, SopE, SopE2, SptP, etc.These effectors are able to induce changes in the cytoskeleton of the non-phagocytic cells, forming ruffles that engulfSalmonellainto the host cells [16-18].In addition to mediating bacterial invasion, SPI-1 effectors are known to activate the immune response of the host [19].The proinflammatory response is thought to promoteSalmonellasurvival and provide a more favorable microenvironment in the guts [19, 20].T3SS-1 effectors also inhibit apoptosis in invaded host cells, providing a longer time forSalmonellato replicate in the cells [21].

SPI-2 expression is activated after internalization of the bacteria into the host cell [22].T3SS-2 is encoded by SPI-2 and is used to inject SPI-2 effectors from the interior of SCV into host cells.SPI-2 effectors mainly include SifA, SpIC, SspH2, SseF, SseG, SseI, SseJ, SpvB, SpvC, etc.[23] They play an essential role in the maturation of SCVs and the replication ofSalmonella.In SCVs, the SPI-2 effectors prevent the formation of phagolysosomes, regulate the transportation and maturation of SCV, and promote the survival and replication ofSalmonella[24].Most genes encoding effectors secreted via T3SS-2 are located within the SPI-2 region, but some SPI-2 effectors reside on the chromosome outside the SPI-2 region [25].Although the expression of SPI-1 and SPI-2 are activated at different stages of infection, they are not completely independent of each other.The mutation of T3SS-2 leads to a significant down-regulation of the expression of some genes located in SPI-1, thereby reducing the invasion ofSalmonellainto host cells [26].

Regulation of Salmonella Pathogenicity Islands

Salmonellacan perceive the various environments they are in (e.g.pH value or iron-limited), and perform a variety of functions by regulating the expression of two SPIs.During the infection process, the interaction betweenSalmonellaand the host is complicated.They need to precisely control the expression of virulence factors (SPIs and others) so that they can maximize their virulence effects at the appropriate time [27].

HilA is the master regulator for the SPI-1 gene circuit, which is a transcription factor that contains a DNA-binding motif belonging to the OmpR/ToxR family [28].HilA can directly bind to the promoter regions of theprg/organdinv/spaoperons and activate their expression.Many regulators or environmental signals indirectly affect SPI-1 gene expression by mediating HilA [29].The expression ofhilAis regulated by at least three other transcriptional activators: HilC, HilD, and RtsA [30, 31].These activators can independently bind to the promoter region ofhilA, but none of the three can work alone to sufficiently activate SPI-1 eitherin vivoorin vitro.Deletion of these activators leads to down-regulation ofhilAexpression.HilD plays a more predominant role among the three, and the two-component regulatory systems, SirA/BarA and OmpR/EnvZ, function through HilD [32].HilC, HilD and RtsA form a feedforward loop that controls the expression ofhilAgene.The expression of HilD activated the expression of HilC and RtsA.As a result, the three factors function coordinately to activate the expression of thehilAgene, which in turn activates the gene expression of the SPI-1 cluster.

Inhibition of Salmonella by Chinese herbal medicine

Chinese herbal medicine inhibits Salmonella growth

CHM can affect the growth of many serotypes ofSalmonella, includingSalmonellaTyphimurium (S.Typhimurium),SalmonellaPullorum (S.Pullorum),SalmonellaCholeraesuis (S.Choleraesuis), etc.S.Typhimurium causes acute gastroenteritis in humans and typhoid fever in mice.More importantly,S.Typhimurium is an important Gram-negative bacterial model organism [33].Huangqin (Radix Scutellariae) is the dried root ofScutellaria baicalensisGeorgi, and its main active ingredients are flavonoids [34].Radix Scutellariaeextract has been shown to have an inhibitory effect on the growth ofS.Typhimurium [35].Gulluceet al.found that hexane extracts of sixAstragalusspecies inhibitedS.Typhimurium without genotoxicity and were active ingredients for potential new phytotherapy [36].Zhao investigated the inhibition ofS.Typhimuriumin vitroby sixteen CHMs.Most of these CHMs have shown a certain inhibitory effect onS.Typhimurium.Among them, four CHMs have better antibacterial effectsin vitro: Hezi (Chebulae Fructus), Dahuang (Radix et Rhizoma Rhei), Gaoliangjiang (Rhizoma Alpiniae Officinarum) and Baitouweng (Radix Pulsatillae) [37].Also, a herbal formula has been reported to protect mice infected withS.Typhimurium more effectively by reducing oxidative stress, up-regulating the expression of antioxidant enzymes and reducing apoptosis compared to gentamicin [38].The formula consists of 100 g each of seven herbs: Baitouweng (Radix Pulsatillae), Huangqi (Radix Astragali), Huanglian (Rhizoma Coptidis), Qinpi (CortexFraxini), Huangbai (Cortex PhellodendriChinensis), Yinyanghuo (Folium Epimedii), Shanzha (Fructus Crataegi).Furthermore, Xiaobo (Berberis Poiretii)is a plant of theBerberisgenus in theBerberidaceaefamily.Berberine (1 in Scheme 1) is an active ingredient ofBerberis poiretii[39].Baoet al.found that berberine has an antibacterial effect onS.Typhimurium [40].Berberine can rupture theSalmonellacells by damaging the bacterial membrane.

Chicken diarrhea is one of the most serious bacterial diseases that harm the chicken industry, andS.Pullorum is the most common pathogen causing chicken diarrhea [41].Yanget al.performed bacterial inhibition experiments against ten different herbs via three methods, and the results showed that the Wuweizi (Fructus Schisandrae Chinensis) and the Wumei (Frutus Mume) have stronger inhibition effects [42].Wanget al.screened out nine CHMs that inhibitS.Pullorum through antibacterial experiments, and developed "Bai Li Kang" preparations based on these medicines [43]: Shiliupi (Pericarpium Granati), Diyu (Radix Sanguisorbae),Chebulae Fructus, Zexie (Rhizoma Alismatis),Radix Scutellariae, Jinyinhua (Flos Lonicerae Japonicae), Huangbai (Cortex PhellodendriChinensis), Cangzhu (RhizomaAtractylodis) and Chenpi (Pericarpium Citri Reticulatae).The protection and cure rates ofS.Pullorum infection treated with "Bai Li Kang" were 88-90% and 75-85%, respectively.Daiet al.found that extracts of Huzhang (Radix et Rhizoma Polygoni Cuspidati), Xiakucao (Spica Prunellae) andChebulae Fructushave strongin vitroantibacterial activity againstS.Pullorum [44].WhenChebulae Fructusis used in combination withRadix et Rhizoma Polygoni Cuspidati, Zihuadiding (Herba Violae), Heshouwu (Radix Polygoni Multiflori) andSpica Prunellae, the antibacterial ability is enhanced by the synergistic effect.WhenRadix Polygoni Multiflori was used with Huzhang (Radix et Rhizoma Polygoni Cuspidati),Herba ViolaeandSpica Prunellae, its antibacterial ability was antagonized.This may be due to the chemical reactions that occurred during the decoction of the various drug components.

S.Choleraesuiscauses serious economic losses in the swine industry [45].Xieet al.investigated thein vitroantibacterial effect of seventeen Tibetan medicines againstS.Choleraesuis, and found that Yuganzi(Fructus Phyllanthi)andFructusChebulaehave strong inhibitory effects [46].Dinget al.found that a herbal decoction consisting of seven herbs [Radix Astragali,Radix Scutellariae, Maiya (Fructus Germinatus Hordei), Gancao (Radix et Rhizoma Glycyrrhizae), Dangshen (Radix et Rhizoma Salviae Miltiorrhizae), Fuling (Poria) and Baizhu (Rhizoma Atractylodis Macrocephalae)] has some inhibitory effect onS.Choleraesuis [47].Chenet al.used two classical methods to investigate thein vitroantibacterial effects and minimum inhibitory concentrations (MICs) of twenty-four single herbal medicines, as well as twelve CHMs formulae againstS.Choleraesuis, respectively [48].The results showed that twelve single herbs such as Jinyinhua (Flos Lonicerae Japonicae),Chebulae Fructus,Radix Astragali, and nine formulae such as “San Huang Tang” formula (mainly containingRhizoma Coptidis,Radix ScutellariaeandCortex Phellodendri Chinensis) had significant inhibitory effects onS.Choleraesuis.Yuanet al.carried out the experiment to investigatein vitroinhibition ofSalmonella(bothS.Pullorum andS.Choleraesuis)by the combination of berberine hydrochloride and mucilage sulfate [49].The combined drugs provided synergistic inhibition against most of the tested strains.

Effects of Chinese herbal medicine on multidrugresistant Salmonella

The emergence and spread of multidrug-resistantSalmonellapose a serious challenge to the prevention and treatment ofSalmonellainfections.Antibiotics of fluoroquinolones (e.g.ciprofloxacin), third-generation cephalosporins (e.g.ceftriaxone), and macrolides (e.g.azithromycin) are commonly used for the clinical treatment of severeSalmonellainfections [50].Currently, the isolation rate of drug-resistantSalmonellais increasing globally, especially for ciprofloxacin-resistant strains [51].It is necessary to develop new drugs that are effective againstSalmonellainfections, especially Salmonellosis caused by drugresistant strains.

Many components of CHM have been demonstrated to have antibacterial activity, which provides an important source for the development of new antibiotics.The CHM formula “Lianhuang” has been reported to inhibit drug-resistantSalmonella[52], which containsRadix ScutellariaeandRhizoma Coptidis.The Lianhuang formula shows more than 80% inhibition rate against kanamycin- and gentamicinresistantSalmonellastrains.Rhizoma Alpiniae Officinarumis a medicinal plant widely used in China, India, and many Southeast Asian countries.The main bioactive substance ofRhizoma Alpiniae Officinarumis 1'-acetoxychavicol acetate (2 in Scheme 1).It effectively eliminated the antibiotic-resistant plasmid fromSalmonellaTyphi (75%), thereby greatly reduced the MIC for multiple antibiotics [53].Niuet al.reported that Yuxingcao (Herba Houttuyniae) extract reduced the number of enrofloxacin- and gentamicinresistantSalmonella[54].Wubeizi (Galla Chinensis) andRadix Scutellariaecan also effectively eliminate the drug-resistant plasmids of drug-resistantSalmonella[55].These examples demonstrated that CHM has great potential to treat Salmonellosis, especially those caused by drug-resistant strains.Understanding the mechanisms in which these CHMs function will greatly advance the novel drug development to treat drug-resistantSalmonellainfections.

Scheme 1.Several typical herbal ingredients with antibacterial activity.

Mechanisms of Chinese herbal medicine inhibiting Salmonella growth and affecting its virulence

Although functions of the CHM in a somewhat mysterious way, increasing evidence indicates that they work to inhibit bacteria growth as summarized above.Alternatively, they alterSalmonellavirulence by affecting host immune responses andSalmonellaT3SS effectors, or transcriptional regulators for pathogenicity.

Chinese herbal medicine affects Salmonella T3SS effectors

As shown in Figure 1,Salmonellainfection can be separated into two stages: invasion and replication, which are largely attributed by genes located in SPI-1 and SPI-2.SipA, SipB and SipC are well known T3SS-1 effectors encoded by SPI-1.Figure 1 summarizes CHM that has inhibitory effects onSalmonellaeffectors.

Figure 1.CHM interacts with SalmonellaT3SS-1 effectors.

Figure 2.Part of SPI-1 regulatory network and T3SS-1 regulators interact with CHM.

Bailixiang (Thymus serpyllum L.) is a member of theThymusgenus of aromatic perennial evergreen herbs in the mintLamiaceaefamily [56].It has a variety of potent biological activities, such as antibacterial, antifungal, antiviral, antioxidant, and antispasmodic activities [57].Its active ingredients include terpenoids, flavonoids, glycosides, phenolic acids and so on.Thymol (chemical name: 2-isopropyl-5-methylphenol, 3 in Scheme 1) is one of the main active ingredients of thyme.Thymol interacts directly withSalmonellaSipA, resulting in a conformational change of SipA and subsequent degradation by Lon protease [58].Cheqianzi (Semen Plantaginis) is the mature dry seeds ofPlantago asiaticaL.orPlantago depressaWilld.Semen Plantaginiscontains a variety of chemical components, including iridoids, phenylglycol glycosides, flavonoids, polysaccharides, triterpenes, steroids and volatile oils [59].The extract of Semen Plantaginissignificantly inhibits the invasion ofS.Typhimurium into epithelial cells at a concentration that had no obvious adverse effect on the bacterial growth.Western blot experiments confirmed that the main active component ofSemen Plantaginis, paeroside (4 in Scheme 1), reduced the expression ofSalmonellaSipC.As a result, the ability ofS.Typhimurium to invade epithelial cells was greatly weakened, consistent with the reduced virulence in a mouse infection model [60].Rougui (Cortex Cinnamomi)is from an evergreen plant of theCinnamomumgenus in theLauraceaefamily.It has antibacterial, anti-inflammatory, anti-tumor, antioxidant and anti-gastric ulcer activities [61].Cinnamon oil is the aromatic oil distilled from branches and leaves ofCinnamomoum cassiaaPresl.Western blot experiments showed that Cinnamon oil reduced the secretion ofSalmonellaSipA and SipB leading to reduced invasion into epithelial cells without adversely affecting the growth ofSalmonellain growth media [62].Boluohui [Macleaya Cordata(Willd.) R.Br.] is a perennial plant of theMacleayagenus in the Papaveraceae family.It contains alkaloids such assanguinarine chloride (5 in Scheme 1), chelerythrine, protopine, allocryptopine, bocconine, dihydrosanguinarine, etc.[63] Sanguinarine chloride, an active ingredient ofMacleaya Cordata(Willd.) R.Br., decreased the expression ofSalmonellaSipA and SipB resulting in reduced invasion ofS.Typhimurium without affecting its growth [64].Carvacrol (6 in Scheme 1) is an aromatic phenolic monoterpene, which is commonly found in the volatile oil of aromatic plants such as thyme and oregano.It can be used in the formulation of fragrances, bactericidal and bacteriostatic agents [65].Giovagnoniet al.demonstrated that carvacrol significantly downregulated the major virulence genes ofSalmonella(prgH, invA, sipA, sipC, sipD, sopB and sopE2) by RT-PCR, thereby reducing the pathogenicity ofSalmonellaand acting as a host cell protector [66].

Chinese Herbal medicine affects transcriptional regulators in Salmonella

HilA, HilC and HilD are main transcriptional regulators modulating SPI-1 genes (Figure 2).Thymol, sanguinarine chloride and cinnamon oil mentioned above had been found to inhibit the expression of regulators.Thymol and sanguinarine interact with HilA to down-regulate T3SS-1 effectors [64, 67].Cinnamon oil also reduced the expression of HilA [62].Mudanpi (Cortex Moutan) is the dried root bark of the buttercup plant Paeonia suffruticosa Andr.Paeonol (7 in Scheme 1) is the main active ingredient ofCortex Moutan, which has various pharmacological effects such as antibacterial and anti-inflammatory, anti-arrhythmia, anti-allergy, etc.[68] Paeonol can suppress the expression of SipA by decreasing the transcriptional level ofhilA.It can also significantly reduce the level of cell damage mediated byS.Typhimurium infection [69].In addition, Sulfur-containing spices were found to affect levels ofSalmonellaregulators.For instance, benzyl isothiocyanate (BITC), diallyl disulfides (DADS) and methylfurfuryl disulfides (MFDS) were able to reduce HilA, HilC and HilD expressions in a concentration-dependent manner.Among them, the inhibitory effect of DADS is most dramatic.DADS is the main antibacterial component in the fresh extract of Dasuan (Allium Sativum L.) [70].BITC is a secondary metabolite in Cruciferae and Cauliflower family.BITC from Baijiezi (Semen Sinapis) significantly slowed the growth of both Gram-negative and Gram-positive bacteria [71].Sulfur-containing spices affected the cell morphology ofSalmonella, and they may have the potential to control food-borne diseases caused bySalmonella[72].

Chinese herbal medicine improves host immunity

It is known that CHM is much less likely to produce drug resistance to bacteria comparing to conventional antibiotics.One reason for this is that CHM may enhance host immunity to control the bacterial infection indirectly whereas conventional antibiotics directly kill the bacteria or inhibit their growth.For example, many CHMs mentioned above have the function of improving host immunity.Thymol significantly reduces the presence of the main inflammatory factors such as interleukin 6 (IL-6), interleukin-1β (IL-1β), tumor necrosis factor-α (TNFα) and interferon-γ (IFN-γ) in the cecum tissue of infected mice [64].Licorice polysaccharide, as an important effective ingredient inRadix et Rhizoma Glycyrrhizae, can enhance the phagocytosis of macrophages, promoted the secretion of IL-1β, IL-6, IL-12 and TNF-α, and blocked the secretion of IL-4, IL-10 and TGF-β [73, 74].Houttuyniacin is the main active ingredient inHerba HouttuyniaeHouttuyniacin improves the immune function of splenectomized animals by enhancing the function of lymphocytes and regulating the T-cell population [75].In addition, Yuganzi (Fructus Phyllanthi) is also a CHM that can inhibit the virulence ofSalmonella.Fructus Phyllanthican modulate the host immune system in various ways, such as promoting non-specific cellular immunity, specific cellular immunity and humoral immunity to enhance human immune function [76].

IL-6 is produced promptly and transiently in response to infections and tissue injuries.It contributes to host defense through the stimulation of acute-phase response, hematopoiesis, and immune reactions [77].IL-1β, a key mediator of the inflammatory response, is crucial for host-defense against infection and injury.IL-1β and TNF-α activate transcriptional factors to produce IL-6 [78].TNF-α, a major pro-inflammatory cytokine, has been shown to play a pivotal role in orchestrating the cytokine cascades in many inflammatory diseases and is a “master regulator” of inflammatory cytokine production [79].IFN-γ is a cytokine with important roles in tissue homeostasis, immune and inflammatory responses and tumor immunosurveillance [80].Thus, IL-6, IL-1β, TNF-α, and IFN-γ are all crucial cytokines in immune and inflammatory responses.Therefore, thymol may exert its function by influencing the host inflammatory responses to contain the infections.

Summary and Prospect

Salmonellaspecies cause serious illness in both humans and animals.Progress has been made to understand its virulence mechanisms and in developing novel drugs to treat infections.Recent advances have shown that many CHMs have the ability to inhibitSalmonellaand its virulence.CHM is not only cheap, it is much less likely to produce drug-resistance in bacteria.CHM may also boost the host immunity to combat bacterial infections.Understanding the molecular mechanisms of how CHM functions may lead to novel drug designs to prevent and treat drugresistant bacterial infections.