Herbs, Spices and Medicinal Plants Used In Hispanic Traditional Medicine Can Decrease Quorum Sensing Dependent Virulence in Pseudomonas aeruginosa

International Journal of Applied Research in Natural Products Vol. 1(2), pp. 9-15, June/July 2008 Available online http://www.healthy-synergies.com ©2...
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International Journal of Applied Research in Natural Products Vol. 1(2), pp. 9-15, June/July 2008 Available online http://www.healthy-synergies.com ©2008 Healthy Synergies Publications

Original Article

Herbs, Spices and Medicinal Plants Used In Hispanic Traditional Medicine Can Decrease Quorum Sensing Dependent Virulence in Pseudomonas aeruginosa Huerta V1, Mihalik K2, Crixell SH2, and Vattem, DA2* 1

Department of Biology, Texas State University, 601 University Drive, San Marcos, TX 78666. 2 Molecular and Cellular Nutrition Laboratory, FCS Department, Texas State University, 601 University Drive, San Marcos, TX 78666

Summary: Inhibition of quorum sensing (QS), a cell density dependent regulation of bacterial virulent gene expression by autoinducers (AI) is an attractive strategy for the discovery of novel antimicrobials and overcome antibiotic resistance. Pseudomonas aeruginosa (PAO1), an opportunistic pathogen in immune compromised patients is under the regulation of the LasR-RhlR system for its QS mediated development of virulence. Natural products have recently become a promising source for deriving molecules that can potentially inhibit quorum sensing. Herbs, Spices and Medicinal Plants (HSMP) used in Hispanic cultures have been used for treating common ailments for many centuries. However, few studies have investigated its QS related antivirulent activities. Our objective was to determine the ability of 25 popular Hispanic HSMP on the expression of QS regulated virulence factors in PAO1. Effect of these extracts on QS mediated PAO1 virulent factors pyocyanin, elastase, and total proteolytic activity were quantified by standard protocols. Results indicated that several extracts reduced pyocyanin synthesis, with some extracts completely inhibiting its formation and secretion. The extracts that decreased the pyocyanin formation also decreased the expression and activity of elastase and other proteolytic enzymes important for the virulence. We observed that HSMP from Central/South American countries can inhibit QS dependent and independent virulent processes in PA-O1. Further research into the exact mechanism of action can lead to better understanding and discovery of new category of drugs and strategies for the management of PAO1 infections and antimicrobial resistance. Industrial relevance: Quorum sensing is an important process involved in bacterial survival and infections, recent research has focused on the development of therapeutic agents which prevent or manage bacterial pathogenesis by inhibiting bacterial QS. Inhibition of quorum sensing offers an alternative to antibiotic mediated bactericidal or bacteristatic approach and reduces the risk for development of resistance. Inhibition of bacterial quorum sensing by attenuating the signals can prevent the development of bacterial virulence and successful establishment of infections. Understanding the quorum sensing inhibition activity of natural bioactive phytochemicals can lead to the discovery of novel compounds and development of more effective strategies in preventing and managing microbial infections. Keywords: Quorum sensing, acylated homoserine lactones (AHL), phytochemicals, antimicrobial activity, medicinal plants from India, virulence factors and Pseudomonas aeruginosa,

Introduction For many years, control of bacterial infections by inhibiting microbial growth has been a primary approach of antimicrobial chemotherapy. An emerging problem associated with continual indiscriminant use of this therapeutic strategy is the selection of resistant bacteria with higher levels of tolerance against broad-spectrum antibiotics. Development of novel antibiotics that interfere with metabolism coupled with continued indiscriminant use of antibiotics will only lead to evolution of new resistance mechanisms and pathways by bacteria. Recently, it has been recognized that there is a need for a strategy that can block very basic mechanisms of bacterial communication that appear to control bacterial virulence factors leading to pathogenicity. Emerging research has suggested that functions including swarming, biofilm formation, secretion of virulence factors and acquiring competency play an important role in successful and recurrent establishment of bacterial infections in living systems. These processes common to several bacterial diseases are now shown to be related to a cell density-dependent regulation of gene expression mediated by the recognition of signaling molecules called autoinducers (AI). This process sometimes referred to as ‘quorum sensing’ (QS) has been shown to modulate the expression of genes involved in processes related to survival, virulence and pathogenicity (Schauder & Bassler, 2001; Lyon & Muir 2003; Raffa et al., 2005) and is mediated by small signaling molecules such as *Corresponding Author: Tel: +1-512-245-7655 Fax: +1-512-245-3829 E-mail: [email protected] Accepted 11 March 2008

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Huerta et al acetylated-homoserine lactones (AHLs). Opportunistic Gram negative bacteria including Pseudomonas sp. have been shown to employ this type of AHL-mediated quorum sensing to regulate the expression of genes related to survival, virulence and competency (Faqua et al., 1994; Pesci et al., 1999). Recently, several inhibitors of QS have been discovered from natural sources and have been produced synthetically. Some natural sources include Australian macroalga, Delisea pulchra and green alga, Ulva lactua that produce halogenated furanones and furanone-like pigments which interfere with quorum sensing (Givskov et al. 1996, Egan et al. 2002). These halogenated furanones synthesized in the laboratory have a high degree of AHL inhibition activity and were able to repress a number of genes controlled by QS. In addition, analogs of S-adenosylmethionine (SAM), such as S-adenosylcysteine and sinefungin can inhibit LuxI mediated synthesis of AHL but can interfere with eukaryotic pathways that use SAM. Recent studies have shown that interference with P. aeruginosa quorum signaling by metabolites from Penicillium species can increase susceptibility to these agents (Rasmussen et al., 2005). Unfortunately, all these compounds are highly reactive and toxic to the host which has limited their therapeutic applications (Hentzer & Givskov, 2003) and potential usage in vivo. The current quest for new antimicrobials is therefore aimed at discovering nontoxic inhibitors of QS that can be used for treatment of bacterial infections in humans (Hentzer & Givskov, 2003). Natural products especially plants used in traditional medicines are a promising source for deriving molecules that can potentially inhibit quorum sensing (Cowan 1999; Wallace 2004; Mihalik et al., 2007). These plants can offer a large and attractive repertoire for the discovery of quorum sensing inhibitors. They are of particular importance as these have been used for thousands of years for the treatment and management of diseases and may have few side-effects and toxicity issues as with many antibiotic regimens and currently known QS inhibitors. Herbs, Spices and Medicinal Plants (HSMP) used in Hispanic cultures have been used for several centuries to treat common ailments, are well known for their antimicrobial effects on a variety of human pathogens (Rojas et al., 1992; Quinlan et al., 2002). However, few studies have investigated their QS related antivirulent activities including in P. aeruginosa. The body of literature discussing HSMP from Central/South American continent as sources of QSI is very limited (Huxtable, 1983; Gomez-Beloz & Chavez, 2001; Lewis& Elvis-Lewis 2003; Mikhail et al., 2004). We believe that some of the antimicrobial properties of HSMP may be contributed by the QS inhibiting phytochemicals present in it. Our objective was to investigate the ability of 25 high quality HSMP from Central/South American continent to modulate the expression of QS dependent and QS independent virulence factors in P. aeruginosa. Materials and Methods Samples and Extraction: HSMP from Central/South America were obtained directly from Cantu's Mexican Hierberia Imports (Austin, TX) and are listed inTable 1. Twenty five different herbs spices and and medicinal plants (0.5 g) were suspended in 10 ml of distilled water (0.5 g/10 ml) and extracted on a rotary shaker for 15 min. The samples were then centrifuged (3000 rpm, 10oC, 10 min) and supernatant was sterile filtered and stored at 4oC (Mihalik et al., 2007). Minimal Inhibitory Concentrations (MIC): Determination of Minimal Inhibitory Concentrations (MIC): MIC values were determined by the broth microdilution test on Pseudomonas aeruginosa (PAO1) by modifying a previously described method (Metzler et al., 2004). The MIC value was recorded as the lowest treatment concentration at which there was no visible growth of the organism. All the quorum sensing assays and anti-virulence assays were performed at concentrations lower than the MIC values known as sub-inhibitory concentration. Microorganisms: Pseudomonas aeruginosa (ATCC 15692), Chromobacterium violaceum (ATCC 12472) were purchased from American Type Culture Collection (ATCC, Bethesda, MD) and were subcultured and maintained in LB. Chromobacterium violaceum O26 and Chromobacterium violaceum 31532 kind gifts from Dr. Bob McLean (Texas State University, San Marcos, TX) were also subcultured and maintained in LB Measurement of QS dependent virulence factors in Pseudomonas aeruginosa (PAO1): PAO1 was grown in LB supplemented to contain 1% extract for 24 hrs at 30oC, then centrifuged (8000 rpm, 4oC, 10min) the virulence factors were determined in cell free supernatants using standard protocols.. Total proteolytic activity: The protein concentrations of the cell supernatants were determined by Lowry’s (Lowry et al., 1951) method. Total proteolytic activity of the culture supernatants was estimated according a method described earlier (Wassif et al., 1999). Briefly, to 100 l of culture supernatants 900 l of 0.5% azocasein (Sigma Chemical Co., St. Louis, MO) prepared in 50 mM tris buffer containing 2 mM CaCl2. Samples were incubated at 37oC for 30 min. After this, 15% TCA (100 ml) was added to stop the reaction and centrifuged (8000 rpm, 4oC, 10min). The absorbance of the supernatants from both controls and treatments was measured at 440 nm. The % change in absorbance was then calculated from the absorbance values. Elastase Activity: Elastase activity was measured by the modifying methods described before in literature (JaffarBandjee et al., 1995; George et al., 2005). Briefly, to 200 µl of culture supernatant 1 mL of 0.5% elastin-congo red solution (in 10 mM PBS) (Sigma Chemical Co., St. Louis, MO) and incubated 37°C for 3 hours in a water bath. The samples were then vortexed and centrifuged at 1200g for 10 minutes at 10°C to remove insoluble elastin congo red. The absorbance of the supernatants from both controls and treatments was measured at 494 nm. The % change in absorbance was then calculated from the absorbance values. Pyocyanin Assay: Pyocyanin was extracted from culture supernatants and measured using the method described previously (Cox 1986 and Sanchez et al., 2002). 1.2 ml of chloroform was mixed with 3 ml culture supernatant. The chloroform layer was transferred to a glass cuvette and absorbance was measured at 690 nm. Pyoverdin Assay: Pyoverdin was measured by an assay described previously by Sanchez et al., 2002. Briefly, 0.05 ml of the aqueous phase from the pyocyanin assay was mixed with 2.45 ml of 0.05M Tris hydrochloride buffer (pH 7.4). The fluorescence was determined by excitation 400 nm and emission at 460 nm. 10

Hispanic Medicinal Plants and Pseudomonas quorum sensing

Results and Discussion Minimal Inhibitory Concentration (MIC): Determination of the MIC values of various HSMP indicated different lethality of the extracts against P. aeruginosa. Among all the extracts tested, Anthemis nobilis (AN), Chiranthodendron pentadactylon (CP), Cassia fistula (CF) and Arctostaphylos uva-ursi (AU) had the lowest MIC values which ranged between 7-52 g/ml, suggesting that they had the greatest ability to inhibit the growth of the bacteria (Figure 1). This was followed by Citrus limon (LE), Psacalium decompositum (PD), Crataegus mexicana (CM), Calluna vulgaris (CA), Tanacetum parthenium (TP), Castela texana (CT) and Artemisia vulgaris (MH) whose MIC values were between 85200 µg/ml. Tagetes lucida (TL), Senecio formosus (SU), FA, Ambrosia artemisiaefolia (AR), Cassia obovata Collad (HJ), Echium Sp (EB), Leucophyllum frutescens (V1), Satureja douglasii (YB) and Ocimum basilicum (OB) had their MIC values between 240-348 g/ml. Cordia boissieri (AC), Acalypha alopecuroidea Jacq (AJ), Prunella vulgaris (PV) and Amphipterygium adstringens (AA) were least effecting in inhibiting the growth of the bacteria and their MIC values ranged between 370-495 g/ml. Elastase activity: Further testing with HSMP at sublethal concentrations indicated that they had significant ability to inhibit QS dependent and QS independent virulence factors in P. aeruginosa. Among all the HSMP tested TP, V1, OB, CM, SU and AJ (Figure 2) were most potent in decreasing the elastase activity and did so by 142%, 137%, 120% 100%, 96% and 95% respectively. This was followed by PV, LE, AA, AC, MH, CT, AU, HJ which decreased the activity of elastase by 90%, 85%, 83%, 80%, 78%, 75%, 70% respectively (Figure 2). CF, TL, YB, CS, CP and EB decreased elastase activity by 63%, 61%, 58%, 55%, 53% and 50% respectively (Figure 2). AR (20%), FA (20%) and PD (0%) were the least effective inhibitors of elastase. Total proteolytic activity Surprisingly enough, even though, a majority of extracts were able to inhibit elastase activity, only AJ, AU, SU, PD and EB (Figure 3) had any significant effect on decreasing the total proteolytic activity in the bacterium. They decreased total proteolytic enzyme activity in Pseudomonas cultures by 95%, 41%, 36% 34%, 10%, respectively. All other extracts did not have any significant ability to inhibit total proteolytic activity, there inhibition activity was less than 5%. Table 1. List of HSMP from Central/South America used (Alphabetical) Common Name Texas/Mexican Olive Anacahuita

Scientific Names Amphipterygium adstringens Cordia boissieri

Yerba del Cancer (Foxtail Copper Leaf) Manzanilla

Acalypha alopecuroidea Jacq. Anthemis nobilis

Artemsia

Ambrosia artemisiaefolia

AR

Bearberry Leaves Mexican Heather (Cancerina)

Arctostaphylos uva-ursi

AU CA

Cuachalalate

Golden Shower Mexican Hawthorn Mexican-hat-plant (Flor de manita)

Calluna vulgaris Cassia fistula Crataegus mexicana

Abbreviation AA AC AJ AN

CF CM CP

Red Bark

Chiranthodendron pentadactylon Cinchona succirubra

Castella

Castela texana

CT

Echium (Boraginaceae)) Flor de Arnica

Echium Sp. Arnica montana

EB FA

Hojase

Cassia obovata Collad

HJ

Lemon Mugwort

Citrus limon Artemisia vulgaris

LE MH

Mexican/Cinnamon Basil

Ocimum basilicum

OB

Groundsel/Matarique Heal All (Toronguil Morado)

Psacalium decompositum Prunella vulgaris

PD PV

Suelda

Senecio formosus

SU

Feverfew ( Altamisa) Mayan Mint Marigold (Yerbaniz)

Tanacetum parthenium

TP TL

Texas Sage

Tagetes lucida Leucophyllum frutescens

Yerba Buena

Satureja douglasii

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CS

V1 YB

500 450 400 350 300 250 200 150 100 50

V1 YB

TP

TL

PV SU

H O B PD

M

H J LE

FA

C P C S C T EB

0 A A A C A J A N A R A U C A C F C M

Minimal Inhibitory Concentration of Phenolics (µ g/ml)

Huerta et al

180 160 140 120 100 80 60 40 20

V1 YB

TL TP

SU

PD PV

H O B

M

H J LE

FA

C P C S C T EB

0

A A A C A J A N A R A U C A C F C M

Inhibition of Elastase Activity (%)

Figure 1. Minimal inhibitory concentrations of different HSMP from Central/South America against P. aeruginosa

Figure 2. Effect of sub-lethal concentrations of HSMP from Central/South America on quorum sensing dependent elastase activity in P. aeruginosa.

Inhibition of Proteolytic Activity (%)

120

100

80

60

40

20

V1 YB

TL TP

PV SU

PD

H O B

M

HJ LE

FA

C P C S C T EB

A A A C A J A N A R A U C A C F C M

0

Figure 3. Effect of sub-lethal concentrations of HSMP from Central/South America on quorum sensing dependent total proteolytic enzyme acticity in P. aeruginosa.

Pyocyanin formation: When we measured the effect of the HSMP to alter the pyocyanin flux, we found that among all the extracts tested OB, PV and YB were most effective in reducing the formation of pyocyanin. They decreased the formation of this pigment by 165%, 132% and 112% respectively (Figure 4). CP, LE, FA, TP, MH, SU, CT and CF 12

Hispanic Medicinal Plants and Pseudomonas quorum sensing were also effective reducing the pyocyanin formation by PAOI, they did so by 78%, 63%, 60%, 50%, 47%, 32%, 28% and 25% respectively (Figure 4). All the other remaining extracts were not effective in decreasing the formation of pyocyanin pigment (Figure 4). Pyoverdin formation: When we assessed the effect of HSMP pyoverdin formation in the bacterium, our results indicated that, only AJ, HJ, EB, TL, SU, LE, EB, PD and MH (Figure 5) decreased the formation of pyoverdin by 90%, 56%, 45%, 43%, 43%, 41%, 30% and 25% respectively. All other extracts had no effect on decreasing the flux of pyoverdin formation in PAO1.

Change in Pyocyanin Flux (%)

50

0

-50

-100

-150

-200

Figure 4. Effect of sub-lethal concentrations of HSMP from Central/South America on formation of quorum sensing dependent pyocyanin pigment formation in P. aeruginosa.

Change in Pyoverdin Flux (%)

20 0 -20 -40 -60 -80 -100 -120

Figure 5. Effect of sub-lethal concentrations of HSMP from Central/South America on formation of pyoverdin pigment formation in P. aeruginosa.

Discussion Elastase along with other proteolytic enzymes is one of the most potent QS regulated virulence factors responsible for penetrating into the host tissues and for establishment of infections. In addition to virulence, proteolytic enzymes are required in bacterial metabolism for its growth and reproduction, and therefore some of the proteolytic enzyme genes may have house keeping functions. Some of these genes may not be under complete regulation of QS for expression (Cox 1986; Jaffar-Bandjee et al., 1995; Wassif et al., 1999; Sanchez et al., 2002; George et al., 2005) The differential effect of the HSMP on elastase, total proteolytic activity and pyocyanin (QS dependent antimicrobial pigment) synthesis might provide us insights into their possible mechanisms of action in modulating the virulence factors regulated by quorum sensing in P. aeruginosa. In P. aeruginosa the QS cascade is mediated AHL’s that are synthesized by the products of the lasI/lasR and rhlI/rhlR genes (Pesci & Iglewiski 1997; Pesci et al., 1997). The products of these genes monitor the concentration of AHL’s and serve as transcriptional activators of a variety of products important in P. aeruginosa virulence and pathogenesis. Some important virulent factors regulated this way are 13

Huerta et al elastase, alkaline protease, hemolysin, pyocyanin, and rhamnolipids. These two AHL systems in P. aeruginosa- N-(3oxododecanoyl) homoserine lactone (3O–C12–HSL), (las system) and N-butyryl homoserine lactone (C4–HSL) (Rhamnolipid rhl system) mediate QS in a hierarchical fashion (Pesci & Iglewiski 1997; Pesci et al., 1997).. Where LasR-C12-HSL activates the transcription of rhlR which then induces the transcription of rhlI to synthesize C4-HSL (Pesci & Iglewiski 1997; Pesci et al., 1997). The differential effects of the extracts on modulating different virulence factors in Pseudomonas might suggest that the extracts are acting at different hierarchical positions in the overall QS cascade. This might suggest that these medicinal plants actually contain not a single compound but a group (profile) of QS modulating compounds which give them a broad spectrum capability to modulate the QS cascade at multiple levels mediated by different AHL molecules, which in P. aeruginosa are 3O–C12–HSL and C4–HSL. The phytochemicals from these medicinal plants can also directly inhibit the Pseudomonas Quinolone Signal (PQS) which is mediated by 2heptyl-3-hydroxy-4(1H)-quinolone 4-quinolone which induces expression of lasB (coding for elastase), rhlI and rhlR (Pesci et al., 1999). It is possible that in addition to their well documented antimicrobial activity the phytochemicals could affect microbial virulence by modulating functions that are unrelated to QS. It is well known that P. aeruginosa has a strong iron requirement, to fulfill its iron need the bacterium secretes siderophores, mainly pyoverdin and to a lesser extent pyochelin (Bjorn et al., 1979). These siderophores function as powerful iron chelators, solubilizing and transporting iron through the bacterial membranes via specific receptor proteins at the level of the outer membrane, eventhough this is an essential virulence factor (Bjorn et al., 1979), it is not under QS regulation. Our results indicated that some of the extracts we tested were also capable of decreasing the formation of pyoverdin significantly, which may additionally contribute to discouraging the growth and survival of the bacteria in a QS independent manner. Conclusion Our results for the first time have shown that HSMP from Central/South America can reduce QS dependent and independent virulence factors. A combination of these HSMP can be used to develop therapeutic strategies for managing P. aeruginosa infections. 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