Current Pharmaceutical Design, 2002, 8, 99-110 1

1381-6128/02 $35.00+.00 © 2002 Bentham Science Publishers Ltd.

Antimicrobial Compounds of Low Molecular Mass are ConstitutivelyPresent in Insects: Characterisation of ββββ-Alanyl-Tyrosine

K. Meylears*1, A. Cerstiaens1, E. Vierstraete1, G. Baggerman1, C.W.Michiels2, A. De Loof1, L. Schoofs1.

1Laboratory of Developmental Physiology and Molecular Biology, Zoological Institute,Katholieke universiteit Leuven, Naamsestraat 59, B-3000 Leuven, Belgium.

2Laboratory of Food Microbiology, Katholieke universiteit Leuven, Kard. Mercierlaan 92, B-3001 Heverlee, Belgium.

Abstract: The number of bacterial and fungal strains that have developed resistanceagainst the classical antibiotics continues to grow. The intensified search for newantibiotic lead compounds has resulted in the discovery of numerous endogenouspeptides with antimicrobial properties in plants, bacteria and animals. Their possibleapplications as anti-infective agents are often limited by their size, in reference to production costs andsusceptibility to proteases. In this article, we report recent isolations of antimicrobial compounds from insects,with molecular masses less than 1 kDa. Experimental approaches are discussed and the first data on theantimicrobial properties of β-alanyl-tyrosine (252 Da), one of such low molecular mass compounds isolatedfrom the fleshfly Neobellieria bullata, are presented. We also offer evidence for the constitutive presence ofantimicrobial compounds in insects of different orders, in addition to the previously identified inducibleantimicrobial peptides.

INTRODUCTION

Miracles Don’t Last Forever. The Need for NewAntimicrobial Agents

Reports of infectious micro-organisms having acquiredresistance against antibiotics for which they were previouslysusceptible, have become more and more frequent. Theindiscriminate and excessive use of antimicrobial agents inhuman and animal disease treatment and as growthpromoters in animal feed, has created a continuous selectivepressure, promoting the development of resistant strains.Moreover, the accumulation of spontaneous, resistant-rendering mutations over time within a bacterial populationand the horizontal spreading of these mutations across majortaxonomic barriers, dangerously accelerates the developmentof strains resistant to a wide range of conventionalantibiotics. Confounding were the reports, made for the firsttime in a number of Japanese hospitals in the late 1950s,revealing the accumulation of several bacterial genes codingfor antibiotic resistance on so-called R-plasmids. Theseplasmids seemed to be freely exchangeable within a bacterialpopulation, thus rapidly creating a subpopulation ofmultiple resistant bacteria. Where barely 30 years ago wethought that we had finally won the battle against infectiousdiseases,

*Address correspondence to this author at the Laboratory ofDevelopmental Physiology and Molecular Biology, Zoological Institute,Katholieke universiteit Leuven, Naamsestraat 59, B-3000 Leuven,Belgium; Tel: +32 16 324260; Fax: +32 16 323902; E-mail:Karen.Meylaers@bio.kuleuven.ac.be

today bacteria are steadily forcing us into the defensiveagain. The World Health Organization (WHO, Geneva)estimates today that 1500 people die each hour from aninfectious disease, half of these being children under fiveyears of age [www.phrma.org].

An element that is partly responsible for the current lackof effective antibiotics, is the fact that pharmaceuticalcompanies reduced or even stopped the search for newantibiotic agents during the 1960s, when the traditionalscreening of soil and marine mico-organisms provided nonew results. Instead, efforts were directed to the chemicalmodification of existing antibiotics, to generate second andthird generation compounds with enhanced properties. Themost recent true novel antibiotic class, the fluoroquinones,were introduced already 30 years ago. Pharmaceuticalcompanies have meanwhile reached the limit of possiblechemical modification of existing antibiotics, while theyhave at each new manipulation eventually been bested by abacterial countermeasure. As such, there were no newantibiotics available against the new or re-emerginginfectious diseases caused by resistant strains.

However, intensified research has meanwhile resulted intwo promising new candidates for novel antibiotic classes:The oligosaccharide agents [1 ] and cationic peptideantimicrobials [2] . Especially this last group of bioactivepeptide compounds, derived mainly from animals, has beenintensively studied during the last decade [2,3,4], but manyquestions concerning cytotoxicity, stability and productioncosts remain.

HO

OH

HOS

OO

HO

O

O

O

OH

NH2

NHNH

NH

O

NH2

CHO

OHa b

O

H2N NH

OH

COOH

cFig. (1). Structural formula of antimicrobial compounds with low molecular mass, isolated from insects.a). N-β-alanyl-5-S-glutathionyl-3.4-dihydroxyphenylalanineb). p-hydroxycinnamaldehyde.c). β-alanyl-L-tyrosine.

Insects, a Valuable Source of Antimicrobial Compounds

The search for lead compounds from natural sources hasresulted in the isolation of numerous peptides andpolypeptides with antimicrobial properties from plants [5],bacteria [6 ] , and mostly animals. Almost 50% of thereported antimicrobial substances were identified ininvertebrates and predominantly in insects. The firstantibacterial peptides to be isolated from an insect in 1981were two cecropins [7]. Insects have since then proven to bean important source of antimicrobial peptides. [reviews 8,9].Being a large group of different species (80% of the faunapresently known to man), with representatives in almost allecological niches, insects are the most comprehensive groupamong the animals. The evolutionary success of insects islargely due to their potent innate immune capabilities.Except for the discrimination between antibacterial andantifungal immune responses reported in Drosophila [10],insects do not display any immune specificity orimmunological memory, but their response to invading cellsis immediate and powerful. The first, cellular line of defenceare the circulating haemocytes, the insect blood cells, thatwill isolate the invading cells and initiate fagocytosis[review 11]. In the mean time, a powerful systemic responseis activated, resulting in the synthesis and release of several

antimicrobial peptides into the haemolymph. These peptideswill then work synergistically to destroy the invading cells,by destructing the bacterial cytoplasmic membrane orinhibiting bacterial housekeeping enzymes. Althoughsimilarities are found throughout orders or genera of insects,each species also seems to have developed particularcompounds in response to its specific environmentalconditions.

The Advantages of Antimicrobial Compounds with LowMolecular Mass

Although several interesting antimicrobial compoundshave meanwhile reached the stage of clinical trials [12,13,www.phrma.org], possible applications are often limited totopical treatments. Being mostly peptidergic in nature, thesenatural antimicrobial compounds are susceptible toproteolytic breakdown by proteases, encountered duringinternal digestion in the gastro-intestinal tract or transport inthe bloodstream, and consequently have too limited half-lifeupon systemic application. Moreover, most antimicrobialpeptides have a high content of Lys and Arg, which are themain target of trypsine-like proteases. As such, it isimportant to identify smaller, perhaps even non-peptidergic

substances with antimicrobial activity, as these can beexpected to be more stable. Being small, they are also morelikely to be suitable for production by chemical synthesis,thus bypassing the need for more expensive and labourintensive recombinant expression systems.

Such small (less then 1kDa), possibly non-peptidergiccompounds with antimicrobial activity do indeed exist ininsects. The first report was made by Leem and co-workersin 1996. From immunised adult fleshfly Sarcophagaperegrina , they isolated the inducible antibacterialcompound N-β-alanyl-5-S-glutathionyl-3,4-dihydroxy-phenylalanine (5-S-GAD, Fig. 1a) with a molecular mass of573 Da [14] . Two years later in 1998, the same teamreported the isolation of the antibacterial and antifungalcompound p-hydroxycinnamaldehyde (148 Da, Fig. 1b)from larvae of the saw fly Acantholyda parki [15]. The lattercompound was also present when the larvae where notpreviously immune challenged. In the same year, two smallsubstances were isolated from larvae of the grey fleshflyNeobellieria bullata: β-alanyl-tyrosine (252 Da, Fig. 1c) and3-hydroxykynurenine (224 Da), which exhibited paralyticactivity when injected into adult insects [16]. One of these,β-alanyl-tyrosine, also displays antimicrobial properties, thatwill be presented in this article.

EXPERIMENTAL APPROACHES: THE ISOLATION

Extraction Procedures

When attempting to isolate antimicrobial peptides,researchers often start the purification from the haemolymphin which these peptides are released, or from the fat body orhaemocytes where the synthesis takes place. When lookingfor a new class of small, perhaps non-peptidergicantimicrobial substances, we chose not to limit our search inthis way, bearing in mind that these compounds may verylikely also have a role in processes other than immunity(discussed in a later paragraph), and that their presence andproduction as such may not be restricted to the haemolymphor fat body. When nothing is known about the physiologicallocation of the compounds, or when the insects are small-sized, whole body extracts should be prepared. The insectsare washed with water containing disinfectant, then rinsedwith deionised water and excess water is removed by filterpaper. Next, the insects are thoroughly homogenised inacidified methanol (methanol/water/acetic acid; 90/9/1;v/v/v). It is by using an acidified extraction solvent that weensure that the larger proteins and polypeptides aredenaturated and precipitate. An extraction procedure usingboiling water/1M acetic acid resulted in a decrease ofantibacterial activity and was discarded (results not shown).The homogenate is centrifuged at 10,000g for 30 min at4°C. Methanol is removed from the supernatant byevaporation and the residue is dissolved in 0.1%trifluoroacetic acid (TFA) in deionised water. Lipids willdisturb the interactions with the HPLC column and canblock the passage of the sample. They are removed from thesample by sequential extraction with ethylacetate and n-hexane, each time removing the upper lipophilic layer that isconsequently formed. Traces of these organic solvents areafterwards removed by evaporation and the remaining

hydrophilic fraction is further concentrated under vacuum.The residue is again dissolved in 0.1% TFA in water,filtrated (0.45 µm) and loaded on a solid phase extractioncolumn (Mega Bond Elute C18 cartridges, Varian). Thesample is eluted in three or four crude fractions with 0.1%TFA in rising concentrations of acetonitrile (ACN). Thefractions are dried under vacuum and screened forantimicrobial activity. The active fractions will be submittedto further purification using High Pressure LiquidChromatography (HPLC).

Isolating Small Molecules by HPLC

Most known antimicrobial peptides have been purifiedusing mainly traditional reversed phase chromatography.Some antimicrobial substances, however, are veryhydrophilic and will consequently not be retained onreversed phase columns. Preliminary screening of extractsprepared as described above, also yielded high antimicrobialactivity in fractions eluted with 0.1% TFA in 5% ACN.Table 1 outlines data on the antibacterial activity of the solidphase fractions of a whole body extract of Galleriamellonella larvae. The term “equivalent” is routinely used inliterature on isolations, to indicate the quantity of sampletheoretically derived from an extract of one animal.

Table 1. Antibacterial Activity of a Whole Body Extract ofGalleria Mellonella Larvae. Fractions as Elutedfrom Mega Bond Elute C18 Solid Phase Cartridge(Varian, USA) with 0.1% TFA in ACN. GrowthInhibition Against Bacillus Thuringiensis wasDetermined after 6 Hours of Incubation at 37°C.(eq): Larval Equivalents; (+): Complete GrowthInhibition; (+/-): Growth Reduction; (-): no GrowthInhibition

0.25 eq. 0.5 eq. 1 eq. 2 eq. 4 eq.

0 % ACN - - - + +

5 % ACN + + + + +

10 % ACN - - +/- + +

20 % ACN - - +/- + +

30 % ACN - - - + +

40 % ACN - - - - +/-

50 % ACN - - - - -

90 % ACN - - - +/-

The compounds that elute with 0.1% TFA in 5% ACNbind only weakly to the hydrophobic phase of the columnand cannot be purified by reversed phase chromatography.Logically, one would turn to the opposite separationprinciple, namely normal phase chromatography. But it wasobserved that these hydrophilic substances do often notreadily dissolve in the hydrophobic solvent used for loadingthe sample onto a normal phase extraction column. In such asituation, ion-exchange and gel filtration chromatographycan be used. Reversed phase chromatography can still behelpful as a “subtractive isolation” procedure, in which the

non-binding hydrophilic active compounds are separatedfrom the binding hydrophobic compounds. Based ondifferences in molecular mass or charge, a further purificationmay then be obtained by respectively gel filtration or ionexchange chromatography.

The salt containing solvents, usually applied to elute thesample from an ion exchange column, may interfere with theantimicrobial screening assay and should be removed beforetesting. Therefore, this step should be used only when thesample is almost pure and can be detected by itschromatographic absorbance pattern. Also, salts can disturbthe ionisation of the sample, needed to determine its massby mass spectrometry, although Matrix Associated LaserDesorption/Ionisation (MALDI) and nanoflow ElectrosprayIonisation (ESI) mass spectrometry can tolerate low amountof salts [17] . For the concentration and isolation of small,hydrophilic compounds the following method isrecommended: start with semi-preparative gel filtration;then, subtractive reversed phase isolation can be used; ionexchange chromatography may be the next step; desalt theselected fraction(s) by analytical gel filtration. It isworthwhile to mention the existence of new chromatographycolumns with higher selectivity for hydrophilic compoundssuch as the Hypercarb analytical column (Hypersil, Cheshire,U.K.), in which the solid phase is characterised by carbonsheets causing a polar retention effect. Depending on thenature of the sample, such a column may render the use ofion-exchange chromatography superfluous.

An Appropriate Bioassay

Following each chromatographic separation, the fractionsare screened for antimicrobial activity using a liquid growthinhibition assay. This test is easy, sensitive and iscompatible with large numbers of fractions. The elutedfractions are concentrated under vacuum to remove all tracesof TFA and ACN, after which the residue is dissolved inmicrobial growth medium. The samples are transferred to theflat-bottom wells of a microtiter plate and an inoculum ofthe chosen test organism is added. The inocula are takenfrom a microbial suspension obtained by incubating themicro-organisms overnight (bacteria) or longer (fungi), so asto obtain a dense culture of viable cells, and then diluting asample into fresh medium to a concentration ranging from10_ to 10

5 CFU/ml (colony forming units, representing

viable bacteria). The volume of the inoculum may varybetween 10 to 100 µl. The bioassay incubation time canrange from several hours to overnight, depending on thegrowth rate of the organism. Growth inhibition is evaluatedeither by direct visual observation, under an invertedmicroscope, or by absorbance reading at approximately600nm. In the latter case, microscopic observation remainsadvisable, as a lower absorbance rate may be due to growthreduction, growth inhibition, or even aggregation of thegrowing micro-organisms. This test can be conducted as anend-point determination of activity, if growth is evaluatedonly after a predetermined incubation time. To obtain a moredynamic analysis, and to ensure the detection of possiblesecondary antimicrobial effects as described in the nextparagraph, the samples can be incubated in a microplateabsorbance reader with temperature control and mixing

possibilities. At regular time intervals, the absorbance ismeasured and used to draw a growth curve.

One should be cautious when interpreting the growthinhibiting activity of the screened fractions. Different typesof antimicrobial activity can be distinguished: (1) a completelack of growth. Either the micro-organisms were killed bythe compound, indicating a bactericidal effect, or the onsetof growth was inhibited. In the latter case, the micro-organisms will stay dormant but can resume growth whenthe interfering compound is removed from the medium, thusindicating a bacteriostatic effect. By simple observation oflack of growth in the microtiter plate, the observer cannotdistinguish between the two possible causes. (2) If the activecompound is present in a sub-optimal concentration, or ifthe fraction contains a less active compound, growthreduction will be observed, which may become indiscernibleafter a prolonged incubation time, as the less sensitive cellscontinue exponential growth and the cells will ultimatelygrow to the same density as controls. (3) In some cases, themicro-organisms were observed to start exponential growthas normal, but stop multiplying after a certain period oftime, rendering a culture at a significantly lower cellconcentration than the control cultures i.e. Fig. (3). Apossible explanation for this effect is that the activecompound is interfering with a bacterial pathway, needed forthe synthesis of a growth factor, such as a vitamin or aminoacid. When this growth factor becomes exhausted in themedium, further growth is inhibited. (4) In the literature ithas been reported that some active compounds need severalhours to kill the bacterial cells, i.e. drosocin needs 6-12hours to display its bactericidal effect in vitro [18]. Duringthe isolation the researcher will usually give priority to thefractions with direct and complete growth inhibiting effect,but it may be very rewarding to isolate the compounds thatcause the secondary effects as well.

Usually, fast growing bacteria such as Escherichia coliare used as test organism for the bioassay. As someantibacterial peptides are active only against Gram positiveor Gram negative bacteria, it is advisable to test arepresentative bacterial strain for each class. One may alsochose to include a mould or yeast as test organism to screenthe extracts for antifungal activity. Available time andamount of sample will determine the choice of testorganisms.

EXPERIMENTAL APPROACHES:CHARACTERISATION OF THE ANTIMICROBIALACTIVITY OF ΒΒΒΒ−−−−AY

Once an active compound has been identified, the natureand extent of its antimicrobial activity are determined. Usingthe example of β-alanyl-tyrosine (β−AY), various proceduresof characterisation are presented next.

Measurement of Growth Inhibiting Activity

The compound was screened for activity against a varietyof micro-organisms, including bacteria and fungi, using theliquid growth inhibition assay as described above. Using

Table 2. MIC values for ββββ-AY on Various Test Organisms. MIC is the Minimal Concentration of ββββ-AY that Inhibits Growth forat Least 6 Hours at 36°C (Bacteria) or 20 Hours at 27°C (Fungi). Concentrations as Determined by AbsorbanceMeasurement at 276 nm and Calculation by the Rule of Lambert-Beer, Using an Extincyy;Tion Coefficient of 1412. (-)no Growth Inhibition at 40 mM ββββ-AY; TSB: Tryptic Soy Broth (Sigma); NB: Nutrient Broth n°2 (Oxoid); MB:Sabouraud 2% Maltose Broth (Merck); IM: Grace’s Insect Medium (Gibco) / Basal Medium Eagle (Sigma) (1/1)

Growth medium MIC (mM)

Gram positive bacteria Micrococcus luteus DPMB-B3 TSB 24

Bacillus thuringiensis * DPMB-B1 IM 8

Bacillus cereus DPMB-B2 IM 8

Listeria innocua LMG 11387 TSB 22

Staphylococcus aureus LMM 0237 NB 12

Gram negative bacteria Salmonella Enteritidis ATCC 3076 NB 16

Escherichia coli LMG 1655 TSB 30

Shigella sonnei LMG 10473 NB 15

Fungi Candida utilis IHEM 4005 TSB -

Candida albicans IHEM 3732 TSB >40

Saccharomyces cerevisiae ATCC 7754 MB 12

Beauveria bassiana IHEM 3558 MB -*Note: our Bacillus thuringiensis strain was originally isolated from an infected primary culture of locust neuronal cells.

Fig. (2). Growth curve of Shigella sonnei over a 10 hour time period as measured on a Bioscreen C reader. Each curve is a mean of twosamples. OD: optical density; Co: control; AB: 0.1 mM chloramphenicol; β-AY: 10, 12, 14 mM β-alanyl-tyrosine; Tyr: 15 mMtyrosine; β-Ala: 25 mM β-alanine.

serial dilutions, the minimal inhibitory concentration orMIC was determined. The MIC is defined as the lowest finalconcentration of the compound, at which no growth isobserved during a predetermined period of incubation. Theassay is performed in a microtiter plate, allowing thesimultaneous testing of a large number of samples againstseveral test organisms. Rising concentrations of β-AY weredissolved in 40 µl of suitable microbial growth medium andadded to a 10 µl inoculum of chosen test organisms, whichis taken from a culture prepared by 100-fold dilution of a

stationary phase culture. After a predetermined incubationtime, growth was evaluated microscopically.

β-AY displayed antibacterial activity against both Gramnegative and Gram positive bacteria. Antifungal activity isdemonstrated by complete growth inhibition of the yeastSaccharomyces cerevisiae and a 75% growth reduction of theyeast Candida albicans at 40 mM, but no activity wasdetected for the yeast Candida utilis or the mould Beauveriabassiana up to a concentration of 40 mM. The MIC values

Fig. (3). Growth curve of Streptococcus faecalis over a 10 hour time period as measured on a Bioscreen C reader. Each curve is a meanof two samples. OD: optical density; Co: control; AB: 0.1 mM chloramphenicol ; β-AY: 2, 6, 12, 18 mM β-alanyl-tyrosine.

are in the millimolar range, while most MIC values foundfor the inducible antimicrobial peptides are in themicromolar range. The low molecular mass compoundsisolated by Leem et al. showed an IC50 (50% inhibitoryconcentration) ranging from 0.02 to 0.08 mM [14,15] . Itshould be noted however, that these high concentrations arephysiologically relevant in the insect as β-AY is present invery high amounts in the larvae and prepupae (discussed inlater paragraph).

To obtain a more dynamic analysis of the growthinhibiting action, the test bacteria were incubated over alonger period of time in a Bioscreen C Microbiology reader(Labsystems Oy, Finland). Absorbance was measured with a'wide bandpass' filter at regular intervals. The antibioticchloramphenicol (30 µg/ml) was used as a positive controlfor the bacteria (except for S. aureus, M. luteus and L.innocua which where found not to be susceptible). Theamino acids β-alanine and tyrosine were used as a negativecontrol. The different growth curves as registered by theBioscreen C Microbiology reader revealed mostly anincreased lag-phase and/or a reduced exponential growth rate.In Fig. (2), the growth curve of Shigella sonnei LMG 10473during the first 10 hours of incubation is presented. β-AYcompletely inhibited growth of S. sonnei for 21 hours (14mM), 26 hours (16 mM) and longer depending on theconcentrations applied. Most bacterial strains eventuallystarted growing and reached cell densities equal to that of thecontrol, depending on the concentrations of β-AY applied. Adifferent pattern of growth inhibition was observed forPseudomenas aeruginosa DPMB-B5 and Streptococcusfaecalis DPMB-B4. In Fig. (3) the growth curve of the latteris presented. In this case, rising concentrations of β-AY didnot delay the onset of exponential growth, but reduced thecell density reached in stationary phase.

Bacteriostatic and Bactericidal Effects of ββββ−−−−AY on B.Thuringiensis

In order to determine whether the effect of β-AY wasbacteriostatic or bactericidal, a culture of Baci l lusthuringiensis DPMB-B1, was incubated overnight at 37°Cin Grace’s Insect Medium/Basal Medium Eagle (1/1) (IM).

A 200 µl sample of bacterial culture in stationary phase(approximately 5x10

7 CFU/ml) was centrifuged (2000 g / 5

min.) to collect the bacteria. The supernatant was decantedand the cells were resuspended in 200 µl potassiumphosphate buffer (100 mM; pH 7), with (test samples) orwithout (control samples) a given concentration of β-AY.The samples were then incubated at 37°C. At specific timeintervals, a sample of the bacterial suspension was taken,diluted and plated on tryptic soy agar to count the number ofsurviving cells as colony forming units (CFU) afterincubation at 37°C for 24 hours.

When B. thuringiensis was transferred from IM growthmedium to phosphate buffer (Fig. 4), cells continued todivide slowly during the first hours but then stoppedgrowing and survived at almost constant cell numbers. Thephosphate buffer does not contain the nutritients required forthe exponential growth of B. thuringiensis. The presence of24 mM β-AY seems to eliminate the initial period of celldivision, but causes no bactericidal effect up to 24 hoursincubation. At 32 mM β-AY, the number of viable cellsremains stable during 5 hours of incubation, but thendecrease from 4.4x10

7 to 10

6 CFU/ml in the next 19 hours.

This is an indication of a lethal effect of β -AY atconcentrations equal to 4 times the MIC value.

The test was repeated with a concentration of 32 mM β-AY, replacing the phosphate buffer with IM growth medium

1.0E+05

1.0E+06

1.0E+07

1.0E+08

1.0E+09

0 5 10 15 20

time (hours)

CFU

/ m

l

control 24 mM 32 mM

Fig. (4). The effect of β-AY on stationary phase cells of B. thuringiensis DPMB-B1 in phosphate buffer (100 mM, pH=7). CFU: colonyforming units.

1.0E+00

1.0E+02

1.0E+04

1.0E+06

1.0E+08

0 1 2 3 4

time (hours)

CF

U/m

l

32 mM control

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -Detection level (20CFU/ml)1.0E+00

1.0E+02

1.0E+04

1.0E+06

1.0E+08

0 1 2 3 4

time (hours)

CF

U/m

l

32 mM control

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -Detection level (20CFU/ml)

Fig. (5). The bactericidal effect of β-AY on stationary phase cells of B. thuringiensis DPMB-B1 in Grace’s Insect Medium/BasalMedium Eagle, (1/1). CFU: colony forming units. The dotted line represents the lower detection limit of 20 CFU/ml.

(Fig. 5). The number of viable cells dropped below thedetection limit of 20 CFU/ml within the first hour.

The test was repeated, in IM growth medium, but usingan initial cell density of approximately 5x10

4 CFU/ml

instead of 107 CFU/ml. In these conditions, cells continued

to grow and reached again a cell density of 107 CFU/ml after

4.5 hours of incubation. The bacteriostatic effect of β-AY ata concentration equalling the MIC (8 mM) was confirmed asgrowth was inhibited up to 24 hours (Fig. 6 ). At a

concentration of 24 mM β-AY, viable cells becameundetectable within 30 minutes, providing evidence for astrong and rapid lethal effect at this concentration.

From these experiments we can conclude that lowerconcentrations of β-AY have a bacteriostatic effect on B.thuringiensis while high concentrations have strong lethaleffect. The strong bactericidal effect was observed only forbacteria in microbial growth medium, and not for bacteria inphosphate buffer.

1.0E+00

1.0E+02

1.0E+04

1.0E+06

1.0E+08

0 1 2 3 4

time (hours)

CFU

/ m

l

control 8 mM 24 mM

Detection level (20CFU/ml)- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -1.0E+00

1.0E+02

1.0E+04

1.0E+06

1.0E+08

0 1 2 3 4

time (hours)

CFU

/ m

l

control 8 mM 24 mM

Detection level (20CFU/ml)- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

Fig. (6). The bactericidal effect of β-AY on exponential phase cells of B. thuringiensis DPMB-B1 resuspended at 105 CFU/ml inGrace’s Insect Medium/Basal Medium Eagle, (1/1). CFU: colony forming units. The dotted line represents the lower detection limit of20 CFU/ml.

Table 3. Antimicrobial Activity of ββββ-Alanyl-Tyrosine (ββββ-AY), Alanyl-Tyrosine (AY), Fmoc-ββββ-Alanyl-Tyrosine (Fmoc-ββββ-AY), ββββ-

Alanyl-Serine (ββββ-AS), ββββ-Alanyl-Valine (ββββ-AV), ββββ-Alanyl-Phenylalanine (ββββ-AF). Bacteria were Incubated in TSB at 37°C.The Yeast S. Cerevisiae was Incubated in MB at 27°C. (+) Complete Growth Inhibition; (+/-): Growth Reduction; (-) NoGrowth Inhibition; (nt): Not Tested

Concentrations tested (mM) ββββ-AY AY Fmoc-ββββ-AY ββββ-AS ββββ-AV ββββ-AF

B. thuringiensis* 20 and 40 + +/- and + - - - -

M. luteus 24 and 48 + +/- and + - - - -

S. aureus 24 and 48 + +/- and + - - - -

E. coli 30 and 60 + - - - - -

S. Enteritidis 32 + - - - - -

S. sonnei 25 + nt nt - - -

S. cerevisiae 12 and 24 + - and +/- - - - -

*strain B. thuringiensis LMG 7138.

Also, the MIC value that quantifies the bacteriostaticeffect, is dependent of the growth medium used. When B.thuringiensis DPMB-B1 was incubated in IM growthmedium, the MIC value of β-AY was determined to be 8mM. However, in TSB growth medium, the MIC was 16mM. Similar observations were made for the test strains ofS. aureus, S. sonnei and S. Enteritidis which were alsofound to have MIC values in TSB that were approximatelytwice as high as in NB. Moreover, the MIC value of B.cereus was determined to be 8 mM in IM and NB but 24mM in TSB.

These findings suggest that the antibacterial activity of β-AY depends on the medium composition and/or on thephysiological state of the cells, since the latter can beinfluenced by the medium composition.

Activity of Structural Analogues

Structural analogues of the compound β-AY werescreened for antimicrobial activity using the bioassay asdescribed above. The concentrations tested were equal to andthe double of the MIC value as determined for β-AY on therespective micro-organisms in TSB or MB growth medium.The results are displayed in table 3.

The negative results with β-AS, β-AV and β-AF indicatethat the hydroxylated phenol ring on the second amino acid,is a requirement for activity. The modified amino acid β-alanine also seems to play an important role in retainingactivity since Fmoc-β-AY did not inhibit growth and AYwas much less active.

Stability

Chromatography was used to determine the presence ofβ-AY after incubation at higher temperatures. Comparison ofHPLC profiles indicated that no degradation had occurredduring 1 week of incubation at 36°C in an aqueous solution,or 3 days incubation at 36°C in Ham’s F12 cell culturemedium. After incubation, the solutions of β-AY were driedand resuspended in bacterial growth medium to aconcentration equal to the MIC value of B. thuringiensisDPMB-B1. The endpoint growth inhibition assay asdescribed above, indicated that antibacterial activity hadremained intact. Moreover, an aqueous solution of β-AY,autoclaved at 120°C for 20 minutes, then dried andresuspended in growth medium to MIC concentration, stillinhibited growth of B. thuringiensis DPMB-B1. These dataindicate a heat-stable antimicrobial activity of β-AY.

The chemical structure and low molecular mass of β-AY,make this modified dipeptide an unlikely target forproteases. However, in the feeding larvae and prepupae ofSarcophaga, the concentration of β-AY rises to very highlevels in the haemolymph, which are quickly reduced at theonset of pupariation due to the action of specific hydrolasesfrom the fat body [19]. The identity of these hydrolases andtheir presence in vertebrates has not yet been described.

Cytotoxicity

Although injection of β-AY in adult insects causesparalysis [16], no cytotoxic effect could be observed in vitroon insect neuronal cells. When applied to rat C6 gliomacells, rat primary neurons or human neuroblastoma cell linesIMR32 and SHSY5Y, no cytotoxic effect of β-AY wasdetected up to 10 mM concentrations [16] . Intraspinalinjection in rats gave no significant effect at concentrationsas high as 198 mM [Klement E. et al., unpublished results].However, the effects of β-AY application in vertebrates mustbe further examined. Besides for clinical applications, β-AYmay be used as antimicrobial additive to cell culture growthmedia, or as preservative in foods, pharmaceuticals,cosmetics and other products susceptible to microbialdeterioration.

MODE OF ACTION

When reviewing the thus far elucidated mode of action ofantibacterial peptides isolated from insects [reviews 20,9],roughly two major groups can be considered:

(1) Most of the inducible antibacterial peptides act on thebacterial membrane, either by physical disruption ordisturbing membrane synthesis: I.e Defensins [21]and cecropins [22] kill bacteria by perforating thecytoplasmic membrane. Bacteria are killedimmediately, i.e. one minute of contact with 0.5 µMdefensin will effectively kill M. luteus in the growingand resting phase [23,24]. On the other hand, attacinsonly partially integrate in the outer membrane,disturbing the incorporation of outer membrane

proteins, which results in the inhibition of theirsynthesis at the transcriptional level, and an increaseof the membrane permeability [25].

(2) Another group of antimicrobial peptides act byspecific inhibitory binding of bacterial housekeepingproteins. I.e. drosocin, pyrrhocoricin and apidaecinact only on Gram negative bacteria. Their activity isfound to be stereospecific [18, 26,27] and bacterialcell death in vitro ensues only after several hours.Recent reports show that antimicrobial activity iscorrelated to the specific inhibition of the bacterialheat shock protein DnaK [28,29].

The antibacterial activity of the low molecular masscompound 5-S-GAD can be inhibited by the presence ofcatalase [14], indicating that its activity is dependent on theproduction of H2O2. Later it was suggested that two protonsare generated from the hydroxyl groups of the dopasubstructure, probably via the formation of a quinone andlinked to an oxygen molecule by a bacterial terminal oxidase[30]. The induction of apoptosis on a macrophage cell lineby 5-S-GAD is also thought to be due to the formation ofH2O2 [30]. The observed selective antitumor activity of 5-S-GAD [31] is, however, not inhibited by catalase, and may bebased upon the observed inhibition of theautophosphorylation of protein tyrosine kinase [30,32]. Thecomposing substructures β-alanyl-dopa and gluthatione didnot exhibit this inhibiting effect on tyrosine kinase [33].

The exact mode of action of p-hydroxycinnamaldehydeand β-AY has not been elucidated as yet. However, thecytotoxicity of hydroxylated phenolic compounds and theiroxidation products has been long recognised and is thoughtto be due to the production of free radicals [34,35] and totheir inhibitory interactions with proteins in general[36,37,38] . The inhibitory effect of such compounds onmicro-organisms is, among other things, dependent on therate of hydroxylation [39,40]. The main effectors of activityare quinones, which are aromatic rings with two ketonesubstitutions. Hydroxylated amino acids can be convertedinto quinones by phenoloxidases such as tyrosinase (Fig. 7).

The bactericidal effect of β-AY at concentrations higherthan the MIC value, was rapid for bacterial cells that aresuspended in microbial growth medium but not for bacterialcells in phosphate buffer. The most likely explanation is thatonly actively metabolizing cells are sensitive to thebactericidal effect of β-AY. It is too early to speculate on themode of action of β-AY, though the lack of a bactericidaleffect in phosphate buffer and the small molecular size of thecompound make it unlikely that it would have a membraneperforating activity. Rather, the observation that growingcells are more sensitive, suggests that β-AY may interactwith a vital metabolic process.

Other low molecular mass molecules with antimicrobialactivity from insects remain to be identified. Their smallmolecular size (less than 1 kDa) however, will attribute totheir chances of being able to cross the microbial membraneand exhibit their antimicrobial activity on intracellularprocesses or structures.

HO CH2CHCOOH

NH2

tyrosine

HO CH2CHCOOH

NH2

HO

dopa

CH2CHCOOH

NH2

O

O

o-quinone

Fig. (7). Conversion of tyrosine to quinone by tyrosinase.

Fig. (8). Elemental composition report after acurate mass determination of a 374 Da fragment on ESI-Q-Tof mass spectrometer.

a.

b.Fig. (9). Fragmentation spectra obtained by collision induced dissociation on a Q-Tof mass spectrometer of (a) synthetic riboflavin(376 Da) and (b) the antibacterial compound with a molecular mass of 374 Da.

PAST SUCCESSES AND FUTURE PROSPECTS

Successful Isolations from Diptera

So far, all insect low molecular mass compounds withantimicrobial properties have been isolated from flies. Theantibacterial 5-S-GAD was isolated from adults of thef leshfly S a r c o p h a g a p e r e g r i n a [ 1 4 ] . p-hydroxycinnamaldehyde was isolated from larvae of the sawfly Acantholyda parki S. [15] and β-alanyl-tyrosine has beenisolated from the larvae of the grey fleshfly Neobellieria

bullata [16].Recently, we have isolated and partially identified

another low molecular mass compound (438 Da) withantibacterial activity from larvae of the grey fleshfly. Similarto β -AY, this compound is also present prior toimmunisation of the insects. During the isolation procedure,the original compound of 438 Da was degraded, but adominant fragment of 374 Da retained a significant degree ofactivity. The molecular masses were determined by use of anElectrospray Ionisation-Quadrupole-orthogonal acceleration-Time of Flight (Esi-Q-Tof) mass spectrometer (Micromass,

UK). After accurate mass determination, special software(Micromass, UK) is used to calculate possible elementaryatom compositions of the compound, using a predeterminedset of atomic masses so as to match the determined masswithin an accuracy range of 10 parts per million (ppm). Ariboflavine-like structure was suggested (Fig. 8).

Further fragmentation by ESI-Q-Tof in MS_ mode of the374 Da compound and commercial available riboflavin (376Da) resulted in similar fragmentation spectra, as presented inFig. (9). The dominant 242 Da fragment that is clearly incommon, was in turn subjected to elemental masscomposition and a known structure was calculated:dimethylalloxazine, which is the basic substructure of theflavines (Fig. 10). Several fragments obtained from the 374Da compound (spectrum b) have masses that are exactly 2Da less than those obtained from synthetic riboflavine(spectrum a). As the mass of the dimethylalloxazinefragment is identical, this mass difference may be due to anoxidation in the saccharide side chain (Fig. 10).

H OH

H OH

H OH

CH2OH

CH2

N O

O

CH3

CH3

Fig. (10). Structure of riboflavine, and its substructuredimethalloxazine (framed).

Riboflavin itself (376 Da) was also simultaneouslypurified from Neobellieria as a less active fraction. An earlyreport already mentioned an antibacterial activity ofriboflavin analogues [41] . Unfortunately, not enoughmaterial was retained to perform the Nuclear MagneticResonance (NMR) analysis needed to elucidate the completestructure of the original and most active 438 Da compound.Efforts will be made to complete the analysis in the nearfuture.

Prospects for Novel Leads

(a) Constitutive Antibacterial Activity

Are these small molecules with antimicrobial activityonly present only in the order of Diptera? To answer this

question, several crude methanolic extracts from larvae ofinsects of various orders were prepared as described above.These extracts were loaded on solid phase C18 columns andeluted in 4 crude fractions. These crude fractions werescreened for antibacterial activity (Table 4) using the insectpathogen Bacillus thuringiensis LMG 7138 in the liquidinhibition assay as described previously.

Table 4. Antibacterial activity of a whole body extractsof insect larvae. Fractions as eluted from Megabond Elute C18 solid phase cartridge (Varian,USA) with 0.1% TFA in ACN. Growthinhibition against Bacillus thuringiensisLMG 7138 was determined in TSB growthmedium after 6 hours incubation at 37°C. (++)Antibacterial activity when 0.5 larvalequivalent was added to the microbial growthmedium. (+) Antibacterial activity when 5larval equivalents were added to the microbialgrowth medium. (-) No activity when 5 larvalequivalents were added to the microbialgrowth medium. (/) Not tested

Species : 0% 30% 60% 90%

Neobellieria bullata (Diptera) ++ ++ + -

Phormia regina (Diptera) / ++ + -

Tenebrio molitor (Coleoptera) ++ ++ + +

Galleria mellonella (Lepidoptera) + ++ + +

Spodoptera frugiperda(Lepidoptera)

/ ++ ++ ++

Highest antibacterial activity was found in the fractionseluting with 0.1% TFA in 0% or 30% ACN, with theexception of the Spodoptera extract, which displayed highantibacterial activity in all fractions. It is important to note,that the animals were neither previously injured norartifically infected in order to induce an immune responseprior to extraction. Previously known antibacterial peptidesfrom insects have been isolated only after bacterial inductionof the immune response. The data presented in Table 4however, indicate the presence of a constitutive reservoir ofantibacterial substances in unchallenged insect larvae. As thelarvae of insects, and in particular the larvae of Diptera, oftengrow and feed mostly on decomposing material, they areintensely exposed to an environment rich in microbes. Aconstitutive reservoir of antimicrobial compounds may existin parallel to the induced antimicrobial peptides that arereleased in the haemolymph in response to septic injury.Synthesis of these induced peptides occurs mainly in the fatbody (equivalent of the mammalian liver) and in certainhaemocytes, although a more local secondary response inbarrier tissues such as tracheal or gut epithelium [42,43] canalso be induced in addition to the systemic response. It hasbeen reported however that a constitutive expression andstorage of antibacterial peptides, occurs in granular cells ofother invertebrates such as the shrimp [44], crabs [45,46] andthe mussel [47] as part of the innate immunity. In 1998, theconstitutive expression of an antibacterial peptide was

Abs

orba

nce

at 2

14 n

m

AU

FS

Abs

orba

nce

at 2

14 n

m

AU

FS

Fig. (11). Preparative HPLC separation of the 30% ACN solid phase fraction, as prepared from a crude extract of Galleria mellonellalarvae. Elution solvent A was 0.1% TFA in deionised water. Elution solvent B was 0.1% TFA in 50% ACN. A gradient from 100%A to80% B in 90 minutes was applied. Fractions with growth inhibiting activity against Bacillus thuringiensis LMG 7138 (BT),Escherichia coli LMG 1655 (EC) and/or Saccharomyces cerevisiae ATCC 7754 (SC) are indicated. Absorbance at 214 nm.

observed in insects, more particularly in the midgut of thecoconut beetle (Coleoptera) [48] and earlier this year,Lamberty et al. reported the constitutive presence of anantifungal peptide in certain haemocytes and the salivaryglands of the fungus-growing termite Pseudocanthotermesspiniger (Isoptera) [49]. It therefore seems that insects havetwo ways of fighting infection: (1) The infection-inducedtranscription of genes coding for synthesis of antimicrobialpeptides, mainly in the fat body, and their release in thehaemolymph. (2) The constitutive production and storage ofantimicrobial compounds, particularly in granular cells.These compounds may be destined for release from storageinto the haemolymph through exocytosis or celldegranulation, or destined for fusion with microbes-containing phagosomes. Whether an infective induction is anabsolute requirement for eventual release of theseconstitutively synthesised compounds into the haemolymph,or whether some may be continuously secreted, remains tobe elucidated.

The mode of fighting infection by granular storage ofantimicrobial peptides and release upon contact, is part ofthe innate immunity of crustaceans [50] and mussels [47],and has been observed for mammalian granulocytes [51,52].On the other hand, in H. cecropia and G. mellonella it wasdemonstrated that only the granular haemocyte typeinternalise bacterial LPS [53] , making the presence ofstorage granules filled with antimicrobial compounds veryacceptable. It is obvious that there may be a close link to thecellular immune response, during which different types ofhaemocytes will concentrate around invading micro-organisms [12].

(b) Preliminary Screening of an Extract of Galleria

Galleria mellonella larvae were collected at the wanderingstage, when they move away from the food to startpupariation. An acidic methanolic extract was prepared asdescribed above and eluted from solid phase Mega Bondcartridges in four crude fractions. The 30% ACN solid phasefraction was then loaded on a preparative HPLC C18column. The obtained chromatogram is presented in Fig.(11). The fractions were screened for antimicrobial activity asdescribed above using two bacterial strains and a yeast.

The presence of several active fractions was observed. Acompound with activity against both the Gram positive andthe Gram negative bacterial test strain as well as the yeasteluted at approximately 7% ACN. Growth inhibitingactivity was observed against B. thuringiensis and S .cerevisiae in fractions eluting at 12% and 16 to 18 % ACN.The fraction eluting at 20% ACN displayed only antifungalactivity while the fraction eluting at 25% ACN was onlyactive against the Gram negative E. coli. These data indicatethe presence of several different antimicrobial compoundspresent in Galleria larvae, prior to any manual induction ofthe immune response.

A first attempt to further purify certain fractions wastroubled by a persistent loss of activity. Different storageconditions were evaluated and although specific conditionsproved to be slightly more suited for specific fractions, nosignificant differences were observed between storage inacetonitrile or dried, at 4°C or –20°C. We observed severaltimes that activity was lost after further separation onanalytical columns. Interestingly, the original fraction stillinhibited microbial growth. This observation may be due toconformational changes during the chromatographicseparation that render the compound inactive.Stereospecificity of the action of antimicrobial compounds

has been reported [18,26,27]. Another possible explanationcould be the dependence of activity on the presence of acofactor that becomes detached, or a synergic action betweentwo different compounds that are separately not or much lessactive. The observed loss of activity may be a factor toexplain the absence of previous reports of isolations of lowmolecular mass substances with antimicrobial propertiesfrom Galleria.

(c)The Constitutive Presence of AntimicrobialCompounds During Development in Tenebrio Molitor

Extracts were made at different developmental stages ofthe yellow mealworm Tenebrio molitor (Coleoptera), andscreened for antimicrobial activity, using a liquid growthinhibition assay.

Table 5. Minimal Animal Equivalents Needed to InhibitGrowth of BT: Bacillus Thuringiensis LMG 7138,EC: Escherichia Coli LMG 1655 and SC:Saccharomyces Cerevisiae ATTC 7754. TheConcentrations Tested were 0.5, 1, 2 and 4Equivalents. (-): No Growth Inhibition at 4Equivalents; (nt): Not Tested. Fractions as Elutedfrom Mega Bond Elute C18 Cartridges with 0.1%TFA in ACN

Developmental stage

Fraction(% ACN)

Inhibition concentration(equivalents)

BT EC SC

Late larvae 0% 0.5 2 1

30 % 0.5 0.5 0.5

60 % 4 4 4

90 % 4 - -

Late pupae 0% 0.5 1 0.5

30 % 0.5 nt 1

60 % 0.5 4 2

90 % 0.5 - -

Adults day 0 0% 0.5 2 1

30 % 0.5 nt 1

60 % 2 4 4

90 % 2 - -

Adults day 4 0% 2 4 2

30 % 0.5 nt 1

60 % 2 4 4

90 % 2 - -

Table 5 shows that the highest antimicrobial activity isusually found in the 0% and 30% ACN fractions. This couldalso be deducted from the data in table 4. The highestoverall antibacterial and antifungal activity are found inlarvae and pupae. When pupating, the animals arevulnerable, as they cannot physically move away from aninfectious environment. At this point in development,

however, metamorphosing processes take place and manymetabolites are formed, and some of these may be effectorsof the observed antimicrobial activity (see next paragraph).

It is apparent from our data that antibacterial compoundsin Tenebrio molitor are most sensitively detected with B.thuringiensis, which is a known insect pathogen. It followsfrom this observation that this bacterial species represents agood bioassay test organism to monitor the purification ofactive compounds from Tenebrio molitor.

NOT JUST FOR DEFENSE

The physiological functions of these low molecular masscompounds in insects, may be diverse and not restricted toimmunity. Some may be secondary metabolites frompathways directed towards non-immune functions.

This is observed in plants where several secondarymetabolites such as tannins, terpenoids, alkaloids andflavenoids are known to exhibit antimicrobial activity invitro [54]. These metabolites are however also responsiblefor various features such as plant pigmentation (quinonesand tannins), odour (terpenoids) and flavour. The phenoliccompound p-hydroxy-cinnamaldehyde in plants is aprecursor of the cell wall component lignine [13] , but hasalso been demonstrated to have antibacterial and antifungalproperties in the saw fly [ 1 5 ] . Recen t ly ,hydroxycinnamaldehydes have been reported to befungicidal, but not bactericidal [55].

In insects, an injury or invasion of various non-selfcomponents induces a proteolytic cascade in thehaemolymph that will converse the phenoloxidase precursor,present in the haemolymph or released by haemocytes uponinduction, into the active enzyme [ reviewed in 56] .Phenoloxidase catalyses the synthesis of melanin pigmentfrom phenolic substances. Hence, the deposition of darkpigment in injured cuticle and to the formation of brownnodules [57,58], or melanotic encapsulations [59,60] aroundinvading micro-organisms or parasites. The metabolites(quinones) of this pathway are known to exhibit cytotoxicand antimicrobial activity [see “mode of action”,35].

Also, Natori et al. [30] have reviewed several defencemolecules from Sarcophaga, that seem to play a role indevelopment as well as in immunity. Some of theseadditional functions are based on the same biochemicalproperties responsible for the antimicrobial activity, whileothers are surprisingly different, indicating a dual mode ofaction from the same molecule.

β-alanyl-tyrosine has only been detected in Diptera of thegenus Sarcophaga and the tachinid Voria ruralis [61] .Earlier reports [62] considered the high amounts of β-AY inthe prepupae of Sarcophaga as a storage form of tyrosineand β-alanine, which are involved in melanisation andsclerotisation of the cuticle and puparium respectively[63,64 ] . This way the water solubility of tyrosine isincreased. Furthermore, the free amino acids are protectedfrom metabolism by other pathways. Another possiblephysiological role of the β-alanyl-tyrosine present

Fig. (12). Concentrations of β-AY in various developmental stages of the fleshfly Neobellieria bullata. Each point is the mean ofthree separate analysis. (Reprint from ref.16.)

Sarcophaga has been postulated by Chiou et al. [16]. Whenan extract of wandering larvae of Neobellieria bullata wasinjected into adults of the same or other insect orders, aparalysing effect was observed. The two responsiblecompounds were identified as 3-hydroxykynurenine and β-AY. Meanwhile, two more paralytic compounds have beenisolated from Tenebrio molitor [review on paralysins ref.65] . The appearance of these endogenous compounds ininsects was highly related to the onset of metamorphosis.Concentrations of β-AY continue to rise during larvaldevelopment, peak just before pupariation and then quicklydrop to undetectable levels in the emerging adult asillustrated in Fig. (12). These observations led to thepostulation of a possible role of β-AY in the immobilisationof the larvae, as part of the onset of the pupriation process.

The newly discovered antibacterial effect of β-AY nowadds to this range of properties. A single white pupa ofNeobellieria bullata contains at least an amount as large as265 µg β-AY (Fig. 12), as determined by amino acidanalysis of the purified fraction and not taking into accountthe losses during purification, hydrolysis and derivatisation.This high amount corresponds to an approximateconcentration of 20 mM in this insect’s body, thus closelyapproaching or exceeding the MIC value of β-AY against avariety of micro-organisms (Table 2). Apparently, the lowspecificity of the compound is compensated by its very highconcentration present. An in vivo antimicrobial effect of β-AY becomes even more likely if one takes into account thefact that insects contain a reservoir of various antimicrobial

compounds which act synergistically. Hence, the MIC valueof β-AY may be lower in vivo than in vitro.

Which function of β-AY came first in evolution, andwhich function(s) were convenient side-effect(s), isintriguing but must remain unanswered.

CONCLUDING REMARKS / HOPES FOR THEFUTURE

The over-prescription and abuse of antibiotics, that hascreated the selective pressure under which resistantpathogenic micro-organisms have emerged, are beingdiscussed these days. As accumulating evidence and researchreports have at last created an important awareness amongstthe public and governmental health institutions, a change inpractice is being admonished. But in the meantime, thethreat of infectious pathogens that are resistant against all thepresently available antibiotics is very real.

Many efforts are directed towards the betterunderstanding and possible inhibition of the basicmechanisms of the resistance itself [ review 66] , aspharmacologists realize that the effective life span of anyantibiotic will eventually be limited. Through frequentlyoccurring mutations, pathogenic micro-organisms acquiretools to evade total annihilation. The eventual generation ofresistant micro-organisms is unavoidable and the demand fornew anti-infective agents is therefore never-ending. A statusexists of a continuous race between the development of

resistance by the infectious pathogens and the developmentof new drugs by pharmaceutical research and industry.

Since the discovery of the antimicrobial peptide cecropinin 1981, insects have proved to be a very rich source ofsubstances with antimicrobial properties, which can functionas lead compounds for further development of anti-infectiveagents. Being the largest and most diverse class within theanimal kingdom, present in almost all ecologicalenvironments, insects represent an extensive source to befurther explored. Each insect species is a potential source forone or several new antimicrobial agents, as each species hasdeveloped an armory of anti-infective compounds against thespecific pathogenic micro-organisms of its particularenvironment. Using chemical modifications to overcome thelimitations of these natural substances, pharmaceuticalcompanies try to develop optimized derivatives forapplication as new antibiotics. In the mean time, each newdiscovery represents a hope for a new lead.

Also, similarities between the innate defense systemsfound in insects and the innate immunity in humans add anextra dimension to the search. Reactions such asphagocytosis and coagulation, and immune mediators suchas cecropins or peptidoglycan recognition protein, are someelements of innate immunity that are conserved from insectsto mammals [67,68] . Striking structural and functionalparallels exist between the NF-κB transcription pathway,regulating the inducible inflammatory responses inmammals, and the activation of the DORSAL gene encodingsome of the inducible antimicrobial peptides in Drosophila[69]. By unraveling the insect immune response system, wemay find tools to enhance our own.

LIST OF ABBREVIATIONS

ACN = Acetonitrile

β-AY = Beta-alanyl-tyrosine

CFU = Colony forming units

ESI-q-Tof = Electrospray ionisation time of flight

HPLC = High performance liquid chromatography

IM = Grace’s insect medium/ basal medium Eagle

MALDI = Matrix assisted laser desorption/ionisation

MB = Maltose broth

MIC = Minimal inhibitory concentration

NB = Nutrient broth n°2

5-S-GAD = Beta-alanyl-5S-glutathionyl-3.4-dihydroxyphenylalanine

TFA = Triflueroacetic acid

TSB = Tryptic soy broth.

ACKNOWLEDGEMENTS

β−alanyl-tyrosine was synthesized by Dr. J. Drijfhout.Fmoc-β−alanyl-tyrosine was kindly provided by Prof. R. J.Nachman. Mass spectrometric analysis was performed byGeert Baggerman. Research was supported by a grant fromthe Vlaams Instituut voor de bevordering van hetWetenschappelijk-Technologisch onderzoek in de industrie(IWT).

REFERENCES

[1] Zopf D., Roth S., The Lancet, 1996, 347, 1017.

[2] Hancock R.E.W., The Lancet, 1997, 349, 418.

[3] Hancock R.E.W., Lehrer R., TIBTECH, 1998, 16, 82.

[4] Savage P.B., Ann. Med., 2001, 33(3), 167.

[5] Broekaert W.F., Cammue B.P.A., Debolle M.F.C.,Thevissen K., Desamblanx G.W., Osborn R.W., Crit. Rev.Plant Sci., 1997, 16, 297.

[6] Baba T., Schneewind O., Trends Microbiol., 1998, 6(2),66.

[7] Steiner H., Hultmark D., Engstrom A., Bennich H., BomanH.G., Nature, 1981, 292, 246.

[8] Otvos L., J. Pept. Sci., 2000, 6, 497.

[9] Boman H.G., Scand. J. Immunol., 1998, 48, 15.

[10] Lemaitre B., Reichhart J.M., Hoffman J.A., Proc. Natl.Acad. Sci. USA, 1997, 94, 14614.

[11] Gillespie P.G., Kanost M.R., Trenczek T., AR Entomol.,1997, 42(1), 611.

[12] Breithaupt H;, Nat. Biotechnol., 1999, 17, 1165.

[13] Hancock R.E.W., Chapple D.S., AAC, 1999, 43 (6), 1317.

[14] Leem J.Y., Nishimura C., Kurata S., Shimada I., KobayashiA., Natori S., J. Biol. Chem. , 1996, 271 (23), 13573.

[15] Leem J.Y., Jeong I.J., Park K.T., Park H.Y., FEBS Letters,1998, 442, 53.

[16] Chiou S.Y., Kotanen S., Cerstiaens A., Daloze D., PasteelsJ.M., Lesage A., Drijfhout J.W., Verhaert P., Dillen L.,Claeys M., De Meulemeester H., Nuttin B., De Loof A.,Schoofs L., Biochem. Biophys. Res. Com., 1998, 246, 457.

[17] Siuzdak G., Mass spectrometry for biotechnologyy,Academic press: London, 1996.

[18] Bulet P., Urge L., Ohresser S., Hetru C., Otvos L., Eur. J.Biochem., 1996, 238, 64.

[19] Dunn P.E., Fader R.G., Regnier F.E., J. Insect Physiol.,1977, 23, 1021.

[20] Bulet P., Hetru C., Dimarcq J.-L., Hoffmann D., Dev. Comp.Immunol., 1999, 23, 329.

[21] Cociancich S., Ghazi A., Hétru C., Hoffmann J.A., LetelierL. J. Biol. Chem., 1993, 268, 19239.

[22] Lockey T.D., Ourth D.D., Eur. J. Biochem., 1996, 236, 263.

[23] Lambert J., Keppi E., Dimarcq J.L., Wicker C., ReichhartJ.M., Dunbar B., Lepage P., Van Dorsselaer A., HoffmannJ., Fothergill J., Hoffmann D., Proc. Natl. Acad. Sci. USA,1989, 86, 262.

[24] Cociancich S, Goyffon M., Bontems F., Bulet P., Bouet F.,Menez A., Hoffmann J., Biochem. Biophys. Res. Comm.,1993, 104, 17.

[25] Carlsson A., Nyström T., de Cock H., Bennich H.,Microbiology, 1998, 144, 2179.

[26] Casteels P., Tempst P., Biochem. Biophys. Res. Comm.,1994, 199, 339.

[27] Otvos L. Jr., Bokonyi K., Varga I., Otvos B.I., HoffmannR., Ertl H.C.J.,Wade J.D., McManus A.M., Craik D.J., BuletP., Protein Sci., 2000, 9, 742.

[28] Kragol G., Lovas S., Varadi G., Condie B.A., Hoffmann R.,Otvos L. Jr., Biochem., 2001, 40(10), 3016.

[29] Castle M., Nazarian A., Yi S.-S., Tempst P., J. Biol. Chem.,1999, 274, 32555.

[30] Natori S., Shiraishi H., Hori S., Kobayashi A., Dev. Comp.Immunol., 1999, 23, 317.

[31] Akiyama N., Hijikata M., Kobayashi A., Yamori T.,Tsuruo T., Natori S., Anticancer Res., 2000, 20(1A), 357.

[32] Leem J.Y., Park H.Y., Fukazawa H., Uehara Y., Natori S.,Biol. Pharm. Bull., 1998, 21(7), 784.

[33] Hijikata M., Kobayashi A., Leem J.Y., Fukasawa H.,Uehara Y., Natori S. Biochem. Biophys. Res. Comm., 1997,237, 423.

[34] Nappi A.J., Vass E., Frey F., Carton Y., Eur. J. Cell Biol.,1995, 68, 450.

[35] Nappi A.J., Ottaviani E., BioEssays, 2000, 22, 469.

[36] Peter M.G., Angew.Chem., 1989, 101, 572.

[37] Stern J.L., Hegerman A.E., Steinberg P.D., Mason P.K., J.Chem. Ecol., 1996, 22, 1887.

[38] Riley P.A., Int. J. Biochem. Cell. Biol., 1997, 29, 1235.

[39] Scalbert A., Phytochemistry, 1991, 30, 3875.

[40] Urs N.V.R.R., Dunleavy J.M., Phytopathology, 1975, 65,686.

[41] Graham D.W., Brown J.E., Ashton W.T., Brown R.D.,Rogers E.F., Experientia, 1977, 33, 1274.

[42] Manetti A.G.O., Rosetto M., Marchini M., Antibacterialpeptides of the insect reproductive tract in MolecularMechanisms of Immune Response in Insects, Brey P.T.,Hultmark D. Ed., Chapman & Hall: London, 1998.

[43] Tzou P., Ohresser S., Ferrandon D., Capovilla M.,Reichhart J.M., Lemaitre B., Hoffmann J.A., Imler J.-L.,Immunity, 2000.13, 737.

[44] Destoumieux D., Bullet P., Loew D., Van Dorsselaer A.,Rodriguez J., Bachère E., J. Biol. Chem., 1997, 272,28398.

[45] Relf J.M., Chisholm J.R., Kemp G.D., Smith V.J., Eur. J.Biochem., 1999, 264(2), 350.

[46] Shigenaga T., Muta T., Toh Y., Tokunaga F., Iwanaga S., J.Biol.Chem., 1990, 265, 21350.

[47] Mitta G., Vandebulcke F., Hubert F., Roch P., J. Cell Sci.,1999, 112 (Pt23), 4233.

[48] Yang J., Yamamoto M., Ishibashi J., Taniai K., YamakawaM., Eur. J. Biochem., 1998, 255(3), 734.

[49] Lamberty M., Zachary D., Lanot R., Bordereau C., RobertA., Hoffmann J.A., Bulet P., J. Biol. Chem., 2001, 276(6),4085.

[50] Destoumieux D., Munoz M., Cosseau C., Rodriguez J.,Bulet P., Comps M., Bachère E., J. Cell Science, 2000,113, 461.

[51] Martin E., Ganz T., Lehrer R.I., J. Leukocyte Biol., 1995,58, 128.

[52] Zanetti M., Literri L., Griffiths G., Gennaro R., Romeo D.,J. Immunol., 1991, 146, 4295.

[53] Preik-Steinhoff H., Eue I., Trenczek T., Verh. Dtsch. Zool.Ges., 1992, 85, 213.

[54] Cowan M.M., Clin. Microbiol. Rev., 1999, 12, 564.

[55] Barber M.S., McConnell V.S., DeCaux B.S.,Phytochemistry, 2000, 54(1), 53.

[56] Söderhäll K., Cerenius L., Johansson W., Theprophenoloxidase activating system in New Directionsin Invertebrate Immunology, Söderhäll K., Iwanaga S.,Vasta G.R., Ed., SOS publications: Fair Haven, 1996, 229.

[57] Ratcliffe N.A., Gagen S.J., Tissue Cell, 1977, 9(1), 73.

[58] Ratcliffe N.A., Brookman J.L., Rowley A.F., Dev. Comp.Immunol., 1991, 15(1-2), 33.

[59] Götz P., Encapsulation in Arthropods in Immunity inInvertebrates: Cells, Molecules and Defense Reactions,Springer-Verlag: Berlin, 1986.

[60] Carton Y., Nappi A.J., Parasitology today, 1997, 13, 218.

[61] Bodnaryk R.P., Comp. Biochem. Physiol., 1972, 43B, 587.

[62] Levenbook L., Bodnaryk R.P., Spande T.F., Biochem. J.,1969, 113, 837.

[63] Hopkins T. L., Kramer K.J., Annu. Rev. Entomol., 1992, 37,273.

[64] Bodnaryk R. P., J. Insect Physiol., 1971, 17, 1201.

[65] Cerstiaens A., Kotanen S., Vierstraete E., Meylaers K., DeLoof A., Schoofs L., Paralysins : Endogenousneurotoxins in insects and vertebrates in R e c e n tResearch Developments in Neurochemistry, PandalaiS.G., Ed., Research Signpost: Kerala , 2001, vol. 4. Inpress.

[66] Wright G.D., Chem. Biol., 2000, 7(6), R127.

[67] Kang D., Liu G., Lundström A., Gelius E., Steiner H.,Immunology, 1998, 95(17), 10078.

[68] Du Pascuier L., Comp. Biochem. Phys. B, 2001, 129, 1.

[69] Hoffmann J.A., Reichhart J.-M., Trends Cell Biol., 1997,7, 309.