Spectroscopic Investigations into Inactivation of Bacterial Virulence

Chapter 25

Spectroscopic Investigations into Inactivation of Bacterial Virulence Factors 1

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Downloaded by PENNSYLVANIA STATE UNIV on August 6, 2012 | http://pubs.acs.org Publication Date: August 2, 2007 | doi: 10.1021/bk-2007-0963.ch025

K. Brandenburg , J. Howe , M. Rössle , and J. Andrä

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1

Forschungszentrum Borstel, Leibniz-Zentrum für M edizin und Biowissenschaften, Biophysik, D-23845 Borstel, Germany European Molecular Biology Laboratory, c/o DESY, D-22603 Hamburg, Germany 2

Infectious diseases are still one of the leading causes of death worldwide. For an effective therapeutic strategy, the corresponding bacterial virulence factors must be identified and methods to neutralize them must be developed. One important example of such bacterial factors is lipopolysaccharide (LPS), belonging to the most potent classes of triggers of mammal immune systems. A new and promising therapeutical approach to controlling bacterial virulence factors is the design and synthesis of suitable antimicrobial peptides (AMP), based on the LPS-binding domain of natural defense proteins. These have the potential not only to kill bacteria but to bind to and deactivate the virulence factors as well. A combination of spectroscopic methods (infrared, X-ray diffraction, fluorescence resonance energy transfer) used for the analysis of the inactivation mechanism of bacterial LPS is presented here.

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© 2007 American Chemical Society

In New Approaches in Biomedical Spectroscopy; Kneipp, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2007.


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Despite the availability of antibiotics, infectious diseases are an increasing threat for human health worldwide. This is largely due to the development of resistances, partially caused by the misuse of antibiotics in agriculture and animal husbandry. In Gram-negative bacteria, the main virulence (pathogenicity) factor responsible for a severe outcome of the infection is endotoxin (lipopolysaccharide, LPS) a constituent of the bacterial outer membrane (/). LPS consists of a lipid moiety, called lipid A, which anchors it in the membrane, and an oligo- or polysaccharide side chain (2). Since lipid A itself exerts all the LPS-typical biological effects, it is called the 'endotoxic principle' of LPS. Lipid A consists of a diglucosamine backbone substituted with two phosphate groups and up to seven acyl chains in amide- and esterlinkages. LPS may act beneficially in mammals by inducing cell mediators such as interleukins and tumor-necrosis-factor a, at high concentrations, however, the excessive production of cytokines leads to severe health problems such as the septic shock syndrom, which still cannot be treated effectively, and is a major cause of death in critical care stations (some 200,000 death cases in the U.S. annually) (J),. The action of LPS on human cells takes place after its removal from the outer bacterial membrane, and an effective therapeutic treatment would thus afford the neutralization of LPS. One approach is the application of synthetic peptides derived from LPS-binding sequences of defense proteins in humans and animals. For this, we have synthesized peptides derived from the amino acid sequence of L/ww/wi-anti-LPS factor (LALF) from the horseshoe crab (4). In order to gain a greater understanding of LPS neutralization, biophysical and physico-chemical techniques were applied. We have established various spectroscopic techniques such as Fourier-transfer infrared (FT-IR), synchrotron radiation small-angle X-ray scattering (SAXS), and fluorescence resonance energy transfer (FRET) spectroscopy and other physical and biological (cytokine induction in human mononuclear cells) assays to fully characterize the LPS:peptide interaction. In this way, a profound characterization of the interaction processes (aggregate structure, molecular conformation, secondary structures, membrane interaction) and the molecular requirements for effective neutralization was possible. This should allow - in an iterative process - the establishment of a peptide library, which should eventually lead to highly active anti-septic drugs with negligible side effects.

Materials and Methods Lipids and peptide Free lipid A was isolated by acetate buffer treatment of lipopolysaccharide from Salmonella minnesota strain R595. After isolation, the resulting lipid A


412 was purified and converted to its triethylamine salt (5). Results of all the standard assays performed on lipid A (analysis of glucosamine and total and organic phosphate content, as well as the distribution of the fatty acid residues) were in good agreement with the chemical properties expected for lipid A from LPS R595, the molecular structure of which has already been solved (6). The peptide Pep F with a sequence of G C K P T F R R L K W K Y K G K F W C G was synthesized with an amidated C-terminus by the solid-phase peptide synthesis technique on an automatic peptide synthesizer (model 433 A ; Applied Biosystems) on the standard Fmoc-amide resin according to the fastmoc synthesis protocol of the manufacturer. The N-terminal Fmoc-group was removed from the peptide-resin and the peptide was deprotected and cleaved with 90% T F A , 5% anisole, 2% thioanisole, 3% dithiothreitol for 3 h at room temperature. After cleavage the suspension was filtered and the soluble peptide was precipitated with ice-cold diethylether followed by centrifiigation and extensive washing with ether. H P L C purification was carried out on a R P - H P L C using an Aqua-C18 column (Phenomenex), and eluted using a gradient of 0-70% acetonitrile in 0.1% trifluoroacetic acid (TFA). Cyclation via cystein residues was achieved by incubating the peptide in 10 % D M S O for 24 h at room temperature. The peptide was further purified by reverse-phase H P L C to a purity greater than 95%. Purity was determined by matrix-assisted laser-desorptiontime-of-flight mass spectrometry (MALDI-TOF M S , Bruker).


LAL

F T I R spectroscopy The infrared spectroscopic measurements were performed on an IFS-55 spectrometer (Bruker, Karlsruhe, Germany). For phase transition measurements, the lipid samples were placed between CaF windows with a 12.5 μτη Teflon spacer. Temperature-scans were performed automatically between 10 and 70 °C with a heating rate of 0.6 °C/min. Every 3 °C, 50 interferograms were accumulated, apodized, Fourier-transformed, and converted to absorbance spectra. As a sensitive measure of the state of order of the hydrocarbon chains, the peak position of the symmetric stretching vibration v (CH ) of the methylene groups was taken, which is around 2850 cm" in the gel and 2852.5 to 2853.0 cm' in the liquid crystalline phase of the acyl chains (7). 2

s

2

1

1

X-ray diffraction spectroscopy X-ray diffraction measurements were performed at the European Molecular Biology Laboratory ( E M B L ) outstation at the Hamburg synchrotron radiation facility H A S Y L A B using the S A X S camera X33 (8). Diffraction patterns in the


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1

range of the scattering vector 0.1 < s < 4.5 nm" (s = 2 sin θ/λ, 20 scattering angle and λ the wavelength = 0.15 nm) were recorded at in the range 5-60 °C with exposure times of 1-2 min using an image plate detector with online readout (MAR345, MarResearch, Norderstedt/Germany). The s-axis was calibrated with Ag-Behenate which has a periodicity of 58.4 nm. The diffraction patterns were evaluated as described previously (9), assigning the spacing ratios of the main scattering maxima to defined three-dimensional structures. The lamellar and cubic structures are most relevant here. They are characterized by the following features: (1) Lamellar: The reflections are grouped in equidistant ratios, i.e., 1, 1/2, 1/3, 1/4, etc. of the lamellar repeat distance dj (2) Cubic: The different space groups of these non-lamellar threedimensional structures differ in the ratio of their spacings. The relation between reciprocal spacing s ki = 1/dhki and lattice constant a is h

2

2

2

s ki = [(h + k + l ) / a ]

1 / 2

h

(hkl = Miller indices of the corresponding set of plane).

Fluorescence resonance energy transfer spectroscopy (FRET) The peptide-induced inhibition of the intercalation of lipid A into liposomes made from phosphatidylserine (PS) alone or mediated by lipopolysaccharidebinding protein (LBP), was determined by FRET spectroscopy applied as a probe dilution assay (4). First the peptide, then lipid A, followed by LBP (or vice versa) were added to the liposomes, which were labelled with the donor dye NBDphosphatidylethanolamine (NBD-PE) and acceptor dye Rhodamine-PE. The final concentrations were: peptide and lipid 1 μΜ and LBP 0.1 μΜ. Intercalation was monitored as the increase of the ratio of the donor intensity I at 531 nm to that of the acceptor intensity I at 593 nm (FRET signal) with on time. D

A

Results and Discussion The synthetic A M P synthesized here (Pep LF) was derived from the LPSbinding sequence of the Limulus polyphemus protein L/ww/w^-anti-LPS factor (LALF) (10) (sequence see above), which was connected to a cycle along the two cysteins; since it has been shown that cyclic LALF-peptides are better able to inhibit the LPS-induced cytokine secretion in human mononuclear cells than linear peptides (//). Clearly, the peptide is multiply positively charged ( +7 LA



414 corresponding to the number of arginins and lysins) as a prerequisite for an electrostatic interaction with negatively charged lipid A . We have taken the 'endotoxic principle' lipid A from Salmonella minnesota strain R595 as the bacterial virulence factor. The ability of the peptide to inhibit the lipid Α-induced cytokine production of human mononuclear cells (see (12 in biological experiments)) was tested. It was found that at [lipid A] = 100 ng/ml the cytokine (tumor-necrosis-factor-ct) production was 1150 pg/ml, which decreases in the presence of the peptide (10fold molar excess) to 250 pg/ml, i.e., a drastic neutralization takes place. For an understanding of this ability of Pep LF to neutralize lipid A , spectroscopic methods were applied to characterize the binding process. The interaction of lipid A with Pep LF was studied with (a) FTIR by monitoring the gel to liquid crystalline phase transition of the acyl chains of lipid A , (b) S A X S by analyzing the aggregate structure of lipid A , and (c) F R E T by investigating the potential peptide-mediated inhibition of the intercalation of lipid A into model liposomes, induced by lipopolysaccharide binding protein (LBP). The state of order or fluidity of the acyl chains of amphiphilic molecules is known to be a characteristic property of each membrane lipid, and was analyzed by FTIR using the peak position of v (CH ). In Fig. 1, the phase transition behavior of the acyl chains of lipid A is presented, as a plot of the peak position of v (CH ) versus temperature. It can be seen from the change in the wavenumber values, that in the presence of the peptide only a slight shift to lower wavenumbers, and a concomitant increase in the phase transition temperature T takes place. This corresponds to a slight rigidification of the lipid A acyl chains. The aggregate structure of lipid A has been found to be a determinant of biological activity (13). The necessary inactivation of lipid A involving its conversion from a cubic or mixed unilamellar/cubic aggregate structure in pure form into a multilamellar one in the presence of binding structures, which lead to an inhibition of the biological activity, has been described (14). The results of the lipid A : P e p system in the temperature range 5 to 60 °C and in Fig. 2b at 40 °C are presented in Fig. 2a. The diffraction patterns are similar at all temperatures (Fig. 2a), and reflect a multilamellar structure. At some temperatures,e.g. at 40 °C a second periodicity becomes obvious, (Fig. 2b); one periodicity lies at 6.49 nm, and another one (in italics) at 5.21 nm. From earlier investigations, it is known that the periodicity at 5.21 nm corresponds to a pure multilamellar lipid A structure, obtained for example at high M g concentration or low water content (5). The value at 6.49 nm corresponds to a multilamellar stack composed of lipid A + peptide, i.e., the peptide leads to an increase in the thickness of the water layer between neighboring stacks. This takes place, however, only in the gel phase of lipid A (< 45 °C, Fig. 1), whereas in the fluid LA

LA

s

s

2

2

c

LALF

2 +


415 2853.5

cm

2853.0 -

1

[Lipid A]:[Pe molar ratio

PLALF

1

Γ

]

1 :0

2852,5-

•—'

2852,0 (D

Ε

2851,5-

ω

2851,0-


D C

>D C 2850,52850,0 2849,520

ι

1

30

1

1

40

50

60

Temperature (°C) Figure 1. Gel to liquid crystalline phase transition of the acyl chains of lipid A from Salmonella minnesota-LPS in the presence of different amounts ofPepuLF* plotted as peak position of the symmetric stretching vibrational band v^CH^) versus temperature.

phase (> 45 °C) the peak at 6.49 nm vanishes (Fig. 2a). This may be explained by a dipping of the peptide into lipid A thus reducing the length of the water layer between neighboring stacks. The incorporation of lipid A , mediated by lipopolysaccharide-binding protein (LBP) into target cell membranes has been described as a prerequisite for endotoxin action, i.e., for cell signalling (75). In this study we have tested whether or not the peptide influences this intercalation. For this, FRET spectroscopy was applied by labelling liposomes made from phosphatidylserine (PS) with two fluorophores, and adding the peptide, peptide + lipid A , and LBP in different ways (Fig. 3). The addition of buffer alone at 50 s only leads to a slight decrease in the FRET signal due to dilution, and the addition of L B P at 100 s results in a small increase in the signal corresponding to incorporation of LBP as described previously (15). The addition of the peptide alone at 50 s causes a strong increase in the FRET signal, which indicates that the peptide alone can intercalate into PS liposomes. The addition of the peptide at 50 s followed by lipid A at 100 s causes two signal increases, first an intercalation of the peptide and then of lipid A , apparently mediated by the peptide.



416

0,0

0,1

0,2

0.3

0,4

0,5

0,6

0,7

0,8

1

s (nm*) I

.

I

.

I

.

I

[Lipid A ] : [Pep

L A L F

]

3:1 molar

(b)

0,1

0,2

0,3

0,4

0,5

0,6

40 *C

0,7

s (nm* ) 1

Figure 2. Small-angle X-ray diffraction pattern - logarithm of the scattering intensity versus scattering vector s - of lipid A from Salmonella minnesota-LPS in the presence of Pep^u? at 90 % water content and in the temperature range 5-60 °C (a) and at 40 °C (b).


417 Interestingly, lipid A , without preincubation of the peptide, does not incorporate into the membrane (not shown) in accordance with previous findings (16). The addition of LBP at 150 s leads to another signal increase corresponding to a protein-mediated lipid A intercalation, as discussed in earlier reports (75, 16). Finally, the preincubation of the peptide with lipid A (Pep ALF+ lipid A), added at 50 s, leads to a long lasting increase in the FRET signal with no further effect at 100 s when L B P was added. The same experiments were also performed with liposomes corresponding to the phospholipid mixture of macrophages (75). It was found that qualitatively the same effects are observed, but quantitatively to a lower extent (data not shown). The FRET data clearly suggest that the peptide alone as well as in the presence of lipid A incorporates into liposomal membranes, and does not cause an inhibition of the lipid A incorporation. Thus, a scavenger function role of the peptide can be excluded. Rather, the structural change of the aggregate structure of lipid A seems to be the decisive process. These reoriented, lamellarized aggregates are still able to incorporate into the target membrane (Fig. 3). Therefore , the inactivation process of lipid A does not take place outside the cell. The data presented are strongly in favor of our conformational concept of endotoxicity (77). This assumes an incorporation of endotoxin (lipid A, LPS) molecules into the relevant mononuclear cells of the immune system. These can be induced by the binding proteins of the serum or by the membranse such as LBP and CD 14. It was also found that in particular membrane-bound LBP is able to cause an intercalation of endotoxins into the membrane by disrupting part of the aggregates (18). In the membrane, biologically active endotoxins represent a considerable disturbance of the membrane architecture due to the conical shape of the lipid A moiety (corresponding to a non-lamellar cubic aggregate structure), and thus they may interact with signaling molecules such as TLR4 (19) or the MaxiK channel (20) leading to their conformational change with subsequent signal transduction. The addition of the peptide to lipid A leads to a iamellarization of lipid A (Fig. 2) and hence to a conformation which does not represent a steric disturbance within the target cell membrane. Alternate interpretations of the neutralization process of endotoxins, namely that multilamellarization would lead to a strong reduction of the epitopes accessible for binding proteins such as LBP, or that the higher binding energies of the endotoxins within the aggregates would hamper the interaction with these proteins are not supported by our findings (4). It should also be noted that the acyl chain fluidity is not apparently a determinant of biological activity. From Fig. 1 it becomes clear that the overall fluidity changes are only small; in particular, at 37 °C there are only marginal changes. This is in accordance with other findings that the acyl chain fluidity of endotoxins may modulate the bioactivity, but is not a determinant per se (21). In order that this peptide or derivatives thereof can ultimately be used in therapeutic applications for infectious diseases, its non-hazardous effect in


L


418 Pep

(Pep

+ Lipid A + LBP

LALF

+IJpidA) + LBP

5J

S'


_

Spectroscopic Investigations into Inactivation of Bacterial Virulence

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