- Email: [email protected]
Inactivation of penicillin-induced staphylococcal L-forms by human serum high density lipoprotein
FEMS Microbiology Letters 156 (1997) 113^117
Inactivation of penicillin-induced staphylococcal L-forms by human serum high density lipoprotein Osamu Shimokawa, Hiroaki Nakayama * Department of Microbiology, Faculty of Dentistry, Kyushu University, Higashi-ku, Fukuoka 812-82, Japan
Received 11 August 1997; accepted 6 September 1997
Abstract
In penicillin-susceptible bacteria, penicillin causes growth of a small fraction of cells as wall-deficient forms if an appropriate osmoprotection is provided (unstable L-forms). A subfraction of human serum high density lipoprotein (HDL3 ) was shown to have the ability to inactivate unstable L-forms of Staphylococcus aureus. The active principle was distinguishable from the welldocumented trypanosome lytic factor 1 with respect to density, size, and other properties. This L-form cytotoxicity therefore seems to represent a novel antimicrobial entity in human serum. Keywords :
High density lipoprotein; Human serum;
Staphylococcus aureus
1. Introduction
L-forms are wall-de¢cient forms of bacteria that can propagate in nutrient media of an appropriate osmotic pressure. In general, plating penicillin-sensitive bacterial cells on an osmotically adjusted agar medium supplemented with penicillin gives rise to Lform colonies with a low frequency, e.g. 1034 . The L-form thus obtained can be subcultured as such if penicillin is present; otherwise massive reversion to the normal coccal or bacillary form takes place. Hence, the designation of unstable L-forms as opposed to stable L-forms, the latter representing rare mutants that remain L-forms even in the absence of
* Corresponding author. Tel.: +81 (92) 642-6331; Fax: +81 (92) 642-6263; E-mail: [email protected]
; L-form ; Bactericidal e¡ect
penicillin probably due to defects in peptidoglycan biosynthesis. Previous studies have shown that supplementing the plating medium with horse serum preheated for inactivation of complement severely represses colony formation of unstable L-forms [1,2], but not that of stable L-forms [2], of Staphylococcus aureus. Here we report that a subfraction of high density lipoproteins (HDLs) from human serum has the ability to inactivate unstable L-forms of S. aureus. This activity is apparently unrelated to the well-documented trypanosome lytic factor 1 (TLF1) [3,4], which was once regarded as an attribute of human HDL but is now ascribed to a haptoglobinrelated protein associated with a fraction sometimes called very high density lipoproteins [5,6]. Thus, the anti-L-form activity is likely to represent a so far unrecognized antimicrobial capacity attributable to lipoprotein in the strict sense.
0378-1097 / 97 / $17.00 ß 1997 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PII S 0 3 7 8 - 1 0 9 7 ( 9 7 ) 0 0 4 1 1 - 4
FEMSLE 7839 10-11-97
114
O. Shimokawa, H. Nakayama / FEMS Microbiology Letters 156 (1997) 113^117
2. Materials and methods
2.1. HDL preparations
Human HDL preparations purchased from Chemicon International (Temecula, CA) were used (LP3; lots 101OCT, 341OEC05, 312OEC06, and C4F060). 2.2. Fractionation methods
The KBr density gradient centrifugation was carried out on stepwise, preformed gradients. A series of solutions with decreasing densities were made by mixing solutions A (0.15 M NaCl) and B (2.62 M NaCl/2.97 M KBr) at ratios of 1:3, 1:2, 1:1.4, 1:1, 1.4:1, 2:1, 3:1, 5:1, and 11:1. Portions (0.8 ml each) of these mixtures were overlaid on top of another in that order followed by 0.8 ml of solution A in 12 ml centrifuge tubes, and HDL (2 mg of protein) dissolved in 2.4 ml of 0.3 mM EDTA (pH 7.5) was then placed on top of each gradient. After centrifugation at 32 000 rpm and 15³C for 48 h in an RPS40T-140 swinging-bucket rotor of the Hitachi 65P ultracentrifuge, 0.25-ml fractions were collected from the bottom of the tube with a peristaltic pump. The density of a solution was assessed by refractometry. Gel ¢ltration with agarose gel [7] was done with a 2U100 cm column of Biogel A-5m (Bio-Rad Laboratories) and 2 mM sodium phosphate (pH 7.2) at 4³C. The dialyzed sample was concentrated to W3 ml before being applied to the column, and the elu-
Fig. 2. Density gradient centrifugation pro¢le for the anti-L-form activity of HDL. Sample, lot C4F060; bracket, active fractions pooled.
ate was collected in 2.8 ml fractions. A¤nity chromatography on ConA-Sepharose (Pharmacia LKB Biotechnology) was performed at 4³C with a 1-ml column and an equilibration bu¡er consisting of 20 mM Tris-HCl (pH 7.2), 1 M NaCl, 1 mM MgCl2 , and 1 mM CaCl2 [8]. The sample was diluted 4-fold with the bu¡er before application to the column. The unadsorbed fraction was eluted with the same bu¡er, and the retained fraction with the bu¡er made 5 M for methyl-K-D-glucoside. Fractions of 0.2 ml each were collected. A¤nity chromatography on Heparin-Sepharose (Pharmacia LKB Biotechnology) was carried out at 4³C with a 1-ml column and an equilibration bu¡er containing 2 mM sodium phosphate (pH 7.4) and 0.05 M NaCl [9]. The sample was dialyzed extensively against the bu¡er before being applied to the column; the unretained fraction was eluted with the same bu¡er, and the retained fraction with a 0.05^2 M linear NaCl gradient made in the bu¡er. 2.3. Assays of anti-L-form activity
Fig. 1. Demonstration of the anti-L-form activity of human HDL. Lacunae on the lawn of L-form colonies are indicative of the activity. Sample, lot C4F060; numbers, magnitudes of dilution in fold.
S. aureus strain KD101 [2] was used for this purpose throughout the present work. This mutant, a derivative of strain 209P, produces unstable L-form colonies with an e¤ciency of W1032 , a ¢gure two orders of magnitude greater than that for the parent, when plated on a solid medium (BHI/NaCl/Ap agar consisting of 3.7% (w/v) brain heart infusion broth (Difco Laboratories), 0.86 M NaCl, 50 Wg ml31 am-
FEMSLE 7839 10-11-97
O. Shimokawa, H. Nakayama / FEMS Microbiology Letters 156 (1997) 113^117
115
Fig. 3. Chromatographic pro¢les of the anti-L-form activity. Active fractions pooled are bracketed. A: Biogel A-5m gel ¢ltration of the density gradient fraction. Vo , void volume. B: ConA-Sepharose chromatography of the Biogel fraction. Arrow, start of elution with 5 M methyl-K-D-glucoside.
picillin, and 1% (w/v) agar) and incubated at 37³C for 2 days. Spot tests for the detection of anti-Lform activity were performed as follows. A liquid L-form culture of KD101 was obtained by gently shaking BHI/NaCl/Ap broth with agar blocks bearing L-form colonies at 37³C overnight. One-ml portions of the liquid culture thus prepared were poured onto BHI/NaCl/Ap agar plates, the excess liquid was removed, and the plates were dried in air at 37³C for 1 h. They were then spotted with 2.5-Wl portions of 2-fold serial dilutions in BHI/NaCl/Ap broth of test samples, and incubated at 37³C for 2 days. The highest dilution of each sample giving a visible growthinhibitory zone was arbitrarily de¢ned as containing one growth-inhibitory unit (U) per ml. Liquid assays for inactivation of L-forms were carried out as follows. From an overnight liquid culture of KD101 Lforms, a 180-Wl portion visibly free of thread-like cell debris was removed and mixed in an Eppendorf tube with 20 Wl of a test sample or 0.86 M NaCl for a control. The mixture was incubated at 37³C by standing, from which 50-Wl samples were taken at
indicated times and spread directly on BHI/NaCl/ Ap agar plates. The plates were incubated at 37³C for 2^3 days. The untreated culture served as the 0time sample. 2.4. Other methods
Protein and total cholesterol were determined by BCA Protein assay reagent (Pierce, Rockford, IL) and Cholesterol C-test (Wako Pure Chemical, Osaka, Japan), respectively. Neutralization tests were done with anti-human apolipoprotein A-I goat serum (Chemicon International). Delipidation of lipoproteins was e¡ected by the standard method [10]. 3. Results and discussion
Having obtained preliminary results implicating HDL in the anti-L-form activity of horse serum [11], we examined human HDL preparations for a similar inhibition by spot tests to ¢nd that human
FEMSLE 7839 10-11-97
116
O. Shimokawa, H. Nakayama / FEMS Microbiology Letters 156 (1997) 113^117
HDL actually had the anti-L-form activity (Fig. 1). In density gradient centrifugation analyses of those preparations, the anti-L-form activity was detected only in fractions of a density between 1.136 and 1.212 g ml31 , roughly corresponding to HDL3 (Fig. 2). Active fractions were pooled and subjected to gel ¢ltration on an agarose column. The anti-Lform activity was found associated with the lipoprotein of a size (215 kDa) typical of HDL3 (Fig. 3A). The overall recovery of the activity up to this step was 62%, and the speci¢c activity increased 4.6-fold. To further con¢rm the association of the activity with HDL, we analyzed the pooled active fractions from the gel ¢ltration by a¤nity chromatography on concanavalin A agarose, known to adsorb apolipoprotein B-containing lipoproteins [8]. The activity passed through the column accompanying the main body of HDL (Fig. 3B), with little increase in the speci¢c activity. The absence of apolipoprotein B in the active fraction indicates that the very low density, low density, and intermediate density lipoproteins, which might possibly have contaminated the active fraction, are not involved in the anti-L-form activity. Additionally, the active principle from the density gradient did not bind to heparin agarose, said to adsorb the apolipoprotein E-containing class of lipoproteins [9]. It seems thus clear that the antiL-form activity of human HDL can be ascribed to the HDL3 subclass lacking apolipoproteins B and E. The possibility that this activity of human HDL may be attributed to TLF1 is highly unlikely from the following argument. First, TLF1 is localized in a fraction with a density of 1.21^1.24 g ml31 (very high density lipoprotein), but not in the typical HDL fraction with the density of 1.136^1.212 g ml31 [5,6]. Second, the size of TLF1 as estimated by gel ¢ltration is not 215 kDa but 500 kDa [5,6]. Third, dithiothreitol, which inactivates TLF1 [5], did not a¡ect the anti-L-form activity (Shimokawa and Nakayama, unpublished results). On the basis of these observations, we call the active principle anti-L-form factor (ALFF). Although ALFF (Biogel fraction) survived heating at 90³C for 60 min, the protein seems to be essential for the activity of ALFF as judged by its sensitivity to protease treatment. Thus, the Biogel fraction (0.92 mg of protein ml31 ) turned inactive upon incubation with trypsin (100 Wg ml31 ) at pH 7.2 and 37³C for 30
Fig. 4. Inactivation of unstable L-forms by ALFF. The ConASepharose fraction was used at a ¢nal concentration of 240 U ml31 .
min. On the other hand, antiserum against human apolipoprotein A-I did not a¡ect the anti-L-form activity of the Biogel fraction, suggesting that this major apoprotein of HDL per se may not play a crucial role in the anti-L-form activity of ALFF. Delipidation resulted in a loss of anti-L-form activity: neither the lipid extract nor the protein residue was active by itself. While it is clear that the lipid moiety alone was insu¤cient for the manifestation of the anti-L-form action, it is obscure whether the inactivity of the protein residue may have resulted from the removal of the lipid or simply from denaturation of the protein. The mode of action of ALFF seems to be inactivation rather than reversible growth inhibition at least under the conditions used. Thus, incubation of L-form cells with the ConA-Sepharose fraction accelerated the loss of colony-forming capacity (Fig. 4). Post-incubation treatment of the mixture with trypsin under the conditions speci¢ed above was unable to reverse the viability loss. Although the L-form inactivation represents a novel biological activity of human HDL, a number of questions remain to be addressed. First, the exact nature of ALFF is not yet known. It may be intact lipoprotein particles, but the possibility exists that some protein(s) alone su¤ces as in the case of TLF1 [6]. Second, we do not know the mechanism for the inactivation of unstable L-forms. Although gross cell lysis as assessed by the release of UV-absorbing substances was absent (Shimokawa and Na-
FEMSLE 7839 10-11-97
O. Shimokawa, H. Nakayama / FEMS Microbiology Letters 156 (1997) 113^117 kayama, unpublished results), this does not eliminate membrane damage as the cause of the inactivation because colony-forming cells represent only a small fraction of the total wall-less cells existing in the population. Third, it is not at all clear how ALFF distinguishes between the stable and unstable Lforms. This question is apparently related to the preceding one, and the elucidation of the action mechanism will provide an answer. Finally, the full implications
of
the
anti-L-form
activity
in
the
host
defence against infection remain to be seen. Conceivably, the anti-L-form activity of HDL might reduce the possibility that treatment of staphylococcal infection with
L-lactam
antibiotics could result in a per-
Staphylococcus aureus :
117
a note of caution on the use of serum
in cultivation of bacterial L-forms. J. Bacteriol. 176, 2751^ 2753. [3] Rifkin, M.R. (1978) Identi¢cation of the trypanocidal factor in normal human serum : high density lipoprotein. Proc. Natl. Acad. Sci. USA 75, 3450^3454. [4] Raper, J., Nussenzuweig, V. and Tomlinson, S. (1996) The main lytic factor of
Trypanosoma brucei brucei
in normal hu-
man serum is not high density lipoprotein. J. Exp. Med. 183, 1023^1029. [5] Hajduk, S.L., Moore, D.R., Vasudevacharya, J., Siqueira, H., Torri, A.F., Tytler, E.M. and Esko, J.D. (1989) Lysis of
panosoma brucei
Try-
by a toxic subspecies of human high density
lipoprotein. J. Biol. Chem. 264, 5210^5217. [6] Smith, A.B., Esko, J.D. and Hajduk, S.L. (1995) Killing of trypanosomes by the human haptoglobin-related protein. Science 268, 284^286.
sistent infection by L-forms. This point would be
[7] Rudel, L.L., Lee, J.A., Morris, M.D. and Felts, J.M. (1974)
more relevant if ALFF should be active on L-forms
Characterization of plasma lipoproteins separated and puri-
of other bacterial species as well. In this connection, it is interesting to note that horse serum inhibits the formation of unstable L-form colonies in
cus faecalis
Enterococ-
([1] ; Shimokawa and Nakayama, unpub-
¢ed by agarose-column chromatography. Biochem. J. 139, 89^ 95. [8] McConathy, W.J. and Alaupovic, P. (1974) Studies on the interaction of concanavalin A with major density classes of human plasma lipoproteins. Evidence for the speci¢c binding of lipoprotein B in its associated and free forms. FEBS Lett.
lished results).
41, 174^178. [9] Nakai, T., Oida, K., Tamai, T., Kutsumi, Y., Hayashi, T.,
References
Takeda, R. and Yamada, S. (1982) Heparin-Sepharose 4B a¤nity chromatography of plasma very low density lipoprotein from streptozotocin-induced diabetic rats. Artery 10, 202^
[1] Kalmanson, G.M., Hubert, E.G., Montgomerie, J.Z. and Guze, L.B. (1968) Serum bactericidal activity against protoplasts. In : Microbial Protoplasts, Spheroplasts and L-forms (Guze, L.B., Ed.), pp. 293^305. Williams and Wilkins, Balti-
221. [10] Scanu, A. (1966) Forms of human serum high density lipoprotein protein. J. Lipid Res. 7, 295^306. [11] Ikeda, M. (1993) Serum inhibition of antibiotic-dependent Lform growth in
more, MD. [2] Shimokawa, O., Ikeda, M., Umeda, A. and Nakayama, H. (1994) Serum inhibits penicillin-induced L-form growth in
Staphylococcus aureus :
likely involvement of
serum high-density lipoprotein. Fukuoka Acta Med. 84, 402^ 410.
FEMSLE 7839 10-11-97
Inactivation of penicillin-induced staphylococcal L-forms by human serum high density lipoprotein Osamu Shimokawa, Hiroaki Nakayama * Department of Microbiology, Faculty of Dentistry, Kyushu University, Higashi-ku, Fukuoka 812-82, Japan
Received 11 August 1997; accepted 6 September 1997
Abstract
In penicillin-susceptible bacteria, penicillin causes growth of a small fraction of cells as wall-deficient forms if an appropriate osmoprotection is provided (unstable L-forms). A subfraction of human serum high density lipoprotein (HDL3 ) was shown to have the ability to inactivate unstable L-forms of Staphylococcus aureus. The active principle was distinguishable from the welldocumented trypanosome lytic factor 1 with respect to density, size, and other properties. This L-form cytotoxicity therefore seems to represent a novel antimicrobial entity in human serum. Keywords :
High density lipoprotein; Human serum;
Staphylococcus aureus
1. Introduction
L-forms are wall-de¢cient forms of bacteria that can propagate in nutrient media of an appropriate osmotic pressure. In general, plating penicillin-sensitive bacterial cells on an osmotically adjusted agar medium supplemented with penicillin gives rise to Lform colonies with a low frequency, e.g. 1034 . The L-form thus obtained can be subcultured as such if penicillin is present; otherwise massive reversion to the normal coccal or bacillary form takes place. Hence, the designation of unstable L-forms as opposed to stable L-forms, the latter representing rare mutants that remain L-forms even in the absence of
* Corresponding author. Tel.: +81 (92) 642-6331; Fax: +81 (92) 642-6263; E-mail: [email protected]
; L-form ; Bactericidal e¡ect
penicillin probably due to defects in peptidoglycan biosynthesis. Previous studies have shown that supplementing the plating medium with horse serum preheated for inactivation of complement severely represses colony formation of unstable L-forms [1,2], but not that of stable L-forms [2], of Staphylococcus aureus. Here we report that a subfraction of high density lipoproteins (HDLs) from human serum has the ability to inactivate unstable L-forms of S. aureus. This activity is apparently unrelated to the well-documented trypanosome lytic factor 1 (TLF1) [3,4], which was once regarded as an attribute of human HDL but is now ascribed to a haptoglobinrelated protein associated with a fraction sometimes called very high density lipoproteins [5,6]. Thus, the anti-L-form activity is likely to represent a so far unrecognized antimicrobial capacity attributable to lipoprotein in the strict sense.
0378-1097 / 97 / $17.00 ß 1997 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PII S 0 3 7 8 - 1 0 9 7 ( 9 7 ) 0 0 4 1 1 - 4
FEMSLE 7839 10-11-97
114
O. Shimokawa, H. Nakayama / FEMS Microbiology Letters 156 (1997) 113^117
2. Materials and methods
2.1. HDL preparations
Human HDL preparations purchased from Chemicon International (Temecula, CA) were used (LP3; lots 101OCT, 341OEC05, 312OEC06, and C4F060). 2.2. Fractionation methods
The KBr density gradient centrifugation was carried out on stepwise, preformed gradients. A series of solutions with decreasing densities were made by mixing solutions A (0.15 M NaCl) and B (2.62 M NaCl/2.97 M KBr) at ratios of 1:3, 1:2, 1:1.4, 1:1, 1.4:1, 2:1, 3:1, 5:1, and 11:1. Portions (0.8 ml each) of these mixtures were overlaid on top of another in that order followed by 0.8 ml of solution A in 12 ml centrifuge tubes, and HDL (2 mg of protein) dissolved in 2.4 ml of 0.3 mM EDTA (pH 7.5) was then placed on top of each gradient. After centrifugation at 32 000 rpm and 15³C for 48 h in an RPS40T-140 swinging-bucket rotor of the Hitachi 65P ultracentrifuge, 0.25-ml fractions were collected from the bottom of the tube with a peristaltic pump. The density of a solution was assessed by refractometry. Gel ¢ltration with agarose gel [7] was done with a 2U100 cm column of Biogel A-5m (Bio-Rad Laboratories) and 2 mM sodium phosphate (pH 7.2) at 4³C. The dialyzed sample was concentrated to W3 ml before being applied to the column, and the elu-
Fig. 2. Density gradient centrifugation pro¢le for the anti-L-form activity of HDL. Sample, lot C4F060; bracket, active fractions pooled.
ate was collected in 2.8 ml fractions. A¤nity chromatography on ConA-Sepharose (Pharmacia LKB Biotechnology) was performed at 4³C with a 1-ml column and an equilibration bu¡er consisting of 20 mM Tris-HCl (pH 7.2), 1 M NaCl, 1 mM MgCl2 , and 1 mM CaCl2 [8]. The sample was diluted 4-fold with the bu¡er before application to the column. The unadsorbed fraction was eluted with the same bu¡er, and the retained fraction with the bu¡er made 5 M for methyl-K-D-glucoside. Fractions of 0.2 ml each were collected. A¤nity chromatography on Heparin-Sepharose (Pharmacia LKB Biotechnology) was carried out at 4³C with a 1-ml column and an equilibration bu¡er containing 2 mM sodium phosphate (pH 7.4) and 0.05 M NaCl [9]. The sample was dialyzed extensively against the bu¡er before being applied to the column; the unretained fraction was eluted with the same bu¡er, and the retained fraction with a 0.05^2 M linear NaCl gradient made in the bu¡er. 2.3. Assays of anti-L-form activity
Fig. 1. Demonstration of the anti-L-form activity of human HDL. Lacunae on the lawn of L-form colonies are indicative of the activity. Sample, lot C4F060; numbers, magnitudes of dilution in fold.
S. aureus strain KD101 [2] was used for this purpose throughout the present work. This mutant, a derivative of strain 209P, produces unstable L-form colonies with an e¤ciency of W1032 , a ¢gure two orders of magnitude greater than that for the parent, when plated on a solid medium (BHI/NaCl/Ap agar consisting of 3.7% (w/v) brain heart infusion broth (Difco Laboratories), 0.86 M NaCl, 50 Wg ml31 am-
FEMSLE 7839 10-11-97
O. Shimokawa, H. Nakayama / FEMS Microbiology Letters 156 (1997) 113^117
115
Fig. 3. Chromatographic pro¢les of the anti-L-form activity. Active fractions pooled are bracketed. A: Biogel A-5m gel ¢ltration of the density gradient fraction. Vo , void volume. B: ConA-Sepharose chromatography of the Biogel fraction. Arrow, start of elution with 5 M methyl-K-D-glucoside.
picillin, and 1% (w/v) agar) and incubated at 37³C for 2 days. Spot tests for the detection of anti-Lform activity were performed as follows. A liquid L-form culture of KD101 was obtained by gently shaking BHI/NaCl/Ap broth with agar blocks bearing L-form colonies at 37³C overnight. One-ml portions of the liquid culture thus prepared were poured onto BHI/NaCl/Ap agar plates, the excess liquid was removed, and the plates were dried in air at 37³C for 1 h. They were then spotted with 2.5-Wl portions of 2-fold serial dilutions in BHI/NaCl/Ap broth of test samples, and incubated at 37³C for 2 days. The highest dilution of each sample giving a visible growthinhibitory zone was arbitrarily de¢ned as containing one growth-inhibitory unit (U) per ml. Liquid assays for inactivation of L-forms were carried out as follows. From an overnight liquid culture of KD101 Lforms, a 180-Wl portion visibly free of thread-like cell debris was removed and mixed in an Eppendorf tube with 20 Wl of a test sample or 0.86 M NaCl for a control. The mixture was incubated at 37³C by standing, from which 50-Wl samples were taken at
indicated times and spread directly on BHI/NaCl/ Ap agar plates. The plates were incubated at 37³C for 2^3 days. The untreated culture served as the 0time sample. 2.4. Other methods
Protein and total cholesterol were determined by BCA Protein assay reagent (Pierce, Rockford, IL) and Cholesterol C-test (Wako Pure Chemical, Osaka, Japan), respectively. Neutralization tests were done with anti-human apolipoprotein A-I goat serum (Chemicon International). Delipidation of lipoproteins was e¡ected by the standard method [10]. 3. Results and discussion
Having obtained preliminary results implicating HDL in the anti-L-form activity of horse serum [11], we examined human HDL preparations for a similar inhibition by spot tests to ¢nd that human
FEMSLE 7839 10-11-97
116
O. Shimokawa, H. Nakayama / FEMS Microbiology Letters 156 (1997) 113^117
HDL actually had the anti-L-form activity (Fig. 1). In density gradient centrifugation analyses of those preparations, the anti-L-form activity was detected only in fractions of a density between 1.136 and 1.212 g ml31 , roughly corresponding to HDL3 (Fig. 2). Active fractions were pooled and subjected to gel ¢ltration on an agarose column. The anti-Lform activity was found associated with the lipoprotein of a size (215 kDa) typical of HDL3 (Fig. 3A). The overall recovery of the activity up to this step was 62%, and the speci¢c activity increased 4.6-fold. To further con¢rm the association of the activity with HDL, we analyzed the pooled active fractions from the gel ¢ltration by a¤nity chromatography on concanavalin A agarose, known to adsorb apolipoprotein B-containing lipoproteins [8]. The activity passed through the column accompanying the main body of HDL (Fig. 3B), with little increase in the speci¢c activity. The absence of apolipoprotein B in the active fraction indicates that the very low density, low density, and intermediate density lipoproteins, which might possibly have contaminated the active fraction, are not involved in the anti-L-form activity. Additionally, the active principle from the density gradient did not bind to heparin agarose, said to adsorb the apolipoprotein E-containing class of lipoproteins [9]. It seems thus clear that the antiL-form activity of human HDL can be ascribed to the HDL3 subclass lacking apolipoproteins B and E. The possibility that this activity of human HDL may be attributed to TLF1 is highly unlikely from the following argument. First, TLF1 is localized in a fraction with a density of 1.21^1.24 g ml31 (very high density lipoprotein), but not in the typical HDL fraction with the density of 1.136^1.212 g ml31 [5,6]. Second, the size of TLF1 as estimated by gel ¢ltration is not 215 kDa but 500 kDa [5,6]. Third, dithiothreitol, which inactivates TLF1 [5], did not a¡ect the anti-L-form activity (Shimokawa and Nakayama, unpublished results). On the basis of these observations, we call the active principle anti-L-form factor (ALFF). Although ALFF (Biogel fraction) survived heating at 90³C for 60 min, the protein seems to be essential for the activity of ALFF as judged by its sensitivity to protease treatment. Thus, the Biogel fraction (0.92 mg of protein ml31 ) turned inactive upon incubation with trypsin (100 Wg ml31 ) at pH 7.2 and 37³C for 30
Fig. 4. Inactivation of unstable L-forms by ALFF. The ConASepharose fraction was used at a ¢nal concentration of 240 U ml31 .
min. On the other hand, antiserum against human apolipoprotein A-I did not a¡ect the anti-L-form activity of the Biogel fraction, suggesting that this major apoprotein of HDL per se may not play a crucial role in the anti-L-form activity of ALFF. Delipidation resulted in a loss of anti-L-form activity: neither the lipid extract nor the protein residue was active by itself. While it is clear that the lipid moiety alone was insu¤cient for the manifestation of the anti-L-form action, it is obscure whether the inactivity of the protein residue may have resulted from the removal of the lipid or simply from denaturation of the protein. The mode of action of ALFF seems to be inactivation rather than reversible growth inhibition at least under the conditions used. Thus, incubation of L-form cells with the ConA-Sepharose fraction accelerated the loss of colony-forming capacity (Fig. 4). Post-incubation treatment of the mixture with trypsin under the conditions speci¢ed above was unable to reverse the viability loss. Although the L-form inactivation represents a novel biological activity of human HDL, a number of questions remain to be addressed. First, the exact nature of ALFF is not yet known. It may be intact lipoprotein particles, but the possibility exists that some protein(s) alone su¤ces as in the case of TLF1 [6]. Second, we do not know the mechanism for the inactivation of unstable L-forms. Although gross cell lysis as assessed by the release of UV-absorbing substances was absent (Shimokawa and Na-
FEMSLE 7839 10-11-97
O. Shimokawa, H. Nakayama / FEMS Microbiology Letters 156 (1997) 113^117 kayama, unpublished results), this does not eliminate membrane damage as the cause of the inactivation because colony-forming cells represent only a small fraction of the total wall-less cells existing in the population. Third, it is not at all clear how ALFF distinguishes between the stable and unstable Lforms. This question is apparently related to the preceding one, and the elucidation of the action mechanism will provide an answer. Finally, the full implications
of
the
anti-L-form
activity
in
the
host
defence against infection remain to be seen. Conceivably, the anti-L-form activity of HDL might reduce the possibility that treatment of staphylococcal infection with
L-lactam
antibiotics could result in a per-
Staphylococcus aureus :
117
a note of caution on the use of serum
in cultivation of bacterial L-forms. J. Bacteriol. 176, 2751^ 2753. [3] Rifkin, M.R. (1978) Identi¢cation of the trypanocidal factor in normal human serum : high density lipoprotein. Proc. Natl. Acad. Sci. USA 75, 3450^3454. [4] Raper, J., Nussenzuweig, V. and Tomlinson, S. (1996) The main lytic factor of
Trypanosoma brucei brucei
in normal hu-
man serum is not high density lipoprotein. J. Exp. Med. 183, 1023^1029. [5] Hajduk, S.L., Moore, D.R., Vasudevacharya, J., Siqueira, H., Torri, A.F., Tytler, E.M. and Esko, J.D. (1989) Lysis of
panosoma brucei
Try-
by a toxic subspecies of human high density
lipoprotein. J. Biol. Chem. 264, 5210^5217. [6] Smith, A.B., Esko, J.D. and Hajduk, S.L. (1995) Killing of trypanosomes by the human haptoglobin-related protein. Science 268, 284^286.
sistent infection by L-forms. This point would be
[7] Rudel, L.L., Lee, J.A., Morris, M.D. and Felts, J.M. (1974)
more relevant if ALFF should be active on L-forms
Characterization of plasma lipoproteins separated and puri-
of other bacterial species as well. In this connection, it is interesting to note that horse serum inhibits the formation of unstable L-form colonies in
cus faecalis
Enterococ-
([1] ; Shimokawa and Nakayama, unpub-
¢ed by agarose-column chromatography. Biochem. J. 139, 89^ 95. [8] McConathy, W.J. and Alaupovic, P. (1974) Studies on the interaction of concanavalin A with major density classes of human plasma lipoproteins. Evidence for the speci¢c binding of lipoprotein B in its associated and free forms. FEBS Lett.
lished results).
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