Phospholipase Activity in Bacteriophage-Infected Escherichia

Transcription

1 JouRNAL of VIROLOGY, May 1975, p American Society for Microbiology Vol. 15, No. 5 Printed in U.S.A. Phospholipase Activity in Bacteriophage-Infected Escherichia coli II. Activation of Phospholipase by T Ghost Infection C. S. BULLER,* M. VANDER MATEN, D. FAUROT, AND E. T. NELSON Department of Microbiology, University of Kansas, Lawrence, Kansas 6605 Received for publication 9 December 197 The release of free fatty acids from the phospholipids of Escherichia coli is initiated immediately after the attachment of T ghosts. A similar accumulation of free fatty acids is observed if the cells are infected with T phage in the presence of chloramphenicol or puromycin. An early accumulation of free fatty acids, however, is not observed in T infections in which chloramphenicol or puromycin are not present, nor does it occur if the E. coli are infected with T phage before ghost infection, suggesting that phage products can prevent the phospholipid deacylation. If E. coli is infected with T ghosts before T phage infection, the accumulation of free fatty acids is not suppressed. When phospholipase-deficient E. coli are infected with T ghosts the appearance of free fatty acids is not observed, suggesting that T ghost attachment can activate the phospholipase of wild-type E. coli. Although the formation of free fatty acid apparently is a consequence of activation of the detergent-resistant phospholipase of the outer membrane, it is not observed in mutants deficient in the detergent-sensitive phospholipase. A variety of agents, including heat, solvents, and detergents can be used to perturb bacterial membrane functions, and one of the consequences is the activation of phospholipase (, 8), resulting in the release of free fatty acids (FFA) from the bacterial phospholipids. Colicins interact with specific receptors in the Escherichia coli cell surface and some can activate host phospholipase (). The major phospholipase of E. coli is membrane bound (, 31), and being latent normally it is likely that its activation signals some structural change or damage to the cell envelope. The attachment of T or T phage to E. coli results in a temporary efflux of cations from the cell (6, 7, 3, 33), also indicative of a change in the cell membrane. Although it has been demonstrated that phage infection leads to phospholipid deacylation before or at the time of lysis (3, 8, 17), it has not previously been shown that the phage attachment process itself can initiate phospholipid deacylation. That the attachment process can alter the membrane is indicated by the immediate block.in transport after T ghost attachment to E. coli (38). Since infection with intact T does not result in blocked transport (38), it is apparent that phage products modify or repair the damage incurred during attachment. In this communication it is demonstrated that the attachment of T ghosts to E. coli results in the deacylation of host phospholipids, and that T phage gene expression is required to suppress the activity. MATERIALS AND METHODS Bacteria and phage. E. coli strains B and K-1 (A) IYMel and wild-type T phage were obtained from L. Astrachan. Timm and Timm-s were received from H. Bernstein and Ts from J. Emrich. The imm locus specifies immunity against superinfecting ghosts and phage in T-infected E. coli (35, 36), and the s gene specifies for resistance to lysis from without (1). E. coli B fad, a mutant which lacks the capability of,-oxidation of fatty acids (3), was obtained from J. Cronan and E. coli S/5,6 from W. Bode. E. coli K-1 DR-DS-, DS-, DR-, and their wild-type parent were obtained from S. Nojima. DR and DS refer, respectively, to detergent-resistant phospholipase A and detergent-sensitive phospholipase A (9,1). High titer phage stocks were prepared from phage lysates either by differential centrifugation or by polyethyleneglycol sedimentation (39). T ghosts were prepared by an osmotic shocking procedure (10) which usually reduced plaque-forming ability by 99.9% or more, and titers were established as described by Duckworth and Bessman (13). Bacteriophage were assayed by the methods described by Adams (1). Media and reagents. Tryptone broth, containing 1% tryptone (Difco) and 0.1 M NaCl, was used as 111

2 11 BULLER ET AL. J. VIROL. growth media. Soft and hard agar for plating phage and bacteria contained 0.6 and 1.5% agar (Difco), respectively. All reagents used in phospholipid extraction and thin layer chromatography were analytical grade. Silica gel, Camag-type DO, without binder, was obtained from Arthur H. Thomas (Philadelphia, Pa.). [1,-10 ]sodium acetate (56. mci/mm) was obtained from New England Nuclear (Boston, Mass.). Chloramphenicol and puromycin were purchased from Sigma Chemical Co. (St. Louis, Mo.). Labeling of phospholipids and assay for FFA. The acyl groups of E. coli phospholipids were labeled by growth of the organisms in tryptone broth containing 0. MCi of [1CJlacetate/ml. The cultures were incubated at 37 C, with aeration, until mid-log phase growth was reached. The cells were collected by centrifugation, and after washing to remove unincorporated [10Jacetate, were suspended in tryptone broth prewarmed to 37 C. These cells were then infected with either T phage or T ghosts and incubated at 37 C with aeration. At various times after infection, 1.0-ml samples were removed for estimation of FFA by using a modification of the procedure of Cronan and Wulff (8). These samples were transferred to screw-cap tubes containing 6 ml of chloroform-methanol (1:, vol/vol) and 0.6 ml of carrier cells (10 mg dry weight/ml). After incubation for at least 1 h at room temperature, the mixture was centrifuged, and the residue was again extracted with 6 ml of chloroform-methanol (:1, vol/vol). The supernatants from the two extractions, containing the E. coli phospholipids and FFAs, were combined and separated into two phases by the addition of 3.8 ml of water, and the chloroform phase was removed by aspiration. After concentration to dryness in a stream of N, the lipids were redissolved in 100 Ml of chloroform and were used for thin layer chromatography. Thin layer chromatography was performed by the methods described by Skipski and Barclay (3). End plates (5 by 0 cm) were coated with Camag gel type DO, without binder. For separation of phospholipid classes, plates were developed in chloroformmethanol-water (65:5:, vol/vol). The FFA were separated from phospholipids by developing the plates in a solvent system containing isopropyl ether/ acetic acid (96:, vol/vol). Lipids were detected by exposing the developed plates to iodine vapors. The radioactivity in the individual spots was determined by quantitatively transferring the gel to vials for counting in a Tri-Carb liquid scintillation spectrometer. To assure complete recovery, all of the gel in a lane was routinely assayed by this procedure. The scintillation fluid used contained 5 g of,5- diphenyloxazole and 0.1 g of dimethyl 1,-bis-- (5-phenyloxazolyl)-benzene in 1 liter of toluene. The identity of the individual phospholipids was established as previously described (). In the FFA assays, the lipid extracts applied to the thin layer chromatography plates contained between 10,000 and 15,000 counts/min of 1C radioactivity. The extent of FFA formation was expressed as the percent of total radioactivity in the free-lipid fraction converted to FFA. In these experiments no attempts were made to assay for the presence of lysophospholipid products. RESULTS Phospholipid hydrolysis by T ghost-infected E. coli. Some of the consequences of T ghost infection of E. coli are suggestive of membrane alterations. It is well known that ghost attachment results in a immediate block in active transport by the host (38) and that the attachment process results in an efflux of cations from the cell (3). Additionally, host cell respiration and macromolecular synthesis are impaired (10, 15, 18). Since many of the enzymes of the membrane are thought to be allotopic (30), it could be expected that degradation of membrane phospholipids might result in membrane dysfunction. Figure 1 illustrates that T ghost attachment to E. coli results in the accumulation of FFA, whereas cells infected with T at multiplicities sufficient to cause lysis inhibition do not demonstrate FFA formation. Although the accumulation of FFA can occur in T-infected cells, it is not observed until just before or during lysis from within (8, 17), or if E. coli B is infected with T rapid-lysis mutants the FFA appearance occurs substantially before lysis (3). In contrast, Fig. 1 illustrates that the appearance of FFA can be observed within min after infection by T ghosts. The assay is based on the appearance of 'C-labeled FFA in cells containing phospholipids with acyl groups labeled before infection by growth in media containing [1C ]acetate. Because unincorporated [1C]acetate is removed before infection, the accumulation of [1C ]FFA cannot be attributed to de novo synthesis after ghost infection. An alternate explanation that [I IFFA are synthesized from acyl thioester precursors derived by p3-oxidation of degradation products of phospholipids can be ruled out by the inability of T ghost-infected E. coli to 10. CL IL 8- - FIG. 1. FFA formation in T ghost-infected E. coli. E. coli B cells with "C-labeled phospholipids were infected with either T phage or T ghosts, at input multiplicities of three. At the indicated times samples were removed and FFA assayed, as described in Materials and Methods. Symbols: 0, ghosts; *, T.

3 VOL. 15, 1975 T GHOST ACTIVATION OF E. COLI PHOSPHOLIPASE incorporate ["CC]acetate into cellular lipids (unpublished data). Effect of chloramphenicol and puromycin on FFA accumulation in T-infected E. coli. The results shown in Fig. 1 indicate that unlike ghost infection, T infection of E. coli does not result in an early formation of FFA. Since T phage and ghosts attach to the host cell surface by the same mechanism, and in doing so induce the same initial type of damage (3), this difference suggests that phage gene expression can result in a modification in the events which are triggered by attachment and with ghosts lead to FFA formation. Alternatively, it is possible that the passage of DNA and/or internal proteins through the membrane in some way prevents FFA formation. T ghosts are devoid of both DNA and internal protein. To distinguish between these alternatives, host cells were incubated with chloramphenicol or puromycin before and during infection with T. Figure indicates that under these conditions an early initiation of FFA formation, similar to that observed in ghost-infected cells (Fig. 1), occurs in T-infected cells. Since the initiation of FFA formation in ghost-infected cells must occur as a consequence of cell surface alterations, these results suggest that phage attachment can produce the same change, but that it can be repaired or modulated by phage gene expression. LL IL -, ) U L. ) A I I I I w 0-- %A A FIG.. FFA formation in T-infected, chloramphenicol- or puromycin-treated E. coli. E. coli B cells, containing "C-labeled phospholipids, were infected with T at input multiplicities of three. Chloramphenicol (CAM), 100 Ag/ml, and puromycin, 00 /g/ml, were added to cultures 10 min before infection and were present throughout the infection. At indicated times samples were removed and FFA assayed as described in Materials and Methods. Symbols: 0, CAM + T; A, puromycin + T; A, puromycin, no T; *, CAM, no T; 0, uninfected and no CAM or puromycin. I i.1p.. T phage suppression of T-ghost induced FFA formation. E. coli cells infected with T ghosts, and subsequently with T phage, are unable to produce progeny (15). If the cells are first infected with T, they become resistant to the killing action of ghosts and progeny phage are produced (1,, 35, 36). The effect of T-phage gene expression on the ghost-induced activation of FFA formation in E. coli is shown in Fig 3. As expected from the results of experiments shown in Fig., the primary infection with T prevented early FFA accumulation after ghost infection. These results indicate that the phage products responsible for the suppression are synthesized within 5 min after T infection. The ghost activated release of FFA, however, cannot be suppressed by superinfection with T. It has been reported that mutations in the imm (35, 36) and s (6, ) genes of T result in decreased immunity of T-infected E. coli against the lethal effects of T ghost attachment. E. coli infected with Timm do not form FFA before the time of lysis, indicating that the imm gene product is not directly involved in the suppression of early FFA formation (Fig. ). If E. coli is infected with a double mutant Timm-s, FFA formation is observed beginning at about 10 min after infection. In other experiments it was observed that Ts-infected cells also demonstrated initiation of FFA formation at 10 min (data not shown). FFA formation has been observed at about 10 min after infection of E. coli by Trll (3). Ts mutants, in addition to being unable to cause resistance to 10 <: 8 IL c6 L 0~L r I 0w W Minutes after Secondary Infection 113 FIG. 3. Effect of T phage on FFA formation in ghost-infected cells. E. coli B, containing "IC-labeled phospholipids, was infected with either T or T ghosts, at input mutiplicities of three. Superinfection, with either T or ghosts as indicated, at input multiplicities of three, was performed at 5 min after primary infection. At designated times samples were removed and FFA assayed as described in Materials and Methods. Symbols: 0, ghosts, then T; 0, ghosts only; 0, T only; *, T, then ghosts.

4 11 BULLER ET AL. J. VIROL. LL LL L) u L. 0) w I -. FIG.. FFA formation in E. coli infected with T imm -s. Wild-type E. coli K-1, with phospholipids labeled before infection by growth in media with [1C]acetate, as described in Materials and Methods, were infected with indicated phage at input multiplicities of eight. At indicated times samples were removed and assayed for FFA, as described in Materials and Methods. Symbols: *, T imm -s; 0, T imm ; and A, wild-type T. lysis from without, phenotypically resemble Tr in being rapid lysis mutants (1). Effect of T ghost infection of phospholipase-deficient E. coli. The preceding experiments (Fig. 1,, and 3) indicate that T ghost, and under certain conditions T phage attachment to E. coli, result in an early accumulation of FFA. Since ["C ]acetate is not present during the infection, it can be concluded that the "C-labeled FFA observed are derived from lipids synthesized before infection. If derived from phospholipids it would require that ghost attachment activates host phospholipase A. Although it is clear that E. coli contain phospholipase A activity (, 9, 1,, 5, 31), the low level of FFA, normally about 1% of the total free lipid (8), suggests that the activity may be suppressed. To determine if the FFA accumulation could be attributed to activation of phospholipase A, we compared the levels of FFA in wild-type and phospholipase deficient E. coli K-1 strains after T ghost infection. The principal phospholipase activities of E. coli have been characterized as DR and DS (9, 1). The DR phospholipase is capable of deacylating phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and cardiolipin, whereas the DS enzyme has specificity for PG. FFA formation begins by min after T ghost infection of wild-type E. coli, but none is observed in phospholipase-deficient DR-DS -, DS-, or DR- (Fig. 5). Although the DR-DS- mutant is not completely devoid of phospholipase (0,1), these experiments indicate that the FFA formation observed after ghost infection is dependent upon the presence of the DR and DS enzymes. Table 1 represents the results of experiments in which the free lipids were extracted and fractionated into FFA and the phospholipid U- -ti (9) L. (L) FIG. 5. FFA formation in wild-type and phospholipase-deficient E. coli K-1. Cultures of wild-type and DS-, DR-, and DR-DS- strains of E. coli K-1, each containing equivalent numbers of log-phase cells with phospholipids labeled to approximately the same extent by growth in ["C]acetate media, were infected with T ghosts at input multiplicities of four. At indicated times samples were removed and assayed for FFA, as described in Materials and Methods. Symbols: A, wild type; A, DR-DS-; 0, DR-; 0, DS-. TABLE 1. Effect of T ghost infection or heat on free lipid composition of E. coli K-1a Strain AA.. % of total FFA CLb PE PG Wild type Controlc Ghost infectedd Heatede DR-DS- Controlc Heatedd a Phospholipids of wild-type E. coli K-1 and DR-DS- were labeled by growth in media containing [1C Jacetate, as described in Materials and Methods. At 30 min after ghost infection or after heating, free lipids were extracted and fractionated as described in Materials and Methods. Results are an average of two independent experiments. b CL, Cardiolipin. c Control, uninfected, incubated at 37 C. d Infected with T ghosts at input multiplicity of three, incubated at 37 C. euninfected, but incubated 30 min at 55 C to activate phospholipase. A

5 VOL. 15, 1975 T GHOST ACTIVATION OF E. COLI PHOSPHOLIPASE 115 classes by thin layer chromatography. The infection of wild-type E. coli with T ghosts resulted in a decrease in the amount of label in PE and PG and an increase in FFA. Since there are no known pathways in E. coli in which PE is a precursor for other phospholipids, and since the DR enzyme is its only known phospholipase capable of PE deacylation, we conclude that the DR phospholipase is activated after ghost attachment. Although the decrease in PG is in agreement with the activation of the DR enzyme, it may also occur as a consequence of its conversion to cardiolipin (7). This is further suggested by the decrease in PG and increase in cardiolipin after incubation of DR-DS - at 55 C for 30 min. Although Table 1 indicates that the DR enzyme is activated, the possibility that the DS enzyme is also involved cannot be excluded. This is further suggested by the absence of FFA formation in ghost-infected DS- (Fig. 5), suggesting that the DR phospholipase is not activated unless the DS enzyme is present. Since the DR enzyme is the major phospholipase, and since PG comprises only 15 to 0% of the phospholipids of E. coli (), it is likely that DS activity would be obscured by that of the DR enzyme. DISCUSSION It is well known that the attachment of T ghosts to E. coli results in the inhibition of active transport (38), synthesis of macromolecules (11), and of respiration (15,18). It is difficult to visualize that all of the membrane proteins involved in these diverse activities could be primary targets of ghost action. Since many membrane proteins may be allotopic (30) it is likely that their activity is altered by changes in their lipid environment. Accordingly, we have examined the effect of ghost attachment on the metabolism of E. coli phospholipids. Our experiments indicate that T ghost attachment results in deacylation of E. coli phospholipids, a process that is initiated within min after infection. The production of FFA is not observed in the infection of an E. coli mutant deficient in both the detergent-resistant and detergent-sensitive phospholipase A activities. Although the DR-DS- mutant is not completely devoid of phospholipase (0, 1), heating cultures of it at 50 C for 1 h, to activate any residual phospholipase, does not result in a decrease in the amount of "C radioactivity in PE (Table 1). Yet the 1CJ activity in PE is clearly reduced after infection of wild-type E. coli with T ghosts (Table 1). Although PG can be used as a precursor for CL synthesis (7), there are no known pathways in E. coli whereby PE is involved as a precursor in the synthesis of other phospholipids. Whereas changes in the relative amounts of each of the phospholipid classes synthesized after infection have been observed in T- or Tr-infected E. coli (16, ), these depend upon phage gene expression and therefore cannot account for the changes observed with ghost-infected E. coli. We therefore conclude that the FFA formation observed after ghost attachment occurs as a consequence of activation of a phospholipase A. We have not at this time made attempts to determine if lysophospholipids also are formed. Other strains of E. coli, including BB, S/5,6, and a B fad mutant were also examined. Early FFA formation was observed in all strains after ghost infection and the levels obtained were approximately the same in each (data not shown). The absence of early FFA formation after ghost attachment to E. coli previously infected with T (Fig. 3) suggests that a phage product(s) can prevent the activation of phospholipase. This is further suggested by the rapid appearance of FFA in cells infected with T phage in the presence of puromycin or chloramphenicol. The formation of FFA in chloramphenicol-treated, T-infected E. coli has been reported (8). Their experiments differed from ours in that they did not measure FFA until a late stage in the infection, and in contrast to our results, they found the puromycin-treated, T- infected E. coli did not accumulate FFA. We have no explanation for this difference, nor do we have evidence that a phage product directly inhibits the host phospholipase. It is quite plausible that phage gene products, which normally interact with the membrane, stabilize it in such a way as to keep the phospholipase suppressed. We have previously reported that T phage and ghosts themselves contain a phospholipase activity (0). The experiments described in Table 1 indicate that PE, and possibly PG, are deacylated after ghost infection. The phageassociated phospholipase has specificity for PG (0), and therefore it is not the main activity leading to FFA formation after ghost infection. Figure 5 indicates that very little FFA formation is observed in T ghost-infected E. coli K-1 DS-, suggesting that the DS enzyme of the inner membrane is activated first, and that this in turn leads to the activation of the DR enzyme. For example, the products of phospholipase activity (FFA or lysophospholipids) themselves, by acting as detergents, may activate other phospholipases. It is not likely that

6 116 BULLER ET AL. J. VIROL. the DR enzyme activity is dependent upon cofactors or subunits of the DS enzyme since the former is localized in the outer membrane fractions (), whereas the DS activity is found in supernatant fractions (1). Additionally, sonicates of the DS- cells retain DR activity (1). Since the DR enzyme, found exclusively in the outer membrane (), apparently is activated, it may account for the release of outer membrane components which is observed immediately but transitorily after T phage infection (19). We are currently investigating this, using wild-type E. coli K-1 and its phospholipase deficient mutants. Phospholipid hydrolysis, occurring as a secondary event related to membrane changes during normal lysis of E. coli K-1 by X phage, has been observed and it was concluded that the S cistron is responsible for the membrane alteration (9). We have not examined the effects of T ghosts on E. coli K-1 strains cured of X. Since FFA formation is not observed in X lysogenized E. coli K-1 DR-DS-, DS-, or DR- (Fig. 5), we believe it unlikely that X phage gene expression is involved in the phospholipase activation observed in T ghost infection of wild-type E. coli K-1. While this manuscript was in preparation it was reported that T ghost-infected E. coli BB, and chloramphenicol treated T phage-infected E. coli BB, were more sensitive to sodium dodecyl sulfate lysis than either untreated cells, or those infected with T in the absence of chloramphenicol. One of the changes observed in ghost-infected cells was an increase in lysophosphatidylethanolamine (5), suggestive of phospholipase activity. At this time we have no data which indicate that the activation of phospholipase is the primary cause of the membrane-associated functional changes occurring after ghost infection. That it is not the primary cause of cell death occurring after ghost attachment is indicated by experiments which demonstrate that phospholipase-aeficient E. coli DR-DS- are killed by T ghost infection (37). ACKNOWLEDGMENTS This investigation was supported by Public Health Service research grant AI from the National Institute of Allergy and Infectious Diseases. E. T. Nelson was supported by Public Health Service predoctoral training grant GM-703 from the National Institute of General Medical Science. M. Vander Maten was supported by National Defense Education Act Title IV predoctoral Fellowship We are indebted to Leonor Valencia for her expert technical assistance. LITERATURE CITED 1. Adams, M. H Bacteriophages, p. Interscience Publishers, Inc., New York Wiley-. Bell, R. M., R. D. Mavis, J. J. Osborn, and P. R. Vagelos Enzymes of phospholipid metabolism: localization in the cytoplasmic and outer membrane of the cell envelope of Escherichia coli and Salmonella typhimurium. Biochim. 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