INFECTION AND IMMUNITY, Sept. 1991, p. 3086-3093 0019-9567/91/093086-08$02.00/0

Vol. 59, No. 9

Heat Shock Proteins and Antigens of Mycobacterium tuberculosis DOUGLAS B. YOUNG* AND THOMAS R. GARBE

Medical Research Council Tuberculosis and Related Infections Unit, RPMS, Hammersmith Hospital, Ducane Road, London W12 OHS, United Kingdom Received 26 December 1990/Accepted

5

June 1991

The heat shock response of Mycobacterium tuberculosis has been characterized in detail by one- and two-dimensional polyacrylamide gel electrophoresis after metabolic labeling with [35S]methionine and "4Camino acids. A temperature increase from 37 to 42°C induced elevated synthesis of three major proteins corresponding to the DnaK, GroEL, and GroES proteins of M. tuberculosis previously identified as prominent antigens. At higher temperatures (45 to 48°C), synthesis of GroEL decreased and novel heat shock proteins with molecular masses of 90, 28, 20, and 15 kDa were observed. These new proteins did not comigrate with known antigens during two-dimensional gel electrophoresis. The heat shock response is discussed with regard to the possible importance of transcriptional regulation of mycobacterial genes in vivo.

Diseases caused by pathogenic mycobacteria remain a major cause of human morbidity and mortality, with tuberculosis alone currently estimated to account for almost 7% of deaths in developing countries, representing approximately 26% of all avoidable adult deaths worldwide (24). With the goal of developing novel strategies for prevention and control of mycobacterial infections, a major research effort has been directed towards elucidation of molecular mechanisms involved in interactions between mycobacterial pathogens and their mammalian host cells. As reviewed extensively elsewhere (13, 38, 41, 42), sequence analysis of several prominent protein antigens of Mycobacterium tuberculosis and Mycobacterium leprae has resulted in their identification as members of highly conserved heat shock protein families, related to the Escherichia coli proteins DnaK, GroEL, and GroES. As a further stage in characterization of these antigens, and as an initial approach to investigating mechanisms of gene regulation in mycobacteria, we report here on a detailed analysis of the effect of heat shock on protein synthesis in M. tuberculosis. Members of the major heat shock protein families from bacteria to humans share a very high degree of sequence identity and represent a significant proportion of the total protein content of all living cells (17, 23). The primary function of these proteins is thought to be as "molecular chaperones," mediating folding and translocation of other proteins within the cell by virtue of their activity as polypeptide chain binding proteins (6, 29). Characteristically, the level of their synthesis is subject to regulation in response to a variety of environmental stimuli in addition to heat shock itself, and this property is reflected in their general description as stress proteins (17, 23). Regulation of the heat shock response in E. coli has been extensively characterized and shown to be mediated by changes in the level of specific RNA polymerase sigma subunits, c32 and SE (8, 10). A link between stress protein synthesis and survival of bacterial pathogens within the mammalian host during infection has been suggested by studies on Salmonella typhimurium, which resembles pathogenic mycobacteria in its ability to survive within host phagocytic cells. Buchmeier and Heffron (3) demonstrated that S. typhimurium DnaK and GroEL are among the most prominent proteins induced *

following entry into host macrophages in a tissue culture system, while Johnson et al. (12) reported that a periplasmic protease (HtrA) identified as a heat shock protein in E. coli plays an essential role in in vivo survival and pathogenicity. Similarly, studies on the intracellular parasite Listeria monocytogenes have led to the suggestion that factors important in host cell interactions may be induced by heat shock (32), and a role for C&32 in the complex regulation of expression of cholera toxin has been uncovered (28). In addition to its relevance with respect to antigen characterization, therefore, it is possible that investigation of the heat shock response in M. tuberculosis will provide information of general importance in understanding host-parasite interactions. MATERIALS AND METHODS

Bacterial growth and heat shock. M. tuberculosis H37Rv (provided by B. W. Allen, Royal Postgraduate Medical School, London) and Mycobacterium bovis bacillus Calmette-Guerin (BCG, Glaxo strain) were grown in Middlebrook 7H9 medium with ADC supplement (Difco Laboratories) with continuous stirring at 37 or 30°C. Cultures used for heat shock experiments were taken from the exponential phase of growth approximately 2 weeks after inoculation of bacterial cultures grown on Middlebrook 7H11 agar with OADC supplement (Difco) into fresh medium. Heat shock was carried out by dispensing 1 ml of culture directly into microcentrifuge tubes which were placed in water baths at the appropriate temperature. After 1 min, 10 ,uCi of [35S]methionine (specific activity 1,00OCi/mmol; Amersham plc) was added to each tube and incubation was continued at the same temperature for 90 min. Unlabeled methionine was then added to a final concentration of 10 mM, and labeled pellets were collected by microcentrifugation for 5 min at 13,000 rpm and washed three times with phosphate-buffered saline. Pellets were resuspended in 50 [lI of deionized water and disrupted by vigorous vortex mixing for 5 to 10 min with an equal volume of 0.1-mm-diameter glass beads. For analysis of the time course of labeling, 10 p.Ci of [35S]methionine was added to a series of samples at 10-min intervals. After a 10-min labeling period, cold methionine was added and samples were transferred immediately to a temperature of 0 to 4°C before being processed for electrophoretic analysis. The response to other stress stimuli was similarly analyzed

Corresponding author. 3086

M. TUBERCULOSIS HEAT SHOCK

VOL. 59, 1991

90-min continuous labeling period after addition of toxic reagents to cultures grown in modified Youman's medium (L-asparagine, 5 g/liter; K2HPO4. 3H20, 6.6 g/liter; citric acid, 1.5 g/liter; MgSO4 7H20, 0.5g/liter; glycerol, 20 g/liter; pH adjusted to 7 with NaOH and ferric ammonium citrate added at 40 mg/liter). Labeling with a '4C-amino acid mixture (specific activity, 50 mCi/matom) was carried out in Middlebrook 7H9 medium with ADC as described above except that the isotope solution was initially freeze-dried to remove ethanol preservative prior to resuspension and addition of the solution to cultures at 10 ,uCi/ml. Electrophoretic analysis. For analysis by polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS), disrupted bacterial pellets were mixed with an equal volume of sample buffer (14) and incubated in a boiling-water bath for 5 min. Discontinuous PAGE was carried out as described by Laemmli (14) by using gels containing 12 or 15% (wt/vol) acrylamide in a minigel system provided by Hoefer Scientific. Proteins were transferred to nitrocellulose filters by electroblotting at 50 V for 1 h as described by Towbin et al. (33), and autoradiographs of the blots were prepared by exposure to X-ray film (Kodak X-Omat AR) for 1 to 3 days at -70°C. Densitometric analysis of autoradiographs was carried out by use of a Shimadzu CS-9000 dual-wavelength, flying-spot scanner. Incorporation of label into different proteins was assessed by measuring the area under individual peaks expressed in absorbance units. For two-dimensional PAGE, urea was added to bacterial extracts to a final concentration of 9 M, followed by addition of lysis buffer containing urea and Nonidet P-40 as described by O'Farrell (27). Samples were separated initially by isoelectric focusing in tube gels containing 4% ampholytes in the pH range of 5 to 7 and 1% ampholytes in the pH range of 3.5 to 10 (27) and then by SDS-PAGE in the second dimension as described above. Autoradiographs were then prepared from nitrocellulose blots. Measurement of the pH in slices prepared from representative gels showed that isoelectric focusing occurred predominantly within the pH range of 5 to 7 under these conditions. Antibody analysis. Defined protein antigens of M. tuberculosis were identified by immunoblot analysis of nitrocellulose filters prepared from one- and two-dimensional polyacrylamide gels by using procedures described previously (19). The following monoclonal antibodies were used: cosll (71-kDa antigen; DnaK) (19), TB78 (65-kDa antigen; GroEL) (4), TB71 (38-kDa antigen) (4), H61.3 (35-kDa antigen) (5), HYT27 (30/31-kDa antigen) (1), D2D (23-kDa antigen) (40), HYT6 (19-kDa antigen) (1), TB68 (14-kDa antigen) (4), and SA12 (12-kDa antigen; GroES) (21).

3087

over a

o9OkD 65kD

-

RESULTS Heat shock response of M. tuberculosis. Metabolic labeling with [35S]methionine was used to monitor the heat shock response of M. tuberculosis cultures exposed to a variety of temperature changes. Analysis of protein synthesis by SDSPAGE and autoradiography clearly demonstrated the induction of a limited set of heat shock proteins but revealed quite distinct differences in the patterns of proteins induced at different temperatures (Fig. 1). Under mild heat shock conditions (37 to 42°C), strong induction of 70- and 65-kDa proteins was evident, while under more severe temperature stress (37 to 48°C), the 65-kDa band was very much less prominent and additional protein bands at 90, 20, and 15-kDa were strongly induced. Although M. tuberculosis cannot

-0-

_

7OkD

(GroEL)

(DnaK)

.

id

-..-2OkD -15kD 4 (dye front) _42 450 480

370

(incubation temperature) FIG. 1. SDS-PAGE analysis of the M. tuberculosis heat shock The effect of heat shock on M. tuberculosis grown at 370C was analyzed by [35S]methionine labeling followed by SDS-PAGE and autoradiography. Mild heat shock (37 to 42°C) resulted in induction of prominent bands at 65 and 70 kDa. At higher temperatures, the 65-kDa band was less prominent and other heat shock proteins were observed with molecular masses of 90, 20, and 15 kDa.

response.

elevated temperatures, it has previously been shown that 90 min of exposure to temperatures up to 500C can be tolerated without loss of viability (36). A quantitative analysis of the heat shock response is shown in Table 1, which records the percentage of total methionine incorporation into each of the bands as measured by a scanning densitometer. Again the biphasic nature of the response is seen in the rise and subsequent decline in incorporation into the 65-kDa band as the temperature is increased, in contrast to the progressive rise, for example, for the 15-kDa band. We have found this pattern to be highly reproducible in many such experiments, although we have observed variations in the actual percentage of incorporation into individual proteins. In some experiments, the characteristic changes in labeling were observed at temperatures 1 to 20C lower than those shown in Table 1, and, even in control cultures at 370C, labeling of the 65-kDa band was found to range from less than 3% to as much as 10% of total incorporation in different experiments. It is possible that these variations reflect some heterogeneity in heat shock protein synthesis and temperature sensitivity of mycobacteria at different stages of their growth cycle. A difference in temperature sensitivity between old and young cultures of M. tuberculosis has been reported (36).

grow at

TABLE 1. Effect of temperature on labeling of individual heat shock proteins Heat shock

protein (kDa)

37°C

90 70 65 20 15

1.6 1.6 3.8 0.0 1.4

% Total label incorporated at:a 45°C 42°C

2.1 9.6 13.4 0.2 2.8

4.3 13.6 12.0 0.3 3.8

48°C

6.8 20.6 2.9 16.1 10.2

Results are expressed as the percentage of the total label incorporated into individual protein bands measured by densitometric analysis of autoradiographs prepared from SDS-polyacrylamide gels as shown in Fig. 1.

3088

INFECT. IMMUN.

YOUNG AND GARBE

A

Western Blot S.: _-

(kD)

_..._.

B (kD) 66-

66_ 36 45

45 36 -

2925 -

29-

dye -

023~~~7

023

-~)Gai 14

dye

acidic

C

400C

19 430 C

basic

acidic'

D

430 C

(kD)

(kD) 66 45

-

6645 36 29 25 -

.0

36 29 25 14 _

.4. I

_

14 Abh

dye basic

dye-

acidic

E (kD)

45 36 29 25

%__

350

25 14 -

14

66

370C

_

~~~~6 C -

.4

basic

acidic

F

480C

(kD)

° -

66 45 36 29 25

-

14

-

0

f 70

62 0

28 *0 -

15

20>% 9

14

dye

0

dye

-

basic

basic

acidic

acidic

FIG. 2. Two-dimensional PAGE analysis of the heat shock response. Extracts from M. tuberculosis labeled with [35S]methionine at different temperatures were analyzed by two-dimensional PAGE and autoradiography of nitrocellulose blots. The blots were also stained with a panel of monoclonal antibodies directed to defined antigens of M. tuberculosis. (A) A representative immunoblot recording the migration positions of antigens with molecular masses (in kilodaltons) as indicated. (B to F) Autoradiographs with the migration positions of six major heat shock proteins (90, 70, 65, 28, 20, and 15 kDa) indicated by arrowheads. Different proteins are prominent at different temperatures. A marked increase in the 15-kDa heat shock protein is seen between panels C and D, for example, and the 20-kDa heat shock protein makes its appearance in panel E. For reference, positions of the major antigens (determined by superimposing autoradiographs with the corresponding immunoblots) are shown by circles on panels B and F.

To determine whether or not the bands observed by SDS-PAGE corresponded to individual proteins, and to establish the relationship between these heat shock proteins and the defined antigens of M. tuberculosis, the heat shock response was analyzed in greater detail by two-dimensional PAGE and immunoblotting (Fig. 2). After electrophoresis, each gel was blotted onto a nitrocellulose filter and the migration positions of defined antigens of M. tuberculosis were determined by staining with monoclonal antibodies (Fig. 2A). The prominent heat shock proteins at 70 and 65 kDa precisely matched the antigens identified by sequence analysis as the mycobacterial DnaK and GroEL homologs (39). The 20-kDa heat shock protein did not overlap, how-

with the 19-kDa antigen (7), and the 15-kDa heat shock protein was significantly more basic than the 14-kDa antigen (7). A further prominent heat shock protein with a molecular mass of 28 kDa was evident on two-dimensional gels, although this was often masked by other similarly sized proteins on a single SDS-PAGE analysis. Although we have detected several novel heat shock proteins in M. tuberculosis, therefore, none of them are included in the panel of antigens recognized by monoclonal antibodies. GroES of M. tuberculosis. Analysis by [35S]methionine labeling failed to show incorporation into the 12-kDa antigen of M. tuberculosis, identified as a GroES homolog by sequence analysis (2, 31). This is consistent with the fact that ever,

VOL. 59, 1991

M. TUBERCULOSIS HEAT SHOCK 4

1.0 -

A

70C

37°C

70kD

37

C

42 OC

B

412

1.5 -

C-12

70kD

0.5-

12kD

_ 65kD

t

3089

u

!2 o

1.0I I

I

45 "C

C

D

48 °C

i

77 66 45

30 17 12 dye molecular weight markers (kD)

,1~ ~ 1

A 15

12613

45°C

--0-70kD 1.5

I I

II

17 12 30 dye molecular weight markers (kD)

1.0 I-

480C -|---70kD

12kD 15kD

/ l2kD

2010 0.5

0.5-

I-4-- 65kD

u

c

-

--12

(OH')

77 66 45

1.0 -

*

65kD

(H)

A 15

(OH-)

A 20

c.-12

(H+)

FIG. 4. Two-dimensional PAGE analysis of 12-kDa heat shock protein. Extracts of M. tuberciulosis cultures labeled with 14C-amino acids were analyzed by two-dimensional PAGE and immunoblotting. Relevant sections from autoradiographs, corresponding to the molecular mass range of 10 to 25 kDa, are shown in panels A to D. A single major protein spot (indicated by arrowheads) was found in the 12-kDa molecular mass position, and this overlapped precisely with the antigen recognized by monoclonal antibody SA12 (GroES). The 15-kDa (C and D) and 20-kDa (D) heat shock proteins are also indicated.

t ~~~15kD

i

D .e. D

I

0

0

11 77 66 45

17 12 30 dye molecular weight markers (kD)

II

1

17 12 dye 30 molecular weight markers (kD)

77 66 45

FIG. 3. Analysis of heat shock response by 14C-amino acid labeling. The effect of heat shock on M. tiubercullosis was analyzed as described in the legend to Fig. 1, except that a "4C-amino acid mixture was used in place of [35S]methionine. Patterns of protein synthesis at different temperatures were compared by SDS-PAGE and autoradiography, with the results presented as densitometric scans. In addition to the heat shock proteins seen in Fig. 1, a major band at 12 kDa was prominent under these conditions.

with that of the antigenically related attenuated vaccine strain M. bov'is BCG. The effect of different induction temperatures was identical in the two strains, with the same characteristic rise and fall of incorporation into GroEL being observed in M. boris BCG (data not shown). The effect was dependent on the extent of heat shock rather than on the absolute temperature, with cultures grown at 30°C showing a heat shock response at 37°C which matched that of cultures

30 c

M. tluberculosis GroES contains no methionine residues, with the initial amino acid (encoded by a GTG codon) being absent from the mature protein (37). Metabolic labeling with a 14C-amino acid mixture, however, clearly demonstrated the presence of a major 12-kDa protein band in autoradiographs prepared from control cultures and during heat shock (Fig. 3). Analysis by two-dimensional PAGE and immunoblotting showed that this band corresponded to a single spot with a migration position overlapping the 12-kDa antigen (Fig. 4). The extent of incorporation of radiolabel into this low-molecular-weight protein suggests that it is being synthesized in very large amounts in molar terms. It was of interest to determine whether the expression of M. tuberculosis GroES resembled the pattern of DnaK with regard to temperature dependence or whether it followed the rise and fall of GroEL. Expression of densitometric results as a percentage of incorporation in fact indicated an intermediate pattern, with a plateau in labeling of GroES at temperatures above 42°C (Fig. 5). Heat shock response of M. bovis BCG. In light of results that indicated a difference in heat shock protein expression in clinical isolates of Listeria monocytogenes differing in virulence properties (32), it was of interest to compare the heat shock response of the virulent M. tiubercullosis H37Rv

0

co a

20

1-

0 -

10

0 0

36

38

40

42 44 46 48 50 temperature FIG. 5. Effect of temperature on DnaK, GroEL, and GroES. Incorporation of radiolabel into M. tuberculosis proteins with molecular masses of 70 (DnaK), 65 (GroEL), and 12 (GroES) kDa was monitored after heat shock at different temperatures in the presence of "4C-amino acids. The results are presented with incorporation into each protein expressed as a percentage of the total incorporation of label as measured by scanning densitometry. Increased labeling of all three proteins was observed during heat shock from 37 to 42°C. At higher temperatures, incorporation into DnaK increased still further while GroES labeling leveled off and GroEL labeling declined.

3090

YOUNG AND GARBE

INFECT. IMMUN.

A40

7OkD

420C

.2 0 m30. 0 C) L. Q 0

2070k D

2OkD

(dye front)

_ 100

0

I

C

I

I

I

.

*

-1

10-20 60-70 labelinig period (mins) 1-11

.40-0

B50

M. bovis BCG

0

0 0.50

M. tuberculosis

0 66

45 35 24 18.4 14.3 molecular weight markers (kD)

FIG. 6. Heat shock response of M. tuberculosis and M. bovis BCG. Cultures of M. tuberculosis and M. bovis BCG grown at 37°C were exposed to heat shock at 48°C in the presence of [35S]methionine. Protein extracts were analyzed by SDS-PAGE, autoradiography, and scanning densitometry. The major heat shock proteins (90, 70, 20, and 15 kDa) overlapped exactly between the two strains, although some differences were seen in minor peaks in the molecular mass range of 26 to 30 kDa.

37°C that were shocked at 42°C. The major proteins of M. bovis BCG induced by severe heat shock (37 to 48°C or 30 to 45°C) showed complete overlap with those of M. tuberculosis, although some differences were noted in the molecular mass range of 26 to 30 kDa (Fig. 6). Analysis by two-dimensional PAGE indicated that several distinct proteins contribute to the apparently simple SDS-PAGE profile seen in this molecular mass range, and further detailed experiments will be required to determine whether or not there is a significant difference in minor heat shock proteins between M. tuberculosis and M. bovis BCG. Kinetics of the heat shock response. Maximum induction of heat shock protein synthesis in E. coli occurs within a few minutes of exposure to heat shock, with a subsequent decline to a steady-state level which reflects the new growth temperature (10, 25). At lethal temperatures, preferential synthesis of heat shock proteins continues in E. coli until the cells die (25). M. tuberculosis and M. bovis BCG have a very slow growth rate (doubling time, 24 h), and a prolonged incubation period of 90 min was required for optimal metabolic labeling. To monitor changes in protein synthesis patterns within this period, cultures were analyzed by a series of 10-min labeling reactions during the period of heat shock. Figure 7 shows a representative example of such an experiment carried out with a culture of M. tuberculosis grown at 37°C and labeled at 42 or 48°C. At 42°C, induction of the 65- and 70-kDa heat shock proteins was observed

grown at

c O

2 40 0

0

o

0

0 C

1 - 11 10 - 20 labeling period (mins)

60 - 70

FIG. 7. Time course of the heat shock response. A culture of M. tuberculosis grown at 37°C was exposed to heat shock at 42 or 48°C, and [35S]methionine was added to different samples at the times shown in the figure. "CC" on the x axis denotes control samples maintained at 37°C. After a 10-min labeling period in each case, extracts were analyzed by SDS-PAGE, autoradiography, and densitometry. At 42°C (A), incorporation of label into the 70- and 65-kDa proteins increased over the first 20 min and continued at the same level during the remainder of the experiment. After transfer to 48°C (B), an initial increase in labeling of the 65-kDa protein was followed by a decline, while the relative incorporation into the 70and 15-kDa proteins showed a progressive increase with time.

during the first 10-min labeling period but was markedly stronger in the second labeling period after 10 min of exposure to the elevated temperature (Fig. 7A). As seen in the continuous-labeling experiments, incorporation into the 65-kDa protein was higher than that into the 70-kDa protein at this temperature. The enhanced synthesis of the two heat shock proteins continued at the same level over a 2-h period at 42°C. After transfer from 37 to 48°C, elevated synthesis of the 65-kDa protein was observed during the first labeling period but was found to decline during further incubation at the high temperature (Fig. 7B). In contrast, incorporation into the 70-kDa protein increased with time at 48°C, and synthesis of the 15- and 20-kDa heat shock proteins became apparent only after about 20 min of exposure to heat shock.

VOL. 59, 1991

M. TUBERCULOSIS HEAT SHOCK

TABLE 2. Induction of heat shock proteins by other stress stimulia Stimulus

Concn (mM)

Cadmium sulfate

0.3 1.2

300RiM Cd2+

% Incorporation in test sample/% incorporation in control 70 kDa

2.2 1.8

3091

65 kDa

1.9 1.8

Cupric chloride

0.3 1.2

1.8 1.6

1.8 1.5

Sodium arsenite

0.3 1.2

3.2 5.0

1.0 0.7

Cumene hydroperoxide

0.1 0.2 0.3

2.8 3.8 3.6

2.0 1.4 1.1

(kD)

-

66 45 36 29 25

9%,

70

O-

665 lo

-

350

230

., _

..

.t e38

28la

20 '

14 dye cd,

Labeling of 70- and 65-kDa proteins was estimated as a percentage of incorporation into total protein by densitometer scanning of SDS-polyacrylamide gels as for Table 1. Results are expressed as the percentage of incorporation in the test sample divided by the percentage of incorporation into the same proteins in a control without addition of stress stimuli. Addition of zinc acetate (0.3 mM) or hydrogen peroxide (0.3, 0.6, and 0.9 mM) failed to stimulate any detectable increase in incorporation into the 70- or 65-kDa protein.

Preferential synthesis of the heat shock proteins (expressed as a percentage of incorporation of label) was maintained over a 2-h period at 48°C, although total protein synthesis declined under these conditions. A similar time course was observed for the heat shock response of M. bovis BCG. In experiments involving cultures grown at 30°C, no elevation in heat shock protein synthesis was detectable during the first 10-min labeling period after transfer to 42°C, indicating an even longer time frame for the heat shock response under these conditions. Other stress stimuli. Many of the E. coli proteins induced by heat shock have also been found to be up-regulated in response to other stress stimuli (34). The extent of induction of the M. tuberculosis 70- and 65-kDa proteins by a variety of toxic metals and oxidative reagents is recorded in Table 2. We were unable to demonstrate induction of heat shock proteins in M. tuberculosis after addition of hydrogen peroxide, although cumene hydroperoxide was found to be an effective inducing agent. We have not excluded the possibility that induction by hydrogen peroxide may actually occur with a time course longer than the 90-min labeling period used in these experiments. Differences in the regulation of DnaK and GroEL were seen in response to some stimuli, with increasing concentrations of sodium arsenite and cumene hydroperoxide preferentially inducing incorporation into DnaK, for example. Induction of low-molecular-weight proteins was also observed with other stress stimuli. Figure 8 shows a two-dimensional PAGE analysis of the effect of cadmium on protein synthesis by M. tuberculosis, with an acidic 15-kDa stress protein being particularly prominent under these conditions. DISCUSSION In spite of the interest generated by the finding of sequence homology between mycobacterial antigens and heat shock proteins, only limited attempts have been made to address the question of whether this structural similarity is reflected by similarities at the level of protein expression. By

basic

acidic

FIG. 8. Cadmium stress of M. tuberculosis. M. tuberculosis was stressed at 37°C by addition of 300 ,uM cadmium sulfate in Middlebrook 7H9 medium with ADC supplement in the presence of [35S]methionine. Analysis of bacterial extracts by two-dimensional PAGE showed prominent labeling of DnaK (70 kDa) and GroEL (65 kDa). A major spot was found at 15 kDa and is labeled 15a to denote its relatively acidic migration position in comparison to that of the 15-kDa heat shock protein. Migration positions of major antigens and heat shock proteins are shown by circles and arrowheads, respectively, along with their molecular masses in kilodaltons.

metabolic labeling, Shinnick et al. (30) showed heat shock induction of GroEL in a fast-growing strain of mycobacteria (Mycobacterium smegmatis), and Mehlert and Young (19) provided similar evidence for induction of DnaK in the attenuated vaccine strain M. bovis BCG. Finally, in a study based on Western blot (immunoblot) analysis of total protein content, Lamb et al. (15) reported evidence for temperature regulation of an 18-kDa protein in Mycobacterium habana. An important aspect of the present study is, therefore, the demonstration that predictions based on sequence analysis of genes encoding the 70-, 65-, and 12-kDa antigens of M. tuberculosis have in fact been verified experimentally. In addition, we have provided evidence of several other novel heat shock proteins in M. tuberculosis. Although immunoblot analysis of two-dimensional gels indicates that these proteins do not correspond to defined antigens identified by monoclonal antibodies, it remains possible that some of these additional heat shock proteins do play a role in the immune response to mycobacteria. If there is an overlap between the proteins induced in response to heat shock and those required for in vivo survival (as suggested by studies of Salmonella and Listeria pathogens [12, 22, 32]), then it might be predicted that some heat shock proteins will be immunogenic during live infection even though they induce little or no response during experimental immunization with killed bacteria or bacterial extracts. It is of interest, therefore, to try to identify these new heat shock proteins. We have previously reported the presence of an open reading frame encoding a putative DnaJ protein of M. tuberculosis (16) and have recently expressed this protein in E. coli and used it to prepare monospecific antisera. The DnaJ protein migrates with a molecular mass (41 kDa) distinct from that of the major heat shock proteins, however, and appears to be only a minor component of control or heat-shocked cells. An open reading frame encoding a GrpE-like protein of M. tuberculosis (predicted molecular mass, 26 kDa) could account for the 28-kDa heat shock protein, and we are cur-

3092

YOUNG AND GARBE

rently pursuing this possibility. It has been suggested that an 18-kDa antigen recognized in M. leprae and M. habana by species-specific monoclonal antibodies belongs to a family of low-molecular-weight heat shock proteins (15, 26), and the 20-kDa protein described here may represent the corresponding M. tuberculosis antigen. By analogy with other bacteria, the prominent high-molecular-weight 90-kDa heat shock protein may be related to the ATP-dependent protease encoded by the lon gene in E. coli (9, 23). In the present study we have focused on the major heat shock proteins of M. tuberculosis, but careful analysis of two-dimensional PAGE separations indicates that there are several other proteins which show distinct but less-dramatic changes in response to temperature increase and these may also provide interesting targets for further investigation. Similarly, proteins induced by other laboratory stress stimuli may also overlap to some extent with those involved in adaptation to intracellular survival in vivo. The pattern of heat shock protein synthesis in M. tuberculosis is dependent on the severity of the temperature stress, with two distinct phases being discernable in the heat shock response. The response to mild heat shock (30 to 37°C or 37 to 42°C) involved induction of DnaK, GroEL, and GroES. In response to higher temperature (30 to 45°C or 37 to 48°C), there was further induction of DnaK, and additional heat shock proteins were found at 90, 28, 20, and 15 kDa. Induction of the GroE proteins was less marked in the second, high-temperature, phase of the response, with labeling of GroEL showing a progressive decrease above 42°C. In view of the 90-min labeling period used in these experiments, it is possible that, in addition to alterations in the rate of synthesis, changes in protein half-life at the different temperatures also make some contribution to these observations. An analogous biphasic response was also seen in time course experiments. Exposure to 48°C resulted in an initial induction of GroEL, with prolonged incubation causing a decline in GroEL and induction of the 15- and 20-kDa heat shock proteins. Studies on the heat shock response in E. coli have focused on genes which are under the transcriptional control of the 32-kDa sigma subunit of RNA polymerase (C432) (10), but other important heat shock genes (including the gene for (32 itself) are transcribed by a second heatshock-related 24-kDa sigma factor (uE) (8, 18). It is possible that the temperature-dependent and time-dependent phases in the M. tuberculosis heat shock response may prove to be analogous to the different o32- and uE-regulated layers of the heat shock response in E. coli. Further analysis of the phenomena described here at the transcriptional level in M. tuberculosis will clearly be required to advance this line of investigation. The mechanism by which the change in temperature is sensed by the organism and the pathway involved in translation of this signal into an alteration in the level and activity of particular transcription factors remain to be clearly defined. It has been proposed that accumulation of abnormal proteins plays a key role in stimulation of the stress response (9), and such a model is particularly attractive in view of the functional role of heat shock proteins in the management of unfolded polypeptides within the cell (29). Abnormal polypeptides may be generated by defective ribosomal activity under stress conditions (35), and the relatively slow kinetics of the heat shock response in M. tuberculosis would be consistent with a lower rate of accumulation of such products, reflecting the lower rate of protein synthesis in a slow-growing organism. The doubling time of M. tuberculosis is approximately 50 times longer than that of E. coli, and

INFECT. IMMUN.

study of such slow-growing bacteria may have an advantage in providing an extended time period in which to analyze the intermediate steps involved in induction of the prokaryotic heat shock response. The second phase of the mycobacterial heat shock response, induced by higher temperatures and by longer incubation times, may be triggered by further increases in intracellular levels of denatured polypeptides resulting from a higher frequency of ribosomal errors. However, it has been shown that alterations affecting chromatin structure can also play a role in temperature-dependent changes in the expression of particular bacterial genes (11), and the possibility of a link between these observations and the different layers of the mycobacterial heat shock response has not been excluded. For many bacterial pathogens it has been demonstrated that proteins which play a key role in host-parasite interactions are coregulated at a transcriptional level in response to environmental signals encountered during infection (20). It is probable that the same considerations apply to mycobacteria, and we suggest that heat shock represents a simple laboratory model for studying coordinated transcriptional regulation in M. tuberculosis. In addition to the interest in heat shock proteins as antigens, therefore, we propose that study of the heat shock response will contribute to an understanding of fundamental aspects of mycobacterial molecular genetics and of host-parasite interactions. REFERENCES 1. Andersen, A. B., Z.-L. Yuan, K. Haslov, B. Vergmann, and J. Bennedsen. 1986. Interspecies reactivity of five monoclonal antibodies to Mycobacterium tuberculosis as examined by immunoblotting and enzyme-linked immunosorbent assay. J. Clin. Microbiol. 23:446-451. 2. Baird, P. N., L. M. C. Hall, and A. R. M. Coates. 1988. A major antigen from Mycobacterium tuberculosis which is homologous to the heat shock protein GroES from Escherichia coli and the htpA gene product of Coxiella burnetii. Nucleic Acids Res. 16:9047. 3. Buchmeier, N. A., and F. Heffron. 1990. Induction of Salmonella stress proteins upon infection of macrophages. Science 248:730732. 4. Coates, A. R. M., J. Hewitt, B. W. Allen, J. Ivanyi, and D. A. Mitchison. 1981. Antigenic diversity of Mycobacterium tuberculosis and Mycobacterium bovis detected by means of monoclonal antibodies. Lancet ii:167-169. 5. Damiani, G., A. Biano, A. Beltrame, D. Vismara, M. F. Mezzopreti, V. Colizzi, D. B. Young, and B. R. Bloom. 1988. Generation and characterization of monoclonal antibodies to 28-, 35-, and 65-kilodalton proteins of Mycobacterium tuberculosis. Infect. Immun. 56:1281-1287. 6. Ellis, R. J. 1990. The molecular chaperone concept. Semin. Cell Biol. 1:1-9. 7. Engers, H. D., and Workshop Participants. 1986. Letter to the editor. Results of a World Health Organization-sponsored workshop to characterize antigens recognized by mycobacteriumspecific monoclonal antibodies. Infect. Immun. 51:718-720. 8. Erickson, J. W., and C. A. Gross. 1989. Identification of the (rE subunit of Escherichia coli RNA polymerase: a second alternate sigma factor involved in high-temperature gene expression. Genes Dev. 3:1462-1471. 9. Goff, S. A., and A. L. Goldberg. 1985. Production of abnormal proteins in E. coli stimulates transcription of Ion and other heat shock genes. Cell 41:587-595. 10. Gross, C. A., D. B. Straus, J. W. Erickson, and T. Yura. 1990. The function and regulation of heat shock proteins in Escherichia coli, p. 167-189. In R. I. Morimoto, A. Tissieres, and C. Georgopoulos (ed.), Stress proteins in biology and medicine. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 11. Hulton, C. S. J., A. Seirafi, J. C. D. Hinton, J. M. Sidebotham, L. Waddell, G. D. Pavitt, T. Owen-Hughes, A Spassky, H. Buc,

M. TUBERCULOSIS HEAT SHOCK

VOL. 59, 1991

12.

13. 14. 15.

16.

17. 18.

19.

20. 21.

22.

23.

24. 25.

26.

and C. F. Higgins. 1990. Histone-like protein Hi (H-NS), DNA supercoiling and gene expression in bacteria. Cell 63:631-642. Johnson, K., I. Charles, G. Dougan, D. Pickard, P. O'Gaora, G. Costa, T. Ali, and C. Hormaeche. 1991. The role of a stressresponse protein in bacterial virulence. Mol. Microbiol. 5:401407. Kaufmann, S. H. E. 1990. Heat shock proteins and the immune response. Immunol. Today 11:129-136. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685. Lamb, F. I., N. B. Singh, and M. J. Colston. 1990. The specific 18-kilodalton antigen of Mycobacterium leprae is present in Mycobacterium habana and functions as a heat-shock protein. J. Immunol. 144:1922-1925. Lathigra, R., D. B. Young, D. Sweetser, and R. A. Young. 1988. A gene from Mycobacterium tuberculosis which is homologous to the DnaJ protein of Escherichia coli. Nucleic Acids Res. 16:1636. Lindquist, S. 1986. The heat shock response. Annu. Rev. Biochem. 55:1151-1191. Lipinska, B., S. Sharma, and C. P. Georgopoulos. 1989. Sequence analysis of the htrA gene in Escherichia coli: a cr32 independent mechanism of heat-inducible transcription. Nucleic Acids Res. 16:10053-10057. Mehlert, A., and D. B. Young. 1989. Biochemical and antigenic characterization of the Mycobacterium tuberculosis 7lkD antigen, a member of the 7OkD heat shock protein family. Mol. Microbiol. 3:125-130. Miller, J. F., J. J. Mekalanos, and S. Falkow. 1989. Coordinate regulation and sensory transduction in the control of bacterial virulence. Science 243:916-922. Minden, P., P. J. Kelleher, J. H. Freed, L. D. Nielsen, P. J. Brennan, L. McPherson, and J. K. McClatchy. 1984. Immunological evaluation of a component isolated from Mycobacterium bovis BCG with a monoclonal antibody to M. bovis BCG. Infect. Immun. 46:519-525. Morgan, R., M. Christman, F. Jacobson, G. Storz, and B. Ames. 1986. Hydrogen peroxide-inducible proteins in Salmonella typhimurium overlap with heat shock and other stress proteins. Proc. Natl. Acad. Sci. USA 83:8059-8063. Morimoto, R. I., A. Tissieres, and C. Georgopoulos. 1990. The stress response, function of the proteins and perspectives, p. 1-36. In R. I. Morimoto, A. Tissieres, and C. Georgopoulos (ed.), Stress proteins in biology and medicine. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Murray, C. J. L., K. Styblo, and A. Rouillon. 1990. Tuberculosis in developing countries: burden, intervention and cost. Bull. Int. Union Tuberc. Lung Dis. 65:6-24. Neidhardt, F. C., and R. A. VanBogelen. 1987. Heat shock response, p. 1334-1345. In F. C. Neidhardt, J. L. Ingraham, K. B. Low, B. Magasanik, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella typhimurium: cellular and molecular biology. American Society for Microbiology, Washington, D.C. Nerland, A. H., A. S. Mustafa, D. Sweetser, T. Godal, and R. A. Young. 1988. A protein antigen of Mycobacterium leprae is

27. 28.

29. 30.

31.

32.

33.

34.

35. 36.

37.

38.

39. 40.

41.

42.

3093

related to a family of small heat shock proteins. J. Bacteriol. 170:5919-5921. O'Farrell, P. H. 1975. High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250:4007-4021. Parsot, C., and J. J. Mekalanos. 1990. Expression of ToxR, the transcriptional activator of the virulence factors in Vibrio cholerae, is modulated by the heat shock response. Proc. Natl. Acad. Sci. USA 87:9898-9902. Rothman, J. E. 1989. Polypeptide chain binding proteins: catalysts of protein folding and related processes in cells. Cell 59:591-601. Shinnick, T., M. Vodkin, and J. Williams. 1988. The Mycobacterium tuberculosis 65-kilodalton antigen is a heat shock protein which corresponds to common antigen and to the Escherichia coli GroEL protein. Infect. Immun. 56:446-451. Shinnick, T. M., B. B. Plikaytis, A. D. Hyche, R. M. van Landingham, and L. L. Walker. 1989. The Mycobacterium tuberculosis BCG-a protein has homology with the Escherichia coli GroES protein. Nucleic Acids Res. 17:1254. Sokolovic, Z., A. Fuchs, and W. Goebel. 1990. Synthesis of species-specific stress proteins by virulent strains of Listeria monocytogenes. Infect. Immun. 58:3583-3587. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76:4350-4354. Van Bogelen, R. A., P. M. Keiley, and F. C. Neidhardt. 1987. Differential induction of heat shock, SOS, and oxidative stress regulons and accumulation of nucleotides in Escherichia coli. J. Bacteriol. 169:26-32. Van Bogelen, R. A., and F. C. Neidhardt. 1990. Ribosomes as sensors of heat and cold shock in Escherichia coli. Proc. Natl. Acad. Sci. USA 87:5589-5593. Wallace, J. G. 1961. The heat resistance of tubercle bacilli in the lungs of infected mice. Am. Rev. Respir. Dis. 83:866-871. Yamaguchi, R., K. Matsuo, A. Yamazaki, S. Nagai, K. Terasaka, and T. Yamada. 1988. Immunogenic protein MPB57 from Mycobacterium bovis BCG: molecular cloning, nucleotide sequence and expression. FEBS Lett. 240:115-117. Young, D., T. Garbe, R. Lathigra, and C. Abou-Zeid. 1990. Protein antigens: structure, function and regulation, p. 1-35. In J. McFadden (ed.), Molecular biology of the mycobacteria. Surrey University Press, Guildford, United Kingdom. Young, D., R. Lathigra, R. Hendrix, D. Sweetser, and R. Young. 1988. Stress proteins are immune targets in leprosy and tuberculosis. Proc. Natl. Acad. Sci. USA 85:4267-4270. Young, D. B., M. J. Fohn, S. R. Khanolkar, and T. M. Buchanan. 1985. Monoclonal antibodies to a 28,000-mol.wt. protein antigen of Mycobacterium leprae. Clin. Exp. Immunol. 60:546-552. Young, D. B., A. Mehlert, and D. F. Smith. 1990. Stress proteins and infectious diseases, p. 131-165. In R. I. Moromoto, A. Tissieres, and C. Georgopoulos (ed.), Stress proteins in biology and medicine. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Young, R. A. 1990. Stress proteins in immunology. Annu. Rev. Immunol. 8:401-420.

Heat shock proteins and antigens of Mycobacterium tuberculosis.

The heat shock response of Mycobacterium tuberculosis has been characterized in detail by one- and two-dimensional polyacrylamide gel electrophoresis ...
2MB Sizes 0 Downloads 0 Views