Vol. 55, No. 12

Mycobacterium leprae Surface Components Intervene in the EarlyPhagosome-Lysosome Fusion Inhibition Event

CLAUDE FREHELL* AND NALIN RASTOGI2Unite de Microscopie Electronique, Departement de Biologie Mole'culaire,' and Unite de la Tuberculose et desMycobacteries, Departement de Bacteriologie et Mycologie,2 Institut Pasteur, 75724 Paris Cedex 15, France

Received 8 June 1987/Accepted 19 August 1987

Bone marrow-derived cultured macrophages were infected with Mycobacterium leprae. The bacteria wereeither used as freshly isolated organisms or incubated with M. leprae antiserum (1:5) for 30 min prior tophagocytosis. Immediately after inoculation (1 to 4 h) and at 1 to 8 days later, macrophages were stained foracid phosphatase activity to assess fusions between phagosomes and lysosomes. Inhibition of fusions wasessentially apparent as an early event, which was partially reversed by antiserum treatment of the bacteria,suggesting a role for M. leprae immunogenic surface components in this early phenomenon. Later incubationtimes (1 to 8 days) did not show any considerable difference between antiserum-treated and nontreatedbacteria. The formation of an electron-transparent zone around phagocytized bacteria and its role inphagosome-lysosome fusion was investigated, and a direct relationship could not be established.

All pathogenic mycobacteria are capable of intracellulargrowth inside the phagosomes of macrophages; however,the mechanisms involved in their intracellular survival differgreatly from one pathogenic species to another (5). It isknown that Mycobacterium tuberculosis mainly escapeshost defense mechanisms by phagosome-lysosome fusioninhibition (1, 2, 10), whereas Mycobacterium avium survivesby both fusion inhibition and electron-transparent zone(ETZ) formation around phagocytized bacteria, the latterlessening the diffusion of lysosomal enzymes towards thebacterial surface (7, 8). The mechanism by which Mycobac-terium leprae resists destruction remains poorly understood.Although it has been stated that M. leprae is capable ofsurvival within the phagolysosomes (5) or of escaping thephagosomal environment (12), the phagosome-lysosome fu-sion inhibition was only recently quantified in the residentperitoneal macrophage system (19). The latter work bySibley et al. (19) emphasized that viable M. leprae hinderedphagosome-lysosome fusion in infected resident peritonealmacrophages; however, contrary to the earlier observationsof Armstrong and d'Arcy Hart (1) with M. tuberculosis,these authors showed that antibody coating of the bacteriaprior to phagocytosis did not revert the phagosome-lysosome fusion inhibition.

Independently of the research by Sibley et al. (19), wehave been actively engaged in elucidating the role of myco-bacterial surface components in their intracellular survival(7, 8, 17). While Sibley's group was engaged in M. lepraeinteractions with resident peritoneal macrophages, utilizingThoria-Sol labeling of secondary lysosomes to assess phago-some-lysosome fusions, we were simultaneously investigat-ing M. leprae-mouse bone marrow-derived macrophage in-teractions, using acid phosphatase (AcPase) cytochemistryto assess phagosome-lysosome fusions (7). The AcPasemethod has an efficacy rate of 80% positive fusions in thecase of macrophages loaded with nonpathogenic bacteria (7).

In a previous study (17), we defined the intracellularsurvival of M. leprae within bone marrow macrophages andassigned a protective role to the ETZ which surrounded thephagocytized bacteria. In the present investigation, we dem-

* Corresponding author.

onstrate that M. leprae surface components intervene in theearly phagosome-lysosome fusion inhibition, and, contraryto the report by Sibley et al. (19), we hereby show that, inour experimental model, specific antiserum coating of M.leprae prior to phagocytosis partially reverts the earlyphagosome-lysosome fusion inhibition. In addition, the useof protein A-gold immunocytochemistry (16) permitted us tofollow the formation of ETZ and to investigate whether arelationship between ETZ formation and phagosome-lysosome fusion inhibition could be established.

MATERIALS AND METHODS

Cells and culture medium. Bone marrow macrophageswere obtained by seeding 2 x i05 bone marrow cells,isolated from 6- to 8-week-old C57BL/6 female mice, per35-mm tissue culture dish (Falcon; Becton DickinsonLabware, Oxnard, Calif.). The culture medium consisted ofDulbecco modified Eagle medium with low glucose (1 g/liter)and high carbonate (3.7 g/liter) concentrations and enrichedwith 10% fetal calf serum heat inactivated for 40 min at 56°C,10% L-cell-conditioned medium, and 2 mM L-glutamine. At4 to 5 days after seeding, the attached cells were rinsed withHanks balanced salt solution and refed with fresh medium.

Bacteria. M. leprae was freshly isolated from an experi-mentally infected armadillo. Infected tissues were manuallyground, using mortar and pestle in the presence of sterilizedsand. To facilitate grinding, Spizizen's salt solution (20) wasadded in small quantities to obtain at the end of grinding notmore than twice the volume of original tissues. The mixturewas centrifuged at 4°C and 800 x g to remove the largerfragments, which were treated similarly two to three times tofurther extract the bacteria. The pooled supernatants werethen centrifuged at 800 x g and 3,000 x g successively toremove the sand plus tissue debris and the larger bacterialclumps, respectively. The bacilli from the 3,000 x g super-natant were recovered by centrifugation at 10,000 x g and40C and washed three times with 250 volumes of Spizizen'ssalt solution to remove the tissue debris. The bacteria werethen treated with 4% (vol/vol) H2SO4 for 10 min at roomtemperature, neutralized with NaOH, and then washed threetimes with 250 volumes of the salt solution. The quality ofthe bacteria at this step was controlled by Ziehl-Neelsen

2916

INFECTION AND IMMUNITY, Dec. 1987, p. 2916-29210019-9567/87/122916-06$02.00/0Copyright © 1987, American Society for Microbiology

on Novem

ber 29, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

PHAGOSOME-LYSOSOME FUSION INHIBITION BY M. LEPRAE 2917

staining and electron microscopy, and the absence of anymicrobiological contamination was verified by plating thebacterial suspension on the usual growth medium.

Antibody-coating experiments. Specific rabbit antiserumagainst total M. leprae antigens prepared and characterizedas reported earlier (13) was a kind gift from F. Papa, Unite dela Tuberculose et des Mycobacteries, Institut Pasteur. Thebacteria were washed and suspended in phosphate-bufferedsaline at an optical density of 0.3 (650 nm), as measured witha Coleman Junior II spectrophotometer, and were thenincubated for 30 min at 20°C with M. leprae antiserum (1:5).A parallel control was performed by incubating the bacteriawith nonimmune rabbit serum (1:5). In both cases, thebacteria were washed twice with phosphate-buffered salineand either used immediately for macrophage infection orpostincubated with protein A-gold as described below.

Infection of macrophage monolayers. The 10-day-old mac-rophages were overlaid with M. Ieprae suspensions (anti-body coated or nonimmune serum coated) to give a bacteria/macrophage ratio of 50 to 100:1. Macrophages were infectedfor 1 to 4 h for assessing the early phagosome-lysosomefusion events. For late events (1 to 8 days), macrophageswere infected for 4 h, washed twice with phosphate-bufferedsaline, and refed with fresh medium. The medium wasrenewed every 5 days.AcPase cytochemistry. Macrophage monolayers were fixed

for 1 h at 4°C (in the culture dishes) with 1.25% glutaralde-hyde in 0.1 M cacodylate buffer (pH 7.2) containing 0.1 Msucrose. They were washed overnight with the same buffer,rinsed once with 0.1 M acetate buffer (pH 5.0), and incu-bated for 30 min at 37°C in prewarmed Gomori reactionmedium (9). Macrophages were rinsed twice with acetatebuffer and once with cacodylate buffer and prepared forelectron microscopy as follows. Cells were fixed for 1 h atroom temperature with 1% osmium tetroxide in 0.1 Mcacodylate buffer. Cells were then scraped off the culturedishes with a rubber policeman, concentrated in agar, andtreated for 1 h with 1% uranyl acetate in Veronal buffer(Winthrop Laboratories, Div. Sterling Drug Co., New York,N.Y.) at a final pH of 5.0. Samples were dehydrated in agraded series of acetone and embedded in Epon. Thinsections were stained with 2% uranyl acetate and leadcitrate. Control experiments performed in the presence of 10mM NaF were negative.

Assessment of fusion. The presence of electron-densematerial within phagosomes after AcPase cytochemistryidentified fusions with lysosomes. To statistically assess thephagosome-lysosome fusion, about 50 macrophages wereexamined per sample, corresponding to 200 to 400 phago-somes in each experiment. The results were calculated as themean of three different experiments. To avoid any statisticalerror, we carefully avoided serial sections, and only profilesexhibiting a nucleus were examined.The respective percentage of phagosomes containing ul-

trastructurally intact (I) and degraded (D) bacteria wascalculated as follows: I bacteria were those maintainingrod-shape, electron opacity, and normal cytoplasmic orga-nization with unbroken cytoplasmic membranes and cellwalls, whereas D bacteria lacked at least one of thesecriteria.

Protein A-gold labeling of antibody-coated bacteria. Anti-body-coated bacteria were incubated with a protein A-goldsuspension (5 nm) in a 1:1 ratio. The protein A-gold suspen-sion was prepared as described earlier (16) and used inexcess.

After 30 min of incubation at 20°C, bacteria were washed

twice with phosphate-buffered saline and either used imme-diately for macrophage infection or processed for electronmicroscopy. Macrophage infection was performed as de-scribed above, and cells were fixed for electron microscopyat 1 to 24 h.

Fixation and embedding for morphological studies. Bacteriawere fixed overnight at 4°C with a mixture of 1.25% (wt/vol)paraformaldehyde and 2.5% (wt/vol) glutaraldehyde incacodylate buffer (pH 7.2; 0.1 M) containing 5 mM Ca2+ andMg2 . Bacilli were then washed for 4 h at 4°C with samebuffer and successively postfixed overnight at 20°C with 1%(wt/vol) OS04 in cacodylate buffer, followed by uranylacetate (1%, wt/vol, in Veronal buffer) for 1 h at 20°C.Dehydration was done in a graded acetone series, followedby embedding in Epon 812. Thin sections mounted onFormvar-coated grids were stained with 2% (wt/vol) uranylacetate and lead citrate and observed under a Siemens 101microscope.Ruthenium red staining of the bacteria was performed as

reported earlier (14) by adding 0.05% (wt/vol) ruthenium redduring fixation and dehydration steps.A similar fixation and embedding procedure was used for

infected macrophages, except that cacodylate buffer alsocontained 0.1 M sucrose and osmium postfixation was per-formed only for 1 h.

Chemicals. Protein A, ,B-glycerophosphate, and glutaral-dehyde grade I were from Sigma Chemical Co., St. Louis,Mo. Paraformaldehyde was from Serva, Heidelberg, FederalRepublic of Germany. Fetal calf serum was from GIBCOEurope, Paris, France. Dulbecco modified Eagle mediumand glutamine were from Biopro, Mulhouse, France.

RESULTS

Quantitative assessment of phagosome-lysosome fusion dur-ing M. leprae infection. As viable counts of M. leprae cannotbe obtained, the ultrastructural intactness and the ATPcontent of these bacilli prior to phagocytosis are a goodindication of their probable viability. Observed under theelectron microscope (Fig. 1A), our M. leprae preparationwas devoid of host tissue debris and also preserved thetypical polysaccharide outer layer (14). Moreover, ourmethod of M. leprae isolation yields bacilli which containedabout 400 pg of ATP per 106 organisms (4), quite similar tothat obtained by Sibley et al. (19).Under the electron microscope, D and I bacteria could be

distinguished from each other, as reported in Materials andMethods. As phagosomes contained clumps of essentiallyheterogeneous M. leprae (three to six bacilli), for eachphagosome the numbers ofD and I bacilli as well as the onessurrounded by electron-dense AcPase deposit were tabu-lated. Figure 1B and C shows AcPase cytochemistry in M.leprae-infected macrophages at 4 and 24 h, respectively.Our results concerning the phagosome-lysosome fusion

during M. leprae infection are presented in Table 1. Phago-some-lysosome fusion inhibition is evident in particular inthe early steps of infection by M. leprae, as the positivephagosome-lysosome fusion index was only about 25%. Thisearly fusion inhibition was independent of the morphologicalstage of the bacteria. Starting from day 1, the positive fusionindex increased to about 60% and remained constant for upto 3 days. However, it decreased to about 40% at day 8 ofinfection.

Effect of antiserum coating on phagosome-lysosome fusion.Figure 2A shows antiserum coating of M. Ieprae by theprotein A-gold method. All bacteria, irrespective of their

VOL. 55, 1987

on Novem

ber 29, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

2918 FREHEL AND RASTOGI

4 r

.-r? -, P ';9 _. '*s'w .d ;

r. ffa

_: ..

jer *s w_E) ';., *, zt* Xs : i

t;k X 8'

i' s >*$' ' s

*;t<'' ', a=T,

:. ;.- ;.: Ent \

,\s .ss 5

.* I"-46

4*

*

A B

FIG. 1. Electron microscopy. (A) Thin section of purified M. leprae suspension stained by the ruthenium red method. The total cell surfacein all bacteria is covered by an ruthenium red-positive deposit. (B) Phagocytized M. Ieprae observed after 4 h of infection in bone marrowmacrophages. AcPase cytochemistry shows the presence of lysosomal enzymes as electron-dense deposits (arrows) around both I and Dbacilli. (C) AcPase cytochemistry of macrophages 24 h after ingestion of M. leprae. AcPase deposits are visible around bacilli (arrows). I,Intact bacteria; D, damaged bacteria; L, lysosomes. Bar, 0.2 ,um.

9

C.

a

INFECT. IMMUN.

v.

on Novem

ber 29, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

PHAGOSOME-LYSOSOME FUSION INHIBITION BY M. LEPRAE 2919

TABLE 1. Phagosome-lysosome fusion in macrophagesinfected by M. lepraea

% Fusionb

Time Control Antiserum

I D T I D T

Phagocytosis1 h 23.8 26.0 24.9 49.1 43.5 46.22 h 23.5 34.8 28.9 50.0 53.9 52.04 h 37.9 37.4 36.1 51.2 54.3 55.0

Postinfection1 day 60.1 59.0 59.2 56.1 53.2 53.53 days 58.2 59.1 58.4 53.1 46.0 53.18 days 43.1 41.2 42.0 44.8 40.1 42.8a Control values correspond to M. leprae treated with nonimmune serum,

whereas the test values (antiserum) correspond to M. leprae treated withspecific antiserum. The percentage represents the number of AcPase-positivephagosomes containing I or D bacteria compared with the total number of I-or D-containing phagosomes of phag-lys fusion. For T bacilli, on the contrary,the percentage is tabulated irrespective of the morphological state of thebacteria. The data represent means of two or three different experiments.

b Control and test values are significantly different during early steps (1 to4 h event; P < 0.005, x2 test) but not during the postinfection period (1 to 8days; P > 0.5, x2 test).

morphological stage of preservation, were found to be cov-ered by specific antiserum. The fate of antiserum-coatedbacilli in the macrophages after infection was verified byelectron microscopy. A typical observation is represented inFig. 2B, showing that most of the phagocytized bacilli werestill covered by the antiserum at 4 h of infection, which isimportant concerning the early phagosome-lysosome fusioninhibition. The initial gold labeling of the M. leprae surface is

6,, ,-.I.,:rf.

located at the periphery of the ETZ (Fig. 2B), an observationsimilar to earlier results of Tereletsky and Barrow (21) withantiserum-coated M. intracellulare.AcPase cytochemistry of the antiserum-coated M. leprae

in macrophages is illustrated in Fig. 3, which shows theabundance of the electron-dense AcPase deposit aroundphagocytized bacteria at 4 h and 3 days of infection.Table 1 shows a quantitative assessment of the phago-

some-lysosome fusion by antiserum-coated M. leprae. Com-pared with the control (Table 1), a partial reversal of theearly fusion inhibition event is demonstrated, as the positivefusion index was about 50% with antiserum-coated bacilliversus about 25% observed with control experiments. Nosignificant variation for D and I bacilli could be observed. Atlater steps of infection (1 to 8 days), this reversal of phago-some-lysosome inhibition was not evident.

Relationship between ETZ formation and phagosome-lysosome fusion. As phagosome-lysosome fusion inhibitionwas an early event, we chose the 1-h infection point todetermine whether a relationship exists between the bacteriacapable of forming ETZ within 1 h of phagocytosis and theessentially early phagosome-lysosome fusion inhibitionevent. No such relationship could be established.

DISCUSSION

It is well established that host protection against myco-bacterial diseases, including leprosy, and recovery aremainly dependent on cell-mediated immunity, in whichT-lymphocyte-macrophage interactions predominate (15).As far as humoral immunity to mycobacterial infections isconcerned, no protective role has yet been assigned toantibodies, and their presence is not always relevant to the

= ;' F. *: g x

W8-' jMjC

4 F w.,. &)

..

.X

X ..... oPS

;w.\. S*)

,+ss; i.- ..., .+.- .B-5 t

_o;

.t EsY.S

AyF.eebP- e ---

JF , R

B- ;1* r % -..-

'ETZ-1,B

FIG. 2. Same bacterial suspension as Fig. 1A, observed after (A) antiserum-coating and subsequent revelation by the protein A-goldmethod and (B) 4 h of phagocytosis by mouse bone marrow macrophages. (A) All bacilli were effectively covered by antiserum (arrows). (B)Gold particles (arrows) are visible around the periphery of bacterial ETZ. Bar, 0.2 p.m.

VOL. 55, 1987

s.-

v. ... ., I

i..AL t

on Novem

ber 29, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

2920 FREHEL AND RASTOGI

rta.-- .ii D s.

t'me' -*

:;

J&.

.

U:A

A"a J

Fl.. *'4.:

."Ad

FIG. 3. AcPase cytochemistry of macrophages infected by antiserum-coated M. leprae after 4 h (A) and 3 days (B) of infection. In bothcases one can observe numerous AcPase-positive deposits around both I and D bacilli. Bar, 0.2 ,um.

stage of infection or to diagnosis (18). Nonetheless, as earlyas 1975, Armstrong and d'Arcy Hart (1) emphasized thatexposure of M. tuberculosis to specific antiserum prior tophagocytosis reverted the usual nonfusion pattern of phago-some-lysosome interactions. This observation has sincebeen limited to M. tuberculosis.For M. leprae, in addition to a recent study that used

Thoria-Sol labeling (19), we hereby demonstrate with theAcPase probe that M. leprae inhibited phagosome-lysosomefusions in bone marrow macrophages. This fusion inhibitionwas essentially an early event (1 to 4 h) and diminishedduring late postinfection (1 to 8 days). These results are

similar to those we observed for M. avium (7), except thatwith M. leprae the early fusion inhibition was independent ofthe morphological state of the bacteria: D bacteria were aseffective in phagosome-lysosome fusion inhibition as I bac-teria. Consequently, our report differed from the recentwork of Sibley et al. (19) on two accounts. First, D bacilli,which actually represented nonviable bacteria, were asefficient as I bacilli in inhibiting phagosome-lysosome fu-sions. Second, this inhibition was observed during the firsthours of phagocytosis (1 to 4 h) and not in later steps (1 to 8days), probably corresponding to the "plateau" period inSibley's data. As both I and D bacteria impaired 1- to 4-hphagosome-lysosome fusions, it can be assumed that in ourmodel system the early fusion inhibition was independent ofbacterial viability.The second part of this study evaluates the influence of

specific antiserum coating of M. leprae prior to phagocytosison phagosome-lysosome fusion and ETZ formation. Ourresults showed that antiserum coating of M. leprae partiallyreversed the early phagosome-lysosome fusion inhibition.As antiserum was used to coat the bacterial surfaces onlyprior to their engulfment, surface components are implicated

in this phenomenon and are probably immunogenic. Thepaper by Sibley et al. (19), on the other hand, does notdemonstrate a significant reversal of phagosome-lysosomefusion inhibition when the bacilli are coated with a specificantiserum. Once again (Table 1), this reversal is only evidentin the early steps of infection, not in the later steps (1 to 8days). Another benefit of the antiserum-coated M. lepraeand their subsequent revelation by protein A-gold labelingwas the observation that, once phagocytized, these bacilliwere still covered by the specific antiserum, as observedearlier for M. avium (21). Although the saturation of cellsurface antigens caused the reversal of phagosome-lysosomefusion inhibition, it did not impair ETZ formation. Conse-quently, it appeared that distinct bacterial components in M.leprae determined phagosome-lysosome fusion inhibitionand ETZ formation. It was earlier postulated that mycobac-terial cell wall components such as mycoside C might beimplicated in ETZ formation and subsequent bacterial pro-tection against host defense mechanisms (6). This study,moreover, suggests that ETZ formation in phagocytized M.leprae is not due to a specific recognition of bacterial surfaceantigens by host macrophages, as ETZ was formed even ifM. Ieprae were antiserum coated prior to phagocytosis.

Despite the limitations of the in vitro macrophage-bacteriainteraction model used in this study, our findings are prom-ising as an aid to understanding host-M. leprae interactions.This investigation suggests not only the role of M. Iepraesurface components in the phagosome-lysosome fusion inhi-bition, but also that specific antibodies might contribute to atleast partial host protection against leprosy by facilitatinghigher phagosome-lysosome fusion in compromised hosts.The mechanism(s) involved in the phagosome-lysosome

fusion inhibition remains to be established for M. leprae andmay be due to one or more of the following: diminished

INFECT. IMMUN.

.1

.r" ...

'c,.

awor

I

.91

4*A Av

on Novem

ber 29, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

PHAGOSOME-LYSOSOME FUSION INHIBITION BY M. LEPRAE 2921

activity or change of enzyme profiles in lysosomal bodies(11); impairment of membrane fusions between differentvacuolar compartments (7); altered intracytoplasmic move-ment of lysosomes (3). We are presently continuing withmacrophage-M. leprae interactions to elucidate this mecha-nism(s) and to identify the mycobacterial components in-volved.

ACKNOWLEDGMENTS

We thank H. L. David and A. Ryter for constructive criticism ofour work and F. Papa for the kind gift of M. leprae antiserum. Theexcellent technical help of R. Daty and S. Cadou is gratefullyacknowledged. We also thank C. Pham for typing this manuscript.

This work was supported by grants from the Institut National dela Sante et de la Recherche Medicale (contract 0620) and the CentreNational de la Recherche Scientifique (UA 04-1148).

LITERATURE CITED1. Armstrong, J. A., and P. d'Arcy Hart. 1975. Phagosome-

lysosome fusion interaction in culture macrophages infectedwith virulent tubercle bacteria. J. Exp. Med. 142:1-16.

2. d'Arcy Hart, P. 1982. Lysosome fusion responses of macro-phages to infection: behaviour and significance, p. 437-447. InM. L. Karnovsky and L. Bolis (ed.), Phagocytosis: past andfuture. Academic Press, Inc., New York.

3. d'Arcy Hart, P., M. R. Young, M. M. Jordan, W. J. Perkins, andM. J. Geisow. 1983. Chemical inhibitors of phagosome-lysosome fusion in cultured macrophages also inhibit saltatorylysosomal movements. A combined microscopic and computerstudy. J. Exp. Med. 158:477-492.

4. David, H. L., N. Rastogi, C. Frehel, and M. Gheorgiu. 1982.Reduction of potassium tellurite and ATP content in Mycobac-terium leprae. Ann. Microbiol. (Paris) 133B:129-139.

5. Draper, P. 1981. Mycobacterial inhibition of intracellular killing,p. 143-164. In F. O'Grady and H. Smith (ed.), "Microbialperturbation of host defences: the Beecham Colloquia. Aca-demic Press, Inc. (London), Ltd., London.

6. Draper, P., and R. J. W. Rees. 1970. Electron-transparent zoneof mycobacterium may be a defence mechanism. Nature (Lon-don) 228:860-861.

7. Frehel, C., C. de Chastellier, T. Lang, and N. Rastogi. 1986.Evidence for inhibition of fusion of lysosomal and prelysosomalcompartments with phagosomes in macrophages infected withpathogenic Mycobacterium avium. Infect. Immun. 52:252-262.

8. Frehel, C., A. Ryter, N. Rastogi, and H. L. David. 1986. Theelectron-transparent zone in phagocytized Mycobacterium

avium and other mycobacteria: formation, persistence and rolein bacterial survival. Ann. Inst. Pasteur (Paris) 137B:239-257.

9. Gomori, G. 1939. Microtechnical demonstration of phosphatasein tissue sections. Proc. Soc. Exp. Biol. Med. 42:23-25.

10. Goren, M. B., P. d'Arcy Hart, M. R. Young, and J. A.Armstrong. 1976. Prevention of phagosome-lysosome fusion incultured macrophages by sulfatides of Mycobacterium tubercu-losis. Proc. Natl. Acad. Sci. USA 73:2510-2514.

11. Katoh, M. 1981. Studies of lysosomal enzymes in macrophagesIII lysosomal enzymes activities in cultured macrophages in-fected with some species of mycobacteria. Hiroshima J. Med.Sci. 30:99-106.

12. Levy, L., N. G. Herman, M. J. Evans, and J. L. Krahenbuhl.1975. Susceptibility of thymectomized and irradiated mice tochallenge with several organisms and the effect of dapsone oninfection with Mycobacterium leprae. Infect. Immun. 11:1122-1130.

13. Papa, F. P., J. Y. Rauzier, and H. L. David. 1984. Occurrence ofantigen BCG160 in leprosy-derived coryneforms. Proceedings ofthe WHO-ESM-SFM Joint Meeting on Mycobacterium leprae,Institut Pasteur. Acta Leprol. (Geneva) 2:351-358.

14. Rastogi, N., C. Frehel, and H. L. David. 1986. Triple-layeredstructure of mycobacterial cell wall: evidence for the existenceof a polysaccharide-rich outer layer in 18 mycobacterial species.Curr. Microbiol. 13:237-242.

15. Rook, G. A. W. 1983. The immunology of leprosy. Tubercle64:297-312.

16. Roth, J., M. Bendayan, and L. Orci. 1978. Ultrastructurallocalization of intracellular antigens by the use of protein A goldcomplex. J. Histochem. Cytochem. 26:1074-1081.

17. Ryter, A., C. Frehel, N. Rastogi, and H. L. David. 1984.Macrophage interaction with mycobacteria including M. leprae.Proceedings of the WHO-ESM-SFM Joint Meeting on Myco-bacterium leprae, Institut Pasteur. Acta Leprol. (Geneva)2:211-226.

18. Sansonetti, P., and P. H. Lagrange. 1981. The immunology ofleprosy: speculations on the leprosy spectrum. Rev. Infect. Dis.3:422-469.

19. Sibley, L. D., S. G. Franzblau, and J. L. Krahenbuhl. 1987.Intracellular fate of Mycobacterium leprae in normal and acti-vated mouse macrophages. Infect. Immun. 55:680-685.

20. Spizizen, J. 1958. Transformation of biochemically deficientstrains of Bacillus subtilis by deoxyribonucleate. Proc. Natl.Acad. Sci. USA 44:1072-1078.

21. Tereletsky, M. J., and W. W. Barrow. 1983. Post-phagocyticdetection of glycopeptidolipids associated with the superficialL1 layer of Mycobacterium intracellulare. Infect. Immun. 41:1312-1321.

VOL. 55, 1987

on Novem

ber 29, 2020 by guesthttp://iai.asm

.org/D

ownloaded from