ELECTRON MICROSCOPE STUDY OF MYCOBACTERIUM LEPRAE ANDITS ENVIRONMENT IN A VESICULAR LEPROUS LESION

TAMOTSU IMAEDA AND JACINTO CONVIT

Instituto Venezolano de Investigaciones Cientificas and Divisi6n de Lepra, Caracas, Venezuela

Received for publication June 19, 1961

ABSTRACT

IMAEDA, TAMOTSU (Instituto Venezolano deInvestigaciones Cientificas, Caracas, Venezuela)AND JACINTO CONVIT. Electron microscope studyof Mycobacterium leprae and its environment in avesicular leprous lesion. J. Bacteriol. 83:43-52.1962.-Biopsied specimens of a borderlineleprosy lesion were observed with the electronmicroscope. In this lesion, the majority of Myco-bacterium leprae were laden with cytoplasmiccomponents. The bacilli were separated from thecytoplasm of host cells by an enclosing mem-brane, thus differing from the environment of well-developed lepra cells in lepromatous lesions.The cell wall is composed of a moderately dense

layer. A diffuse layer is discernible outside thecell wall, separated from it by a low density space.It is suggested that the cell wall is further coatedby a low density layer, although the nature of theoutermost diffuse layer has not yet been deter-mined.The plasma membrane consists of a double

layer, i.e., dense inner and outer layers separatedby a low density space. The outer layer is closelyadjacent to the cell wall. In the region where theouter layer of the plasma membrane enters thecytoplasm and is transformed into a complexmembranous structure, the inner layer enclosesthis membranous configuration. Together theyform the intracytoplasmic membrane system.

In the bacterial cytoplasm, moderately dense,presumably polyphosphate bodies are apparent.As neither these bodies nor the intracytoplasmicmembrane system are visible in the degeneratingbacilli, it seems probable that these two compo-nents represent indicators of the state of bacillaryactivity.

Submicroscopic structures of Mycobacteriumleprae have been studied with the use of bothultrathin sections of leprous lesions and bacilliseparated from lesions (Bishop, Suhrland, andCarpenter, 1948; Malfatti, 1951; Yamamoto

et al., 1958a; Chatterjee, Das Gupta, and De,1959; De Souza-Araujo, 1960; McFadzean andValentine, 1960). In none of these cases, however,was the cytoplasmic structure of the bacilliclearly defined.

Recently, Brieger, Glauert, and Allen, (1959)observed an intracytoplasmic membrane in thisbacillus, using organ culture of lepromas.

Fortunately, we encountered a biopsied tissuewhich contained many bacilli and disclosed theirfine structure. The present study was undertakento elucidate the submicroscopic structure ofM. leprae and its bacillary environment, whichhave never been observed before in biopsiedspecimens.

MATERIALS AND METHODS

The material employed in this study was abiopsied specimen from a case of borderlineleprosy which had been diagnosed both clinicallyand histopathologically. This lesion showedreddish infiltration and a small blister.The specimen was fixed with 1% osmium

tetroxide, buffered with 2,4 ,6-collidine (0.2 M) atpH 7.35, for 6 hr (Hama, 1959). Tissue chips weredehydrated with acetone and embedded in a mix-ture of n-butyl (80%) and methyl methacrylate(20%). Ultrathin sections were obtained bymeans of an ultramicrotome (Leitz Ultra-Miko-tom, Fernindez-Monin, Germany) equippedwith a diamond knife. After sectioning, some ofthe ultrathin sections were treated for 15 minwith 2% uranyl acetate solution (modificationof the method of Watson, 1958). The electronmicroscope used was a Siemens Elmiskop I at60 kv.To demonstrate M. leprae in a leproma, one of

the nodules of a lepromatous patient was ex-

amined in the same manner as mentioned above.

RESULTS AND DISCUSSION

Bacillary environment in the host cell. Electronmicrographs of this lesion show many bacilli dis-tributed in the cytoplasm of the histiocytic cells

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44 IMAEDA AND CONVIT

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FIG. 1. Mycobacterium leprae abundantly distributed in the cytoplasm of the host cell. These bacilli are

separated from the cytoplasm by the enclosing membrane (EM). A homogeneously dense substance, sup-

posedly the remnant of opaque droplets, attaches to the bacilli (arrow). In the lower part of this picture, a

bacillus (Bd) shows the cytoplasmic condensation.

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FIG. 2. Cell wall (CW) consisting of a moderately dense layer. The plasma membrane (PM) appears tobe composed of a double layer separated by a low density space. The inner layer here seems to recede withthe bacterial cytoplasm. The outer layer of the plasma membrane adheres closely to the cell wall. In a regionindicated by arrows, the plasma membrane enters the bacterial cytoplasm and is transformed into an intra-cytoplasmic membrane system, generally limited by the inner layer of the plasma membrane. Nuclear ap-paratus (Ar) is visible in a low density area. PB: polyphosphate body.

FIG. 3. Slightly dense, diffuse layer (double arrow) is separated from the cell wall by a low density layer.The latter is also apparent between the cell wall and the clinging dense substance (arrow). CM: intracyto-plasmic membrane system.

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an irregular shape near the nuclear region. The remnant of the opaque droplet does not attach directly to the

cell wall, but is separated by a low density space (arrow).

(Fig. 1). The bacilli are not surrounded by amoderately dense substance, which was called"opaque droplet" by Yamamoto et al. (1958a, b),but are enveloped by a i-embrane separatingthem from the cytoplasm of the host cells. Thisenclosing membrane, clearly distinguishable fromthe bacterial cell wall and sometimes includingseveral bacilli (Fig. 1 and 3), may be the remnantof the host cell membrane taken into the cyto-plasm when the bacilli were phagocytized, assuggested by Chapman, Hanks and Wallace(1959) in their study of -11. lepraemurium. The

space inside the enclosing membrane is usuallyelectron transparent, but occasionally containsfine granules.

It should be noted that the enclosing membraneobserved in this lesion does not frequentlyappear in the case of lepra cells. In lepromas, ahost-cell reaction to bacillary reproduction occursinactively and, in consequence, the bacilli pro-liferate rapidly, forming glomerations termed"globi". Furthermore, a single bacillus, or agroup of bacilli, is surrounded by an opaquedroplet and foamy structure (Fig. 9) , probably of

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M. LEPRAE IN LEPROUS LESION

FIG. 7. Intracytoplasmic membrane system composed of the outer layer of the plasma membrane (doublearrow) and the enclosing inner layer of the plasma membrane. A low density space (arrow) is visible be-tween the cell wall and the remnant of the opaque droplet.

FIG. 8. Parallel arrangement of the intracytoplasmic membrane system (CM). In the region indicated byan arrow, both the inner and outer layers of the plasma membrane are taken into the cytoplasm.

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IMAEDA AND CONVIT

..............42gS............

FIG. 9. A lepra cell in a well-developed leproma of another lepromatous patient. The majority of thebacilli show cytoplasmic condensation, and consequently their fine structures are not clearly observed. Thesebacilli are partially enclosed by opaque droplets (OD) and foamy structures (FS). Note the difference be-tween the bacillary environment of this lesion and that of the borderline lesion shown in other figures.

lipid nature, as a result of the abnormal lipidmetabolism of the cell in lepromatous leprosy(Imaeda, 1960).An osmiophilic substance, thought to be the

remnants of opaque droplets, was found clingingto the bacilli within one enclosing membrane(Fig. 1, 3, 6, and 7). From this evidence it is sur-mised that the cells containing the bacilli,wrapped in opaque droplets, may be broken downthrough a destructive process, and that the bacillireleased to extracellular spaces may be phago-cytized again by the histioeytie cells.

In other words, one may be able to observe theinitial stage of cytoplasmic changes after phago-cytosis. During a secondary stage these enclosingmembranes may disappear and opaque dropletsmay take their place by wrapping around thebacilli. Of course, this process occurs only inborderline and lepromatous leprosy lesions. Thebacillary environment in this lesion is quite dif-ferent from that of any other well-developedleproma, although the process is the same.

Bacterial cell wall and plasma membrane. It hasalready been reported bv Yamamoto et al.

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M. LEPRAE IN LEPROUS LESION

(1958a) that the cell wall of M. leprae consists ofthree layers. In the studies of other mycobacteria,such as M. avium, M. tuberculosis var. hominisH37Rv and B.C.G., and M. leprafmurium, it hasbeen shown that their cell walls are also composedof three layers (Fukushi, 1959; Chapman et al.,1959; Shinohara et al., 1959; Takeya, 1959).In the present study, the cell wall, which ap-

pears to be 6 to 10 m,A wide, shows a moderatelydense layer (Fig. 2). Adhering closely to the cellwall, a dense layer approximately 3 m,u thick isvisible. This dense layer is believed to be theouter layer of the plasma membrane, as discussedin a later paragraph.

Outside the cell wall, a slightly dense, diffuselayer, separated from the cell wall by a lowdensity space, is frequently discernible (Fig. 3and 7). When moderately dense droplets attachto the bacilli, a low density space is always visiblebetween the cell wall and the droplets (Fig. 1, 3,6, and 7). Since these droplets are closely adjacentto the outermost diffuse layer of the cell wall, thespace between the cell wall and the droplets maynot be simply a shrinkage space. Therefore, thisfinding may be indicative of the fact that the cellwall is coated by a nonosmiophilic substance,supposedly of a waxy nature.

It is difficult to determine whether the slightlydense diffuse layer covering the outermost surfacearound the bacillus belongs to one of the com-ponents of the bacillary surface, or whether it isa host-cell product absorbed on the coating sub-stance of the cell wall.Many bacilli in the lepromas (Fig. 9) and also

in the later stage of reactional tuberculoid lesions(Nishiura, 1960) show a cytoplasmic condensa-tion which is believed to be a sign of bacillarydegeneration. In the initial stage of the bacillarydegeneration, the coating substance is still ob-served, although the bacilli already show thecytoplasmic condensation. However, this coatingsubstance is no longer visible in the degeneratedbacilli in either the old leproma or the later stageof reactional tuberculoid lesions. Based on thesefacts, it would seem that the coating substancearound the bacterial cell wall may disappear in thelast stage of bacillary degeneration.The plasma membrane is composed of a double

dense layer of about 30 A in thickness, separatedby a low density space of about 30 A in width(Fig. 2, 4, 5, 7, and 8). The outer dense layer isin the most intimate contact with the cell wall,

and consequently no space is apparent betweenthem. The inner layer of the plasma membraneborders on the bacterial cytoplasm. In some re-gions, two layers of the plasma membrane enterthe cytoplasm and are transformed into the mem-branous configuration (Fig. 2, 4, 5, and 7).

In studies of the Mycobacterium Jucho strain(Koike and Takeya, 1961) and Bacillus medusa(Fitz-James, 1960), the plasma membrane ofthese bacilli also appears to be a double layerand has similar dimensions to M. leprae, but it isclearly separated from the cell wall by a- lowdensity space. On the other hand, Van Iterson(1961) reported in her study of B. subtilis thatthe outer layer of the plasma membrane is adja-cent to the cell. This being so, the submicroscopicstructure of the plasma membrane of M. lepraeresembles that of B. subtilis rather than those ofthe Jucho strain and B. medusa.

Nuclear apparatus. Electron microscopicstudies of bacterial cells have established that thebacterial nucleus includes threads of dense ma-terial, 30 to 60 A in diameter, when especiallyrefined fixation and embedding methods are em-ployed (Ryter and Kellenberger, 1958; Takeya,1959; Glauert, Brieger, and Allen, 1961; VanIterson and Robinow, 1961). On the other hand,Chapman et al. (1959) failed to demonstrate thesefine threads in the nucleus of M. lepraemurium.

Figures 2, 3, 6, 7, and 8 reveal an irregularlyshaped, dense material in the transparent vacu-oles. This is considered to represent the nuclearapparatus, which, however, does not correspondto its true visualization because of an artifactcaused by the shrinkage of the nuclear materialduring preparation, as described by Takeya(1950). From this point of view, the preparationused in our study is inappropriate for a discussionof the fine structure of nuclear apparatus.

Polyphosphate body. Homogeneous, dense sub-stances are seen in the bacterial cytoplasm. Thesesubstances appear to be round or oval, sometimesas irregular masses (Fig. 1, 2, 3, 6, and 7). Theyare not delimited by any membrane and ap-parently have no internal structures, althoughthey are easily distinguishable from the granularmatrix of the cytoplasm. There is often visible agranular appearance caused by electron bom-bardment.As these substances never evidence any feature

showing shrinkage, it is assumed that they arecomposed of osmiophilic matter which is not de-

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IMAEDA AND CONVIT

formed through preparation for electron micro-scope examination, thus differing from thenuclear substance.

It is well known that M. tuberculosis also con-tains a dense substance, called "A-Body", whichis easily vacuolated by electron bombardment.Takeya et al. (1954), Mudd, Takeya, andHenderson (1956), Mudd, Yoshida, and Koike(1958), and Takeya (1959) concluded that thisbody represents neither the nuclear apparatusnor the mitochondria, but rather a polyphosphateas an energy source of the bacilli.The morphological appearance of the dense

substance in M. leprae shows a close similarity tothat of M. tuberculosis. It is therefore supposedthat the dense substance in M. leprae also com-prises a polyphosphate, although this has not yetbeen proved. Drews (1960) suggested in his studyof M. phlei that the polyphosphate body mightoriginate from irregularly shaped intermediatestages or from an agglomeration of the micro-granules. However, the fact that irregularlyshaped dense bodies generally appear near thenuclear region suggests that they may probablybe related to the metabolism of the bacillarynuclei.

It should be emphasized that polyphosphatebodies never appear clearly in the degeneratingbacilli contained in lepra cells (Fig. 9). In thelesion observed in this study, the bacilli were inan active condition, as discussed earlier. Basedon this evidence, it is suggested that the presenceof the polyphosphate body may be one of the

indicators of the activity of M. leprae in hostcells.

Intracytoplasmic membrane system. Complex,membrane-limited structures are found in thebacterial cytoplasm. They show various configu-rations, some beehivelike, others convoluting orlamellar, consisting of a dense layer approxi-mately 30 A thick (Fig. 2, 3, 4, 5, 6, and 7). Asseen in Fig. 2, 5, and 7, this membrane systemshows a close connection with the outer layer ofthe plasma membrane.

In some regions, the enclosing membrane andthe convoluted membrane are seen to be parallel,being separated by a low density space of 30 Ain width (Fig. 8), and appear similar to the unitmembrane described by Robertson (1959) in hisstudies on animal cells. The parallel membranesystem observed by Brieger et al. (1959) in thecytoplasm of M. leprae may correspond to theparticular arrangement of these two membranes.From their studies of other bacilli, such as B.

medusa, B. megaterium, and MycobacteriumJucho, Fitz-James (1960), Giesbrecht (1960), andKoike and Takeya (1961) showed that the intra-cytoplasmic membrane system is derived mainlyfrom a branching of the plasma membrane. Onthe other hand, Van Iterson (1961) observed thatthe membranous organelles of B. subtilis are incontact with the plasma membrane or with thecell wall. From our studies with methacrylate-embedded material, it would seem highly proba-ble that the intracytoplasmic membrane systemof M. leprae is composed of the outer layer of the;; .;., ........- . i ; t* rt<. *..;w*.ir;b -tsg,* 4-v.. I6".1 ..............................................DLMi :-.-.*u 1W..

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FIG. 10. A hypothetical scheme showing the relationship between the intracytoplasmic membrane systemand the plasma membrane. DL, diffuse layer outside the cell wall; CW, cell wall; PM, plasma membrane;BC, bacillary cytoplasm; and CS, coating substance.

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M. LEPRAE IN LEPROUS LESION

plasma membrane, which is surrounded by theinner layer (Fig. 10). Thus, the intracytoplasmicmembrane system of M. leprae is quite similar tothose of B. subtilis, B. medusa, B. megaterium,and the Jucho strain of Mycobacterium. However,further studies using other preparative methodswill be necessary to clarify the relationship be-tween the intracytoplasmic membrane systemand the plasma membrane or cell wall.The intracytoplasmic membrane system may

represent one of the sites of energy-yielding en-zyme systems in this bacillus, since it consists ofthe plasma membrane in which bacterial enzymesmay be located, as postulated by Weibull andBergstrom (1958) and Mitchell (1959). It is be-lieved, therefore, that this structure may be oneof the bacterial organelles related to metabolism,as observed in other mycobacteria and strepto-myces (Shinohara, Fukushi, and Suzuki, 1957;Shinohara et al., 1958a, b, 1959 Takeya et al.,1958; Niklowitz, 1958; Toda, Takeya, and Koike,1958; Takeya, 1959; Fukushi, 1959; Chapmanet al., 1959; Zapf, 1959; Drews, 1960; Glauertand Hopwood, 1960; Koike and Takeya, 1961).Furthermore, this structure is not revealed in thedegenerating bacilli showing cytoplasmic con-densation. From this evidence, it may be con-concluded that such a membrane system appearsonly in active bacilli.

ACKNOWLEDGMENT

The authors wish to thank the members of ourelectron microscopy group for their helpful dis-cussions, and J. Busquets for his photographicassistance.

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