Cell-free extracts of tissue-derived cells of Mycobacterium lepraemurium TMC 1701 have been found to have mycobacterial catalase which is of the T type. Immunological distance measurements of this catalase against three reference mycobacterial systems, as determined by a micro-immunoprecipitation technique, showed that catalase from M. lepraemurium TMC 1701 is most closely related to that of Mycobacterium avium but is still distinct from it. In this respect, this strain is uniquely positioned between Mycobacterium tuberculosis and M. avium

Diaz, G. A.

Katoch, V. M.

Wayne, L. G.

English

Publications of the IAS Fellows

INTERNATIONAL JOURNAL OF SYSTEMATIC BACTERIOLOGY, OCt. 1982, p. 416-418 
0020-7713/82104041603$02.00/0 
Copyright 0 1982, International Union of Microbiological Societies 
Vol. 32, No. 4 
Characterization of Catalase by Micro-Immunoprecipitation in 
Tissue-Derived Cells of Mycobacterium lepraemurium TMC 
1701 
V. M. KATOCH,l L. G. WAYNE,1,2 AND G. A. DIAZ' 
Tuberculosis Research Laboratory, Veterans Administration Medical Center, Long Beach, California 90822l 
and Department of Medical Microbiology, California College of Medicine, University of California at Irvine, 
Irvine, California 926642 
Cell-free extracts of tissue-derived cells of Mycobacterium lepraemurium TMC 
1701 have been found to have mycobacterial catalase which is of the T type. 
Immunological distance measurements of this catalase against three reference 
mycobacterial systems, as determined by a micro-immunoprecipitation tech- 
nique, showed that catalase from M .  lepraemurium TMC 1701 is most closely 
related to that of Mycobacterium avium but is still distinct from it. In this respect, 
this strain is uniquely positioned between Mycobacterium tuberculosis and M .  
avium. 
Mycobacterial catalases have been broadly 
grouped into two major classes, T and M (lo), 
depending upon their physical properties, sus- 
ceptibility to various inhibitors, and kinetics of 
activity (1, 2, 10). In earlier studies (8-lo), 
measurement of the differences in their serologi- 
cal specifications has proven to be a useful tool 
for taxonomic or (indirectly) evolutionary stud- 
ies of mycobacteria. Immunological distance 
correlates well with the structural divergence of 
proteins having substitutions of up to about 40% 
of the component amino acids (4). However, 
studies on tissue-derived mycobacteria are diffi- 
cult because of low yields of organisms as well 
as contamination with host enzymes (11). Previ- 
ous studies (3,5) have described the presence of 
peroxidase in the Antwerp strain of Mycobac- 
terium lepraemurium grown in vivo and of cata- 
lase in Keishicho-strain cells grown in vitro. 
This study was undertaken to confirm the myco- 
bacterial origin of the catalase present in tissue- 
derived M. lepraemurium TMC 1701 suspen- 
sions and also to evaluate its taxonomic 
importance. 
MATERIALS AND METHODS 
The strain of M. lepraemurium used in this study 
was the Hawaiian strain (TMC 1701) grown in ABG6 
hybrid mice (supplied from the Trudeau Institute Inc., 
Saranac Lake, N.Y.). Cells were harvested and puri- 
fied by the method of P. Draper (personal communica- 
tion). The two-polymer aqueous-phase system consist- 
ed of 0.50 g of 1% polyethylene glycol palmitate 
(supplied by the National Institute of Medical Re- 
search, London), 0.245 g of polyethylene glycol 6000 
(Union Carbide Corp., New York, N.Y.; recently 
named 8000), 0.35 g of Dextran T500 (Pharmacia Fine 
Chemicals, Piscataway, N.J.; lot no. 11648), 0.025 ml 
of 2 M NaC1, 0.10 ml of 0.05 M phosphate buffer (pH 
6.9), and 3.28 ml of distilled water; this was sufficient 
to purify 0.2 ml of packed bacterial cells in the final 
stages of purification. The cells were suspended in 
low-salt buffer and were sonicated by a method de- 
scribed previously (2). Sonic extracts were added to a 
suspension of DEAE-cellulose (Sigma Chemical Co., 
St. Louis, Mo.) and were washed in low-salt buffer; 
the product was then eluted in 0.5 M NaCl for use in 
this study (9). 
The catalase preparation was first assayed both in 
the presence and the absence of 3-amino-l,2,4-triazole 
(AT). Equal volumes of catalase preparation and AT 
(final concentration, 10 mg/ml) were incubated at 37°C 
for 2 h and then were analyzed for catalase activity by 
the zero-order TiC14 assay of residual H202 as de- 
scribed previously (10). 
Untreated cell-free extracts were then incubated 
with serial dilutions of reference sera specific for 
mycobacterial catalases from different mycobacterial 
species, and the unreacted catalase was assayed by a 
microenzyme titration technique which is a modifica- 
tion of the method described earlier (9). Briefly, 10 p1 
of catalase preparation (0.01 U) was mixed with 10 pl 
of each of the serum dilutions in polyethylene centri- 
fuge tubes and was incubated at 37°C for 1 h, followed 
by overnight incubation at 5°C. The contents were 
then drawn into 20-4 microcapillary tubes (Corning 
Glass Works, Coming, N.Y.; catalog no. 70995-20). 
The lower ends of these capillary tubes were sealed 
with putty and the tubes were then centrifuged at 900 
x g for 20 min. The capillary tubes were cut at a 
distance from the meniscus corresponding to 15 pl of 
contents, and this supernatant was discharged and 
analyzed for catalase as described earlier (9), except 
that the volumes of H202 and titanium tetrachloride- 
sulfuric acid used were reduced proportionately. The 
first-order calculations of the T catalase activity and 
immunological distance were based upon earlier publi- 
cations (8-10). The reference antisera to T catalases 
were prepared as also described previously (8-10); the 
VOL. 32, 1982 CHARACTERIZATION OF M .  LEPRAEMURZUM CATALASE 417 
sera included in the study were RS 75-32726 (anti- 
Mycobacterium avium TMC 716 catalase), RS 114- 
30180 (anti-Mycobacterium kansasii TMC 1201 cata- 
lase), and RS 120-10381 (anti-Mycobacterium 
tuberculosis H3,Rv catalase). The micro method was 
compared with the standard method (9) and was found 
to give comparable immunological distances for sever- 
al mycobacterial catalase titration systems. 
RESULTS 
It was found that AT had no significant effect 
on catalase activity in the cell-free preparation 
of M. lepraemurium sonicates; the mean 
changes in absorbance at 410 nm observed in the 
assays were 0.200 and 0.204 with and without 
AT treatment, respectively. This resistance to 
AT indicated that the catalase in the preparation 
was of the T type and that there was no signifi- 
cant M-type catalase activity, either of host or 
bacterial origin, in these sonicates. 
The immunological distance experiments (Ta- 
ble 1) revealed that the catalase from M. leprae- 
murium TMC 1701 preparations was most close- 
ly related to M. avium catalase. The standard 
and micro methods showed a good correlation, 
the immunological distance between M. avium 
and M .  lepraemurium TMC 1701 being 24 k 4 by 
the standard method and 24 5 6 by the micro 
method. It was found that the M. lepraemurium 
TMC 1701 catalase was positioned between 
those of M. avium and M .  tuberculosis and was 
quite distant from that of M. kansasii. That is, 
the sum of the distances from M. lepraemurium 
TMC 1701 to M. tuberculosis and to M .  avium is 
almost identical to the reciprocal distances be- 
tween M. avium and M .  tuberculosis reported 
previously (9). Furthermore, the relative dis- 
tances seen among three reference systems ex- 
hibited a high order of complementarity to the 
relative values previously reported between 
these three reference systems (8-10). This zero- 
order micro technique required only 0.01 U per 
test as compared with 0.05 U per test by the 
zero-order standard method (8) or 0.5 U per test 
by the first-order macro method used in the 
earliest studies (2). 
DISCUSSION 
Stanford (6) noted a marked similarity be- 
tween M. avium and M .  lepraemurium strains in 
terms of numbers of shared antigens as deter- 
mined by immunodiffusion, and Stanford and 
Grange later (7) suggested that M .  lepraemurium 
should be recognized as a subspecies of M. 
avium. The present study, with a different pa- 
rameter, supports the findings of Stanford (6) 
that M. lepraemurium is more closely related to 
M. avium than to any other species tested, but 
there does appear to be a distinct immunological 
distance between catalases of the two species. 
TABLE 1. Immunological distances of T catalase of 
M .  lepraemurium TMC 1701 from three standard T 
catalase reference systems 
Immunological distances from:" 
Strain tested M .  M .  
tuberculosis kansasii 
H3mf  TMC 716 TMC 1201 
M .  tuberculosis (97 k 5) (145 * 9) 
M. lepraemurium 68 * 8 24 k 6 70 2 6 
M .  avium TMC (98 * 6) (15 2 7) 
M .  kansasii (143 * 16) (29 k 3) 
H37RV 
TMC 1701 
71 6 
TMC 1201 
a Values show immunological distance standard 
deviation (3 replications). Numbers in parentheses are 
data from previously published studies (8-10). 
In a previous study, Wayne and Diaz suggested 
a correlation between the position of species on 
a diagram of divergence of their T catalases and 
the evolution of these species in terms of their 
saprophytic and pathogenic natures (10). In the 
present study, catalase from M. lepraemurium 
TMC 1701 was found to be uniquely positioned 
between those of M. avium and M .  tuberculosis, 
providing further support for a correlation be- 
tween the evolution of species and their patho- 
genicity. So far, no other mycobacterial species 
have been described to be positioned like this on 
the scale of divergence. 
It can be concluded from the present study 
that the catalase detected in M .  lepraemurium 
TMC 1701 preparations is a mycobacterial T 
catalase rather than a host enzyme and that it 
has a position between the catalases of M. avium 
and M .  tuberculosis. Furthermore, the facts that 
this micro technique needs 5 times less enzyme 
than the semimicro standard technique (8) and 
50 times less enzyme than the macro method (2) 
and is equally sensitive and specific make it 
highly suitable where a very small amount of 
material, e.g., tissue-derived bacteria, is avail- 
able. Thus, in this study five mice yielded about 
0.5 ml of packed cells of M. lepraemurium TMC 
1701 and enough mycobacterial catalase for 
about 50 serum titrations. This kind of serologi- 
cal approach may be useful for studying other 
enzyme systems as well. 
ACKNOWLEDGMENTS 
The authors thank F. M. Collins and P. J.  Patel (Trudeau 
Institute Inc.) for providing a culture of M .  Iepraemurium 
TMC 1701; we thank Philip Draper (National Institute of 
Medical Research, London) for advice on purifying the myco- 
bacterial cells. 
V.M.K. was working as an International Tropical Disease 
Research Fellow of the National Institutes of Health (grant no. 
1 F05  TW02973-01) on sabbatical leave from Central JALMA 
Institute for Leprosy, Agra, India. 
418 KATOCH, WAYNE, AND DIAZ 
REPRINT REQUESTS 
Address reprint requests to: Dr. L. G. Wayne, Veterans 
Administration Medical Center (151), Long Beach, CA 90822. 
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Characterization of catalase by micro-immunoprecipitation in tissue-derived cells of Mycobacterium lepraemurium TMC 1701

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Characterization of catalase by micro-immunoprecipitation in tissue-derived cells of Mycobacterium lepraemurium TMC 1701

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