CELLULAR IMMUNOLOGY 82, 88-97 (1983)

Mycoplasmas: The Pathogens’ Pathogens*

WALLACEA.CLYDE,JR., AND GERALDW.FERNALD

Departments of Pediatrics, and Microbiology and Immunology, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27514

Received February 16, 1983

It is a singular honor to have this opportunity to address an audience composed of friends, students, and colleagues of Lewis Thomas. A topic for this presentation was easily identified when review of the roster showed Lewis to be the only other mycoplasmologist in the group. His interest in these unique microorganisms spans many years, and his contributions, adding insight to our knowledge of their biology, are numerous. My purpose will be to review some of the information on the pathogenic properties of mycoplasmas to illustrate why they hold so much fascination. This presentation is in two parts: the first is subtitled “Notes of a Biology Watcher Watcher,” by Wallace A. Clyde, followed by “Mycoplasmas: the Pathogens’ Pathogens,” by Wallace A. Clyde and Gerald W. Femald.

NOTES OF A BIOLOGY WATCHER WATCHER

Although I have known Lewis for many years, the opportunity to watch the biology watcher in his natural habitat came in 197 1 when he allowed me into his laboratory for a sabbatical year. This was a most eventful year for Lewis as he moved from his second chair of pathology to his second medical school deanship at Yale University. Much more important, however, was that this was the year he rediscovered Bach (i.e., Johann Sebastian). Strains of Brandenburg Concerti and all the rest often came wafting under his office door as he worked alone at his desk.

Orientation to the Thomas laboratory required a certain amount of facility since the place literally was a zoo. On any given day one or combinations of the species listed in Table 1 might be the objects of experimentation. Simultaneous ongoing studies included dissection of Mycoplasma gallisepticum encephalopathy in turkeys, Mycoplasma neurofyticum rolling disease in mice, the effects of penicillin on guinea pig gut flora, Mycoplasma pneumoniae infections in Syrian hamsters, and Shwartzmann reactions in rabbits. And, that was only during the daytime. The place was characterized by an air of enthusiastic expectation. The very sound of the word “fibrinoid!” was sure to cause a rush to the nearest microscope, where heavy breathing might be heard as the cerebral arteries of a turkey poult with mycoplasma-induced fibrinoid necrosis were inspected.

* This paper was presented at the Symposium, “Infection, Immunity and the Language of Cells: A

Meeting in Honor of Lewis Thomas,” held at New York University Medical Center, on November 22, 1982.

88

0008-8749/83 $3.00 Copyright 0 1983 by Academic Press. Inc. All rights of reproduction in any form reserved

MYCOPLASMAS: THE PATHOGENS’ PATHOGENS 89

TABLE 1

Experimental Subjects

Gallus domesticus Mus Musculis Cavia cobaya Mesocricetus auratus Oryctologus cuniculus

During the course of my year in New Haven, I learned many valuable lessons from Lewis. So that these experiences will not be lost, I would like to share three of the more important lessons with you. Particularly in the field of mycoplasmology one strains to find the organisms in biological preparations because of their very small size. Shown in Fig. 1 is a phase-contrast micrograph of a mycoplasma-infected cell culture; but which are the mycoplasmas, and which are the cellular organelles? The lesson is clear (Fig. 2). The desired distinction is impossible, since Mother Nature’s building blocks very often are round.

The second lesson concerned the planning of laboratory experiments (Fig. 3). The point is that running controls is a waste of time if you cannot first produce an experimental effect. Having obtained the desired effect, it can then be reproduced with all proper controls. Now, this approach had an interesting effect on the atmosphere in the laboratory, as illustrated in Fig. 4. An average week is plotted in the figure by the daily relative mood. Note that the baseline on Monday is at 1, since as stated

FIG. 1. Phase-contrast micrograph of a mycoplasma-infected cell culture (bar = 10 pm).

90 CLYDE AND FERNALD

ON MORPHOLOGIC INTERPRETATION “Nature abounds with little round things.”

-L. Thomas, 1971

FIG. 2. Lesson 1.

before this was an upbeat operation. The experiment saris controls performed on Monday is analyzed Tuesday, with rising euphoria as the experimental effect is pro- duced. This level is sustained on Wednesday while the replicate experiment, this time with controls, is initiated. On Thursday gloom follows discovery that the tantalizing experimental effect is shown also in the control preparations. Friday is a day of recovery and going back to the drawing board before coasting through the weekend and starting the process again on Monday. Actually, this rarely happened; Lewis would come in around midnight Thursday and redesign the experiment using the same data to provide answers to a completely different question.

The final lesson to be shared displays the true intellect and imagination of the man being honored today (Fig. 5). The idea is to enter a new area of investigation free of the biases of all who have gone before. The value of the information base available is more than offset by the opportunity to try new things that would appear illogical based on the state of knowledge. A corollary to this lesson is the advice that every investigator should have a small, private place to work at the bench so that he will not be seen doing apparently foolish things. I have found all of these lessons useful in my daily work, and perhaps others may, as well.

MYCOPLASMAS: THE PATHOGENS’ PATHOGENS

For purposes of the remaining discussion, mycoplasmas have been termed the pathogens’ pathogens because of their ability to base their unusual mechanisms of virulence on very limited genetic information (Table 2). With a genome size of only 5 X 10’ Da, roughly one-third that of Escherichia cob, mycoplasmas represent an amazing conservation of gene expression in their survival, replication, and patho- genicity. Although bound only by a trilayered unit membrane, the organisms are by no means simply “little round things” whose shape is determined by the physical properties of their surroundings. Many pathogenic species have distinct morphology under optimal conditions of growth: Mycoplasma gallisepticum, the poultry pathogen, is ovoid, M. pulmonis of rodents is globular with a stalk, M. pneumoniae of man is a filament with a bulbous tip; Spiroplasma citri, the agent of citrus stubborn disease, resembles a spirochete. Maintenance of the characteristic morphology could reside in molecular configurations, much like the changing shape of erythrocytes in different forms of hemoglobinopathy. Alternatively, structural units could be involved; recently, evidence of a cytoskeleton in M. pneumoniae has been put forward.

The four pathogens used as illustrations have other points in common. All of them are motile, based on possession of an actin-like material which is incompletely defined

ON EXPERIMENTAL DESIGN Never include controls in the first experiment.”

-L. Thomas, 1971

FIG. 3. Lesson 2.

MYCOPLASMAB THE PATHOGENS PATHOGENS 91

DAY

FIG. 4. An average week in the Thomas laboratory.

at present. Another important component of pathogenicity is the ability of these species to attach to host cells. Through this mechanism colonization of mucous surfaces, or of the phloem tubes of plants, is permitted. In the case of M. pneumoniae, mediation of attachment is via a large surface protein (190K Da) which is present only on the specialized terminal organelle but not over the remainder of the cell membrane.

Once attached to host cells, the organisms mediate their disease processes through production of both extracellular and cellular products. A neurotoxin with highly selective tropism is produced by M. neurolyticum, mediating the classic rolling disease of mice. The agent of bovine pleuropneumonia, 44. mycoides, secretes a capsule-like galactan important to its pathogenicity. Hydrogen peroxide production by M. pneu- moniae is potentially injurious to host cell membranes. In some situations mycoplasma membranes or fragments of them are mediators of disease: the pulmonary lesions of rodents caused by hf. pulmonis can be reproduced by introducing only the membranes into the respiratory tract. hf. gullisepticum, which lacks demonstrable extracellular products, may affect brain capillaries with cellular components moving distally from infected foci in cerebral arterial walls.

A curious feature of many pathogenic mycoplasmas is their ability to evade host defense mechanisms in such a way as to establish persistent infections, which may span the life of the host, and to produce chronic, progressively destructive processes. Bovine pleuropneumonia, rodent bronchiectasis, and murine arthritis are good ex- amples. Information being developed on these diseases suggests that two factors may be involved: biological mimicry, and ability of the pathogens to cause perturbations

ON SCIENTIFIC LITERATURE “Avoid it when entering a new field.”

-L. Thomas, 1971

FIG. 5. Lesson 3.

92 CLYDE AND FERNALD

TABLE 2

Special Features of Pathogenic Mycoplasmas

1. Procaryotic cells with small genome 2. Specific morphology

M. gallisepticum M. pulmonis M. pneumoniae S. citri

3. Motility 4. Host cell attachment mechanisms 5. Extracellular products

Neurotoxin Gala&n Hz02

6. Cellular products 7. Biological mimicry 8. Host immune modulation

of the hosts’ immune mechanisms. These new findings will be discussed in some detail since they serve to illustrate best the unique features of mycoplasmas. Study of these diseases, particularly human atypical pneumonia, may provide a means to further our understanding of autoimmune processes.

The peculiar immunology of mycoplasma diseases was fust noted with the de- scription of streptococcus MG agglutinins in human atypical pneumonia cases (1) (Table 3). (You may note recurrences of the name “Thomas” as this subject is discussed.) In 1943, of course, the etiology of atypical pneumonia was uncertain. Although Monroe Eaton had just published his pioneering studies on production of pneumonia in cotton rats inoculated with human sputum, these studies were not widely accepted until many years later; additional time was required to establish that Eaton’s agent was a new species of mycoplasma. The possibility that the purported mycoplasma was really an L-phase variant of the “indifferent streptococcus” was dismissed with one of the earliest DNA homology studies (2) the publication of which was sponsored by Thomas, leaving the question about the origin of streptococcal

TABLE 3

Evidence of Immune Perturbations during Mycoplasma pneumoniae Disease

Observation Mechanism Reference

Streptococcus MG agglutinins

Lung-reactive antibody, brain, other tissues

Cold hemagglutinins

Tuberculin anergy

Prolonged colonization

Glucosyl diglyceride cross- reactivity

Unknown

I-antigen cross-reactivity

Unknown

Unknown

Thomas et al. (I) Plackett et al. (3)

Thomas et al. (4) Biberfeld (6)

Peterson et al. (5) Feizi and Taylor-Robinson (7)

Biberfeld and Sterner (8)

Multiple reports

MYCOPLASMAS: THE PATHOGENS’ PATHOGENS 93

MG antibodies unanswered. Later Plackett provided evidence that the MG strep- tococcus and M. pneurnoniae contained a glycosyl diglyceride in common, suggesting that antigenic cross-reactivity was the responsible mechanism (3).

Other significant observations that can be traced to 1943 are the occurrence of antitissue antibodies in some patients with M. pneumoniae disease. The development of an immune response detectable by a complement fixation procedure using normal lung tissue as the antigen was reported by Thomas and co-workers (4). Peterson et al. observed that some patients with atypical pneumonia produced antibodies which caused agglutination of human erythrocytes when serum and cells were refrigerated (5). These findings were extended subsequently by Biberfeld who described the pro- duction of antibodies to human nerve and cardiac tissues (6). The mechanism by which these autoantibodies are stimulated remains unknown but will be the object of speculation later in this discussion. In the case of cold hemagglutinins there appears to be cross-reactivity between M. pneumoniae and the I antigen of the human eryth- rocyte membrane which may offer one explanation (7). The glycolipids of the organism have some similarity of sphingomyelin, to cardiolipin, and to substances in such disparate life forms as streptococci and spinach. Since the complement fixation test in popular use for diagnosis of M. pneumoniae disease employs lipid extracts of the organism as antigen, it is obvious that specificity of the reaction is problematic. Improved serodiagnosis may be possible with use of other organism antigens, for instance, a membrane surface component such as the attachment protein mentioned earlier.

The possibility that M. pneumoniae infections may actually suppress the host im- mune response is suggested by recent work of Biberfeld and Sterner (8). Universal administration of BCG vaccine in Sweden provided the opportunity for these inves- tigators to study tuberculin sensitivity during atypical pneumonia in their country. Anergy to tuberculin which they demonstrated lasted from 3 to 6 weeks in most patients, although rarely this effect persisted up to 5 months. The mechanism involved has not been determined at present, but the possibilities are intriguing based on newer knowledge of cellular immunology.

The prolonged colonization of the respiratory mucosa by M. pneumoniae following host recovery from disease suggests inadequacy of local immune mechanisms. In patients the organisms may be recovered for 4-6 weeks, even when appropriate antibiotic therapy has been given. The same effect is seen in experimental models: hamsters and cotton rats remain infected for about 6 weeks; the organisms can be recovered from inoculated guinea pigs as long as 6 months. A similar situation exists with infections by M. mycoides and M. suipneumoniae, which may last up to 10 months in cattle or swine, respectively. Rodent and poultry mycoplasma infections can persist for the life of the host. Experimental studies have provided some clues about the ways mycoplasma infections modulate host immune mechanisms, as sum- marized in Table 4.

The observations to be described include nonspecific suppression and stimulation of both humoral and cell-mediated immunity, effects somehow inexorably intertwined with specific host immune responses to the mycoplasmas. The first study of im- munosuppressive effects was reported in 1972 (9); however, it should be recognized that this work was initiated in the laboratory of Lewis Thomas by Kaklamanis in 1966. Rats inoculated simultaneously with M. arthritidis and pseudomonas phage 4 5 showed suppression of antibody production to the phage. In the rat model, the

CLYDE AND FERNALD

TABLE 4

Experimental Evidence of Immune Perturbations due to Mycoplasma-Host Interactions

Observation Reference

Suppression of humoral antibody response M. anhritidis in rats M. arthritidis in rabbits

Suppression of cell-mediated immunity M arthritidis in rats M. mycoides in cattIe M. pneumoniae in man

Polyclonal activation of lymphocytes M. pulmonis mitogenic effect M. pneumoniae ? IgM-plasmacytosis in lungs

Ir gene control of lymphocyte cytotoxicity for M. arftrrifidis-infected cells

RakIamanis and Pavlatos (9) Bergquist et al. (10)

Kaklamanis and Pavlatos (9) Roberts et al. (I I) Biberfeld and Sterner (8)

Ginsberg and Nicolet (12) Biberfeld and Gronowicz (14) Femald ef al. (I 5)

Cole et al. (I 7)

Ir gene control of interaction between M. hyorhinis and mouse B cells

Stanbridge er al. (18)

responsiveness of lymphocytes to PHA also was diminished, indicating concurrent suppression of cell-mediated immunity. Working with rabbits, Bergquist et al. showed that inoculation of the animals with only the membranes of M. arthritidis suppressed antibody response to an E. coli antigen (10). The suggestion is provided that the immunosuppressive mediator is on the organism surface, and that its expression does not require the presence of viable mycoplasmas. Further evidence for mycoplasma suppression of cell-mediated immunity includes reports of depressed lymphocyte responsiveness to PHA in cattle infected with M. mycoides (1 I), and the tuberculin anergy during M. pneumoniae pneumonia mentioned earlier (8).

In addition to the nonspecific immunosuppression by mycoplasma infections, there also is evidence of nonspecific stimulation. The first report concerned a PHA-like mitogenic effect of M. pulmonis on rat lymphocytes (12). This effect was unrelated to prior exposure of the rats to the organisms, since the same response could be obtained in germ-free animals. The mitogenic activity of the mycoplasma resided in the membrane fractions; evidence that it is a protein was suggested by its heat lability and inactivation by proteolytic enzymes. Further studies by Naot ei al. have indicated that the M. pulmonis substance is mitogenic for both T and B lymphocytes of the rat (13). Other examples of lymphocyte stimulation are provided by M. arthritidis which affects mouse T lymphocytes and M. neurolyticum which is mitogenic for mouse B lymphocytes.

A recent finding of special relevance to this discussion is evidence that M. pneu- moniae causes polyclonal B-cell activation. Initial work by Biberfeld and Gronowicz showed that this organism was mitogenic for mouse B lymphocytes in vilro (14). That the effect is nonspecific polyclonal stimulation is supported by the demonstration that antibodies were produced simultaneously to several different hapten-conjugated erythrocyte antigens. Human B, but not T, lymphocytes obtained from excised tonsils and adenoids also arc stimulated nonspecifically by the M. pneumoniae mitogen. In

MYCOPLASMAS: THE PATHOGENS’ PATHOGENS 95

the hamster model of infection with this organism, peribronchial round cell infiltration is seen, as in the natural human disease. Fernald and co-workers demonstrated that these cells predominately were IgM-producing plasmacytes (15). This finding was unexpected, given the state of knowledge in the early 1970s since it was anticipated that IgA or IgG plasmacytes would dominate the infiltrates, in specific response to the mycoplasmas on the epithelial surface of the bronchi. The specificity of the IgM antibodies being produced is unknown, but it is possible that the hamster cell response is an in vivo demonstration of polyclonal B-cell activation. The likelihood that the phenomenon occurs in natural disease is enhanced by recent reports that peripheral blood lymphocytes from patients with pneumonia spontaneously produce antibodies to unrelated agents such as measles, rubella, and herpes simplex during cultivation in vitro ( 16).

Other studies in vitro of the interactions between mycoplasmas and lymphocytes indicate, as with other microbial systems, that specific receptors on the cell membranes are involved. Since these receptors are under genetic control, a handy explanation for the host specificity of certain mycoplasma infections is suggested. Further, the variable disease expression resulting from infection could be based on this mechanism. Working with M. arthritidis and various congenic and H2 recombinant mouse strains, Cole and co-workers have shown that control of the degree of lymphocyte respon- siveness was determined by the H2 gene complex (17). In this instance the response measured was mitogenesis, inducing previously unsensitized cells to become cytotoxic for syngeneic fibroblast target cells. Analysis of the results suggested that the region controlling induction of cytotoxic lymphocytes was probably the I-E and/or I-C subre- gions of the major histocompatibility complex. It was concluded that the results could be connected to the previously observed variability in susceptibility of different mouse strains to the pathogenic effects of M. arthritidis.

Stanbridge and co-workers observed that M. hyorhinis behaves as a multivalent ligand during interaction with mouse B lymphocytes, attaching to the cell membrane and then becoming redistributed to one pole, forming a “cap” (18). This activity was accompanied by co-capping of certain surface antigens of the lymphocyte preceding blastogenesis. Mouse splenic B lymphocytes expressing the Thy-l antigen were those infected by the mycoplasma, suggesting this or an associated surface material is a requisite receptor site. Additional experimentation with cells from various mouse strains again demonstrated a controlling effect from the H2 complex in the I subregion. In addition to their connections with host specificity of different mycoplasma infections, the findings of Cole and of Stanbridge may be useful for understanding genetic sim- ilarities and differences in instances where the same mycoplasma infects more than one host species. Humans, guinea pigs, hamsters, cotton rats, and chick embryos apparently share similar receptor sites since all can be infected with M. pneumoniae. The outcome varies in these situations, however, because only man demonstrates clinical illness. Both mice and rats can be infected with M. neurolyticum, but only mice experience rolling disease. Turkeys and chickens can be infected with M. gal- lisepticum, but turkeys alone develop the encephalopathy syndrome.

CONCLUSIONS

From the information that has been summarized it is possible to derive several conclusions based on the idea that one facet of the pathogenicity of mycoplasmas

96 CLYDE AND FERNALD

TABLE 5

Speculations on Mycoplasma Pathogenicity Based upon Alterations of Host Immunity

1. Prolonged colonization and a. Biological (antigenic) mimicry chronic disease b. Suppression of specific antibodies by nonspecific activation of

lymphocyte pool

2. Production of autoantibodies a. Cross-reactive antigens b. Polyclonal B-cell activation c. Alteration of T-suppressor cells

3. Variable expression of disease a. Genetic variation in receptors for attachment proteins in the host b. Variable recognition of foreign antigens

c. Variation in immunosuppressive effects on host immune response

relates to their ability to modulate host immunity (Table 5). First, the prolonged colonization characteristic of many mycoplasma diseases, and chronic pathological processes, could occur through a degree of biological mimicry on the part of the organisms so that they are not recognized as foreign by the host’s immune system. Alternatively, or in addition, nonspecific activation of the resident lymphocyte pool may inhibit or delay initiation of a specific immune response to the organisms. Second, several possibilities may underlie the production of antibodies directed against host tissues. The idea of biological mimicry implies occurrence of similar or shared antigens in host and parasite, and cross-reacting antibodies may be generated. Perhaps more attractive is the possibility that nonspecific activation of normally quiescent lymphocyte clones leads to production of antibodies recognizing host antigens. An influence of infection on suppressor T lymphocytes, occurring directly or through lymphokines resulting from B-cell mitogenesis, would be another way that clones able to make antitissue antibodies could emerge. Lastly, recent work provides possible mechanisms for variation in mycoplasma disease expression in different host species, or even within one particular species. Factors involved could include variation in the receptor sites for mycoplasma attachment proteins, since receptors are under genetic control. The ability of different hosts to recognize mimicking antigens as foreign may vary in addition. The immuno-stimulating and/or suppressive effects on individual immune responses may be different as well.

In closing, I recall Lew Thomas’ visit to our University in Chapel Hill during October 1982. His talk, on the subject of future directions in medical education, made use of a new hobby he had developed. Information from his studies on linguistics formed the basis of the address. Now, I cannot compete with so sophisticated a hobby, but I have a new one, as well: the composition of scientific limericks. Two on the subject of mycoplasmas may have some pertinence here, to wit:

The members of Class Mollicutes Are little microbial beauties:

Globe, filament, spiral- Unlike objects viral-

They’re among the procaryotes.

It’s almost like a theology- This mycoplasma biology.

For the beasts are so small, You can’t see them at all.

Have faith in Mycoplasmology!

MYCOPLASMAS: THE PATHOGENS PATHOGENS 97

REFERENCES

1. Thomas, L., Mirick, G. S., Cumen, E. D., Ziegler, J. E., and Horsfall, F. L., Science 98, 566, 1943. 2. McGee, Z. A., Rogul, M., Falkow, S., and Wittler, R. G., Proc. Natl. Acad. Sci. USA 54, 451, 1965. 3. Placket& P., Mannion, B. P., Shaw, E. J., and Lemcke, R. M., Aust. J. Exp. Biol. Med. Sci. 47, 17 1,

1969. 4. Thomas, L., Cumen, E. C., Mirick, G. S., Ziegler, J. E., and Horsfall, F. L., Jr., Proc. Sot. Exp. Bioi.

Med. 52, 121, 1943. 5. Peterson, 0. L., Ham, T. H., and Finland, M., Science 97, 167, 1943. 6. Biberfeld, G., Zentralbl. Bakteriol. Hyg. 241, 236, 1978. 7. Feizi, T., and Taylor-Robinson, D., Immunology 13, 405, 1967. 8. Biberfeld, G., and Sterner, G., Stand. J. Infect. Dis. 8, 7 1, 1976. 9. Kaklamanis, E., and Pavlatos, M., Immunology 22, 695, 1972.

10. Bergquist, L. M., Lau, B. H. S., and Winter, C. E., Infect. Zmmun. 9, 410, 1974. 11. Roberts, D. H., Windsor, R. S., Masiga, W. M., and Kariavu, C. G., Infect. Immun. 8, 349, 1973. 12. Ginsburg, H., and Nicolet, J., Nature (London) 246, 143, 1973. 13. Naot, Y., Tully, J. G., and Ginsburg, H., Infect. Immun. 18, 3 10, 1977. 14. Biberfeld, G., and Gronowicz, E., Nature (London) 261, 238, 1976. 15. Femald, G. W., Clyde, W. A., Jr., and Bieninstock, J., J. Zmmunol. 126: 16 1, 1967. 16. Biberfeld, G., Stand. J. Immunol. 6, 1145, 1977. 17. Cole, B. C., Daynes, R. A., and Ward, J. R., J. Immunol. 128, 922, 1981. 18. Stanbridge, E. J., Bretzius, K. A., and Good, R. F., Zsr. J. Med. Sci. 17, 628, 198 1.