Article Published in the Author Account of

Gerald J Domingue

Demystifying Pleomorphic Forms in Persistence and Expression of Disease: Are They Bacteria, and Is Peptidoglycan the Solution?

Abstract: There is considerable circumstantial evidence linking tissue pleomorphic forms of unknown origin with idiopathic chronic inflammatory, collagen, lymphoproliferative, nephro-urological (including interstitial cystitis and prostatodynia), and neoplastic diseases. Although these forms have been observed in stained tissue histopathologic specimens for many decades, most are ignored and generally regarded as diagnostically insignificant staining artifacts or debris. It is hypothesized that these pleomorphic forms are not staining artifacts/cellular debris, but instead represent various stages in the life cycle of stressed bacteria: cell wall-deficient/defective (often called L-forms) that are difficult-to-culture or nonculturable. Essential to the thesis is that small, electron dense, non-vesiculated L-forms are the central (core) element in bacterial persistence. Depending on the stimulus received, these dense forms might be considered as undifferentiated cells, with the capacity to develop along several different routes. Hence, these altered forms created in vivo take up intracellular and/or extracellular residence; possibly establishing a sort of immune protected parasitic relationship, resisting/surviving phagocytic action, and creating subtle pathologic changes in the host during a prolonged period of tissue persistence. This might translate into an etiology for chronic inflammatory diseases, when the stressed bacteria increase in numbers and overwhelm the normal biological functions of the host. In the last few decades, an increasing percentage of the population has become immunosuppressed. Some mechanisms for this increase are aging; autoimmunity; congenital, metabolic and degenerative disorders; and AIDS. The life of a patient so affected is prolonged by therapy with hormones, antimicrobials, and immunosuppressants. It is therefore not surprising that pleomorphic, dormant, and mutant bacterial populations arise in vivo when bacteria are exposed to agents that interfere with structural components and metabolic processes necessary to survival of the microbe. Recent provocative, microbiological data lend credence to the hypothesis and corroborate the multiplicity of pleomorphic forms that develop during reproduction of L forms in vitro. It is proposed that in vivo persistence of these bacterial elements escape immune surveillance partially, completely, or may integrate with host cell organelles to create bacteria-host-cell-antigen complexes which could provoke immunopathologic consequences. Highly relevant, newly published data on modifications of gene expression, modes of division for stressed bacteria, and the paradoxical finding of peptidoglycan in L-forms are pertinent to the hypothesis that atypical, pleomorphic bacteria are the organisms operative in persistence and expression of pathology over a wide spectrum of diagnostically troublesome human diseases.



Rationale for Hypothesis Theory

For many decades, atypical bacteria have been suspected of being persistent pleomorphic vehicles contributing to subsequent relapse in infectious diseases and as etiologic agents in chronic inflammatory conditions of unknown origin. The central thesis of this medical hypothesis is that nonculturable, pleomorphic forms (atypical bacteria-like, cell wall-deficient/defective bacteria, L-forms) observed for decades in tissue biopsies obtained from patients suffering from various idiopathic diseases (lymphoproliferative, collagen, nephro-urological) and even neoplasms are bacterial forms and not staining artifacts or host cellular debris (Figure 1). These bodies observed in stained histologic sections (periodic acid-Schiff, acid-fast, Brown and Brenn stains), with further study by phase microscopy, transmission and scanning electron microscopy, and fluorescent stains, are of varying sizes and shapes: small, filterable (0.22 micron) and non-filterable electron dense bodies, vesiculated large globose forms (mother cells), and long and short filaments attached and unattached to larger round forms (Figures 1-3). It is believed that these atypical forms represent various stages in the life cycle of stressed bacteria, partially or completely cell wall-deficient (often called L-forms), that take intracellular residence in the host, persist unrecognized, and are tolerated by the host cellular and humoral immune systems because of their aberrant shapes, alterations in antigenic structure, and unique biochemical/physiological characteristics unlike those of the parent, classical, ordinary wall-containing bacteria from which they were derived. It is not surprising that pleomorphic, dormant, and mutant bacterial populations arise in vivo when bacteria are stressed by antimicrobials, hormones, and immunosuppressants that interfere with structural components and metabolic processes essential to survival of the microbe. These altered, pleomorphic, aberrant forms may be of serious pathologic consequence to the host as a cause of persistent infection. The altered shapes of these prokaryotic forms mimicking host cell architecture along with their membrane antigens, and genetically changed states, may facilitate non-recognition by host immune defenses, creating an ideal environment for persistence of these atypical bacteria in a metabolically privileged site. Their ultimate acceptance by host cells could lead to prolonged residence in human tissues creating only subtle pathologic changes in the host, often characteristic of a number of chronic inflammatory and so-called autoimmune diseases of humans (Domingue, 1995; Domingue and Woody, 1997). Is it possible that such prokaryotic intracellular persisters after prolonged interaction with host cells could undergo an exchange of genes leading to a malignant condition? Some possible mechanisms for diseases caused by dormant bacteria are the transient immune dysfunctions that can be brought about by severe physical or emotional stress. In addition, the introduction of a co-infecting symbiotic organism can adversely alter the relationship of host and microbe. It is unwise to dismiss the pathogenic capacities of any microbe in a patient with a mysterious illness (Domingue, 1995; Domingue and Woody, 1997).

Figure 1. Cyst within a hypertrophied renal pelvic epithelial cell from a patient with Fanconi syndrome, showing a large spheroidal body with a dense core, a slender relatively lucent cortex, and a hematoxylin-staining outer membrane. Note clusters of small spheroidal bodies associated with the cyst membrane as well as the outer membrane of the large spheroid. Periodic acid-Schiff stain. Bar, 10 μm. (Reprinted with permission from Domingue and Woody, 1997.)

Figure 1. Cyst within a hypertrophied renal pelvic epithelial cell from a patient with Fanconi syndrome, showing a large spheroidal body with a dense core, a slender relatively lucent cortex, and a hematoxylin-staining outer membrane. Note clusters of small spheroidal bodies associated with the cyst membrane as well as the outer membrane of the large spheroid. Periodic acid-Schiff stain. Bar, 10 μm. (Reprinted with permission from Domingue and Woody, 1997.)

Defining the role of these aberrant organisms in human host-pathogen interactions could lead to a clarification of their function as cryptic persisters in vivo and latency and chronicity in a wide spectrum of inflammatory and autoimmune diseases, and could possibly extend to the etiology of certain cancers.

Experimental Basis for the Hypothesis: Demonstration of the Phenomena of Bacterial Persistence and Reversion in Human Embryonic Kidney (HEK) Fibroblasts

Many life forms such as Chlamydiae, the malaria parasite, and trypanosomes have been known for a very long time to have complex intracellular/extracellular life cycles with remarkable pleomorphism of the phases. The truism that nature’s successful survival tricks tend to be repeated was confirmed when we were able to demonstrate that this strategy is also used by cell wall deficient/defective bacteria (Green et al., 1974a). In a series of experiments, human embryonic kidney fibroblasts (HEK) were infected with Enterococcus faecalis (formerly Streptococcus faecalis) relatively stable L-forms that were capable of reverting to a wall-containing form, i.e., an unstable L-form under defined growth conditions (Green et al., 1974a). After introduction of the inoculum into the cell culture system, the cells appeared normal by EM, even though they contained phagocytosed vesicular L-forms. Nonetheless, a series of subsequent EMs revealed autolysis of these bodies and the generation of many elementary bodies that were diverse in size and morphology but still no evidence of injury to the HEK cells. With the appearance of transitional forms containing some cell wall fragments, injury and death of the HEK cells occurred along with complete reversion of many of the transitional variants to bacteria with complete walls. Reversions were sporadic, occurring in various experiments on days 4, 5, 14, 25, 29, 30, 56, and 63 post infection. These findings documented for the first time that an unstable L-form was linked with the phenomena of persistence and reversion in a cell culture system. Survival of the unstable L form over long periods of time in the HEK system was particularly intriguing in view of the fact that the unstable L-form survived only 14 to 48 hours in nutrient media in the absence of HEK cells (based on EM visualization of dead forms and inability to culture any organisms). It therefore appeared that the HEK cells were providing some mode of protection.

On the other hand, a stable L-form (incapable of reverting to a parent form) derived from another E. faecalis strain inoculated in HEK cells resulted in positive cultural findings for up to 73 days post infection. The specific fluorescence of persisting stable L-form dense bodies was confirmed by an indirect immunofluorescence test that utilized specific antibodies to the stable L-form. Differences in the cultivation of the stable and unstable L-forms in infected HEK cells at various time intervals post infection may have reflected their basic morphological diversity: namely, the prevalence of dense opaque forms and numerous free-floating dense bodies in the stable L-form cultures in contrast to a preponderance of the vesiculated forms containing dense bodies within the vesicles of the unstable L-form.

Figure 2. Surgical specimen from a patient with nephrotic syndrome, showing a hypertrophied renal tubular epithelial cell within a renal tubule. Note the intense silver staining of the contained intracellular pleomorphic bacteria-like forms. Jones methenamine silver stain. Bar, 10 μm. (Reprinted with permission from Domingue and Woody, 1997.)

Figure 2. Surgical specimen from a patient with nephrotic syndrome, showing a hypertrophied renal tubular epithelial cell within a renal tubule. Note the intense silver staining of the contained intracellular pleomorphic bacteria-like forms. Jones methenamine silver stain. Bar, 10 μm. (Reprinted with permission from Domingue and Woody, 1997.)

These data clearly indicated that the stable L-forms could persist for prolonged periods in cells, remained viable, and were culturable from these cells as L-forms per se. Because the stable L- form appeared to coexist with human cells, causing no classical pathology, its fate (assuming that it could never regain the ability to revert in this HEK cell system) remained unclear. Therefore, it would have been worthwhile to prolong the experimentation beyond the points reported to determine whether organisms survive and whether prolonged survival causes pathology in host cells. These findings with unstable and stable L-forms have broad implications for the field of bacterially caused persisting and relapsing infectious diseases.

Could the phenomena of persistence and reversion observed with the group D relatively stable enterococcal L-form in a cell culture system be extrapolated to in vivo situations involving other kinds of bacteria? It is hypothesized that the occurrence of viable persisting bodies are very likely a universal feature of bacterial L-forms. Therefore the chemical and immunologic nature of these bodies warrants complete exploration. These points are pertinent since a theme of persistence and relapse runs throughout the wealth of literature on the otherwise diverse diseases with which L forms have been associated. In an effort to provide supporting data to the production and universality of dense bodies in L-forms, a most expedient approach would be to attempt to understand the reproductive cycle of the L-form in order to gain insight into clinical management of patients suspected for harboring cell wall deficient/defective bacteria.

The Small, Dense, Nonvesiculated Form as Central (Core) Element in Persistence

We organized into a logical sequence the observations made on growth characteristics, morphology, and ultrastructure of the unstable L-form of E. faecalis used in the HEK system previously described. We hypothesized that a reproductive cycle in which small, dense nonvesiculated L-forms are the central (core) element. These forms divide and bud rapidly. Additionally, they appear to be capable of growth and development within vesicles of mature mother forms. When these forms are released from the vesicles into the surrounding fluid medium, further growth occurs, resulting in the development of immature and ultimately large, mature mother Forms (Figures 4-6). Under conditions unfavorable for L-form growth, these dense forms develop first into transitional forms and then into wall-containing organisms. We reasoned that these dense forms might be considered undifferentiated cells with the capacity to develop along several different routes, depending upon the stimulus received. The pathology observed in the HEK cell system with this relatively stable L-form was triggered when the L-form was reverting to the wall-containing bacterial form. We assumed from our morphological data that the accumulation of electron-opaque material and the formation of mesosome-like structures were synonymous with aging and death as well as being prerequisites for reversion to the bacterial form. Hence, reversion to the wall-containing form as well as aging, with subsequent death, of the L-form may be the effect of common causes, namely, the depletion of available nutrients and the accumulation of toxic products in the growth medium (Green et al., 1974a).

Figure 3. Acridine orange stain of a renal tubular epithelial cell in the urine of a nephrotic patient. Note the yellow-green fluorescence (DNA) of the contained pleomorphic bacteria-like forms. Oil immersion. Bar, 10 μm. (Reprinted with permission from Domingue and Woody, 1997.)

Figure 3. Acridine orange stain of a renal tubular epithelial cell in the urine of a nephrotic patient. Note the yellow-green fluorescence (DNA) of the contained pleomorphic bacteria-like forms. Oil immersion. Bar, 10 μm. (Reprinted with permission from Domingue and Woody, 1997.)

Reversion of the L-form may aptly illustrate one of the immutable laws of nature: when faced with unfavorable environmental conditions, an organism must adapt or die. This may raise the question of how a compromised cell wall deficient/defective bacterium mobilizes the energy necessary for reversion to the bacterial walled phase. We speculated that these atypical forms are genetically programmed to develop a cell wall when nutrients and energy sources are wanting. Undoubtedly there is an evolutionary advantage to being an independent, free-living form able to forage for the best nutrients. The unstable L-form has probably taken up its parasitic residence within the host cell only because there are hazards in the extracellular milieu (Green et al., 1974a; 1974b).

Proposed Developmental Stages in a Stressed Pathogenic Bacterium

The hypothesized reproductive cycle for a relatively stable L-phase variant of enterococcus suggested that tiny dense bodies are capable of developing into undifferentiated cells and have the potential for unlimited growth and division. We proposed that one large, vesiculated, mature, parent L form may develop into many elementary bodies that become undifferentiated dense forms that may then be extruded from the parent large bodies as propagating organisms. Such forms might retain the ability to mature within the vesicle of the parent or even outside of it after rupture as long as they remain attached to it. Such extruded bodies contain a bacterial genome and minimal metabolic capability (i.e., enzymes and cofactors) sufficient to initiate energy production and biosynthesis. They may reproduce as dormant forms without cell walls and may revert to cell wall-containing bacteria, or they may do both. As a result, production of the dense bodies within the vesiculated parent would represent still another type of bacterial differentiation (Figure 6).

Our observations of electron-dense cytoplasmic bodies within parent cells of wall-defective enterococci might suggest that symptom reappearance in certain chronic bacterial infectious diseases, including spirochetoses (syphilis; Lyme disease) and tuberculosis is related to bacterial differentiation into resistant forms (elementary dense cytoplasmic bodies) that persist in tissue. We have shown that elementary bodies derived from L-forms of at least 0.24 μm may, in fact, grow into undifferentiated forms. Smaller dense bodies within vesicles are assumed not to play a major role in reproduction, possibly because they are either deficient or devoid of DNA. It is our belief that these dense bodies might be capable of maturation within the parent L form vesicle or even after its rupture. In any case, the role of dense bodies as resistant forms of pathogenic bacteria deserves further investigation. Regarding reversion to a wall-containing bacterium, we hypothesized that the L-form does not separate completely from the newly formed classical bacterium but that, instead, components of the L-form are incorporated into the walled forms. The failure to observe transformation of autonomous dense bodies into bacterial forms with cell walls supports this interpretation (Green et al., 1974b).

Current Corroborative Experimental Evidence

Peptidoglycan in L-Forms: An Innovative Finding

Figure 4. Various hypothesized stages of development (A-D) from dense form (A) to mature vesiculated mother form (D). Note elementary bodies within vesicles as well as those arising out of cytoplasm of mother form (arrows). Note the presence of dense granular cores within both D1 and D2. x 12,188. (Reprinted with permission from Green, Heidger and Domingue, 1974b.)

Figure 4. Various hypothesized stages of development (A-D) from dense form (A) to mature vesiculated mother form (D). Note elementary bodies within vesicles as well as those arising out of cytoplasm of mother form (arrows). Note the presence of dense granular cores within both D1 and D2. x 12,188. (Reprinted with permission from Green, Heidger and Domingue, 1974b.)

An intriguing, surprising, paradoxical finding by Joseleau-Petit et al. (2007) revealed that Escherichia coli L-forms undergo residual peptidoglycan synthesis indispensable for growth and probably required for cell division. Experiments were performed to conclusively prove that peptidoglycan synthesis was required for the survival of the L-forms by removing the supply of peptidoglycan precursors. Inhibiting any of the three different cytosolic steps in the synthesis of peptidoglycan destroyed L-form growth, and provided corroborating evidence that L-forms did not tolerate complete loss of peptidoglycan. Additionally, it was shown that the L-forms retained 7% of the amount of peptidoglycan produced by the rod-shaped E. coli grown in parallel cultures without the inhibiting agent (cefsulodin). The peptidoglycan produced by the L-forms was normal in structure. Heretofore, bacterial L-forms were considered as cell wall-less prokaryotes, completely devoid of peptidoglycan. Their mutant studies indicated that colony formation required D-glutamate, diaminopimelate, and MurA enzyme activity, which are all specific for synthesis of peptidoglycan. Additionally, they devised a simplified, rich, hypertonic broth culture method incorporating the beta lactam antibiotic, cefsulodin, a specific inhibitor of penicillin-binding proteins (PBPs) 1A and 1B that rapidly converted, within 24 hours, all E. coli cells into heterogeneously sized, spherical, osmosensitive L-forms. These produced colonies on agar surfaces (unlike their past ancestors which grew as embedded colonies in a specific concentration of agar), and could be continually propagated in liquid medium. This procedure achieved a proper balance between the set of reaction to be inhibited versus those that must be retained (Young, 2007).

Since L-forms were first described in 1935, generating L-forms in vitro was cumbersome and time consuming because it required incubating cells in complex media in the presence of elevated concentrations of penicillin in agar (gradient plates) with continual passage for many years to maintain viability as L-forms per se. The cleverly designed study by Joseleau-Petit et al. (2007) offers challenging new ways for experimental advances on bacterial L-form reproduction and the overall genetics of atypical forms from various bacterial genera. The methodology presented on the ease and rapidity of producing L-forms in the laboratory is a most useful achievement in itself, and should facilitate further experimentation on the role of pleomorphic forms in pathogenesis.

Life Without a Cell Wall or Division Machine in a Bacillus

This renewed interest on the basic biology of bacterial L-forms as exemplified in the Joseleau-Petit et al. study, along with several other recent fundamental revelations, including those of Leaver et al. (2009) who reported in Nature, on the development of a controllable system for generating B. subtilis L-forms is welcomed, and sets the stage for melding basic science with clinical relevance in an area of medical research which has for too long been forgotten and neglected. Leaver et al. used genome sequencing for identifying a point mutation which predisposed cells to grow without a cell wall. The L-forms propagated did not require the normal FtsZ-dependent division machine (FtsZ: Filamenting temperature-sensitive mutant Z: prokaryotic homologue to the eukaryotic protein tubulin). Instead, an extrusion-resolution mechanism was revealed and confirmed by time lapse imaging of B. subtilis L-forms. This mechanism of proliferation is similar to the proposed reproductive cycle in persistence and reversion of L-forms in a human embryonic kidney tissue culture system which we investigated utilizing transmission electron microscopic techniques for Enterococcus faecalis L-forms (Green et al., 1974a; 1974b). Leaver et al (2009) speculate that this type of propagation might shed light into how early cellular life forms (devoid of cell walls) may have flourished.

L-Forms Respond to Cell Wall Deficiency by Modifying Gene Expression and Mode of Division

Another recent publication on Listeria monocytogenes L-forms (Dell’Era et al., 2009) demonstrated that these L-forms responded to cell wall deficiency by modifying gene expression and the mode of division. Peptidoglycan precursors were still produced in the wall-less forms. They, too, confirmed via time-lapse microscopy of fluorescently labeled L-forms, a switch to a novel transcriptomics of parent and L-form cells in which manifold differences in expression of genes were associated with morphological and physiological functions. As with our transmission electron microscopy findings on a developmental cycle for E. faecalis (Green et al., 1974b), the L. monocytogenes L-forms showed genome-containing membrane vesicles forming within enlarged L-forms (mother forms), and subsequently released by collapse of the mother cell. These authors also reported that the L. monocytogenes L-forms featured down-regulated metabolic functions correlating with the dramatic shift in surface to volume ratio, whereas upregulation of stress genes reflects the difficulties in adapting to this unusual, cell wall-deficient lifestyle.

Figure 5.  (Top) Mature vesiculated mother form with collapsed peripheral vesicle (horizontal arrow). Note swirled arrangement of dense, irregularly constricted filaments (vertical arrow). (Bottom) Immature dense form with expanded nucleoid and a single vesicle containing elementary particles. x 16,250.  (Reprinted with permission from Green et al., 1974b.)

Figure 5. (Top) Mature vesiculated mother form with collapsed peripheral vesicle (horizontal arrow). Note swirled arrangement of dense, irregularly constricted filaments (vertical arrow). (Bottom) Immature dense form with expanded nucleoid and a single vesicle containing elementary particles. x 16,250. (Reprinted with permission from Green et al., 1974b.)

These recent findings may help to stimulate further research directed towards a better understanding of how atypical, stressed bacteria (L-forms, pleomorphic bodies, dense forms, filaments, filterable bacterial forms — all derived from ordinary wall-containing bacteria) survive in vivo. This could lead to clearer perceptions of cellular and molecular changes in host pathogen-interactions, of antibiotic resistance, and of bacterial pathogenesis.

Molecular Mechanisms Essential for Formation and Survival of L-Forms

Newly published research addresses molecular mechanisms essential for formation and survival of L-forms. Glover et al. (2009) in Johns Hopkins University conducted whole genome transcriptome analyses, mutant library screens, and complementation experiments to explore the molecular origin of E. coli L-form formation, identifying stress genes and pathways overlapping with persisters and biofilm bacteria. This study provided a framework for future research on the interaction of identified genes and pathways that lead to L-form development. Of special relevance were the mutant screens which identified three mutant groups with varying degrees of defects in L-form formation or survival in comparison to the wall-containing classic E. coli. Those mutants exhibiting complete lack of L-form growth fit in to pathways related to cell envelope stress, DNA repair, iron regulation, and outer membrane biogenesis. They were able to restore the mutants to L-form growth by their respective wild type genes, confirming their role in L-form formation or survival. An analogous situation might well exist in a variety of infectious diseases that lead to in vivo persistence of atypical, pleomorphic bacterial forms. Research should be aimed at understanding how the identified pathways and genes interact and lead to the surface characteristics of L-forms. These provocative findings also have implications for insight on the emergence of antibiotic resistance, molecular clarifications on persistence, and the possible design of novel drugs and vaccines for interrupting the host-pathogen interaction in persistent infections and expression of disease.

Survival of E. coli Under Lethal Heat Stress by Converting to L-Forms

Markova et al. (2010) recently reported intriguing findings on the survival of E. coli under lethal heat stress by converting to L-forms. Their results provoke a re-examination of the traditional view of killing strategies against bacteria. They provided evidence of paradoxical survival through L-form conversion of E. coli high cell density populations after lethal treatments by boiling or autoclaving. More astonishing is that the authors state that the dense confluent growth of E. coli in solid medium before lethal treatments harbored a smaller L-form subpopulation when the culture entered stationary phase (which can also occur under conditions of nutritional starvation). It is known that when bacterial cells reach stationary phase, growth practically ceases and cells find themselves under unfavorable conditions. Non-growing cells become tolerant to killing by lethal factors. These authors believe that their evidence of crystalline structures and some regular patterns found both in “starvation” and “lethal heat treatment” experiments appear to be closely linked to the processes of L-form conversion. Similar pseudo-crystalline aggregates were observed in streptococcal L-forms by Cole (1968). DNA protection by biocrystallization may be crucial and widespread in prokaryotes. Markova et al. (2010) stated that the observed phenomenon of biocrystallization in L-form cultures presents a provocative stimulus for further investigation, giving the opportunity to clarify the unique survival strategy of a bacterial population under lethal conditions, in which normal, wall-containing bacteria cannot survive. The authors emphasized that association of this phenomenon with L-form conversion is best understood when the unusual life style that L-forms exhibit is considered. They affirmed that L- forms behave like an entire population within which the role of individual organisms and organelles is difficult to ascertain. Markova et al further elaborated that the L cycle, either syncytia designed as “symplasm” consisting of numerous nuclei embedded in a cytoplasm within one L-body, or the smallest and most resistant to environmental stresses, filterable L-granules consisting mainly of DNA and most likely exerting nuclear functions, are the most remarkable (probably same as electron dense bodies in our study, Green et al., 1974a; 1974b). Moreover, they concluded that chromosomal DNA, especially in L-forms, should be regarded as a substantial mass — the nucleoid body, which can vibrantly interact with other components. They went on to emphasize that the dynamic character of morphogenesis of L-form populations and the various ‘disintegrating’ or ‘reintegrating’ processes taking place, suggest that the preservation of the most important component of the bacteria, namely the nucleoid (by formation of crystalline assemblies) is compatible with the L-form life cycle, and as a possible mode of protection against lethal heat factors. Certainly, these results contradict the traditional view that autoclaving kills all bacterial cells, and should encourage further studies on the dynamics of L-form survival in vitro and in vivo.

Figure 6. Electron micrograph of mature vesiculated mother L-form with full peripheral vesicle just prior to extrusion.  Note the similarity between the dense form within the vesicle and that from outside, with reference to peripheral cytoplasm with expanded nucleoid (arrows). x 13,750. (Reprinted with permission from Green et al., 1974b.)

Figure 6. Electron micrograph of mature vesiculated mother L-form with full peripheral vesicle just prior to extrusion. Note the similarity between the dense form within the vesicle and that from outside, with reference to peripheral cytoplasm with expanded nucleoid (arrows). x 13,750. (Reprinted with permission from Green et al., 1974b.)

Is Hodgkin’s Disease a Human Counterpart of Bacterially Induced Crown-Gall Tumors in Dicotyledons?

In a letter to The Lancet, Sauter (1995) pointed to a bacterial etiology for Hodgkin’s disease (also known as Hodgkin’s lymphoma). Sauter’s hypothesis implicates a botanic analogue with a specific and unusual pathogen interacting with the host. Briefly, he proposes that Hodgkin’s disease is a human counterpart of bacterially induced crown-gall tumors provoking malignant tumors in dicotyledons in which Agrobacterium tumefaciens (a plant pathogen) transfers its DNA to the plant’s DNA (exchange of genetic material — oncogenic plasmids from a prokaryote to a eukaryote resulting in crown- gall tumors of plants). He further proposed that bacterial DNA sequences should be looked for in Reed-Sternberg cells in human tissues, and if detected, might explain how Hodgkin’s disease has features of a bacterial infection. He implicates Bartonella species as the etiologic agent of Hodgkin’s disease because it belongs to the alpha-2 subgroup of proteobacteria as does Agrobacterium tumefaciens, which is also classified within this nomenclature. He further speculates that antibiotic treatments of very early Hodgkin’s disease may be successful before the genetic exchange between prokaryotic and eukaryotic cells has occurred. Sauter and colleagues were able to demonstrate regression of Hodgkin’s disease in the lung of a patient by prolonged treatment with ciprofloxacin and clarithromycin. Although Sauter and Blum (2003) implicate Bartonella as a possible etiologic agent in Hodgkin’s disease, no cultural, serological, or molecular bacteriologic data are presented to substantiate their claim. They did show in Periodic acid-Schiff stained sections the presence of intracellular rod- and sphere-like forms in all tissues from Hodgkin’s patients and in all patients suffering from sclerosing mediastinal B-cell lymphomas, but not in sections of non-Hodgkin’s lymphomas (Sauter and Kurrer, 2002; Sauter and Blum, 2003). These pleomorphic forms morphologically resemble the elementary and dense bodies that develop during the reproductive cycle of stable and unstable L-forms in vivo and in vitro (Green et al., 1974a 1974b). Therefore, identification of peptidoglycan in pleomorphic bodies in tissues obtained from Hodgkin’s and sclerosing mediastinal B-cell lymphomas may help to further implicate bacteria as etiologic agents in these diseases.

Pleomorphic Bacteria and Cancer

Pleomorphic bacteria and cancer have a long, complicated, and controversial history (Hess, 1997). Since the 1960s, Cantwell (1982) is among the most fervent, and dedicated supporters for the role of bacteria as primary pathogens and etiologic agents in certain neoplastic, lymphoproliferative and collagen diseases. He is a strong advocate for use of the acid-fast stain (a mycobacterial stain) for demonstrating pleomorphic bodies in malignant tissues and especially for those cancers that metastasize to the skin. Despite a voluminous literature on the subject (Hess, 1997; Wainwright, 1999) and general non acceptance of the role of persisting forms (bacteria?) in cancerous tissues, the time may now be propitious to mobilize and utilize modern molecular microbiological technology to define the nature of pleomorphic bodies in a variety of cancers. Is bacterial peptidoglycan detectable in pleomorphic bodies in tissue biopsies from cancer patients? Are bacterial DNA sequences detected in situ in tissues harboring pleomorphic forms? Affirmative answers to these questions would undoubtedly create quite a spark of interest and should be a motivating force for stimulating new, innovative, molecular experimentation on an otherwise moribund subject. One should be reminded that it is currently well accepted that Helicobacter pylori is the cause of gastritis and peptic ulceration and that there is a positive association between H. pylori infection and the development of gastric and pancreatic cancers. This host-pathogen interaction is cleverly engineered: H. pylori produces the enzyme urease which helps to reduce stomach acidity creating a more sustaining milieu for the organism, while ineffective white blood cells responding to the infectious site, die, releasing nutrients that feed the H. pylori, favoring persistence. This astonishing and significant discovery by Marshall and Warren was first published in The Lancet (1983), and in ensuing years, many scientists and clinicians were skeptical, finding it difficult to believe that bacteria could grow in the stomach’s acidic, pH 2 environment. This garnered a Nobel Prize in medicine for Marshall and Warren’s remarkable discovery in 1995.

Bacteriologic Advances in Laboratories: Testing of Hypothesis

In this age of molecular microbiology, we are now able to appreciate the world-wide range and multiplicity of bacteria that was not fully attainable by culture methods of the past. Bacteriologic advances in laboratories which include the broad-range polymerase chain reaction and other sophisticated technology such as representation difference analysis, expression library screening, and host gene expression profiling have revealed an increasing number of previously unidentifiable organisms in a variety of pathologic conditions (Domingue and Woody, 1997; Relman, 1999). The ultrastructural localization of peptidoglycan in pleomorphic forms utilizing techniques that incorporate highly specific antipeptidoglycan antibodies for targeting peptidoglycan (as marker), such as fluorescein labeled monoclonal antibodies, should be explored on tissue biopsies. Current advances in cryoelectron tomography have opened new windows in prokaryotic and eukaryotic ultrastructure (Steven et al., 2003; Fernandez et al., 2004; Jensen et al., 2007; Li and Jensen, 2009; Vollmer and Seligman, 2010). It can reveal the structure of prokaryotic and eukaryotic cells in their native states in three dimensions at molecular resolutions. This technique offers promise for resolving many fundamentally important questions about bacterial ultrastructure (pleomorphic bodies persisting in human tissues). Recent developments in electron microscopy help to alleviate and correct wrong views caused by the introduction of artifacts in EM preparations, imaging, and image evaluation procedures (low temperature embedding, cryo-techniques, computer averaging). Additionally, these improvements in EM methodology offer promise for the detection of so far unidentified cytological properties of the bacterial cell (pleomorphic forms, partially or completely cell wall deficient as persisting agents in tissues and body fluids).

These methodologies are having a positive impact on clinical microbiology and infectious diseases for identification of bacteria. Because it is estimated that less than 10% of all bacteria have been identified, it is inevitable that the past systems of microbial classification will become increasingly cumbersome and counterproductive clinically. A correlating transition to one with more potential should definitely be explored. Therefore the present trend toward using sequence-based identification of difficult-to-culture and nonculturable organisms should successfully achieve this end. It is hoped that clinicians frustrated by negative cultures in obviously infected patients will encourage clinical microbiology laboratories to expand their diagnostic capabilities so that the role of these more fastidious microorganisms can be appreciated (Domingue and Woody, 1997).

Concluding Remarks on Bacterial Persistence and Expression of Disease

The interaction of microbes within the host can lead to the enhancement or depression of their individual properties. Clinical expression of their presence in the host depends on the genetic vulnerability of the host, the particular environmental stresses, and the number and location of such consortia. The clinician who faces this tangled scenario must quantitate and define the dynamic that has led to the patient’s illness.

Many bacteria-like elements can be visualized at the ultrastructural level, but cannot be grown in culture. Nucleic acid analyses in vitro can approximate the locations of these bacteria on the phylogenetic tree. Unfortunately, none of these sophisticated laboratory procedures are consistently successful in identifying pleomorphic organisms persisting in tissues, nor are they guides to optimal therapy. Pleomorphic, cryptic organisms, whether intra- or extracellular, are ubiquitous. A first step would be to demonstrate their presence in tissue samples (as previously described) when laboratories report culture negative findings in patients suspected of having a bacterial infection; or to attempt to grow them in culture. Quantifying and identifying the cells most parasitized are impractical routine clinical approaches. Koch’s postulates cannot be fulfilled, because it is impossible to precisely duplicate all the variables that are involved in disease expression. Any patient with a history of recurrent infections and persistent disability is sending signals that this phenomenon is occurring. The autoimmune disorders, in which no organisms can be identified by routine techniques, are suspect in this regard. The selection of antimicrobial agents for patients with cryptic infections can be quite frustrating. Even if an organism grows in vitro, it may not represent the primary pathogen. In addition, drug susceptibility testing fails to reveal the action of the agent on the infecting organism’s toxicity and capacity to adhere to cell membranes in vivo.

Although physicians are discouraged from the indiscriminate use of antimicrobial agents without strong cultural, immunologic, or molecular evidence that the therapy is appropriate and that the severity of the illness justifies the risk of side effects, it is, nevertheless, a common clinical practice and undoubtedly contributes to the development of pleomorphic, persisting bacterial forms and mutants in vivo.

Survival of a species requires that a reasonable identity be maintained. Over time, mechanisms to maintain “self” have evolved. Many such relationships have been so successful that both host and invading organism benefit. Such a process, which transiently reduces immune competence, can occur episodically in healthy subjects in association with various stresses or in particular diseases, such as rubeola. As one ages, there tends to be an insidious accumulation of intracellular microbial forms. Such quiescent organisms tend to be activated to a more toxic form when homeostatic disturbances threaten their cellular loci. The numbers and locations of cells involved with one or more types of organisms determine the clinical reaction. It can be very difficult to decide whether a new illness is due to a new organism or to an interaction with one or more pleomorphic cryptic organisms. These interactions can be as complex as the well-known increase in toxicity of Corynebacterium diphtheriae when this bacterium is infected by bacteriophage. The distinction between phage genes and bacterial genes is blurred with respect to both function and reality. It is conceivable that much of the heredity of bacteria is of viral origin, because many unknown defective proviruses may exist in nature; on the other hand, phages may be fragments of bacterial DNA that have acquired the capacity for independent reproduction. Indeed, with a history of mutual interaction of viruses and bacteria over the course of evolution, the endeavor of sharply distinguishing their genes must be meaningless. These philosophic concepts are implicit in any discussion of the role of dormant, persistent, difficult-to-culture, and impossible-to-culture bacteria in disease (Domingue and Woody, 1997).

From the evidence available in the literature, it seems that mycoplasmas are a diverse group of wall-less prokaryotes derived from various bacteria. It has been convincingly demonstrated by immunologic methodology that acholeplasmas are descended from streptococci, specifically from groups N and D. It therefore seems logical to conclude from molecular and immunologic data that mycoplasmas are not a true phylogenetic class and that they are not descended from one single common ancestor. A teleologic approach to the evolutionary relationship between mycoplasmas and cell wall-defective/deficient bacteria should consider the survival advantage of an organism with a cell wall in a hostile primordial environment. Only after the appearance of higher life forms was there a protective niche for mutant microorganisms (Domingue and Woody, 1997).

If we are to extend these findings to clinical relevance, it is tempting to speculate that in vivo genetic events may lead to development of bacteria with aberrant cell wall morphology and physiology and may involve complex interactions among a variety of bacteria and host cells. Such interactions might lead to persistence of a dormant bacterial phase in patients with infectious diseases. This may be a continual biologic process in all living hosts, with the host environment serving as the determinant for evolution, persistence, and survival of morphologically altered microbes. Previously described, newly published, provocative, molecular microbiological data lend credence to the hypothesis and corroborate the multiplicity of pleomorphic forms that develop during reproduction of L-forms in vitro. Recent studies on modifications of gene expression and modes of division for stressed bacteria are highly relevant to the hypothesis. It is proposed that in vivo persistence of these bacterial elements escape immune surveillance partially, completely, or may integrate with host cell organelles to create bacteria-host-cell-antigen complexes which could provoke immunopathologic consequences. To speculate further, bacterial persisters in a scenario of molecular mimicry might possess peptide sequence similarities with self peptides sufficient to result in cross-activation of autoreactive T or B cells by pleomorphic form derived peptides. Might there also be an analogous situation to that of H. pylori growth in the human stomach: Do persistent atypical bacterial forms produce enzymes which neutralize hostile host factors creating a more hospitable tissue environment; and are there microbial factors antagonistic to white blood cells preventing their migration to the infectious site or scene of pleomorphic form persistence? Furthermore, there may be an exchange of genetic material between the persisting prokaryote nucleoid (oncogenic plasmids or unknown nuclear interactions) and host eukaryotic chromosome creating cellular alterations adequate to initiate neoplastic growths.

Scientists skilled in disciplines such as cellular adhesion, transposition of genetic elements, and microbial reassembly as mechanisms for the genesis of unusual organisms should be able to design, execute, and interpret experimentation that will confirm or refute, unambiguously, the proposed hypothesis. If pleomorphic forms in tissues are confirmed as bacteria by sensitive and specific methodology, the clinical and research implications are unlimited; and have the potential for clarifying the mysterious and poorly understood host-pathogen interactions in persistent infections and expression of innumerable idiopathic diseases.

Acknowledgments

This work was supported over a period of thirty years (1967-1997) by The Hume Research Fund, Department of Urology, Tulane University School of Medicine; grants from NIH (1 RO 1 DK44812-08), Veterans Administration, Schlieder Foundation, Cadwallader Family Foundation, Interstitial Cystitis Association; and a donation from the Harp Family.

Disclosure

The author reports no conflicts of interest.

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[Discovery Medicine; Discov Med 10(52):234-246, September 2010.]

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