**6. Clinical significance and role of mycobacterial L-forms**

Arguments for and against significance of L-forms as infecting and persisting agents, respectively their role in human and animal diseases, are limited because of difficulties in their isolation, cultivation and identification. However, a lot of papers, reviews (Allan et al., 2009; Beran et al., 2006; Domingue and Woody, 1997; Domingue, 2010; Gumpert & Taubeneck, 1983; Onwuamaegbu et al., 2005; Zhang, 2004) and several monographs (Domingue, 1982; Mattman, 2001; Prozorovski et al., 1981), support the concept that L-forms can be induced *in vivo*, can persist there for a significant span of time and can be the cause for latent, chronic and relapsing/recurrent infections, as well as for diseases of unknown infectious-allergic or autoimmune origin.

However, of all the bacteria, L-forms predominate and are crucial to the survival of *M. tuberculosis in vivo.* Therefore they are thought of as carriers of a tubercular constitution (Mattman, 2001). The understanding of cell wall deficiency in *M. tuberculosis* may occur as consequence of a long-lasting interaction with the host and as a strategy to ensure its survival and persistence *in vivo* is still limited, as mycobacteria are quite difficult to detect, especially when in their viral-like, cryptic state. Although the mechanisms of spontaneously occurring *in vivo* cell wall deficient forms are difficult to explain, many authors have considered that mycobacteria, undergoing L-form transformation, are of clinical significance

under favorable for the pathogen circumstances. Mattman defined the essential factors for reversion, the most popular of which are omission of the inducing agent, changes in nutrition, concentrating populations, inoculation in to experimental animals and others (Mattman, 2001). Of special interest is the reversion stimulated by products from microbes. Rathham & Chandrasekhar reported about reversion of filterable variants of tubercle bacillus from sputum by culturing with Freund's adjuvant (Rathham & Chandrasekhar, 1976). Although atypical forms are genetically programmed to develop a cell wall, it is not yet clear how compromised cell wall deficient bacteria mobilize the energy necessary for reversion to bacterial walled phase. It is interesting to note that the reversion of mycobacterial L-forms to normal TB bacilli appeared to be more difficult and slower,

There is a widespread assumption, which perceives the dormant state of *M. tuberculosis* as a *reversible* state or as ability of mycobacteria to reverse into active state and to reactivate the disease (Shleeva et al., 2010). Recently, it has been found that bacteria possess a specific system for autoregulation of growth and development, which participates in control of cell differentiation at the level of regulation of the functional activity of subcellular components and of the cell as a whole (Shleeva et al., 2010). Resuscitation-promoting factors have also been identified and their role in latency and reactivation of tuberculosis have been investigated (Biketov et al., 2007; Zhang et al., 2001;). Five genes encoding Rpf-like proteins have been found in *M. tuberculosis* genome, which may act in reactivation of "nonculturable " forms of *M. tuberculosis* (Kana et al.*,* 2008; Mukamolova et al., 2002; Tufariello et al., 2004)*.* Shleeva et al. (2003) found that cell-free culture liquid of an exponential-phase *Mycobacterium tuberculosis* culture or the bacterial growth factor Rpf exerted a resuscitating effect, substantially increasing the growth capacity of the nonculturable cells in liquid medium*.* During resuscitation of nonculturable cells, a transition from ovoid to rodlike cell

Arguments for and against significance of L-forms as infecting and persisting agents, respectively their role in human and animal diseases, are limited because of difficulties in their isolation, cultivation and identification. However, a lot of papers, reviews (Allan et al., 2009; Beran et al., 2006; Domingue and Woody, 1997; Domingue, 2010; Gumpert & Taubeneck, 1983; Onwuamaegbu et al., 2005; Zhang, 2004) and several monographs (Domingue, 1982; Mattman, 2001; Prozorovski et al., 1981), support the concept that L-forms can be induced *in vivo*, can persist there for a significant span of time and can be the cause for latent, chronic and relapsing/recurrent infections, as well as for diseases of unknown

However, of all the bacteria, L-forms predominate and are crucial to the survival of *M. tuberculosis in vivo.* Therefore they are thought of as carriers of a tubercular constitution (Mattman, 2001). The understanding of cell wall deficiency in *M. tuberculosis* may occur as consequence of a long-lasting interaction with the host and as a strategy to ensure its survival and persistence *in vivo* is still limited, as mycobacteria are quite difficult to detect, especially when in their viral-like, cryptic state. Although the mechanisms of spontaneously occurring *in vivo* cell wall deficient forms are difficult to explain, many authors have considered that mycobacteria, undergoing L-form transformation, are of clinical significance

**6. Clinical significance and role of mycobacterial L-forms** 

infectious-allergic or autoimmune origin.

when compared to other bacteria.

shape occurred.

for the incidence of relapses and are a prognostic unfavorable indicator (Berezovski & Salobai, 1988; Dorozhkova. et al., 1989, Dorozhkova et al., 1990; Khomenko et al., 1980). Observation of atypical, non-acid fast and cell wall deficient forms of *M. tuberculosis* in patient specimens suggests their occurrence *in vivo*. Kochemasova succeeded in isolating *M. tuberculosis* L-forms from cerebrospinal fluid, from resected sections of different organs of tuberculosis patients, as well as from urine of patients with renal tuberculosis during long lasting chemotherapy (Berezovski & Golanov, 1981; Kochemasova et al., 1970; Kochemasova, 1975). L-variants of *M. tuberculosis* were observed during antibacterial therapy of tuberculosis meningitis by Kudriavtsev et al. (1974). Of special interests were the reports by different authors about isolation of *Mycobacterium tuberculosis* L-forms from sputum and caverns of patients with pulmonary tuberculosis (Takahashi, 1979a, 1979b; Tsybulkina, 1979;). Zhu et al. (2000) found cell wall deficient forms of *M. tuberculosis in*  biological material,particularly sputum and blood from patients with pulmonary tuberculosis. The first report of L-forms from *Mycobacterium scrofulaceum* infection, occurring in an 11 –year- old boy, was made by Korsak (1975). L-colonies consisting of non- acid fast coccoids and large spheres grew from autopsy materials (dermal lesions, brain, spleen, kidney, lung and intesties), sometimes making syncitya and reverting to acid fast bacilli.

Regardless of the huge progress in TB research and the development of new molecular technologies, pathogenesis of latent tuberculosis is still not well understood. The dynamic hypothesis of Cardona (2009) suggests that latent tuberculosis infection is caused by the constant endogenous reinfection of latent bacilli. Considering this hypothesis, constant "escape" of bacilli from granulomas before fibrosis is the primary source of bacteria, reactivation would never occur after a specific time period, unless the host suffered an immunosuppressive episode (Cardona & Ruiz-Manzano, 2004). Of special interest is the finding that foamy macrophages are able to maintain a stressful environment that keeps the bacilli in non-replicating state, but on the other hand, allow them to escape from granulomas, making them more resistant to future stressful conditions (Cardona et al., 2000; Cardona et al.; 2003; Cardona, 2009).

Currently, asymptomatic latent tuberculosis is defined not by identification of bacteria, but by host immune response tests. Although individuals with latent tuberculosis harbor viable bacteria, it is difficult to identify them (Young et al., 2009; Manabe & Bishai, 2000). Among the unresolved mysteries of latent tuberculosis is the nature and anatomical situation of persisting tubercle bacilli (Grange 1992). The common observation that acid-fast bacilli are frequently absent in smears is an indication that pathology may result from *in vivo* propagation of cell wall deficient mycobacteria (Domingue, 1982; Judge& Mattman, 1982). Thus, if diagnosis by finding these forms (cell wall free, non acid-fast persisting bacilli) becomes practice, it may have valuable application in diagnosis of latent tuberculosis.

There are many tuberculous syndromes in which the aetiology is occult or imitative of other diseases (Domingue, 1982; Judge & Mattman, 1982). Traditional concept of the mycobacterial aetiology of sarcoidosis and especially the assumption that cell wall deficient forms rather than bacillary are involved has been supported by several reports. Varying acid fast spindle-shaped or yeast-like structures, termed *pleomorphic chromogens*, and cell wall deficient forms of *M. tuberculosis* complex were detected in lymph node tissue from subjects with sarcoidosis (Alavi and Moscovic, 1996; Moscovic,1978). Cantwell also suggested that acid-fast organisms, found in skin lymph nodes and lung tissue from patients with

Cell Wall Deficiency in Mycobacteria: Latency and Persistence 209

In conclusion, tubercle bacilli may use L-form conversion as unique adaptive strategy to survive and reproduce under unfavorable conditions in hosts. Possibility for persistence and reversion of L-forms to classical TB bacilli *in vivo* elaborates on some specific aspects of Lconversion phenomenon and link them to the mechanisms at play in latent tuberculosis. Morphologically modified and non-acid fast L-forms of mycobacteria are difficult to identify and often remain unrecognized, or are mistaken for contaminants. "L-form persistence phenomenon" of actively growing and propagating by unusual modes cell wall deficient cells differs definitively from the current understanding for latency as persistence of a few''non-replicating'' or ''dormant'' bacteria. Mycobacterial L-forms give rise to many unsolved questions concerning their biology and behavior *in vivo*, as well as about the genetic regulatory mechanisms leading to their appearance. Cell wall deficiency in mycobacteria remain an interesting topic that needs to be re-examined in the context of

This work was supported by grant ID № 02/27 of the National Scientific Fund in Bulgaria.

Alavi, H. & Moscovic, E. (1996). Immunolocalization of cell-wall-deficient forms of

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Armbruster, C.; Junker, W.; Vetter, N.& Jaksch, G. (1990). Disseminated bacilli Calmette-

*Infectious Diseases,* Vol. 162, No5 (November 1990), pp.1216, ISSN: 0022-1899 Belianin, I.; Nikolaeva, G. & Martynova, L. (1997). Action of dissolved ozone on

Beran, V.; Havelkova, M.; Kaustova, J.; Dvorska, L. & Pavlik I. (2006). Cell wall deficient

Berezovski, B. & Golanov, V. (1981). *Mycobacterium tuberculosis* L forms in patients with

Berezovskii, B. & Salobai, R. (1988). The role of L variants of Mycobacteria in the

Biketov, S.: Potapov, V.; Ganina, E.; Downing, K., Kana, B. & Kaprelyants, A. (2007). The role

Boris, M.; Teubner, D. & Shinefield, H. (1969). Bacterial interference with L-forms. *Journal of Bacteriology*, Vol.100, No2, (November 1969), pp.791-795, ISSN: 0021-9193

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development and clinical course of recurrences of pulmonary tuberculosis.

of resuscitation promoting factors in pathogenesis and reactivation of Mycobacterium tuberculosis during intra- peritoneal infection in mice. *BMC* 

**7. Conclusion** 

modern molecular biology.

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0032-953

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**8. Acknowledgment** 

**9. References** 

sarcoidosis, were mycobacterial cell wall deficient forms (Cantwell, 1982a,b). Judge & Mattman grew mycobacterial cell wall deficient forms (predominantly coccoid forms, larger L forms and short acid-fast rods) from blood of patients with sarcoidosis (Judge & Mattman, 1982). Polymerase chain reaction (PCR) was used to detect mycobacterial DNA in clinical samples from patients with sarcoidosis and in half the sarcoidosis patients was found *M tuberculosis* DNA (Saboor et al, 1992). A report, describing the molecular characterization of *M. tuberculosis* complex isolates from patients with sarcoidosis and tuberculosis, showed that half of the isolates from sarcoidosis patients did not resemble the spoligotypes of the isolates from patients with tuberculosis (Gazouli et al., 2005). Cell wall-defective mycobacteria were isolated also from skin lesions and cerebrospinal fluid of patients with sarcoidosis and identified to be *M. a. paratuberculosis* or other *M. avium-intracellulare* complex members (El-Zaatari et al., 1996). A relationship between cell wall deficient forms of *M. a. paratuberculosis* and Crohn's disease has been found by some authors, although this aetiological agent has not yet been conclusively proven (Hermon-Taylor and Bull, 2002; Hulten et al., 2001 a, b, c; Sechi et al. ,2001; Schwartz et al., 2000).

Future research in the field of cell wall deficiency in mycobacteria promises an increased accent on its association with latent and persistent bacterial state, which should be supported with modern molecular biological evidences. In order to better understand the nature of L-conversion phenomenon, it will be important to correlate *in vitro* with *in vivo* (experimental animals and patients) findings.

Since many researchers do not believe in existence of L-conversion phenomenon in mycobacteria, molecular genetic studies are relatively scarce (Hulten et al., 2000a,b; Lu et al, 2009; Melnikoava &, Mokrousova, 2006; Vishnevskaia et al. , 2001; Wang et al., 2007; Wall et al., 1993). On the other hand, L-forms are "difficult-to-identify" by most of the standard DNA-based tests, probably due to their unusual life style and irregular division. The relative scarcity and rather inaccessibility of the genetic material in L-forms make them generally difficult for genetic studies. De Wit & Mitchison (1993) indicated that mycococci derived from mycobacteria did not exist. The authors examined stored cultures of the mycococcus form of *M. bovis* BCG and *M. phlei* which were prepared by Csillag in 1972 and 1969 and found that restriction fragment patterns of the DNA of the variant forms and the parent mycobacteria were not similar. Traag et al (2009) also found no evidence that mycobacteria produced free-living "spores" (i.e cocci). However, the verification of L-forms isolated from experimental animals as genuine *M. tuberculosis* but not as contaminating bacteria became possible in our study, with species - specific spoligotyping test (spacer oligonucleotide typing technique) and after some modification of the initial steps in preparing the L-form cultures (Markova et al., 2008a). Spoligotyping results provided interesting insight into the occurrence of certain polymorphisms, *i.e.* insertion or deletion of spacer signals in some of the L-form isolates. In our laboratory, we have also gained much experience in experiments to obtain stable mycobacterial L-forms *in vitro* and have already developed a reproducible protocol, which allows obtaining sufficient biomass of L-cultures to get enough DNA. Under screening is a spectrum of the most examined genes for detection, identification and characterization of *Mycobacterium tuberculosis* complex in stable mycobacterial L-form cultures. The next necessary step after gene screening would be the sequencing analysis, in order to understand what kind of genetic events happen during L-transformation and which mechanisms lead to cell wall deficiency.

## **7. Conclusion**

208 Understanding Tuberculosis – Deciphering the Secret Life of the Bacilli

sarcoidosis, were mycobacterial cell wall deficient forms (Cantwell, 1982a,b). Judge & Mattman grew mycobacterial cell wall deficient forms (predominantly coccoid forms, larger L forms and short acid-fast rods) from blood of patients with sarcoidosis (Judge & Mattman, 1982). Polymerase chain reaction (PCR) was used to detect mycobacterial DNA in clinical samples from patients with sarcoidosis and in half the sarcoidosis patients was found *M tuberculosis* DNA (Saboor et al, 1992). A report, describing the molecular characterization of *M. tuberculosis* complex isolates from patients with sarcoidosis and tuberculosis, showed that half of the isolates from sarcoidosis patients did not resemble the spoligotypes of the isolates from patients with tuberculosis (Gazouli et al., 2005). Cell wall-defective mycobacteria were isolated also from skin lesions and cerebrospinal fluid of patients with sarcoidosis and identified to be *M. a. paratuberculosis* or other *M. avium-intracellulare* complex members (El-Zaatari et al., 1996). A relationship between cell wall deficient forms of *M. a. paratuberculosis* and Crohn's disease has been found by some authors, although this aetiological agent has not yet been conclusively proven (Hermon-Taylor and Bull, 2002;

Future research in the field of cell wall deficiency in mycobacteria promises an increased accent on its association with latent and persistent bacterial state, which should be supported with modern molecular biological evidences. In order to better understand the nature of L-conversion phenomenon, it will be important to correlate *in vitro* with *in vivo*

Since many researchers do not believe in existence of L-conversion phenomenon in mycobacteria, molecular genetic studies are relatively scarce (Hulten et al., 2000a,b; Lu et al, 2009; Melnikoava &, Mokrousova, 2006; Vishnevskaia et al. , 2001; Wang et al., 2007; Wall et al., 1993). On the other hand, L-forms are "difficult-to-identify" by most of the standard DNA-based tests, probably due to their unusual life style and irregular division. The relative scarcity and rather inaccessibility of the genetic material in L-forms make them generally difficult for genetic studies. De Wit & Mitchison (1993) indicated that mycococci derived from mycobacteria did not exist. The authors examined stored cultures of the mycococcus form of *M. bovis* BCG and *M. phlei* which were prepared by Csillag in 1972 and 1969 and found that restriction fragment patterns of the DNA of the variant forms and the parent mycobacteria were not similar. Traag et al (2009) also found no evidence that mycobacteria produced free-living "spores" (i.e cocci). However, the verification of L-forms isolated from experimental animals as genuine *M. tuberculosis* but not as contaminating bacteria became possible in our study, with species - specific spoligotyping test (spacer oligonucleotide typing technique) and after some modification of the initial steps in preparing the L-form cultures (Markova et al., 2008a). Spoligotyping results provided interesting insight into the occurrence of certain polymorphisms, *i.e.* insertion or deletion of spacer signals in some of the L-form isolates. In our laboratory, we have also gained much experience in experiments to obtain stable mycobacterial L-forms *in vitro* and have already developed a reproducible protocol, which allows obtaining sufficient biomass of L-cultures to get enough DNA. Under screening is a spectrum of the most examined genes for detection, identification and characterization of *Mycobacterium tuberculosis* complex in stable mycobacterial L-form cultures. The next necessary step after gene screening would be the sequencing analysis, in order to understand what kind of genetic events happen during L-transformation and

Hulten et al., 2001 a, b, c; Sechi et al. ,2001; Schwartz et al., 2000).

(experimental animals and patients) findings.

which mechanisms lead to cell wall deficiency.

In conclusion, tubercle bacilli may use L-form conversion as unique adaptive strategy to survive and reproduce under unfavorable conditions in hosts. Possibility for persistence and reversion of L-forms to classical TB bacilli *in vivo* elaborates on some specific aspects of Lconversion phenomenon and link them to the mechanisms at play in latent tuberculosis. Morphologically modified and non-acid fast L-forms of mycobacteria are difficult to identify and often remain unrecognized, or are mistaken for contaminants. "L-form persistence phenomenon" of actively growing and propagating by unusual modes cell wall deficient cells differs definitively from the current understanding for latency as persistence of a few''non-replicating'' or ''dormant'' bacteria. Mycobacterial L-forms give rise to many unsolved questions concerning their biology and behavior *in vivo*, as well as about the genetic regulatory mechanisms leading to their appearance. Cell wall deficiency in mycobacteria remain an interesting topic that needs to be re-examined in the context of modern molecular biology.
