**4. Formation and persistence of mycobacterial L-forms in vivo**

Animal models for the study of tuberculosis include guinea pigs, mice, rabbits and nonhuman primates. Despite the difficulty in modeling human latency in experimental animals, the understanding of both host and microbial factors that contribute to the establishment and maintenance of a persistent *M. tuberculosis* infection has progressed and the information gathered is pertinent to human latent tuberculosis (Flynn & Chan, 2001). Formation of *M. tuberculosis* L-forms *in vivo* were demonstrated by means of biological experiments on guinea pigs (Li, 1990; Markova et al., 2008b ; Ratnam &Chandrasekhar, 1976; Snitinskaia et al., 1990;), mice (Belianin et al., 1997) and rats (Markova et al., 2008a).

In our study, we established a rat model of experimental tuberculosis that produces mycobacterial cell-wall deficient forms *in vivo* (Markova et al, 2008a). Although rats are not a common animal model for TB research, we attempted, on basis of our previous experience with other bacterial L- form experimental infections (Markova et al, 1997; Michailova et al., 2000), to use the capability of these animals to exhibit high innate resistance to infections, thus ensuring inhibition of classical bacterial forms and inducing the occurrence of cell-wall deficient forms. After intraperitoneal and intranasal infection with *M. tuberculosis*, samples from lung, spleen, liver, kidney, mesenterial and inguinal lymph nodes and bronchoalveolar and peritoneal lavage liquid were taken and plated simultaneously on Löwenstein-Jensen medium or inoculated into specially supplemented for L-forms Dubos broth at weekly intervals over five weeks. Mycobacterial L-form cultures were isolated throughout

Cell Wall Deficiency in Mycobacteria: Latency and Persistence 205

*in vivo* for a long time. As far as cell wall deficiency facilitates the bacterial survival under unfavorable conditions, L-forms of different bacterial species have been shown to survive and persist for an extended period inside macrophages due to the ineffectual phagocytosis, digestion and clearance (Markova et al., 1997; Michailova et al., 1993; Michailova et al., 2000b; Michailova et al., 2007). The finding that of all the bacteria, L-forms predominate and are crucial to the survival of mycobacteria *in vivo* (Mattman, 2001; Michailova et al., 2005) needs to be taken into account when developing and putting in use new viable mycobacterial vaccines, especially considering that L-forms of *M. bovis* BCG bacilli have been found in the blood of persons vaccinated against TB with BCG vaccine (Xalabarder, 1958). This provides us with insight of the importance of L-form conversion phenomenon

for the behavior, persistence and safety of live BCG vaccines.

a b c

d e f

Fig. 9. Formation of BCG L-form cells (L) within the vacuoles in guinea pigs peritoneal macrophages**: a, b , c** -at day 1 after BCG installation, the interactions of BCG bacilli (**\***) with peritoneal cells demonstrated initial phases of phagocytosis including attraction, adhesion and attachment of bacteria to the phagocytes and processes of bacterial enclosing and engulfment; **d, e, f**- at day 45 after BCG installation, formation of BCG L-form cells (L) within the vacuoles near to mitochondria (M); **d**, **e** - L-form multiplication inside

macrophages; **f**- BCG L-form large bodies , surrounded by multi membranes; Bar = 0.5 μm

Reversion of L-forms to normal parental bacteria is an important property, which is inducible by changing the condition of cultivation *in vitro* or occurs spontaneously *in vivo*

**5. Reversion of mycobacterial L-forms to classical TB bacilli** 

(Markova et al., 2008 )

the whole period of the experiment, including the last two weeks, when typical mycobacterial colonies consisting of classical bacilli were not isolated on Löwenstein-Jensen medium. If we had used only the classical isolation procedure with Löwenstein-Jensen media alone, we would have been led to falsely believe that mycobacteria were completely eliminated. However, mycobacteria continued to persist as L-forms at the late stage of infection. We believe that the established by us rat model of experimental tuberculosis can mimick latent infection.

Mycobacteria can convert to cell wall deficient forms (L-forms) inside macrophages. After intraperitoneal administration of BCG, samples of peritoneal lavage fluid from guinea pigs were obtained at day 1, 14 and 45. In order to study whether and how *M. bovis* BCG can transform in L-forms and persist *in vivo*, series of events during interaction of live BCG bacilli with peritoneal macrophages in guinea pigs were evaluated and observed by transmission electron microscopy (Markova et al, 2008b). At the late intervals of infection, an interesting phenomenon of L-form formation inside macrophages was observed. The percent of the formed L-forms at day 14 was about 15% and at day 45, we did not find any BCG bacilli with normal morphology - all observed bacteria were in L-form state. Examination of BCG bacilli inside macrophages revealed morphological peculiarities typical of cell wall deficient bacterial L-forms, as well as different modes of L-form multiplication. As shown in Fig. 9 (d, e, f), pleomorphic and relatively large BCG L-form bodies were found inside vacuoles which were found to persist for a long time inside macrophages due to the ineffectual phagocytosis, digestion and clearance**.** Fusion of small phagosomes containing Lforms and formation of larger ones was seen as well . Additional point of interest was the observation that many mitochondria (M) with enlarged size and endoplasmic reticulum dilation (ER) were clustered closely and around L-forms . The observed process of organelle translocation appeared to be related to the intracellular life of L- forms - survival and multiplication. Microbial digestion, respectively a process of complete phagocytosis of Lforms, was not observed. Some intra-phagosomally located L-forms inside macrophages were surrounded by multi-membranes (Fig. 9 f) and so packed within membranes they were released to the extracellular space. The observed cycle of L-form attachment and engulfment by new phagocytes at the late stage of infection suggests that L-forms probably exploit apoptotic-like pathway as means of returning to the extracellular environment and for subsequent rounds of new entry and uptake by macrophages. Obviously, such apoptosislike pathway may protect L-forms from humoral and cellular host defense factors during their trafficking from intracellular to extracellular compartment and vice versa. It is generally assumed that apoptosis has developed as a host defence mechanisms against infection, but it is not completely clear what advantages apoptosis can provide to bacteria (Keane et al., 2000; Riendeau & Kornfeld, 2003; Rosenberger & Finlay, 2003). A number of authors have presented evidence that cell-wall defective variants can be formed within macrophages (Mattman, 2001; Michailova et al., 2000a; Thacore & Willett, 1966). Thacore and Willett (1966) have reported about formation of spheroplasts of *M. tuberculosis* within tissue culture cells.

Since *M. bovis* BCG is an attenuated live strain, little is known about how long it can survive in the vaccinated individuals . Reports about detection and isolation of BCG bacilli from patients with AIDS many years after vaccination (Armbruster et al., 1990; Reynes et al., 1989; Smith et al., 1992) give rise to questions about the mechanisms by which BCG bacilli persist

the whole period of the experiment, including the last two weeks, when typical mycobacterial colonies consisting of classical bacilli were not isolated on Löwenstein-Jensen medium. If we had used only the classical isolation procedure with Löwenstein-Jensen media alone, we would have been led to falsely believe that mycobacteria were completely eliminated. However, mycobacteria continued to persist as L-forms at the late stage of infection. We believe that the established by us rat model of experimental tuberculosis can

Mycobacteria can convert to cell wall deficient forms (L-forms) inside macrophages. After intraperitoneal administration of BCG, samples of peritoneal lavage fluid from guinea pigs were obtained at day 1, 14 and 45. In order to study whether and how *M. bovis* BCG can transform in L-forms and persist *in vivo*, series of events during interaction of live BCG bacilli with peritoneal macrophages in guinea pigs were evaluated and observed by transmission electron microscopy (Markova et al, 2008b). At the late intervals of infection, an interesting phenomenon of L-form formation inside macrophages was observed. The percent of the formed L-forms at day 14 was about 15% and at day 45, we did not find any BCG bacilli with normal morphology - all observed bacteria were in L-form state. Examination of BCG bacilli inside macrophages revealed morphological peculiarities typical of cell wall deficient bacterial L-forms, as well as different modes of L-form multiplication. As shown in Fig. 9 (d, e, f), pleomorphic and relatively large BCG L-form bodies were found inside vacuoles which were found to persist for a long time inside macrophages due to the ineffectual phagocytosis, digestion and clearance**.** Fusion of small phagosomes containing Lforms and formation of larger ones was seen as well . Additional point of interest was the observation that many mitochondria (M) with enlarged size and endoplasmic reticulum dilation (ER) were clustered closely and around L-forms . The observed process of organelle translocation appeared to be related to the intracellular life of L- forms - survival and multiplication. Microbial digestion, respectively a process of complete phagocytosis of Lforms, was not observed. Some intra-phagosomally located L-forms inside macrophages were surrounded by multi-membranes (Fig. 9 f) and so packed within membranes they were released to the extracellular space. The observed cycle of L-form attachment and engulfment by new phagocytes at the late stage of infection suggests that L-forms probably exploit apoptotic-like pathway as means of returning to the extracellular environment and for subsequent rounds of new entry and uptake by macrophages. Obviously, such apoptosislike pathway may protect L-forms from humoral and cellular host defense factors during their trafficking from intracellular to extracellular compartment and vice versa. It is generally assumed that apoptosis has developed as a host defence mechanisms against infection, but it is not completely clear what advantages apoptosis can provide to bacteria (Keane et al., 2000; Riendeau & Kornfeld, 2003; Rosenberger & Finlay, 2003). A number of authors have presented evidence that cell-wall defective variants can be formed within macrophages (Mattman, 2001; Michailova et al., 2000a; Thacore & Willett, 1966). Thacore and Willett (1966) have reported about formation of spheroplasts of *M. tuberculosis* within

Since *M. bovis* BCG is an attenuated live strain, little is known about how long it can survive in the vaccinated individuals . Reports about detection and isolation of BCG bacilli from patients with AIDS many years after vaccination (Armbruster et al., 1990; Reynes et al., 1989; Smith et al., 1992) give rise to questions about the mechanisms by which BCG bacilli persist

mimick latent infection.

tissue culture cells.

*in vivo* for a long time. As far as cell wall deficiency facilitates the bacterial survival under unfavorable conditions, L-forms of different bacterial species have been shown to survive and persist for an extended period inside macrophages due to the ineffectual phagocytosis, digestion and clearance (Markova et al., 1997; Michailova et al., 1993; Michailova et al., 2000b; Michailova et al., 2007). The finding that of all the bacteria, L-forms predominate and are crucial to the survival of mycobacteria *in vivo* (Mattman, 2001; Michailova et al., 2005) needs to be taken into account when developing and putting in use new viable mycobacterial vaccines, especially considering that L-forms of *M. bovis* BCG bacilli have been found in the blood of persons vaccinated against TB with BCG vaccine (Xalabarder, 1958). This provides us with insight of the importance of L-form conversion phenomenon for the behavior, persistence and safety of live BCG vaccines.

Fig. 9. Formation of BCG L-form cells (L) within the vacuoles in guinea pigs peritoneal macrophages**: a, b , c** -at day 1 after BCG installation, the interactions of BCG bacilli (**\***) with peritoneal cells demonstrated initial phases of phagocytosis including attraction, adhesion and attachment of bacteria to the phagocytes and processes of bacterial enclosing and engulfment; **d, e, f**- at day 45 after BCG installation, formation of BCG L-form cells (L) within the vacuoles near to mitochondria (M); **d**, **e** - L-form multiplication inside macrophages; **f**- BCG L-form large bodies , surrounded by multi membranes; Bar = 0.5 μm (Markova et al., 2008 )

#### **5. Reversion of mycobacterial L-forms to classical TB bacilli**

Reversion of L-forms to normal parental bacteria is an important property, which is inducible by changing the condition of cultivation *in vitro* or occurs spontaneously *in vivo*

Cell Wall Deficiency in Mycobacteria: Latency and Persistence 207

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;

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

Cardona et al.; 2003; Cardona, 2009).

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, when compared to other bacteria.

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 shape occurred.
