Egresos para la operación del servicio de acueducto

Among the changes that accompany the shift of aerobic cultures to the hypoxic NRP state is induction of the alpha crystallin homologue protein, which has not been detected outside the TB complex {M. tuberculosis, M.africanum M. microti M. cannetti, M. bovis and M. bovis BCG) (Yuan, etal., 1996). The alpha crystallin homologue, also referred to as URB-1 antigen; 14K antigen; the small heat shock protein sHsp 16; Hsp 16.3; 16kDa and acr or hspX gene, is produced when the bacilli encounter hypoxic conditions (Wayne, 1994, Yuan, et ai., 1996) or on exposure to nitric oxide (Garbe, etal., 1999).

It has been suggested that the 16kDa protein plays a protective role as a chaperone based on its ability to prevent thermal dénaturation of alcohol dehydrogenase and aggregation of citrate synthase in vitro (Garbe, et ai., 1999), Over-expression of acr in MTB resulted in slower growth and a slower decline in viability after growth stopped on entry into stationary phase (Yuan, et ai., 1996). Yuan and colleagues also demonstrated synthesis of 16kDa at a low level in logarithmic phase cultures, and a marked increase in synthesis during the transition from log phase to stationary phase.

One of the protective mechanisms of 16kDa protein could be correlated with thickening of the cell wall during late stationary phase. When subjected to anaerobic stress, MTB and M. bovis BCG induce a massive up-regulation of the production of this protein, which is associated with a thickened cell wall

(Cunningham and Spreadbury, 1998). The 16kDa protein was strongly associated with the cell envelope, fibrous peptidoglycan-like structures, and intracellular and peripheral clusters. A thickened cell wall may play a role in stabilising cell structures during long-term survival, thus helping the bacilli survive the low oxygen tension in granulomas. It would be interesting to know the acid- fast nature of those bacilli with a thickened cell wall.

Recently, regulators of 16kDa expression have been documented. Using whole genome microarray, 100 genes have been identified whose expression is rapidly induced in response to hypoxic conditions. Among the induced genes is an apparent operon that includes the putative two-component response regulator pair Rv3133c/Rv3132c. When the expression of this operon is disrupted by targeted disruption of the upstream gene, the hypoxic regulation of acr is eliminated. These results suggest a possible role for Rv3132c/3133c/3134c in mycobacterial latency (Sherman, etal., 2001).

One recent study has revealed the essential role of 16kDa for the survival of bacilli within the macrophages in vitro, acr expression was found to be induced during the course of in vitro infection of macrophages. The mutant strain for this gene was shown to be equivalent to wild-type H37Rv in in vitro growth rate and infectivity but was significantly impaired for growth in both mouse bone marrow derived macrophages and THP-1 cells (Yuan, et ai., 1998). In addition to its proposed role in maintenance of long-term viability during latent, asymptomatic infections, these results establish a role for the Acr protein in replication during initial MTB infection. The immunodominance of the 16kDa antigen in both the murine and human systems has been documented (Jackett, et ai., 1988, Lee, et ai., 1992, Vordermeier, et ai., 1993, Friscia, et ai., 1995, Wilkinson, et ai., 1998). Antibody to 16kDa is found in a high percentage of TB patients, suggesting hypoxic shift down of tubercle bacilli to the NRP state in vivo. In this thesis, antibody response to 16kDa antigen was investigated in people presumed to be latently infected to evaluate the sero diagnostic value of this antigen for diagnosis of latent infection.

Reactive nitric oxide intermediates (RNI) stress also induces 16kDa protein expression, importance of RNI in the control of MTB in the murine systems has been well documented by the demonstration of reactivation of MTB growth in mice that do not produce nitric oxide (Flynn, et a/., 1998). There are still controversial views regarding the role of RNI in human TB. Nevertheless, nitric oxide synthase has been recently detected in alveolar macrophages fixed immediately upon isolation from TB patients. Exposure of MTB to a panel of five structurally diverse NO donors collectively known as Nox, resulted in induction of 16kDa protein (Garbe, et a/., 1999). Furthermore, mutation in the acr gene impaired survival of MTB in macrophages. Coincidental expression of 16kDa and the respiratory type of nitrate reductase, suggests a role for 16kDa in a protective mechanism against RNI.

Another group of genes that are found to be associated with adaptation of the bacilli to various environmental conditions are alternative sigma factors. The sigF

gene is induced under a variety of stress conditions, such as cold/heat shock; nutrient depletion; exposure to antibiotics, in particular to ethambutol; rifampin; streptomycin; and cycloserine; entry into macrophages, and on entry into stationary phase in vitro (DeMaio, et ai., 1996, DeMaio, et ai., 1997, Michele, et ai., 1999, Chen, et ai., 2000).

SigF gene of MTB shares significant homology with the sigF of Streptomyces coelicoiour and Baciilus subtiiis, which is a sporulation-specific sigma factor and to sigB of Staphyiococccus aureus, a sigma factor that participates in methicillin resistance (Chen, et ai., 2000). It is not clear yet, whether sigF oi MTB also has a similar function. However, it was found to be involved in the expression of hspX

gene, coding 16kDa protein, (Manabe, et ai., 1999), which in turn contributes to the thickening of cell wall of MTB and M. bovis BCG during the late stationary phase (Cunningham and Spreadbury, 1998). Chen and colleagues also noted that the 16kDa protein was completely absent in s/gF knockout mutant stationary phase cultures suggesting that alpha crystallin expression requires s/gF (Chen, et ai., 2000). s/gF knockout mutants did not show any difference either in short term intracellular growth or susceptibility to lymphocyte-mediated inhibition of intracellular growth compared to the wild type, suggesting that sigF is not

necessary for intracellular survival of MTB (Chen, et al., 2000). This latter observation is surprising as it was shown that the 16kDa protein is important for survival in a human macrophage-like cell line and in primary mouse macrophages (Yuan, at a!., 1998). Nevertheless, other studies have shown that

SigF may not be the sole regulator of hspX expression and many of the conditions that up-regulate sigF expression has no effect on the expression of

hspX (Yuan, at ai., 1996). s/gF knockout mutants were not as virulent as the wild type of bacilli, because death of BALB/c mice infected by the mutant was significantly delayed (Chen, at ai., 2000).

Expression of all the above-mentioned genes favours the bacilli over the host, and enables them to survive during the chronic phase of the tuberculosis infection. However, it appears that not all the proteins of MTB that are expressed/induced under stress favour the bacilli. Over-expression of Hsp70, a stress response-related heat shock reduced the survival of an Hsp70 repressor mutant (AhspR) in a murine model (Stewart, at ai., 2001). Even though the mutant was as virulent as the wild type during the initial stage of the disease, it showed impaired survival during the subsequent plateau phase of the infection. This may be due to the early recognition of secreted proteins by the host immune system.