Mycobacterial isocitrate lyase gene and uses thereof

Details Mycobacterial isocitrate lyase gene and uses thereof Mycobacterial isocitrate lyase gene and uses thereof

1998-04-03

2002-05-14

Duffy, Patricia A. (Department: 1645)

Chemistry: molecular biology and microbiology

Vector, per se

C435S183000, C435S232000, C536S023100, C536S023200, C536S023700

Reexamination Certificate

active

06387694

ABSTRACT:

BACKGROUND OF THE INVENTION
Pulmonary tuberculosis initiates with the inhalation and retention in the lung alveoli of a “droplet nucleus” containing from 1-10 tubercle bacilli. Most cases of human tuberculosis originate from a single primary lesion in the lung parenchyma; the number of bacilli initiating an infection is therefore extremely small (Medlar). Patent tuberculous disease develops only after expansion of this initially small bacillary population by replication within host macrophages. In order to grow, persist, and cause disease, tubercle bacilli must obtain nutrients from the parasitized host. Little is known, however, of the mechanisms involved in nutrient acquisition by tubercle bacilli in vivo. Writing in 1976, Ratledge opined that “[T]he entire subject of in vivo nutrition of bacteria when within the phagocytic cells of the host is probably the largest single area of ignorance in the whole of our knowledge concerning the physiology of the mycobacteria. Clearly this is a crucial area where knowledge should be sought as it is only by understanding the true behavior and requirements of the bacteria when growing in vivo that we shall learn how to prevent their multiplication and, hopefully, how to cause their death” (Ratledge, 1976) Unfortunately, the intervening decades have marked little progress in this area. With the advent of molecular genetic tools for the manipulation of the pathogenic mycobacteria, a genetic approach to this problem is now feasible.
In the infected host,
M. tuberculosis
bacilli replicate within host macrophages. Following phagocytosis, tubercle bacilli reside within modified phagosomes that apparently fuse with vacuoles derived from the endosomal compartment (Sturgill-Koszycki et al., 1996) but that fail to acidify fully or to fuse with lysosomes (reviewed in Clemens, 1996). As an intracellular parasite,
M. tuberculosis
would potentially have access to a variety of nutrients that are abundant within the host cell cytoplasm (Wheeler and Ratledge, 1994). The enclosure of tubercle bacilli within tightly apposed membranous vacuoles (Moreira et al., 1997) might, however, limit access to cytoplasmic constituents. This idea was supported by the recent demonstration that a leuD auxotroph of the attenuated bacille Calmette-Guerin (BCG) strain of tubercle bacillus was incapable of replicating in mice (McAdam et al., 1995) or in cultured macrophages (Bange et al., 1996). Although
M. tuberculosis
is not a nutritionally fastidious organism, bacillary growth does require a carbon substrate(s) to provide building blocks for biosynthetic reactions and energy for metabolism. In vitro,
M. tuberculosis
is capable of utilizing a wide range of carbon substrates, including carbohydrates, amino acids, and C2 carbon sources such as acetate and fatty acids (Wayne, 1994). It is not known which of these substrates are available to
M. tuberculosis
replicating within the confines of the phagosomal compartment.
Extensive biochemical studies have been made of tubercle bacilli isolated directly from the lungs of chronically infected mice (reviewed in Segal 1984). Using manometry, Segal and Bloch (1956) showed that these “in vivo grown” bacilli displayed a vigorous respiratory response to fatty acids but failed to respond to a variety of other substrates. In contrast, respiration of tubercle bacilli grown in vitro was readily stimulated by both glucose and glycerol, which are the preferred substrates for in vitro cultivation of tubercle bacalli. These observations suggested that tubercle bacilli in vivo may be adapted to utilization of fatty acids and may repress pathways for utilization of other carbon sources. Later studies revealed that in vivo grown bacilli retained the ability to oxidize
14
C-glucose to
14
C—CO
2
, but that addition of exogenous glucose suppressed the respiration of endogenous substrates presumably including fatty acids (Artman and Bekierkunst, 1960).
Two specialized pathways are required for utilization of fatty acids as sole carbon source. The b-oxidation pathway catalyzes the breakdown of fatty acids to assimilable acetyl CoA units, which are further oxidized via the Krebs cycle (Clark and Cronan, 1996). The glyoxylate shunt is an anaplerotic pathway for replenishment of essential Krebs cycle intermediates consumed by biosynthetic pathways during growth on C
2
carbon sources such as fatty acids and acetate (Cronan and LaPorte, 1996). This anaplerotic function is subsumed by pyruvate carboxylase when cells are grown on carbohydrates. Wheeler and Ratledge (1988) found that in vivo grown mycobacteria readily oxidized [
14
C]-palmitate to [
14
C]—CO2, implying that the enzymes required for b-oxidation of fatty acids were expressed in vivo. (In fact, evolution of [
14
C]—CO2 from [
14
C]-palmitate is the basis of the widely used “BACTEC” system for detection of
M. tuberculosis
in clinical specimens [Heifets and Good, 1994].) In addition, these authors demonstrated expression of both enzymes of the glyoxylate shunt (malate synthase and isocitrate lyase) by in vivo grown mycobacteria. In
Escherichia coli
, expression of the enzymes of the b-oxidation pathway and of the glyoxylate shunt is under transcriptional control: transcription is repressed during growth on carbohydrates and is induced during growth on fatty acids. Although these enzymes and their regulation have been characterized only partially in mycobacteria, their expression by in vivo grown bacilli suggests that fatty acids may be utilized in vivo. If so, then the b-oxidation pathway and the glyoxylate shunt may be essential for in vivo growth or persistence of tubercle bacilli.
SUMMARY OF THE INVENTION
The present invention provides a purified and isolated nucleic acid encoding mycobacterial isocitrate lyase. The present invention specifically provides for nucleic acid sequences encoding mycobacterial isocitrate lyase that are obtained from
M. tuberculosis
and
M. smegmatis
. Also provided by the present invention are mutated nucleic acid sequences encoding mycobacterial isocitrate lyase.
Additionally, the present invention provides vectors which comprises the nucleic acid sequences encoding mycobacterial isocitrate lyase of the present invention, and vectors which comprises the mutated nucleic acid sequences encoding mycobacterial isocitrate lyase of the present invention, as well as host cells containing these vectors.
Further provided by the present invention is an agent that inhibits the activity or expression of a mycobacterial lyase protein, a method of identifying agents that inhibit the activity or expression of a mycobacterial lyase protein, and a method of producing the agents.
Finally, the present invention provides a method of identifying genes required for persistence of mycobacteria.


REFERENCES:
patent: 5504005 (1996-04-01), Bloom et al.
patent: 5686590 (1997-11-01), Jacobs et al.
patent: 5773267 (1998-06-01), Jacobs et al.
patent: 5783386 (1998-07-01), Jacobs et al.
patent: 5854055 (1998-12-01), Bloom et al.
patent: 5976844 (1999-11-01), Kasler et al.
patent: WO 92/22326 (1992-12-01), None
patent: WO 98/41533 (1998-09-01), None
Kaplana et al, PNAS, 88: 5433-5437, Jun. 1991.*
Seshadri et al, Indian J. Biochemistry & Biophysics 13/1: 95-96, 1976.*
Graham et al, PNAS, 96/20 : 11554-11559, 1999.*
Fsihi et al, Molecular Microbiol, 16/5: 909-919, 1995.*
Wayne et al, Infection & Immunity, 37/3: 1042-1049, 1982.*
Cole et al, Nature, 393(6685):537-544, 1998.*
Graham et al, PNAS, 96:11554-11559, 1999.*
Bharadwaj et al, Indian J. Leprosy, 59/2:158-162, 1987.*
Kannan et al, Indian J. Leprosy, 57/3:542-548, 1985.*
Vereecke et al, Gene, 145:109-114, 1994.

Affiliated with

Also associated with

Rating

Mycobacterial isocitrate lyase gene and uses thereof does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Mycobacterial isocitrate lyase gene and uses thereof, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Mycobacterial isocitrate lyase gene and uses thereof will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2866049