text: the present invention will now be described more fully hereinafter with reference to the accompanying drawings , in which preferred embodiments of the invention are shown . unless otherwise defined , all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains . although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention , suitable methods and materials are described below . any publications , patent applications , patents , or other references mentioned herein are incorporated by reference in their entirety . in case of conflict , the present specification , including any definitions , will control . in addition , the materials , methods and examples given are illustrative in nature only and not intended to be limiting . accordingly , this invention may be embodied in many different forms and should not be construed as limited to the illustrated embodiments set forth herein . rather , these illustrated embodiments are provided so that this disclosure will be thorough and complete , and will fully convey the scope of the invention to those skilled in the art . other features and advantages of the invention will be apparent from the following detailed description , and from the claims . with the foregoing in mind , an in vitro dormancy model that can be adapted to drug screening would help to discover antilatency drug candidates , in vitro models suitable for such screening are urgently needed . a number of different stresses have been applied to mtb in vitro in an attempt to generate a dormant state and gene expression changes have been investigated . most of these models involve single stress factors such as oxygen depletion , nutrient deprivation , no treatment and acidic conditions . the gene expression changes during combined nutrient deprivation and 10 % and 0 . 2 % oxygen stress on stationary phase cultures were investigated . some of these stress conditions such as prolonged nutrient starvation caused mtb to become highly rif - resistant but accumulation of storage lipids was not tested . on the other hand hypoxic conditions we used that caused tg accumulation did not develop resistance to 5 μg / ml rif ( unpublished ). the nrp - 1 condition was reported to cause resistance to a lower concentration of rif ( 1 μg / ml ) but lipid accumulation was not tested . we suggest that both rif - resistance and lipid storage are hallmarks of dormancy . since individual stress conditions do not allow the pathogen to fully meet these criteria , we attempted to mimic the in vivo conditions by applying multiple stresses thought to be encountered in vivo by mtb and tested whether the pathogen would accumulate storage lipids and develop rif - resistance . bacilli within granulomas encounter low oxygen ( 5 %) but not hypoxia , high co 2 ( 10 %) concentrations , low nutrient levels and acidic ph . based on these reports , we used 5 % o 2 , 10 % co 2 , ph 5 . 0 and 10 % dubos medium in a multiple stress in vitro model . our preliminary results show that the combination of the four stress factors leads to accumulation of storage lipids ( tg and we ), development of rif - resistance and gene expression changes thought to be associated with dormancy . some of the gene expression changes are similar to those found in the pathogen from infected lungs of hosts , including primates and a human tb patient . both rif - resistance and storage lipid accumulation are associated with dormancy . however , the commonly used in vitro hypoxia model , does not show both of these characteristics . therefore we developed a novel multiple stress model that applies four different stresses that the pathogen is thought to encounter in the host . we grew mtb cultures in low ph ( ph 5 . 0 ), low nutrient ( 10 %) dubos medium without glycerol , with high ( 10 %) co 2 and low ( 5 %) oxygen gas phase . mtb cultures in 10 % dubos ( difco ) medium at ph 5 . 0 at an od 600 of 0 . 2 were maintained under 5 % o 2 + 10 % co 2 + 85 % n 2 by replacing the air phase every other day ; oxygen levels did not change significantly during the two day period . after monitoring the progressive changes that happened to the pathogen , we chose to harvest cells at 3 , 9 and 18 days under such conditions for more detailed studies . these studies included examination of storage lipids , antibiotic ( rif and inh )- resistance , gene expression changes directly relevant to storage lipid synthesis by quantitative real time pcr ( qpcr ), and gene expression profiles by microarray analyses . tlc showed that we and tg accumulated under the multiple stress conditions reaching near maximal levels by 9 days fig1 . the major wax ester was oleyl oleate and the major fatty acids in the tg were c16 and c18 with less c26 ( data not shown ). under these conditions more we accumulated than tg in absolute amounts . control samples at ph 7 . 0 or ph 5 . 0 without additional stress showed no increase in storage lipids . nile red staining revealed storage lipid accumulation under the multiple stress conditions . it is well known that mtb cultures contain a heterogeneous population of cells under different physiological states . as the culture was subjected to multiple stress factors we observed decrease in acid fast staining cells with increasing lipid body staining cells from a barely detectable level to a significant percentage of the total cells by 18 days ( fig2 , 3 ). drug resistance was tested by treatment with 5 μg / ml rif for 5 days followed by serial dilution and plating . by 9 days about 10 % of the cells were found to be rif resistant whereas the starting culture contained about 0 . 03 % rif - resistant cells . rif - resistance increased up to 18 days , sometimes reaching up to 25 % at 5 μg / ml rif . the tgs1 ( rv3130c ) disruption resulted in the loss of rif - resistance which was restored in the complemented mutant ( table 1 ). hypoxic conditions , that were previously found to cause accumulation of storage lipids , did not cause the cells to develop detectable rif - resistance at 5 μg / ml . the tgs1 ( rv3130c ) disruption resulted in loss of tg accumulation under multiple stress . however the complemented mutant showed a level of tg accumulation comparable to the wild type ( fig4 ). gene expression changes directly relevant to storage lipid accumulation were examined by real - time pcr . among all the tgs genes , induction of tgs1 ( rv3130c ) was by far the highest at 9 days under the multiple stress condition , followed by rv3371 and rv3088 ( fig5 ). microarray analysis also indicated upregulation of rv3371 under multiple stress condition ( data not shown ). upregulation of rv3088 probably resulted from the low ph as it has been previously reported to be induced under acidic stress . up - regulation of rv3371 is noteworthy as it was also shown to be up - regulated in human lung granuloma by microarray analysis . the degree of induction of tgs1 ( rv3130c ) was comparable to that of icl and acr ( hspx ), genes previously reported to be induced during persistence . our preliminary experimental results raise the possibility that lipid accumulation under different stress conditions might use different sets of tgs genes . the tigr pathogen functional genomics resource center provided the mtb genome microarray for this study . under our multiple stress condition , genes that encode enzymes involved in glyoxylate cycle such as isocitrate lyase ( acea ) and citrate synthase ( glta1 ) showed significant increase in expression for all time points examined ( fig6 ). from these data we infer that the metabolic regulation of cells adapting to the multiple stresses was similar to that observed in persistent bacilli adapting to the phagosomal environment of a macrophage . under multiple stresses , mtb showed shutdown of both atp / nad energy regeneration systems . while gene expression for anaerobic respiration was continuously increased at the later time points , the aerobic respiration was significantly repressed at all the time points . all the subunits encoding nadh dehydrogenase and the ubiquinol - cytochrome c complex were repressed more than 2 - fold . in addition , the expression of the genes encoding atp synthase subunits was repressed . slowdown of the transcription / translation apparatus was evident during the multiple stresses . many genes related to transcription and translation apparatus were all consistently repressed . genes involved in modification of chromosome and cell division were repressed by the multiple stresses . the expression level of the gene cluster , mas , fad28 , mmpl7 , and ppsa - e , associated with phthiocerol dimycocerosate ( pdim ) synthesis and transport , that was repressed at the beginning of the multiple stress treatment , gradually increased more than two - fold and remained high throughout the period of in vitro multiple stress . further , the mas - like gene pks2 , which is responsible for encoding a hepta / octa - methyl branched fatty acid synthase , was highly expressed . these changes are consistent with the report that dormant cells have thickened walls . we also found significant induction of the genes classified as the stress response genes ( eg . hspx ) that has been suggested to play a role in maintaining long term survival within the host . the gene array analysis results were verified by qpcr analysis of selected test genes . repressed and induced gene transcript level changes indicated by microarray analysis were found to be consistent with the changes indicated by the qpcr method ( data not shown ). we investigated whether the lipid accumulation that occurs as a result of multiple stresses might be reflected in changes in buoyant density . we fractionated the culture on a percoll ® gradient in 10 ml seton easy - seal polyallomer centrifuge tubes with seton noryl crown assembly . this procedure resolved cells based on buoyant density ( fig7 ). the banding pattern changed as the cultures were subjected to multiple stresses for increasing duration . these changes are consistent with the conclusion that application of multiple stresses caused progressive changes in lipid accumulation resulting in increasing percentages of cells in the lighter fractions . auramine - o / nile red staining of the different fractions showed that with increasing periods under the multiple stresses , increasing percentage of cells became lipid - loaded and lost acid - fast staining ( presumably dormant cells ). staining of percoll ® fractions from 18 day stressed cultures showed that the lighter fractions were more enriched in , lipid loaded cells that lost acid - fastness . after this long stress period most cells were in the lighter fractions ( fig7 ). upon percoll ® density gradient fractionation of the 18 - day multiple - stressed culture , the great majority of the cells were distributed in the lighter fractions . when rif - resistance was assessed by the alamar blue dye method the lighter fractions showed a higher percentage of rif - resistant cells ( data not shown ). the small number of heavier cells present in this culture showed rif - susceptibility comparable to that of the starting culture . recent meta - analysis of mtb microarray data from many in vitro and in vivo conditions that are thought to induce dormancy ( murphy and brown , bmc infect . dis . i , 84 - 100 , 2007 ) indicated that a set of genes possibly involved in lipid storage and utilization are highly upregulated . we have determined the transcriptional profile of the genes , selected on the basis of the meta - analysis by real - time pcr using 7500 fast system ( applied biosystem ). detection of transcriptional upregulation of the known dormancy - responsive genes such as hspx , icl and dosr ( fig1 ) is consistent with our conclusion that the multiple stress conditions induce dormancy . seven of the 10 genes in the first priority group , such as rv3130c along with a few other tgs genes ( rv3371 , rv1760 ), a few genes encoding potential hydrolases ( lipx lipy , cut3 ), and fatty acyl - coa reductase gene ( rv3391 ) showed upregulation . three of the 21 genes in the next priority group showed upregulation under the multiple stress condition . one of them showed surprisingly high induction . the other two upregulated genes in this group were cut2 and lipz . only one gene ( rv2285 , a tgs ) in the third priority group showed upregulation . among the tgs products that showed the highest enzymatic activity ( when expressed in e . coli ) only this tgs showed a preference for oleoyl - coa . we already have mutant for this gene and this mutant showed the second most impaired ability to accumulate tg under hypoxia as indicated in the preliminary results presented in the application . it is noteworthy that 7 of the 10 genes in the first priority group and 3 out of 21 genes from the second priority group and only one out of 17 in the third priority group showed upregulation . since the prioritization is based on meta - analysis of the degree of their upregulation under a variety of conditions thought to induce dormancy , our results suggest that the multiple stress model reflects real dormancy and adds validity to our approach . the tuberculous granuloma , which is thought to be a hypoxic environment , consists of a core of mtb - infected macrophages surrounded by lipid - loaded macrophages , mononuclear phagocytes and lymphocytes enclosed by a fibrous cuff . the differentiation of macrophages into lipid - loaded macrophages in tuberculous granulomas is a well - documented observation and the secretion of cytokines by the infected lipid - loaded macrophages probably helps to maintain the granuloma . histological studies revealed the presence of lipid - loaded macrophages in the granulomas of immunocompetent and hiv - 1 infected patients with tb . lipid - loaded macrophages contain abundant cytosolic stores of tg and cholesterol esters . a recent study showed that exposure of human macrophages to hypoxia ( 1 % o2 ) converted them into lipid - loaded cells and m . bovis bcg infection induced the conversion of macrophages into lipid - loaded cells but the nonpathogenic mycobacterium smegmatis failed to induce lipid body formation . thus lipid bodies within mtb - infected macrophages may have important roles in pathogenesis and possibly in latency . human thp - 1 monocytic cell line - derived macrophages ( tdm ) are known to be converted into lipid - loaded macrophages . therefore they can serve as a more convenient experimental model for studies on mtb mutants , because their use can avoid the variability in responses encountered in the use of human peripheral blood monocyte - derived macrophages and provide a readily available uniformly reproducible cell model suitable for high throughput screening of drug candidates . lipid bodies have been found in mtb obtained from patients with active disease . however , the origin of these lipids remains unknown . the pathogen inside the lipid - loaded macrophages might utilize fatty acids derived from the lipid bodies in the host cells to store lipids within mtb for later use . such a possibility was raised by the recent finding that adipocytes might be a home for dormant mtb in humans . in fact , mtb inside adipocytes were found to accumulate lipid bodies while becoming dormant , as indicated by their resistance to killing by drugs . the lipid bodies found in the pathogen from patients probably originate from the lipid bodies in the macrophages . our results indicate that mtb within lipid - loaded macrophages can use the host &# 39 ; s tg to accumulate tg within the pathogen and this mtb becomes rif resistant meeting our criteria for dormancy . herein we disclose a newly developed thp - 1 derived macrophage ( tdm ) system for infection with mtb . thp - 1 cells , differentiated into macrophages by treatment with 100 nm pma for 3 days , were incubated for 3 days in 1 % o 2 and 5 % co 2 . oil red - 0 staining revealed lipid droplet accumulation in such macrophages ( fig2 ). when the tdm were infected with mtb at a multiplicity of infection ( moi ) of 1 . 0 for 4 hr and incubated in 1 % o 2 / 5 % co 2 for 3 days , lipid bodies accumulated in the host cells ( fig3 ). mtb cells within the macrophages showed nile red stained lipid bodies ( fig4 ). most mtb cells showed loss of acid fast staining and thus stained only red ; a few showed some acid fast and lipid staining ( yellow ). we modified our experimental protocol to allow for longer incubation of infected macrophages . we infected tdm with mtb at an moi of 0 . 1 ( 1 bacillus per 10 macrophages ) and extended the incubation of the infected tdm to 7 days under hypoxia . after 7 days , infected tdm were lysed and the cell debris was removed by centrifugation at 300 g for 10 min . the mtb cells were pelleted by centrifugation at 3000 g for 10 min and washed . lipids from the host - lipid bodies were extracted from the supernatant and the lipids from the pelleted to analyze fatty acid composition , host or mtb tg was purified by preparative tlc and the methyl esters generated by bf3 / methanol transesterification , were analyzed by capillary gc . the amount of fatty acids from the tg isolated from mtb , recovered for tdm , is more than enough for such gc analysis ( fig6 ) pathogens were extracted . tlc analysis of the lipids revealed that tg in the host cells was markedly increased by incubation under hypoxia for 7 days and the levels of tg were lower in infected tdm under hypoxia ( fig5 a ). lipids extracted from mtb recovered from infected tdm were also analyzed by tlc . we detected increased tg level in mtb cells recovered from tdm incubated under hypoxic conditions ( fig5 b ). the fatty acid composition of the tg from the pathogen was not identical to that of the host tg . c16 : 0 , c18 : 0 and c18 : 1 fatty acids were the dominant components in both the pathogen and the host . longer chain saturated fatty acids ( c24 . c26 and c28 ) that were present in the pathogen tg were absent in the host tg . we conclude that the tg that accumulated in the pathogen probably consisted of fatty acids from the host and some fatty acids generated within the pathogen . gene expression changes occurring in the pathogen within tdm were examined using a two - step real time pcr method . briefly , total rna was isolated and purified from a mixture of mtb - infected tdm using trizol ( invitrogen ) and qiagen rneasy column purification method . total rna was dnase treated twice , purified through qiagen mini elute rneasy column , purity of rna was checked at every step . controls without reverse transcription ( rt ) verified lack of dna contamination . first - strand cdna , synthesized with exo - resistant random hexamers and superscript iii reverse transcriptase ( invitrogen ) was used for multiplex pcr using many mtb gene specific primer sets . all primers and taqman probes were designed using visualomp6 software from dna software , inc ( ann arbor . mi ). the taqman probes have a fluorescein reporter dye ( fam ) at 5 ′- end and a black hole quencher ( bhq ) at 3 ′- end . each multiplex and real - time pcr primer was checked for specificity and efficiency . differences in mtb specific gene transcripts were quantified by real - time pcr on generated multiplex - pcr products with nested taqman primers and probes . the overall reliability and sensitivity of the two - step rt - pcr method to quantify gene expression profiling has been discussed in detail elsewhere . we have thus far done only a subset of genes thought to be relevant to lipid storage and metabolism ( fig7 ); icl was by far the most induced gene , consistent with the idea that the pathogen in tdm grows on fatty acids . it is noteworthy that lipy , that was previously shown by us to be involved in tg mobilization , was highly induced and some of the other lip genes also showed induction . dosr and tgs genes were also induced probably indicating their involvement in the storage of fatty acids derived from host - lipids as tg resynthesized within the pathogen , consistent with our hypothesis . fatp , that might be involved in fatty acid transport into the pathogen was also induced . putative fabp genes also showed some induction . these results indicate that our hypothesis concerning storage and mobilization of host lipids by the pathogen has real validity . we analyzed the resistance of mtb recovered from tdm after a 7 day incubation under 20 % o 2 or 1 % o 2 to rif and inh by cfu determination . tdm were infected with mtb at an moi of 0 . 1 . mtb cells inside tdm were exposed to antibiotic for 2 days prior to lysis of tdm and recovery of the bacilli . the recovered mtb cells were diluted and plated on agar plates without antibiotic and incubated for 4 weeks after which cfus were enumerated . antibiotic resistance is expressed as percentage of control without antibiotic . as indicated in table 2 , mtb recovered from tdm incubated under 20 % o 2 showed resistance to both antibiotics . others have found development of rif resistance in host cells . we found that rif resistance increased significantly in mtb recovered from tdm incubated under 1 % o 2 for 7 days compared to normoxic conditions . these results indicate support for our hypothesis that lipid - loading of macrophages favor the entry of mtb into dormancy . in making further progress developing the macrophage dormancy model , we tested different moi in the lipid loaded macrophage system . we assessed the viability of mtb - infected lipid - loaded macrophages under hypoxia under different moi . at moi 1 . 0 or higher the host cell viability was seriously compromised . at moi 0 . 1 , after 7 days under 1 % o 2 , 40 % of the original tdm population remained intact as an adhered monolayer and were loaded with lipid droplets . about 94 % of these lipid - loaded tdm cells in the adhered monolayer were viable . these results support the notion that these lipid - loaded tdms provide a tg - enriched sanctuary for mtb , favoring its entry into dormancy . we originally suspected that mtb utilizes the macrophage lipid bodies to acquire fatty acids and store them as tg within the pathogen to enable it to go through dormancy . to test this hypothesis , we labeled tdm lipids by incubating the cells with [ 14 c ] acetic acid or [ 14 c ] oleic acid , under 1 % o 2 for 2 days . these cells were washed three times with sterile phosphate - buffered saline ( pbs ) to remove unincorporated radiolabel . thin - layer chromatographic ( tlc ) analysis of the labeled lipids extracted from tdm showed that major part ( about 60 %) of the radioactivity in the lipids derived from labeled acetate and oleate was in tg that accumulated in tdm under 1 % o 2 . these pre - labeled cells were infected with mtb at an moi of 0 . 1 for 4 h under 1 % o 2 . following infection , extracellular mtb were removed by thoroughly washing the tdm monolayer with sterile pbs . infected tdm were incubated for 5 more days under 1 % o 2 . the host lipids and lipids from mtb recovered from the host cells were obtained and the lipids were analyzed by tlc . the fatty acid composition of the mtb and host tg was analyzed by resolving the intact tg and fatty acid methyl esters derived from tg on reversed - phase silica - tlc and argentation - tlc . analysis of intact tg from [ 14 c ] acetate in tdm was composed of saturated and unsaturated fatty acids . however , the tg of mtb was predominantly composed of saturated fatty acids as indicated by greater mobility on agno 3 - impregnated tlc ( fig8 a ). analysis of fatty acid methyl esters prepared from tg isolated from tdm and mtb indicated that the tg of mtb , recovered from tdm labeled with [ 14 c ] acetate , was composed primarily of saturated fatty acids , mostly 14 c - 16 : 0 and a very small quantity of 14 c - 18 : 0 fatty acids ( fig8 b , c ). see also table 3 , below . mtb recovered from [ 14 c ] oleic acid - labeled tdm had tg that was distinctly different in fatty acid composition from the tg in tdm . while the 14 c in tdm tg was predominantly in 18 : 0 ( about 81 % of total fatty acids ), 14 c in mtb tg was mainly in unsaturated fatty acids ( about 70 % of total fatty acids ). the identity of these fatty acids is to be determined . these results clearly indicate that mtb acquires fatty acids from tdm lipid bodies for synthesizing tg as a potential energy source . the tg stored within the pathogen probably includes the fatty acids from the host lipids and fatty acids generated by modification and / or catabolism and resynthesis . the biochemical processes involved can be deduced only after further characterization of the tg that accumulates in the host and in the pathogen . we also postulated that the mtb tgs gene products may be pivotally involved in synthesizing tg within the mtb cell from fatty acids acquired from host tg . to test this hypothesis , we infected the pre - labeled tdm with wild - type mtb and tgs1 ( rv3130c ) deletion mutant ( δrv3130c ) as described in the methods section . about 1 % of the radiolabel in the tg in tdm was found in the tg isolated from mtb . we quantitated the radioactivity in the tg of mtb and δrv3130c recovered from tdm . the results indicate that tg accumulation by the δ3130c was decreased by 90 - 95 % when compared to the wild - type ( table 3 ). these results additionally suggest that rv3130c plays an essential role in the accumulation of tg by mtb within lipid - loaded macrophages . in order to visualize the mtb inside lipid - loaded host cells , infected tdm after 7 days under hypoxia were fixed with 4 % paraformaldehyde and stained for mtb with carbolfuschin followed by methylene blue or hematoxylin and eosin to stain the host cell . mtb inside tdm were also stained with the mycolic acid - specific fluorescent dye auramine - o followed by nile red which stains neutral lipids . interestingly , as seen in fig9 , the mtb - infected tdm which were incubated under hypoxia for 7 days appeared to be fusing together . it is probable that these tdm are in the process of forming multinucleate giant cells ( asterisk in fig9 a ) which are known to be present in the hypoxic environment of the granuloma in close vicinity to lipid - loaded macrophages . these observations support the hypothesis that this lipid - loaded macrophage system is a good model for in vivo latency . individual mtb cells inside tdm that accumulated neutral lipids , as indicated by nile red staining , lost acid - fastness as shown by weak or total loss of auramine - o staining ( fig1 ). conversely , mtb cells which stained strongly with auramine - o did not accumulate neutral lipids . this accumulation of neutral lipids and loss of acid - fastness by a subset of mtb cells within lipid - loaded tdm under hypoxia , correlates well with our data in the preliminary results , that demonstrated the development of rif resistance by about 25 % of the mtb population and increase in tg within mtb from tdm under the same conditions . since then , we have reconfirmed these results with additional experiments . based on these results , the multiple stress in vitro latency model disclosed herein appears to be the best one available for screening chemicals to discover drug candidates that can eliminate latent pathogen . accordingly , in the drawings and specification there have been disclosed typical preferred embodiments of the invention and although specific terms may have been employed , the terms are used in a descriptive sense only and not for purposes of limitation . the invention has been described in considerable detail with specific reference to these illustrated embodiments . it will be apparent , however , that various modifications and changes can be made within the spirit and scope of the invention as described in the foregoing specification and as defined in the appended claims .