Both proteins are immunodominant antigens that are recognized by the sera in a majority of TB patients A mutation that disrupts both closely linked genes in M. It is not known which of the two genes or both are necessary for the virulence of M.
Rv and Rv are located in the RD1 deletion region, the first deletion found when comparing the genome of a wild-type M. RD1 contains the structural genes for nine proteins, Rv through Rv This region is found in all virulent M. This has now been confirmed by two independent series of experiments. In one case, using a knockout strategy, a deletion of the RDI region was made in M. The other experiments used a knockin approach, in which the RD-1 region was inserted into the chromosome of M.
These studies showed that the complemented strain was much more virulent than its attenuated parent It is not yet known whether any of the other genes of the RD1 region are necessary for virulence. Significantly, the avirulent M.
The Esat6 and CF genes are cotranscribed in M. The members of the M. This suggests that similar complexes will also be formed. Another member of the Esat6 family, Rv, has been identified as being differentially expressed in mouse lungs by using a promoter trap screen, and it is also induced in macrophages, as determined by RT-PCR analysis Dubnau and Smith, unpublished.
The role of the virulence of Rv in virulence has not been analyzed yet, but experiments are in progress. Rv is also found adjacent to another gene, Rv, that encodes a member of the Esat6 family, suggesting a complex of these two proteins. The kDa protein is an immunodominant antigen that is recognized by T cells and sera from TB patients. When M. However, there are confusing and contradictory reports regarding host responses to this protein.
It was originally noted that M. The strain was rapidly cleared from the lungs and spleen of infected mice, a SGIV phenotype. Adding back the wild-type M. Later experiments were less conclusive because was shown that mutations in the gene encoding the kDa protein had no effect on M. In addition, it was found that neither overexpressing the kDa protein nor deleting its structural gene in M. Equally uncertain is the effect of the protein on host responses during M.
It has been reported that addition of the purified kDa protein to human MDMs causes upregulation of the important Th1 cytokine IL Similarly, addition of the kDa protein can activate human neutrophils On the other hand, when the gene for the kDa protein was introduced into M. Interestingly, murine dendritic cells infected with M. The inclusion of glutamine synthase is an operational one, since as discussed above its presence in culture filtrates probably results from cell leakage and lysis.
In addition to its essential role in nitrogen metabolism, MSO inhibition studies have shown that the M. These results suggest that this enzyme is a good target for the development of new drugs with less toxicity than MSO for the mammalian host. As discussed above, the mycobacterial cell wall and envelope is a complex structure containing many proteins, lipids, and carbohydrates, many of which are found only in these bacteria.
Is a subset of these components are unique to pathogenic mycobacteria and thus are expected to be excellent targets for further investigations of M. Erp is a surface-located protein that was originally identified by means of a phoA fusion strategy to identify secreted M.
A similar erp mutation was made in M. The function of the protein is unknown because the mutant shows normal growth in vitro but mutant bacteria recovered from infected mice grow much more slowly that the wild-type and complemented mutant strains. The mas gene was disrupted in M. No data on the virulence phenotypes of the mas mutant were presented, but citation of unpublished experiments mentioned that it was attenuated for growth in macrophages and mice.
This is consistent with the results of other published studies that are discussed in the following sections. The Kolattukudy group has also created mutations in genes encoding related cell wall components, but their virulence phenotypes have not been reported 8 , FadD26 was originally annotated as an acyl coenzyme A acyl-CoA synthetase involved in fatty acid degradation.
It was inactivated by the STM procedure in M. In the same studies, M. Similar mouse results were obtained by another group using STM technology with M. In this latter case, the transposon inserted in the promoter region of fadD26 and the disruption also affected the expression of the downstream ppsA to ppsE operon Rv to encoding a polyketide synthase required for phthiocerol biosythesis 8 , and one of the mutant phenotypes is the absence of PMID.
It is not known whether the phenotypes of the fadD26 transposon insertion mutations are solely due to a polar effect on the downstream pps operon or also reflect a role for FadD26 in these processes. However, the close linkage between fad26 and genes for PMID synthesis, including mas , suggests that FadD26 may have a synthetic rather than a degradative function, as originally annotated. Like the latter gene, it was also annotated as a fatty acid CoA synthase, and the fadD28 mutant has a similar GIV phenotype in mice.
This protein is a member of a large group of related proteins, and one of the phenotypes of the mutant is the failure to transport PMID, although it does synthesize this complex molecule 45 , The mutant is attenuated for growth in mice exhibiting a GIV phenotype. Other members of the mmpL family were identified in one of the transposon searches, as were several other genes but they are not discussed here because they not yet been studied in great detail Mycobacteria have three mycolyl-transferase enzymes, encoded by three genes, fbpA , fbpB , and fbpC , that transfer long-chain mycolic acids to trehalose derivatives, and the proteins can also bind the cell matrix protein fibronectin The Fbp proteins are also found in the culture filtrate and are also known as the antigen 85A, 85B, and 85C complex or the to kDa proteins.
The three fbp genes have been separately inactivated, but only the M. The observation that these proteins are immunodominant has led to the creation of a new live vaccine that was made by introducing the M. This recombinant strain shows better protection against virulent M. This vaccine strategy has not been used with the M.
A gene cluster in M. It was postulated that the reaction catalyzed by the methyltransferase encoded by mmaA4 , i. To verify this and to see the effects of this mutation on M.
The mutant grows normally but does not make the methoxy- and ketomycolates, as originally predicted. In addition, it shows marked cell wall alteration since it is less permeable to various compounds such as glycerol and chenodeoxycholate but is more resistant to oxidative stress than the wild-type M. The mutant strain was attenuated in mice showing a GIV phenotype PcaA is a methyltransferase that forms cyclopropane residues in mycolic acids. It was originally detected on the basis of the unusual colony morphology of an M.
Microscopic examination of the mutant showed a corded structure of the clumped bacterial cells, and sequence analyses of the mutated gene showed it was a member of a family of proteins that introduce cyclopropane residues into mycolic acids. To analyze the role of PcaA in M. The colonial and microscopic phenotypes of the mutant were similar to those of the M.
While the BCG mutant was cleared more rapidly from the lungs than its parent, the growth of the M. However, the M. Microscopic observations showed much less pathology in the lungs of mice infected with the mutant strain than in the lungs of those infected with the wild-type strain.
A porin-like protien, OmpA, has been found in M. This is an extremely interesting result, suggesting that the environment faced by M. One caveat to this hypothesis is that true complementation experiments were not performed in these studies, i. As discussed elsewhere in this review, complementation is essential for concluding that a particular phenotype is caused by a mutation. HbhA is a heparin-binding hemagglutin protein that is localized on the surface of virulent mycobacteria but is not found in M.
The mutant exhibited wild-type ability to be phagocytosed by and to grow in murine or human macrophages. However, it was taken up poorly by pneumocytic cells, although the bacterial generation time was normal intracellularly. The mutant grew normally in the lungs of infected mice but had a longer generation time and reached a lower bacterial load in spleens compared to the wild-type and complemented mutant strains. The unique properties of this mutant indicate that HbhA is important for M.
LAM is included in this list of virulence factors because of its importance as an immunomodulator as detemined by experiments similar to those described above for the kDa protein. LAM, a complex glycolipid that contains repeating arabinose-mannose disaccharide subunits, is a major component of the M. LAM can also scavenge oxygen radicals, in vitro, and inhibits the host protein kinase C. These multiple phenotypes suggest that LAM functions to downmodulate host responses to M. Since many pathogens become starved for certain essential nutrients and cofactors, e.
In addition, mutations have been created in genes encoding respiratory enzymes and enzymes that protect against oxidative stresses occurring during normal aerobic respiration, as well those created by infected hosts.
As discussed above, observations made approximately 50 years ago indicated that M. These old observations are supported by more recent work, e. Icl isocitrate lyase is an enzyme that converts isocitrate to succinate in the glyoxalate shunt.
This allows bacteria and plants to grow on acetate or fatty acids as sole carbon sources since the glyoxalate shunt provides a source of carbon that can enter the Krebs cycle. Initial observations made using an in vitro model to study M. It initially grows normally in mice but stops growing in lungs and is cleared at the time when cell-mediated immunity is initiated, a PER phenotype Additional evidence indicating the importance of isocitrate lyase includes the observation that icl mRNA levels increase in the lungs of M.
It was identified in one of the STM experiments that found fadD26 discussed above and had a similar attenuated phenotype in mice Nothing more is known about the function of this protein or why it is essential for bacterial survival in vivo.
There are 36 genes annotated in the M. Inactivation of fadD33 caused a complex virulence phenotype in mice, since the wild-type parent and fadD33 mutant grew normally in the spleens and lungs of infected mice but the mutant grew less well in livers approximately 1 log unit less. This phenotype was similar to that exhibited by M. The reason for the tissue-specific phenotype of the mutant is not known, and neither is the actual function of the FadD33 protein.
Other annotated fatty acid metabolic genes fadA4 , fadA5 , and echA19 have been identified as being induced in human macrophages by using a promoter trap selection, but only the fadA4 results were tested and validated by mRNA determination Similar promoter trap experiments have now identified nine annotated fad genes as being induced in the lungs of M. Dubnau et al. These mouse results have not been validated yet, and no mutations have been made in any promoter trap-identified genes to determine their virulence phenotypes, but these experiments are under way.
Three of these, plcA , plcB , and plcC , are closely linked to each other, but plcD is not. Disruptions of the plc genes were obtained by screening a transposon mutant library made in M. In addition, some mutations were made by a two-step plasmid procedure Phospholipase C activity was determined in cell extracts of strains that had individual and multiply mutated plc genes, and all individual mutants have lower enzyme activities than that of the wild-type M.
Triple plcABC and quadruple plcABCD mutants have negligible enzyme activity, and strains made by using a plcABC mutant as the recipient for individual plc genes showed that all restore some activity. However, both of these multiple plc mutants are attenuated in mice, showing a GIV phenotype. Pantothenate is essential for for the synthesis of CoA and other important molecules involved in fatty acid biosynthesis and degradation, intermediary metabolism, and other cellular processes.
The mutant was attenuated for virulence, as determined by measuring the survival time of SCID and immunocompetent mice and the bacterial burden in immunocompetent animals In the former experiments, the SCID mice infected with the panCD mutant survived for an average of days while mice infected with the wild-type H37Rv lived an average of 5 weeks.
Similar results were observed for immunocompetent mice. In addition, the histopathologic changes in the lungs of mice infected with the mutant were much smaller than those observed in wild-type infections. Complementation of the mutant with the M. When injected into mice, the mutant was also protective against an aerosol challenge with virulent M. As discussed earlier in this section, initial attempts to isolate attenuated M. The leuD auxotroph was also able to protect against virulent M.
TrpD is anthranilate phosphoribosyl transferase, which is involved in the tryptophan biosynthetic pathway. The mutant was severely attenuated in murine macrophages, hardly grew in SCID mice suggesting a SGIV phenotype , and did not kill any of these mice ProC is a pyrrolinecarboxlate reductase, which is involved in proline biosynthesis, and the M. Its virulence phenotype is intermediate between that of the the wild-type M. It is killed in murine macrophages but not as rapidly as the trpD strain, and it kills SCID mice with median killing time of days, in contrast to the wild-type infection, in which all mice are killed by 29 days.
PurC is 1-phosphoribosylaminoimidazole-succinocarboxamide synthase, which is involved in purine biosynthesis, and it was inactivated in both M. The growth of both mutants in inactivated murine macrophages is attenuated, with the M. Both mutants are severely attenuated in mice with an SGIV phenotype. Magnesium and iron are essential for life, and defects in the uptake of these elements frequently lessen the virulence of bacterial pathogens.
Following this rationale, two mutants have been made in M. Since there is an mgtC ortholog annotated in the M. The mutant is also severely attenuated for growth in mice, exhibiting a SGIV phenotype. However, a similar mgtC mutant made in M. The reasons for these discordant results in two laboratories are not known, but different M.
The actual role of mgtC in M. The mbt operon, consisting of mbtA through mbtJ , encodes enzymes whose function is to synthesize mycobactin and carboxymycobactin 72 , , the major siderophores in M. This regulon is repressed by IdeR in high-iron conditions , MbtB is an enzyme in this pathway and catalyzes the formation of an amide bond between salicylate and serine, a step in mycobactin synthesis. Since iron is essential for most lifeforms but is usually in the form of the largely insoluble ferric salts in the environment, iron uptake systems are required to solubilize these salts and to transport the iron into the cell.
In bacteria, siderophores usually perform this chelation and solubilization function, and the iron they carry is taken into cells by high-affinity transporters. In addition, pathogens require iron acquisition systems, usually siderophores, during infection to obtain iron from host iron-containing proteins such as transferrin and lactoferrin.
In response to infection, the host frequently sequesters iron to prevent bacterial growth Since mutations in siderophore-biosynthetic genes frequently cause attenuation of virulence in bacterial pathogens, the M. The mutant shows wild-type growth in iron-rich media, grows poorly when iron is limiting, and is unable to synthesize the two mycobactin-derived siderophores.
It also grows more slowly than the wild type in human macrophages, as measured by a luminescence assay, indicating the mycophagosome may be low in iron. This latter hypothesis is supported by experiments which show that levels of mbtB and mbtI mRNA are increased during M. There is no published report on the growth phenotype of the mbtB mutant in mice, but several lines of evidence, in addition to its macrophage growth phenotype, suggest that it will be attenuated.
These all indicate that iron is limiting in the M. Timm et al. The identity of this gene is currently under investigation and, when found, should be an important tool with which to study M. Another reason suggesting that iron is limiting for M.
IdeR is the major mycobacterial regulator of iron uptake and storage genes, repressing the former and activating the latter 79 , , , However, it is included in this section on virulence factors because of a report that the presence of a mutated DtxR, which exhibits iron-independent repression in other contexts, decreases the growth of M.
Although it was not demonstrated directly in vitro or during infection, it was postulated that the mutated DtxR is repressing M. Despite certain caveats, this interesting observation suggests that iron acquisition is essential for M. On the other hand, most aerobic organisms, including bacteria, have enzymes that degrade peroxides and H 2 O 2 , which are normal by-products of aerobic respiration and can give rise to toxic ROIs if allowed to accumulate.
These enzymes, generally superoxide dismutases and catalases, as well as related enzymes, are also important for the response to various external oxidative stresses. Since phagocytic cells produce ROIs to kill invading bacteria, it is not surprising that these enzymes are important for M.
NarG is a subunit of the prokaryotic respiratory anaerobic nitrate reductase that plays major role in respiration in the absence of oxygen, and anaerobic nitrate reductase activity increases when M. The mutant had no anaerobic nitrate reductase activity, but its growth under aerobic or anaerobic conditions was unaffected. When the M. When SCID mice were infected, the wild-type parent grew well while the mutant showed no replication but was not cleared.
In normal mice, the wild-type BCG strain did not replicate but the mutant was rapidly cleared from the lungs, livers, and kidneys, exhibiting a SGIV phenotype These results have not been confirmed for M. KatG is a catalase:peroxidase that degrades H 2 O 2 and organic peroxides. It is the only enzyme with catalase activity in M. In addition, an M. Another katG mutant of M. Complementation with the wild-type katG restored enzyme activity and virulence The role of FurA in virulence is not currently known.
AhpC is an alkyl hydroperoxide reductase, and enzymes of this type function to detoxify organic hydroxyperoxides. Attempts to inactivate this gene in the M.
The resulting phenotypic mutant produced less AhpC than did the wild type and was more sensitive to H 2 O 2 and cumene hydroperoxide. The mutant was also much less virulent in a guinea pig model, showing 3 log units fewer CFUs than the wild type. It has been postulated that AhpC can compensate for the lack of lack of catalase:peroxidase activity in M. However, levels of AhpC are not correlated with the virulence of katG mutants SodA is the iron-factored superoxide dismutase that degrades superoxides, which are normal by-products of normal aerobic respiration and are also produced by the phagocytic repiratory burst enzyme.
It is therefore important for the survival of intracellular pathogens during infections. SodA is the major enzyme with this activity in M. To circumvent this problem, as was done with the ahpC gene discussed above, an antisense approach was used to make a phenotypic sodA mutation in M. The phenotypic mutant produced much less SodA protein and was severely attenuated in mice, showing up to 5 log units fewer CFUs than the wild type in lungs and spleens, and it was rapidly cleared, demonstrating an SGIV phenotype.
Recent results have indicated that M. Kernodle, personal communication. SodC is the Cu,Zn-factored superoxide dismutase that is responsible for a small part of total Sod activity in M. Two laboratories have inactivated this gene in M. In one case, sodC was inactivated in M. It was not affected in inactivated murine macrophages or activated macrophages from respiratory burst-deficient mice In the other study, the M. The reasons for the discrepant results are not known, but different M.
Since transcriptional regulators control the transcription of many genes, a directed mutational strategy to inactivate regulatory genes would be expected to find some that are important for M. One of the major strategies used by prokaryotes to radically change their life-style in response to a changed environment involves using RNA polymerase holoenzymes with different promoter specificities.
This is achieved by the formation of new holoenzymes containing different sigma factors, which allows the transcription of genes required for the new conditions. Pathogens, including M.
Sigma A is the essential principal mycobacterial sigma factor and is presumably necessary for most mycobacterial housekeeping gene transcription , Unlike the products of most of the other transcriptional regulatory genes that were directly inactivated, sigma A was identified as a virulence factor by complementation of an attenuated M. The original mutation in sigA that causes attenuation is a partial loss of function that allows the sigma A protein to function in general transcription, since the mutant grows normally in vitro broth cultures and solid media , but it is presumably unable to transcribe at least one virulence gene.
The attenuating mutation is an arginine-to-histidine change at amino acid residue RH of the protein and is localized to a C-terminal domain that, in other sigma factors, interacts with transcriptional activators. This suggested that sigma A must interact with a transcriptional activator that allowed the expression of a gene s necessary for virulence Recent experiments have confirmed this hypothesis since it has been shown that WhiB3 Rv interacts with sigma A This important finding is discussed below.
Hopefully, DNA array analyses will soon compare the global expression profiles of strain ATCC and its complemented derivative strain to see which genes are not transcribed in the mutant strain, as has been done for other M. These analyses should allow the identification of genes in the sigma A regulon that require WhiB3 and should ultimately lead to the identification of those that are essential for virulence. The derived amino acid sequence of M.
It was speculated that the latency of M. The mutant has no macrophage phenotype and is attenuated for virulence in mice, using mortality as a criterion. Recently, direct transcription assays have identified genes transcribed by RNAP-sigma F, and a promoter sequence has been identified that strongly resembles the B. This information and future DNA array analyses should allow the identification of sigma F-dependent M. Interestingly, this latter work also showed that the activity of sigma F is controlled posttranslationally by its binding to an anti-sigma protein Rvc that previously had been identified as having sequence similarity to the anti-sigma F and anti-sigma B proteins of B.
In turn, the activity of the M. Significantly, the function of Rvc is regulated by the redox potential, while it is proposed that Rvc activity is controlled by phosphorylation Since these stresses might be found during M.
To test this idea, sigE was inactivated in M. The mutant is more sensitive to detergent, high temperature, and oxidative stress than is the wild-type parent M. Preliminary results show that the mutant is attenuated in wild-type mice with a GIV phenotype and kills SCID mice more slowly than the wild type does: all mice infected with the mutant were dead by 70 days, while M.
Manganelli et al. DNA array analyses comparing the sigE mutant and the wild-type parent showed that 38 genes required sigma E for their expression during normal growth while 23 other genes in 13 transcription units required this transcription factor for their induction after sodium dodecyl sulfate SDS stress. Nine of these transcription units had a conserved ECF sigma factor-like promoter sequence in the region directly upstream of the first gene in each unit. Among the genes requiring sigma E for their expression during unstressed growth are some encoding proteins involved in translation, transcriptional control mycolic acid biosynthesis, electron transport and the oxidative stress response.
Genes requiring sigma E during SDS stress encode proteins that are involved in fatty acid degradation, some that are heat shock proteins, and several that are putative transcriptional regulators. Gomez and I. Smith, unpublished results , required sigma E under both stressed and unstressed conditions, and recent experiments have shown that RNAP-sigma E can transcribe sigB Rodrigue et al.
This question is currently being investigated. As is the case with many ECF sigma factors, sigma E activity is downregulated by an anti-sigma factor, RseA, that is encoded by a gene, Rv, adjacent to sigE Rodrigue et al. The possible role of RseA in virulence is not known and is also being studied. Sigma H is another member of the ECF family of sigma factors, like sigma E, and is very similar to the sigma R of Streptomyces species.
Sigma R responds to certain types of oxidative stress, such as diamide treatment, that oxidize protein-SH groups, which then form intramolecular disulfide bonds Its promoter recognition activity is blocked by binding of an anti-sigma factor, RsrA, that is encoded by a gene adjacent to sigR. On oxidative diamide stress, key SH groups in RsrA become oxidized and its binding to sigma R is disrupted Sigma R is then able to transcribe several genes such as its own structural gene sigR and the trx operon, encoding thioredoxin and thioredoxin reductase, which can reduce proteins that were oxidized by diamide treatment.
Thioredoxin and its reductase function to return the system to the unstressed state, since the newly reduced RsrA can again bind to sigma R Subsequently, this gene was inactivated in three laboratories , , The phenotype of the sigH mutant is as expected from the Streptomyces experiments and the earlier M.
A combination of DNA array analyses and individual gene expression assays showed that there are no genes that require sigma H during unstressed growth , but genes similar to those transcribed by the Streptomyces RNAP-sigma R, including those encoding thioredoxin reductase and two thioredoxins, are dependent on sigma H for their expression after diamide stress , , Many of these M. Transcription assays have shown that some of these genes are transcribed by mycobacterial RNAP-sigma H The virulence phenotype of the sigH mutant is subtle in that its growth in macrophages and mice is normal in terms of bacterial load , , but there are differences in lung histopathology, including fewer granulomas and a generally delayed pulmonary inflammatory response As is the case for Streptomyces , M.
Biochemical experiments have shown that the purified M. As discussed earlier in this review, bacteria have multiple two-component systems, each responding to different stimuli, and several laboratories have made mutations in M. PhoP shows high similarity to the PhoP response regulator of S. On this basis, phoP was disrupted in a clinical M.
The mutant grows poorly in mouse macrophages and is severely attenuated in mouse organs, where it has an SGIV phenotype.
These results have been confirmed and extended as phoP has been disrupted in M. In vitro experiments have shown that the M. Genes controlled by PhoP are not known yet, including those important for virulence, and this is currently being investigated.
PrrA is one of the 13 annotated response regulators in the M. It had been previously shown that this gene was upregulated during M. The growth of the prrA mutant in mouse primary macrophages was slightly lower than that of the wild-type M. In agreement with these observations, the use of GFP reporter fusions with the prrA promoter showed that this gene was transiently induced in macrophages, with peak levels of gene expression 4 h after infection and with levels declining after that time.
The significance of PrrA to virulence is not clear, given the very subtle attenuation phenotype. Rv is another M. The mutant had an unusal phenotype in that it grew better than the wild type in murine macrophages and human MDMs.
However, it did not persist in the lungs and livers of infected mice, growing initially and being cleared after days, respectively, showing a delayed PER phenotype. Virulence phenotypes of other two-component gene mutations have been measured, e.
No macrophage phenotype was observed, and these genes were not induced in macrophages In agreement with these results, 10 of the 13 two-component response regulator genes in M. Included in this group were mutants with disruptions in regX and trcS. Other two-component systems in M. Another response regulator, DosR Rvc , controls the global response to oxidative stress and low oxygen response, including the expression of the anoxia-induced hspX gene as well as its own expression A proteomic analysis of M.
Thus, DosR is important for the mycobacterial response to various environmental stresses, and a recent report shows that disruption of the M. While a mutation in M. In addition to sigma factors and response regulators, bacteria use other types of transcriptional regulators to control the expression of large groups of genes. As discussed above, there are many ORFs that are annotated as transcriptional regulators in the M. Among the rarely characterized M.
Other transcriptional regulators have been described that are potentially important for virulence, like RelA , since relA mutant shows defects in long-term survival in vitro while its macrophage growth is normal. However, there have been no published reports describing the relA mutant phenotype in animal models. The synthesis of M.
Investigations were carried out to see whether the M. Biochemical studies showed that purified M. Thus, M. While the M. The reason for this attenuation is not known, but it was suggested that the higher levels of HSPs in the mutant may cause increased host immunosurveillence followed by more efficient killing of the pathogen In addition to potential roles in immunosurveillance, M. Recombinant M. It has recently been shown that M.
This suggests a role for GroES in Pott's disease, the extrapulmonary form of TB discussed earlier in this review that is marked by the weakening and resorption of spinal vertebrae.
WhiB was originally described in S. There are seven members of the WhiB family in M. For this reason, studies of mycobacterial WhiB orthologs have been performed, initially with M. The same whiB3 mutation was made in M. This attenuation phenotype is similar to that observed in the original M.
This result indicates significant differences in virulence mechanisms of these two members of the M. Genome comparisons of M. Also not known is the role in virulence of other members of the M. A promoter trap search has identified whiB2 Rvc as being differentially expressed in the lungs of M.
As shown in this review, much work has been performed in the search for M. Various targets have been identified, e. More work has to be performed so that that every potential gene is systematically inactivated in the M. Ideally, the mariner transposon system can be modified to incorporate signature tagging so that many potential mutants can be tested in vivo at the same time. In addition to this global search, there are more specific approaches that can be undertaken.
For example, as mentioned earlier in this review review, some M. The numerous comparative genomic sequencing projects being conducted now may identify potential genes that are responsible for these phenotypes, and these could then be individually characterized. As previously discussed, all M. Of the nine genes in the deleted region, only Rv, encoding Esat6 has been implicated in virulence, since a mutant with a disruption in this gene is attenuated for virulence It is not known whether any of the other genes in the RD1 region are also important for M.
As an intracellular pathogen, M. In the same way, the infected phagocytic cell and surrounding tissue respond in a global sense to the presence of an intruding pathogen. As discussed earlier in this review, DNA arrays have been used to study the global expression of genes in wild-type and mutant M.
Proteomic techniques have also been used to measure the levels of large numbers of bacterial proteins when M. The purpose of this section is to discuss how new global methods are currently being used and could be used in the near future to study the interactions between M. There have been some descriptions of host responses when macrophages are allowed to phagocytose M. However, these macrophage responses have generally been limited to the expression of a small number of genes or the levels of a few proteins, and as discussed earlier in this review, there are often widely discordant results from different laboratories.
Recently, DNA array analyses have been used to study host gene expression during M. This study also used primary macrophages from mutant mice, providing significant information on mechanisms of the host response to infection.
To more completely describe the M. As an example of this combined approach, mutations in the M. Thus, global expression profiling with DNA arrays can now be used to identify the M.
This was recently done with wild-type S. At the same time, it will be possible to globally compare the expression of macrophage genes during the infection with wild-type M. However, since these regulators presumably control many genes that are differentially expressed during macrophage infection, it may not be possible to determine initially the identity of the bacterial effector molecules important for virulence that are missing in the mutants.
Thus, it will be important also to characterize the M. Health systems are important and need to be strengthened. As with other health interventions, the success of tuberculosis treatment and control in a country is often determined by the strength of its health system McKee and Atun ; WHO In a sense, the major risk factor for acquiring TB is breathing. Thus, people of all social and economic statuses are at risk. While TB disproportionately affects the poor, the narrative that TB is a disease only of the poor is misleading and counterproductive, if it leads either to further stigmatization of the disease or to the view that middle- and high-income countries need not worry about the disease.
The analytical framework underlying this chapter defines key functions of the health system, ultimate goals, and contextual factors that affect the health system figure It also draws on earlier studies by Atun ; Atun and Coker ; Atun, Samyshkin, and others ; Samb and others ; and Swanson and others Governance and organization.
The policy and regulatory environment; stewardship and regulatory functions of the ministry of health and its relation to other levels of the health system; and structural arrangements for insurers and purchasers, health care providers, and market regulators. The way funds are collected, funds and risks are pooled, finances are allocated, and health care providers are remunerated. Resource management. The way resources—physical, human, and intellectual—are generated and allocated, including their geographic and needs-based allocation.
Service delivery. Both population- and individual-level public health interventions and health care services provided in community, primary health care, hospitals, and other health institutions. Each of these functions is influenced by the economic, demographic, legal, cultural, and political context. As the framework suggests, health system goals include better health, financial protection, and user satisfaction.
Personal health services and public health interventions should be organized to achieve an appropriate balance of equity including reducing out-of-pocket [OOP] expenditures and impoverishment of individuals and families , efficiency, effectiveness that is, the extent to which interventions are evidence based and safe , responsiveness, equity, and client satisfaction as perceived by the users of services. This chapter is organized as follows. First, we provide a detailed discussion of the global burden of disease and clinical context, followed by a review of approaches to diagnosis, treatment, and prevention.
The aim throughout is to approach TB through a health system lens and, in the latter part of the chapter, to provide recommendations for improving delivery strategies and strengthening health systems, including care, supply chain, and information systems. Because the current tools for combating TB are seriously inadequate, we conclude with sections on critical research and development and economic analyses of new interventions for diagnosis, treatment, and vaccines.
However, in certain TB cases that are caused by pauci-bacillary disease, where only a small number of M. In cases where the culture is negative, the standard used to compare with the diagnostic test could be response to treatment, clinical features, or a positive culture in the future.
A TB diagnosis in this population would likely be achieved on a case-by-case basis and as such, would probably not be reported in many studies 9 , 38 , Diagnosing TB worldwide still consists of methods that are intended to isolate the pathogen, which is a major limitation when the mycobacterial load is low or the site of infection is not easily accessible.
For these reasons, diagnosis of smear-negative TB is often delayed, and such a diagnosis is often made based on the clinical response to empiric anti-TB treatment without microbiological confirmation 38 , 39 , Diagnostic algorithms have been recommended in the absence of rapid, simple, and accurate diagnostic tools for smear-negative pulmonary TB 11 , 38 , 39 , Diagnosing smear-negative pulmonary TB is achieved with at least two adequate expectorated multiple sputum samples, with negative smears, chest radiography findings consistent with TB, and a lack of response to a number of days of broad-spectrum antibiotics while awaiting culture results.
For such patients, sputum cultures should be obtained and active follow-up is needed 25 , There are several limitations to using diagnostic steps based on antibiotics response. Because the fluoroquinolones and aminoglycosides are active against the M. Strict adherence to the sequential steps of the algorithm could delay appropriate treatment in patients with an illness that is worsening rapidly. Finally, studies have shown that patients with TB might respond, at least transiently, to empirical broad-spectrum antibiotics, a frequent element of diagnostic algorithms 7 , A clinically-diagnosed TB case is one that does not fulfill the criteria for bacteriological confirmation but has been diagnosed with active TB by a clinician or other medical practitioner who has decided to give the patient a full course of TB treatment.
Clinically-diagnosed cases subsequently found to be bacteriologically-positive before or after starting treatment should be reclassified as bacteriologically confirmed Clinicians who manage patients with suspected TB should ensure that their diagnostic practices align with the guidelines for TB, and use sputum smear microscopy and culture to investigate adult patients with suspected active TB 3 , 9 , Novel methods allow for rapid diagnosis of active TB in patients with negative sputum smears for AFB and enable prompt, highly accurate identification of drug-resistant strains of M.
Some of the structural limits of current TB diagnostics are likely to be overcome by such new tools, but research is still needed. The success of implementing new technologies and the development of additional diagnostic approaches must take into account the diagnostic needs of each context point-of-need testing approach together with the logistic, economic and technical constraints that are present in the majority of intermediate-TB-burdened settings in Korea.
Conflicts of Interest: No potential conflict of interest relevant to this article was reported. National Center for Biotechnology Information , U. Tuberc Respir Dis Seoul. Published online Apr 2. Yon Ju Ryu , M. Find articles by Yon Ju Ryu. Author information Article notes Copyright and License information Disclaimer.
Corresponding author. Address for correspondence: Yon Ju Ryu, M. Phone: , Fax: , rk. All rights reserved. This article has been cited by other articles in PMC. Abstract Pulmonary tuberculosis TB persists as a great public health problem in Korea. Keywords: Lung, Tuberculosis, Diagnosis. Introduction Tuberculosis TB is a global health concern for both developing and developed countries and has recently become more complex due to persistence in aging populations and the rise of drug-resistant strains, even in Korea 1 , 2.
Diagnostic Methods 1. AFB smear microscopy and culture For pulmonary TB, sputum is the most critical sample for laboratory testing. Molecular methods 1 Nucleic acid amplification testing Nucleic acid amplification NAA tests are a reliable way to increase the specificity of diagnosis, but the sensitivity is too poor to rule out disease, especially in smear-negative paucibacillary disease where clinical diagnosis is equivocal and where the clinical need is greatest 23 , Diagnostic Algorithms of Pulmonary Tuberculosis 1.
Footnotes Conflicts of Interest: No potential conflict of interest relevant to this article was reported. References 1. World Health Organization. Increased tuberculosis burden due to demographic transition in Korea from to Tuberc Respir Dis. Early detection of tuberculosis: an overview of approaches, guidelines and tools [Internet] Geneva: World Health Organization; Update: the radiographic features of pulmonary tuberculosis.
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Definitions and reporting framework for tuberculosis: revision updated December [Internet] Geneva: World Health Organization; National Institute for Health and Clinical Excellence. Tuberculosis: clinical diagnosis and management of tuberculosis, and measures for its prevention and control.
Wilson ML. Recent advances in the laboratory detection of Mycobacterium tuberculosis complex and drug resistance. Clin Infect Dis. Korean guidelines for tuberculosis. Cochrane Database Syst Rev. Adult-onset pulmonary tuberculosis: findings on chest radiographs and CT scans. Clinical and radiographic correlates of primary and reactivation tuberculosis: a molecular epidemiology study. Chest radiographic findings in patients with tuberculosis with recent or remote infection.
Pulmonary tuberculosis: CT findings: early active disease and sequential change with antituberculous therapy. Diagnostic accuracy of same-day microscopy versus standard microscopy for pulmonary tuberculosis: a systematic review and meta-analysis. Lancet Infect Dis. Fluorescence versus conventional sputum smear microscopy for tuberculosis: a systematic review.
Diagn Microbiol Infect Dis. Current evidence on diagnostic accuracy of commercially based nucleic acid amplification tests for the diagnosis of pulmonary tuberculosis.
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