Mycobacterium tuberculosis H37Rv has a single nucleotide polymorphism in PhoR which affects cell wall hydrophobicity and gene expression.

Mycobacterium tuberculosis is a successful pathogen that can adapt to multiple environmental niches. As part of its repertoire of adaptive responses, two-component regulatory systems play a major role in co-ordinating gene expression at the global level. The PhoPR system controls major cellular functions, including respiration, lipid metabolism, the immediate and enduring hypoxic responses, stress responses and persistence. We identified a single nucleotide polymorphism (SNP) found in the sensor kinase (PhoR) of this system between two commonly used strains of M. tuberculosis, H37Rv (PhoR(P152)) and CDC1551 (PhoR(L152)). We constructed an isogenic strain of H37Rv carrying PhoR(L152), as well as strains containing two different copies of the PhoPR locus, to determine the functional consequences of the SNP on phenotypic traits. The previously identified Apr locus was not acid-inducible in H37Rv, although it was in the CDC1551 strain. Surprisingly, the acid-responsive expression was not completely dependent on the PhoR SNP, and the locus remained constitutively expressed even in the isogenic strain H37Rv:PhoR(L152). The pattern of expression in PhoPR merodiploid strains was more complex, with neither allele showing dominance. This suggests that Apr regulation is more complex than previously thought and that additional factors must be responsible for Apr upregulation in response to acid conditions. In contrast, differences we identified in cell hydrophobicity between the two strains were wholly dependent on PhoR, confirming its role as major regulator of cell wall composition. Thus the SNP in the sensor kinase has functional consequences which account for some of the differences between widely used laboratory strains.

Inhibition of the sole type I signal peptidase of Mycobacterium tuberculosis is bactericidal under replicating and nonreplicating conditions.

Proteins secreted by bacteria perform functions vital for cell survival and play a role in virulence in Mycobacterium tuberculosis. M. tuberculosis lepB (Rv2903c) encodes the sole homolog of the type I signal peptidase (SPase). The lepB gene is essential in M. tuberculosis, since we could delete the chromosomal copy only when a second functional copy was provided elsewhere. By placing expression under the control of an anhydrotetracycline-inducible promoter, we confirmed that reduced lepB expression was detrimental to growth. Furthermore, we demonstrated that a serine-lysine catalytic dyad, characteristic for SPase function, is required for LepB function. We confirmed the involvement of LepB in the secretion of a reporter protein fused to an M. tuberculosis signal peptide. An inhibitor of LepB (MD3; a beta-aminoketone) was active against M. tuberculosis, exhibiting growth inhibition and bactericidal activity. Overexpression of lepB reduced the susceptibility of M. tuberculosis to MD3, and downregulation resulted in increased susceptibility, suggesting that LepB is the true target of MD3. MD3 lead to a rapid loss of viability and cell lysis. Interestingly, the compound had increased potency in nonreplicating cells, causing a reduction in viable cell numbers below the detection limit after 24 h. These data suggest that protein secretion is required to maintain viability under starvation conditions and that secreted proteins play a critical role in generating and surviving the persistent state. We conclude that LepB is a promising novel target for drug discovery in M. tuberculosis, since its inhibition results in rapid killing of persistent and replicating organisms.

The arabinosyltransferase EmbC is inhibited by ethambutol in Mycobacterium tuberculosis.

Ethambutol (EMB) is an antimycobacterial drug used extensively for the treatment of tuberculosis caused by Mycobacterium tuberculosis. EMB targets the biosynthesis of the cell wall, inhibiting the synthesis of both arabinogalactan and lipoarabinomannan (LAM), and is assumed to act via inhibition of three arabinosyltransferases: EmbA, EmbB, and EmbC. EmbA and EmbB are required for the synthesis of arabinogalactan, and at least one enzyme (M. tuberculosis EmbA [EmbA(Mt)]) is essential in M. tuberculosis. EmbC(Mt) is also essential for the viability of M. tuberculosis but is involved in the synthesis of LAM. We show that mutations in EmbC(Mt) that reduce its arabinosyltransferase activity result in increased sensitivity to EMB and the production of smaller LAM species in M. tuberculosis. Overexpression of EmbC(Mt) was not tolerated in M. tuberculosis, but overexpression of Mycobacterium smegmatis EmbC (EmbC(Ms)) led to EMB resistance and the production of larger LAM species in M. tuberculosis. Treatment of wild-type M. tuberculosis strains with EMB led to inhibition of LAM synthesis, resulting in the production of smaller species of LAM. In contrast, no change in LAM production was seen in EMB-resistant strains. Overexpression of EmbB(Ms) in M. tuberculosis also resulted in EMB resistance, but at a lower level than that caused by EmbC(Ms). Overexpression of EmbA(Mt) in M. tuberculosis had no effect on EMB resistance. Thus, there is a direct correlation between EmbC activity and EMB resistance, as well as between EmbC activity and the size of the LAM species produced, confirming that EmbC is one of the cellular targets of EMB action.

SOURCES: a code for calculating (alpha,n), spontaneous fission, and delayed neutron sources and spectra.

SOURCES is a computer code that determines neutron production rates and spectra from (alpha,n) reactions, spontaneous fission and delayed neutron emission owing to the decay of radionuclides in homogeneous media, interface problems and three-region interface problems. The code is also capable of calculating the neutron production rates due to (alpha,n) reactions induced by a monoenergetic beam of alpha particles incident on a slab of target material. The (alpha,n) spectra are calculated using an assumed isotropic angular distribution in the centre-of-mass system with a library of 107 nuclide decay alpha-particle spectra, 24 sets of measured and/or evaluated (alpha,n) cross sections and product nuclide level branching fractions, and functional alpha particle stopping cross sections for Z < 106. Spontaneous fission sources and spectra are calculated with evaluated half-life, spontaneous fission branching and Watt spectrum parameters for 44 actinides. The delayed neutron spectra are taken from an evaluated library of 105 precursors. The code outputs the magnitude and spectra of the resultant neutron sources. It also provides an analysis of the contributions to that source by each nuclide in the problem.

The Mycobacterium tuberculosis dosRS two-component system is induced by multiple stresses.

Induction of the Mycobacterium tuberculosis dosR gene, which is known to respond to hypoxia, was measured using RTq-PCR following exposure to different stresses. Increased expression was seen after exposure to S-nitrosoglutathione (GSNO), ethanol and (to a lesser extent) H2O2, but not heat- or cold-shock. We also demonstrated that hspX, which is dependent on dosR for expression, is induced when cultures are left standing for 30 min, while significant but minor induction was seen following a 10 min centrifugation. Microarray analysis was used to compare gene expression in wild-type and deltadosR strains following 30 min standing. Fifty-two genes were significantly up-regulated, and 19 genes were down-regulated. These included genes that had previously been reported as being part of the dosR regulon, and also some novel ones.

amiA is a negative regulator of acetamidase expression in Mycobacterium smegmatis.

BACKGROUND: The acetamidase of Mycobacterium smegmatis is a highly inducible enzyme. Expression of this enzyme is increased 100-fold when the substrate acetamide is present. The acetamidase gene is found immediately downstream of three open reading frames. Two of these are proposed to be involved in regulation. RESULTS: We constructed a deletion mutant in one of the upstream ORFs (amiA). This mutant (Mad1) showed a constitutively high level of acetamidase expression. We identified four promoters in the upstream region using a beta-galactosidase reporter gene. One of these (P2) was inducible in the wild-type, but was constitutively active in Mad1. CONCLUSIONS: These results demonstrate that amiA encodes a negative regulatory protein which interacts with P2. Since amiA has homology to DNA-binding proteins, it is likely that it exerts the regulatory effect by binding to the promoter to prevent transcription.

Characterization of auxotrophic mutants of Mycobacterium tuberculosis and their potential as vaccine candidates.

Auxotrophic mutants of Mycobacterium tuberculosis have been proposed as new vaccine candidates. We have analyzed the virulence and vaccine potential of M. tuberculosis strains containing defined mutations in genes involved in methionine (metB), proline (proC), or tryptophan (trpD) amino acid biosynthesis. The metB mutant was a prototrophic strain, whereas the proC and trpD mutants were auxotrophic for proline and tryptophan, respectively. Following infection of murine bone marrow-derived macrophages, H37Rv and the metB mutant strain survived intracellularly for over 10 days, whereas over 90% of proC and trpD mutants were killed during this time. In SCID mice, both H37Rv and the metB mutant were highly virulent, with mouse median survival times (MST) of 28.5 and 42 days, respectively. The proC mutant was significantly attenuated (MST, 130 days), whereas the trpD mutant was essentially avirulent in an immunocompromised host. Following infection of immunocompetent DBA mice with H37Rv, mice survived for a median of 83.5 days and the metB mutant now showed a clear reduction in virulence, with two of five infected mice surviving for 360 days. Both proC and trpD mutants were avirulent (MST of >360 days). In vaccination studies, prior infection with either the proC or trpD mutant gave protection equivalent (proC mutant) to or better (trpD mutant) than BCG against challenge with M. tuberculosis H37Rv. In summary, proC and trpD genes are essential for the virulence of M. tuberculosis, and mutants with disruptions in either of these genes show strong potential as vaccine candidates.

Use of the mycobacteriophage L5 excisionase in Mycobacterium tuberculosis to demonstrate gene essentiality.

UNLABELLED: SWTTING: Demonstrating that a gene is essential is always difficult, but this is particularly true for a slow-growing organism such as Mycobacterium tuberculosis. One method currently used is to show that homologous recombination leading to gene inactivation only occurs in the presence of a second copy of the gene, but obtaining statistically significant data can be prohibitively difficult. L5-based integrating plasmids have been widely used in the genetic analysis of mycobacteria. The L5 excisionase has been used in Mycobacterium smegmatis to excise and recover these plasmids from chromosome. OBJECTIVE: Our aims were to establish whether the L5 excisionase could function in M. tuberculosis to remove an L5-based integrated plasmid and, if so, to use this technology as the basis for an improved method for determining whether a gene is essential. DESIGN: We took two strains of M. tuberculosis carrying the essential gene glnE integrated into the chromosome on an L5-based plasmid, one of which lacked the functional chromosomal copy of the gene. We transformed these with vectors expressing the L5 excisionase and looked for loss of the integrated plasmid. RESULTS: We obtained efficient excision of an integrated vector from the wild-type strain. However, when the integrated vector carried the only functional copy of the essential gene glnE, the numbers of colonies recovered were reduced to background levels. CONCLUSION: The L5 excisionase does function in M. tuberculosis and can be used to confirm the essentiality of a gene. This technology also allows further analysis of essential genes that is difficult or impossible using current methods.

Gene Replacement using Pretreated DNA.

Gene replacement by homologous recombination (HR) is an invaluable tool in understanding the physiology and the significance of specific genes in the virulence of Mycobacterium tuberculosis. It will also allow for the development of rationally attenuated strains as candidate vaccines to prevent the spread of tuberculosis. Classically, allelic replacement involves the introduction of nonreplicating DNA (suicide plasmids) carrying a mutated copy of the targeted gene, most often disrupted by an antibiotic resistance determinant, into the chromosome. A single recombination event (cross-over) between the two alleles will result in integration of the entire plasmid to generate a single crossover (SCO) strain carrying both wild-type and mutated copies of the gene. If two recombination events occur, a double cross-over (DCO) is generated where the wild-type allele is replaced by the mutant allele. Strains with an SCO can also give rise to DCO strains when a second recombination event takes place (Fig. 1).

glnE is an essential gene in Mycobacterium tuberculosis.

Mycobacterium tuberculosis possesses a homologue of glnE, potentially encoding a regulator of glutamine synthetase activity. We attempted to construct glnE-disrupted mutants using a two-step strategy, whereby a single-crossover strain was first isolated, followed by sacB counterselection to isolate the double-crossover strain. Of 192 sucrose-resistant colonies tested, none were mutants, although the wild-type double crossover could be easily isolated. When a second copy of the wild-type glnE was integrated into the chromosome, we could isolate both wild-type and mutant double-crossover strains. Thus, the chromosomal gene could only be replaced with a disrupted copy when another functional copy of the gene was provided, demonstrating that this gene is essential under the conditions tested.

Mycobacteria: bugs and bugbears (two steps forward and one step back).

The use of molecular techniques to study the mycobacteria has advanced greatly since the first genomic libraries of Mycobacterium tuberculosis and M. leprae were constructed in 1985. However, there are still pitfalls for the unwary. Most of the problems associated with the use of molecular techniques to study mycobacteria can be related to one of the following problems: slow growth rate causing problems with contamination; the formation of macroscopic clumps when grown in culture; resistance to standard chemical lysis procedures; the requirement for containment facilities for pathogenic species; the lack of suitable genetic vectors; and the problems of spontaneous antibiotic resistance. Despite these problems, considerable progress has been made and standard techniques have been developed for the preparation of protein, nucleic acids (DNA and RNA) and cell wall components, chemical and transposon mutagenesis and gene replacement methods, the use of reporter genes and expression vectors, and improved detection and drug sensitivity testing.

An inducible expression system permitting the efficient purification of a recombinant antigen from Mycobacterium smegmatis.

A novel expression vector utilising the highly inducible acetamidase promoter of Mycobacterium smegmatis was constructed. High-level induction of a model antigen, the Mycobacterium leprae 35 kDa protein, was demonstrated in recombinant M. smegmatis grown in the presence of the acetamidase inducer acetamide. The recombinant protein could be simply and efficiently purified from the bacterial sonicate by virtue of a C-terminal 6-histidine tag, demonstrating that this purification strategy can be used for the mycobacteria. The histidine tag had no apparent effect on the protein conformation or immunogenicity, suggesting that the vector described may prove useful for the purification of native-like recombinant mycobacterial proteins from fast-growing mycobacterial hosts.

Inactivation of mycobacteriophage D29 using ferrous ammonium sulphate as a tool for the detection of viable Mycobacterium smegmatis and M. tuberculosis.

There is still an urgent requirement for more sensitive, cost-effective methods for detection and susceptibility testing of mycobacteria in clinical samples. We have been investigating a simple bacteriophage-based system which could be used for both purposes. As this depends upon the detection of phages which have successfully infected cells, a key step is the efficient removal or inactivation of phages remaining free in the culture medium. We demonstrate here the use of ferrous ammonium sulphate as an effective agent for the inactivation of mycobacteriophage D29 without impairing phage replication in previously infected host bacteria. Using this property, we report the detection of viable Mycobacterium smegmatis, M. bovis BCG and M. tuberculosis using simple low-cost technology. The method is highly sensitive, since it is able to detect 10 colony-forming units of M. smegmatis. It is also rapid, with the detection of M. tuberculosis in sputum specimens within 48 h.

The effects of two types of relaxation training on students' levels of anxiety.

In the present study, high school students who received training in either behavioral relaxation or progressive muscle relaxation demonstrated significantly lower state anxiety scores than did those who had not received such training. No significant differences were found on trait anxiety scores. There was no significant effect of gender and also no significant interaction effects between the three groups and gender for either state or trait anxiety. Implications of these findings for high school counselors who work with students dealing with anxiety-producing problems are presented.

A Mycobacterium smegmatis mutant with a defective inositol monophosphate phosphatase gene homolog has altered cell envelope permeability.

A bacteriophage infection mutant (strain LIMP7) of Mycobacterium smegmatis was isolated following transposon mutagenesis. The mutant showed an unusual phenotype, in that all phages tested produced larger plaques on this strain compared to the parent strain. Other phenotypic characteristics of the mutant were slower growth, increased clumping in liquid culture, increased resistance to chloramphenicol and erythromycin, and increased sensitivity to isoniazid and several beta-lactam antibiotics. Permeability studies showed decreases in the accumulation of lipophilic molecules (norfloxacin and chenodeoxycholate) and a small increase with hydrophilic molecules (cephaloridine); taken together, these characteristics indicate an altered cell envelope. The DNA adjacent to the transposon in LIMP7 was cloned and was shown to be highly similar to genes encoding bacterial and mammalian inositol monophosphate phosphatases. Inositol is important in mycobacteria as a component of the major thiol mycothiol and also in the cell wall, with phosphatidylinositol anchoring lipoarabinomannan (LAM) in the cell envelope. In LIMP7, levels of phosphatidylinositol dimannoside, the precursor of LAM, were less than half of those in the wild-type strain, confirming that the mutation had affected the synthesis of inositol-containing molecules. The impA gene is located within the histidine biosynthesis operon in both M. smegmatis and Mycobacterium tuberculosis, lying between the hisA and hisF genes.

Development and use of a conditional antisense mutagenesis system in mycobacteria.

Expression from a 2.3 kb region upstream of the inducible acetamidase gene from Mycobacterium smegmatis was shown to be upregulated by acetamide. A DNA fragment containing the start of the M. smegmatis hisD gene was cloned in front of the promoter, such that the antisense message was produced. When this construct was induced in vivo, the bacteria became phenotypically histidine auxotrophs; this auxotrophy was restored by histidine supplementation. Auxotrophy was not observed under non-induced conditions. Antisense mutagenesis may be useful for observing the phenotypic inactivation of specific mycobacterial genes, and an inducible system such as that described would allow the study of essential genes.

A humanized form of a CD4-specific monoclonal antibody exhibits decreased antigenicity and prolonged plasma half-life in rhesus monkeys while retaining its unique biological and antiviral properties.

Certain monoclonal antibodies (MAbs) directed against CD4 can efficiently block HIV-1 replication in vitro. To explore CD4-directed passive immunotherapy for prevention or treatment of AIDS virus infection, we previously examined the biological activity of a nondepleting CD4-specific murine MAb, mu5A8. This MAb, specific for domain 2 of CD4, blocks HIV-1 replication at a post-gp120-CD4 binding step. When administered to normal rhesus monkeys, all CD4+ target cells were coated with antibody, yet no cell clearance or measurable immunosuppression occurred. However, strong anti-mouse Ig responses rapidly developed in all monkeys. In the present study, we report a successfully humanized form of mu5A8 (hu5A8) that retains binding to both human and monkey CD4 and anti-AIDS virus activity. When administered intravenously to normal rhesus monkeys, hu5A8 bound to all target CD4+ cells without depletion and showed a significantly longer plasma half-life than mu5A8. Nevertheless, an anti-hu5A8 response directed predominantly against V region determinants did eventually appear within 2 to 4 weeks in most animals. However, when hu5A8 was administered to rhesus monkeys chronically infected with the simian immunodeficiency virus of macaques, anti-hu5A8 antibodies were not detected. Repeated administration of hu5A8 in these animals resulted in sustained plasma levels and CD4+ cell coating with humanized antibody for 6 weeks. These studies demonstrate the feasibility of chronic administration of CD4-specific MAb as a potential means of treating or preventing HIV-1 infection.