Paper Versus Digital Data Collection Methods for Road Safety Observations: Comparative Efficiency Analysis of Cost, Timeliness, Reliability, and Results.

BACKGROUND: Roadside observational studies play a fundamental role in designing evidence-informed strategies to address the pressing global health problem of road traffic injuries. Paper-based data collection has been the standard method for such studies, although digital methods are gaining popularity in all types of primary data collection. OBJECTIVE: This study aims to understand the reliability, productivity, and efficiency of paper vs digital data collection based on three different road user behaviors: helmet use, seatbelt use, and speeding. It also aims to understand the cost and time efficiency of each method and to evaluate potential trade-offs among reliability, productivity, and efficiency. METHODS: A total of 150 observational sessions were conducted simultaneously for each risk factor in Mumbai, India, across two rounds of data collection. We matched the simultaneous digital and paper observation periods by date, time, and location, and compared the reliability by subgroups and the productivity using Pearson correlations (r). We also conducted logistic regressions separately by method to understand how similar results of inferential analyses would be. The time to complete an observation and the time to obtain a complete dataset were also compared, as were the total costs in US dollars for fieldwork, data entry, management, and cleaning. RESULTS: Productivity was higher in paper than digital methods in each round for each risk factor. However, the sample sizes across both methods provided a precision of 0.7 percentage points or smaller. The gap between digital and paper data collection productivity narrowed across rounds, with correlations improving from r=0.27-0.49 to 0.89-0.96. Reliability in risk factor proportions was between 0.61 and 0.99, improving between the two rounds for each risk factor. The results of the logistic regressions were also largely comparable between the two methods. Differences in regression results were largely attributable to small sample sizes in some variable levels or random error in variables where the prevalence of the outcome was similar among variable levels. Although data collectors were able to complete an observation using paper more quickly, the digital dataset was available approximately 9 days sooner. Although fixed costs were higher for digital data collection, variable costs were much lower, resulting in a 7.73% (US $3011/38,947) lower overall cost. CONCLUSIONS: Our study did not face trade-offs among time efficiency, cost efficiency, statistical reliability, and descriptive comparability when deciding between digital and paper, as digital data collection proved equivalent or superior on these domains in the context of our project. As trade-offs among cost, timeliness, and comparability-and the relative importance of each-could be unique to every data collection project, researchers should carefully consider the questionnaire complexity, target sample size, implementation plan, cost and logistical constraints, and geographical contexts when making the decision between digital and paper.

Host cell-induced components of the sulfate assimilation pathway are major protective antigens of Mycobacterium tuberculosis.

New therapies to control tuberculosis are urgently required because of the inability of the only available vaccine, BCG, to adequately protect against tuberculosis. Here we demonstrate that proteins of the Mycobacterium tuberculosis sulfate-assimilation pathway (SAP) represent major immunogenic targets of the bacillus, as defined by strong T-cell recognition by both mice and humans infected with M. tuberculosis. SAP proteins displayed increased expression when M. tuberculosis was resident within host cells, which may account in part for their ability to stimulate anti-M. tuberculosis host immunity. Vaccination with the first enzyme in this pathway, adenosine-5'-triphosphate sulfurylase, conferred significant protection against murine tuberculosis and boosted BCG-induced protective immunity in the lung. Therefore, we have identified SAP components as a new family of M. tuberculosis antigens, and we have demonstrated that these components are promising candidate for inclusion in new vaccines to control tuberculosis in humans.

Protective immunity afforded by attenuated, PhoP-deficient Mycobacterium tuberculosis is associated with sustained generation of CD4+ T-cell memory.

Definition of protective immunity induced by effective vaccines is important for the design of new pathogen control strategies. Inactivation of the PhoP response-regulator in Mycobacterium tuberculosis results in a highly attenuated strain that demonstrates impressive protective efficacy in pre-clinical models of tuberculosis. In this report we demonstrate that the protection afforded by the M. tuberculosis phoP mutant strain is associated with the long-term maintenance of CD4(+) T-cell memory. Immunization of mice with SO2 resulted in enhanced expansion of M. tuberculosis-specific CD4(+) T cells compared with vaccination with the BCG vaccine, with an increased frequency of these cells persisting at extended time-points after vaccination. Strikingly, vaccination with SO2 resulted in sustained generation of CD4(+) T cells displaying a central memory phenotype, a property not shared by BCG. Further, SO2 vaccination markedly improved the generation of polyfunctional cytokine-secreting CD4(+) T cells compared with BCG vaccination. The improved generation of functionally competent memory T cells by SO2 correlated with augmented recall responses in SO2-vaccinated animals after challenge with virulent M. tuberculosis. This study defines a mechanism for the protective effect of the SO2 vaccine and suggests that deletion of defined virulence networks may provide vaccine strains with potent immuno-stimulatory properties.

Metabolic syndrome and associated urolithiasis in adults enrolled in a community-based health program.

BACKGROUND: Urolithiasis is a common and recurrent disease, whose prevalence rate has recently increased in parallel to obesity pandemic. OBJECTIVES: To estimate the prevalence of history of urolithiasis in a non-randomized sample of adults assisted by a community-based health program and to analyze its association with metabolic syndrome. METHODS: Cross-sectional study set in Niteroi, Rio de Janeiro, Brazil, including adults (non-diabetic hypertensives, diabetics or controls). Participants were assessed through a standardized questionnaire and underwent clinical and laboratory evaluation, including blood and urine samples. The diagnosis of metabolic syndrome was based on harmonized criteria. RESULTS: A total of 740 adults were enrolled (M: F = 0.85; 43±12 years; 30% white, and 70% non-white). Almost half of subjects (42.5%) had metabolic syndrome. The prevalence of urolithiasis in the sample was 10.1%. White skin colour, family history, and metabolic syndrome were independently associated with urolithiasis (P < 0.05). Subjects with the syndrome (excluding cases on diuretics) had more acidic urine (P = 0.014), increased natriuresis (P = 0.01) and higher uricosuria (P = 0.001) compared with non-affected ones. The prevalence of urolithiasis increased in proportion to the number of criteria for metabolic syndrome (P for trend <0.005). CONCLUSIONS: Metabolic syndrome is a modifiable factor associated with urolithiasis in a way that the frequency of positive history increases proportionally to the number of its diagnostic criteria. These findings reinforce the recent suggested link between urolithiasis and cardiovascular risk factors.

Characterization of the Protective Immune Responses Conferred by Recombinant BCG Overexpressing Components of Mycobacterium tuberculosis Sec Protein Export System.

Mycobacterium bovis Bacillus Calmette-Guérin (BCG) is the only approved vaccine against tuberculosis (TB). However, its efficacy in preventing pulmonary TB in adults is limited. Despite its variable efficacy, BCG offers a number of unique and beneficial characteristics, which make it suitable as a vaccine vehicle to express recombinant molecules. In Mycobacterium tuberculosis, the general Sec pathway is an essential cellular process, and it is responsible for exporting the majority of proteins across the cytoplasmic membrane, including potent immune-protective antigens, such as members of the antigen 85 (Ag85) complex. We engineered BCG to overexpress the M. tuberculosis SecDFG proteins in order to improve the efficiency of the Sec-dependent export system and, thus, enhance the secretion of immunogenic proteins. BCGSecDFG displayed increased intracellular survival within macrophages in vitro and greater persistence in the lymphoid organs of vaccinated mice than parental BCG. In addition, vaccination with BCGSecDFG generated higher numbers of IFN-γ-secreting T cells in response to secreted mycobacterial antigens compared to BCG, particularly members of the Ag85 complex. Furthermore, vaccination with BCGSecDFG significantly reduced the bacterial load in the lungs and spleens of M. tuberculosis-infected mice, which was comparable to the protection afforded by parental BCG. Therefore, the modification of protein secretion in BCG can improve antigen-specific immunogenicity.

Advax adjuvant formulations promote protective immunity against aerosol Mycobacterium tuberculosis in the absence of deleterious inflammation and reactogenicity.

The development of safe and effective adjuvants is a critical goal of vaccine development programs. In this report, we defined the immunostimulatory profile and protective effect against aerosol Mycobacterium tuberculosis infection of vaccine formulations incorporating the semi-crystalline adjuvant δ-inulin (Advax). Advax formulated with CpG oligonucleotide and the QS-21 saponin (AdvaxCpQS) was the most effective combination, demonstrated by the capacity of CysVac2/AdvaxCpQS to significantly reduce the bacterial burden in the lungs of M. tuberculosis-infected mice. CysVac2/AdvaxCpQS protection was associated with rapid influx of neutrophils, macrophages and monocytes to the site of vaccination and the induction of antigen-specific IFN-γ+/IL-2+/TNF+ polyfunctional CD4+ T cells in the lung. When compared to the highly potent adjuvant combination of monophosphoryl lipid A and dimethyldioctadecylammonium bromide (MPL/DDA), AdvaxCpQS imparted a similar level of protective efficacy yet without the profound stimulation of inflammatory cytokines and vaccination site ulceration observed with MPL/DDA. Addition of DDA to CysVac2/AdvaxCpQS further improved the protective effect of the vaccine, which correlated with increased polyfunctional CD4+ T cells in the lung but with no increase in vaccine reactogenicity. The data demonstrate that Advax formulations can decouple protective tuberculosis immunity from reactogenicity, making them ideal candidates for human application.

Protective efficacy of recombinant BCG over-expressing protective, stage-specific antigens of Mycobacterium tuberculosis.

Tuberculosis (TB) remains a major cause of mortality and morbidity worldwide, yet current control strategies, including the existing BCG vaccine, have had little impact on disease control. CysVac2, a fusion protein comprising stage-specific Mycobacterium tuberculosis antigens, provided superior protective efficacy against chronic M. tuberculosis infection in mice, compared to BCG. To determine if the delivery of CysVac2 in the context of BCG could improve BCG-induced immunity and protection, we generated a recombinant strain of BCG overexpressing CysVac2 (rBCG:CysVac2). Expression of CysVac2 in BCG was facilitated by the M. tuberculosis hspX promoter, which is highly induced inside phagocytic cells and induces strong cellular immune responses to antigens expressed under its regulation. Intradermal vaccination with rBCG:CysVac2 resulted in increased monocyte/macrophage recruitment and enhanced antigen-specific CD4+ T cell priming compared to parental BCG, indicating CysVac2 overexpression had a marked effect on rBCG induced-immunity. Further, rBCG:CysVac2 was a more potent inducer of antigen-specific multifunctional CD4+ T cells (CD4+IFN-γ+TNF+IL-2+) than BCG after vaccination of mice. This improved immunogenicity however did not influence protective efficacy, with both BCG and rBCG:CysVac2 affording comparable level of protection aerosol infection with M. tuberculosis. Boosting either BCG or rBCG:CysVac2 with the CysVac2 fusion protein resulted in a similar improvement in protective efficacy. These results demonstrate that the expression of protective antigens in BCG can augment antigen-specific immunity after vaccination but does not alter protection against infection, further highlighting the challenge of developing effective vaccines to control TB.

Delta inulin-based adjuvants promote the generation of polyfunctional CD4+ T cell responses and protection against Mycobacterium tuberculosis infection.

There is an urgent need for the rational design of safe and effective vaccines to protect against chronic bacterial pathogens such as Mycobacterium tuberculosis. Advax™ is a novel adjuvant based on delta inulin microparticles that enhances immunity with a minimal inflammatory profile and has entered human trials to protect against viral pathogens. In this report we determined if Advax displays broad applicability against important human pathogens by assessing protective immunity against infection with M. tuberculosis. The fusion protein CysVac2, comprising the M. tuberculosis antigens Ag85B (Rv1886c) and CysD (Rv1285) formulated with Advax provided significant protection in the lungs of M. tuberculosis-infected mice. Protection was associated with the generation of CysVac2-specific multifunctional CD4+ T cells (IFN-γ+TNF+IL-2+). Addition to Advax of the TLR9 agonist, CpG oligonucleotide (AdvaxCpG), improved both the immunogenicity and protective efficacy of CysVac2. Immunisation with CysVac2/AdvaxCpG resulted in heightened release of the chemoattractants, CXCL1, CCL3, and TNF, and rapid influx of monocytes and neutrophils to the site of vaccination, with pronounced early priming of CysVac2-specific CD4+ T cells. As delta inulin adjuvants have shown an excellent safety and tolerability profile in humans, CysVac2/AdvaxCpG is a strong candidate for further preclinical evaluation for progression to human trials.

Influence of phthiocerol dimycocerosate on CD4(+) T cell priming and persistence during Mycobacterium tuberculosis infection.

The characterisation of mycobacterial factors that influence or modulate the host immune response may aid the development of more efficacious TB vaccines. We have previously reported that Mycobacterium tuberculosis deficient in export of Phthiocerol Dimycocerosates (DIM) (MT103(ΔdrrC)) is more attenuated than wild type M. tuberculosis and provides sustained protective immunity compared to the existing BCG vaccine. Here we sought to define the correlates of immunity associated with DIM deficiency by assessing the impact of MT103(ΔdrrC) delivery on antigen presenting cell (APC) function and the generation of CD4(+) T cell antigen-specific immunity. MT103(ΔdrrC) was a potent activator of bone marrow derived dendritic cells, inducing significantly greater expression of CD86 and IL-12p40 compared to BCG or the MT103 parental strain. This translated to an increased ability to initiate early in vivo priming of antigen-specific CD4(+) T cells compared to BCG with enhanced release of IFN-γ and TNF upon antigen-restimulation. The heightened immunity induced by MT103(ΔdrrC) correlated with greater persistence within the spleen compared to BCG, however both MT103(ΔdrrC) and BCG were undetectable in the lung at 70 days post-vaccination. In immunodeficient RAG (-/-) mice, MT103(ΔdrrC) was less virulent than the parental MT103 strain, yet MT103(ΔdrrC) infected mice succumbed more rapidly compared to BCG-infected animals. These results suggest that DIM translocation plays a role in APC stimulation and CD4(+) T cell activation during M. tuberculosis infection and highlights the potential of DIM-deficient strains as novel TB vaccine candidates.

Mycobacterium tuberculosis components expressed during chronic infection of the lung contribute to long-term control of pulmonary tuberculosis in mice.

Tuberculosis (TB) remains a major cause of mortality and morbidity worldwide, yet current control strategies, including the existing BCG vaccine, have had little impact on disease control. The tubercle bacillus modifies protein expression to adapt to chronic infection of the host, and this can potentially be exploited to develop novel therapeutics. We identified the gene encoding the first step of the Mycobacterium tuberculosis sulphur assimilation pathway, cysD, as highly induced during chronic infection in the mouse lung, suggesting therapies based on CysD could be used to target infection. Vaccination with the composite vaccine CysVac2, a fusion of CysD and the immunogenic Ag85B of M. tuberculosis, resulted in the generation of multifunctional CD4+ T cells (interferon (IFN)-γ+TNF+IL-2+IL-17+) in the lung both pre- and post-aerosol challenge with M. tuberculosis. CysVac2 conferred significant protection against pulmonary M. tuberculosis challenge and was particularly effective at controlling late-stage infection, a property not shared by BCG. CysVac2 delivered as a booster following BCG vaccination afforded greater protection against M. tuberculosis challenge than BCG alone. The antigenic components of CysVac2 were conserved amongst M. tuberculosis strains, and protective efficacy afforded by CysVac2 was observed across varying murine MHC haplotypes. Strikingly, administration of CysVac2 to mice previously infected with M. tuberculosis reduced bacterial load and immunopathology in the lung compared with BCG-vaccinated mice. These results indicate that CysVac2 warrants further investigation to assess its potential to control pulmonary TB in humans.

Homogentisate 1-2-Dioxygenase Downregulation in the Chronic Persistence of Pseudomonas aeruginosa Australian Epidemic Strain-1 in the CF Lung.

Some Pseudomonas aeruginosa strains including Australian Epidemic Strain-1 (AES-1 or AUS-01) cause persistent chronic infection in cystic fibrosis (CF) patients, with greater morbidity and mortality. Factors conferring persistence are largely unknown. Previously we analysed the transcriptomes of AES-1 grown in Luria broth, nematode growth medium for Caenorhabditis elegans assay (both aerobic) and artificial sputum medium (mainly hypoxic). Transcriptional comparisons included chronic AES-1 strains against PAO1 and acute AES-1 (AES-1R) against its chronic isogen (AES-1M), isolated 10.5 years apart from a CF patient and not eradicated in the meantime. Prominent amongst genes downregulated in AES-1M in all comparisons was homogentisate-1-2-dioxygenase (hmgA); an oxygen-dependent gene known to be mutationally deactivated in many chronic infection strains of P. aeruginosa. To investigate if hmgA downregulation and deactivation gave similar virulence persistence profiles, a hmgA mutant made in UCBPP-PA14 utilising RedS-recombinase and AES-1M were assessed in the C. elegans virulence assay, and the C57BL/6 mouse for pulmonary colonisation and TNF-α response. In C. elegans, hmgA deactivation resulted in significantly increased PA14 virulence while hmgA downregulation reduced AES-1M virulence. AES-1M was significantly more persistent in mouse lung and showed a significant increase in TNF-α (p<0.0001), sustained even with no detectable bacteria. PA14ΔhmgA did not show increased TNF-α. This study suggests that hmgA may have a role in P. aeruginosa persistence in chronic infection and the results provide a starting point for clarifying the role of hmgA in chronic AES-1.

Destabilized green fluorescent protein for monitoring transient changes in mycobacterial gene expression.

The green fluorescent protein (GFP) is a useful reporter for the study of gene expression and protein localisation within living cells. The stability of GFP permits its intracellular accumulation and detection, but renders it less useful for assessing transient changes in gene expression. We have developed a destabilized form of GFP for monitoring gene expression in mycobacteria. By fusing to the C-terminal end of GFP an 11 amino acid peptide encoded by the E. coli ssrA gene, we have developed a form of GFP that exhibits gradual, time-dependent degradation within the fast-growing species Mycobacterium smegmatis. This unstable variant of GFP detected transient changes in the activity of the stress-induced Mycobacterium tuberculosis sigE promoter; by contrast, unmodified GFP only detected a delayed 'switch-on' of this promoter upon exposure to acid stress. Both forms of the protein displayed equivalent stability in the slow-growing species Mycobacterium bovis bacille Calmette-Guerin (BCG), suggesting differing recognition of the ssrA-encoded peptides in slow- and fast-growing mycobacteria. This system will facilitate studies exploring dynamic changes in mycobacterial gene expression.

The secreted lipoprotein, MPT83, of Mycobacterium tuberculosis is recognized during human tuberculosis and stimulates protective immunity in mice.

The long-term control of tuberculosis (TB) will require the development of more effective anti-TB vaccines, as the only licensed vaccine, Mycobacterium bovis bacille Calmette-Guérin (BCG), has limited protective efficacy against infectious pulmonary TB. Subunit vaccines have an improved safety profile over live, attenuated vaccines, such as BCG, and may be used in immuno-compromised individuals. MPT83 (Rv2873) is a secreted mycobacterial lipoprotein expressed on the surface of Mycobacterium tuberculosis. In this study, we examined whether recombinant MPT83 is recognized during human and murine M. tuberculosis infection. We assessed the immunogenicity and protective efficacy of MPT83 as a protein vaccine, with monophosphyl lipid A (MPLA) in dimethyl-dioctadecyl ammonium bromide (DDA) as adjuvant, or as a DNA vaccine in C57BL/6 mice and mapped the T cell epitopes with peptide scanning. We demonstrated that rMPT83 was recognised by strong proliferative and Interferon (IFN)-γ-secreting T cell responses in peripheral blood mononuclear cells (PBMC) from patients with active TB, but not from healthy, tuberculin skin test-negative control subjects. MPT83 also stimulated strong IFN-γ T cell responses during experimental murine M. tuberculosis infection. Immunization with either rMPT83 in MPLA/DDA or DNA-MPT83 stimulated antigen-specific T cell responses, and we identified MPT83(127-135) (PTNAAFDKL) as the dominant H-2(b)-restricted CD8(+) T cell epitope within MPT83. Further, immunization of C57BL/6 mice with rMPT83/MPLA/DDA or DNA-MPT83 stimulated significant levels of protection in the lungs and spleens against aerosol challenge with M. tuberculosis. Interestingly, immunization with rMPT83 in MPLA/DDA primed for stronger IFN-γ T cell responses to the whole protein following challenge, while DNA-MPT83 primed for stronger CD8(+) T cell responses to MPT83(127-135). Therefore MPT83 is a protective T cell antigen commonly recognized during human M. tuberculosis infection and should be considered for inclusion in future TB subunit vaccines.

Cutaneous immunosurveillance by self-renewing dermal gammadelta T cells.

The presence of γδ T cell receptor (TCR)-expressing cells in the epidermis of mice, termed dendritic epidermal T cells (DETCs), is well established. Because of their strict epidermal localization, it is likely that DETCs primarily respond to epithelial stress, such as infections or the presence of transformed cells, whereas they may not participate directly in dermal immune responses. In this study, we describe a prominent population of resident dermal γδ T cells, which differ from DETCs in TCR usage, phenotype, and migratory behavior. Dermal γδ T cells are radioresistant, cycle in situ, and are partially depend on interleukin (IL)-7, but not IL-15, for their development and survival. During mycobacterial infection, dermal γδ T cells are the predominant dermal cells that produce IL-17. Absence of dermal γδ T cells is associated with decreased expansion in skin draining lymph nodes of CD4(+) T cells specific for an immunodominant Mycobacterium tuberculosis epitope. Decreased CD4(+) T cell expansion is related to a reduction in neutrophil recruitment to the skin and decreased BCG shuttling to draining lymph nodes. Thus, dermal γδ T cells are an important part of the resident cutaneous immunosurveillance program. Our data demonstrate functional specialization of T cells in distinct microcompartments of the skin.

Cutinase-like protein-6 of Mycobacterium tuberculosis is recognised in tuberculosis patients and protects mice against pulmonary infection as a single and fusion protein vaccine.

Infection with Mycobacterium tuberculosis continues to be a leading cause of death in many regions of the world, and control of this disease is hampered by the lack of a safe and effective vaccine. Secreted proteins of M. tuberculosis are an important group of antigens for subunit vaccines which target this infection. We have tested three secreted members of the cutinase-like protein (CULP) family of M. tuberculosis for their potential as protein vaccine antigens. Culp6 elicited a strong T lymphocyte response in M. tuberculosis infected mice, and importantly, in tuberculosis (TB) patients tested. Culp1, Culp2 and Culp6 when delivered as protein vaccines to mice, induced potent IFN-gamma responses which in turn translated into a significant level of protection against aerosol M. tuberculosis infection. A Culp1-6 fusion protein provided an increased level of protection against infection compared to Culp1 or Culp6 alone. The data presented here may indicate that the cell wall-associated, putatively essential protein Culp6, shown here for the first time to be recognised in TB patients, is an attractive candidate for inclusion in future subunit vaccines.

Sulfite reduction in mycobacteria.

Mycobacterium tuberculosis places an enormous burden on the welfare of humanity. Its ability to grow and its pathogenicity are linked to sulfur metabolism, which is considered a fertile area for the development of antibiotics, particularly because many of the sulfur acquisition steps in the bacterium are not found in the host. Sulfite reduction is one such mycobacterium-specific step and is the central focus of this paper. Sulfite reduction in Mycobacterium smegmatis was investigated using a combination of deletion mutagenesis, metabolite screening, complementation, and enzymology. The initial rate parameters for the purified sulfite reductase from M. tuberculosis were determined under strict anaerobic conditions [k(cat) = 1.0 (+/-0.1) electron consumed per second, and K(m(SO(3)(-2))) = 27 (+/-1) microM], and the enzyme exhibits no detectible turnover of nitrite, which need not be the case in the sulfite/nitrite reductase family. Deletion of sulfite reductase (sirA, originally misannotated nirA) reveals that it is essential for growth on sulfate or sulfite as the sole sulfur source and, further, that the nitrite-reducing activities of the cell are incapable of reducing sulfite at a rate sufficient to allow growth. Like their nitrite reductase counterparts, sulfite reductases require a siroheme cofactor for catalysis. Rv2393 (renamed che1) resides in the sulfur reduction operon and is shown for the first time to encode a ferrochelatase, a catalyst that inserts Fe(2+) into siroheme. Deletion of che1 causes cells to grow slowly on metabolites that require sulfite reductase activity. This slow-growth phenotype was ameliorated by optimizing growth conditions for nitrite assimilation, suggesting that nitrogen and sulfur assimilation overlap at the point of ferrochelatase synthesis and delivery.

Contribution of L-alanine dehydrogenase to in vivo persistence and protective efficacy of the BCG vaccine.

The tuberculosis (TB) vaccine strain Mycobacterium bovis BCG is unable to utilise alanine and this deficiency is thought to inhibit the growth of the vaccine in vivo and limit vaccine efficacy. In this report we demonstrate that L-alanine catabolism can be conferred on BCG by introduction of the gene encoding L-alanine dehydrogenase (Ald) of Mycobacterium tuberculosis. Restoration of Ald activity did not change the in vivo growth of BCG in macrophages or mice, and protection against aerosol M. tuberculosis infection was not altered by addition of ald to the BCG vaccine. These results demonstrate that the inability to utilise L-alanine is not a contributing factor to the attenuated phenotype of BCG and does not influence the protective efficacy of the vaccine against TB.

The trifunctional sulfate-activating complex (SAC) of Mycobacterium tuberculosis.

The sulfate activation pathway is essential for the assimilation of sulfate and, in many bacteria, is comprised of three reactions: the synthesis of adenosine 5'-phosphosulfate (APS), the hydrolysis of GTP, and the 3'-phosphorylation of APS to produce 3'-phosphoadenosine 5'-phosphosulfate (PAPS), whose sulfuryl group is reduced or transferred to other metabolites. The entire sulfate activation pathway is organized into a single complex in Mycobacterium tuberculosis. Although present in many bacteria, these tripartite complexes have not been studied in detail. Initial rate characterization of the mycobacterial system reveals that it is poised for extremely efficient throughput: at saturating ATP, PAPS synthesis is 5800 times more efficient than APS synthesis. The APS kinase domain of the complex does not appear to form the covalent E.P intermediate observed in the closely related APS kinase from Escherichia coli. The stoichiometry of GTP hydrolysis and APS synthesis is 1:1, and the APS synthesis reaction is driven 1.1 x 10(6)-fold further during GTP hydrolysis; the system harnesses the full chemical potential of the hydrolysis reaction to the synthesis of APS. A key energy-coupling step in the mechanism is a ligand-induced isomerization that enhances the affinity of GTP and commits APS synthesis and GTP hydrolysis to the completion of the catalytic cycle. Ligand-induced increases in guanine nucleotide affinity observed in the mycobacterial system suggest that it too undergoes the energy-coupling isomerization.

Regulation of mutY and nature of mutator mutations in Escherichia coli populations under nutrient limitation.

Previous analysis of aerobic, glucose-limited continuous cultures of Escherichia coli revealed that G:C-to-T:A (G:C-->T:A) transversions were the most commonly occurring type of spontaneous mutation. One possible explanation for the preponderance of these mutations was that nutrient limitation repressed MutY-dependent DNA repair, resulting in increased proportions of G:C-->T:A transversions. The regulation of the mutY-dependent DNA repair system was therefore studied with a transcriptional mutY-lacZ fusion recombined into the chromosome. Expression from the mutY promoter was fourfold higher under aerobic conditions than under anaerobic conditions. But mutY expression was higher in glucose- or ammonia-limited chemostats than in nutrient-excess batch culture, so mutY was not downregulated by nutrient limitation. An alternative explanation for the frequency of G:C-->T:A transversions was the common appearance of mutY mutator mutations in the chemostat populations. Of 11 chemostat populations screened in detail, six contained mutators, and the mutator mutation in four cultures was located in the region of mutY at 66 min on the chromosome. The spectrum of mutations and rate of mutation in these isolates were fully consistent with a mutY-deficiency in each strain. Based on PCR analysis of the region within and around mutY, isolates from three individual populations contained deletions extending at least 2 kb upstream of mutY and more than 5 kb downstream. In the fourth population, the deletion was even longer, extending at least 5 kb upstream and 5 kb downstream of mutY. The isolation of mutY mutator strains from four independent populations with extensive chromosomal rearrangements suggests that mutY inactivation by deletion is a means of increasing mutation rates under nutrient limitation and explains the observed frequency of G:C-->T:A mutations in glucose-limited chemostats.

The Mycobacterium tuberculosis cysD and cysNC genes form a stress-induced operon that encodes a tri-functional sulfate-activating complex.

Sulfur metabolism has been implicated in the virulence, antibiotic resistance and anti-oxidant defence of Mycobacterium tuberculosis. Despite its human disease relevance, sulfur metabolism in mycobacteria has not yet been fully characterized. ATP sulfurylase catalyses the synthesis of activated sulfate (adenosine 5'-phosphosulfate, APS), the first step in the reductive assimilation of sulfate. Expression of the M. tuberculosis cysD gene, predicted to encode the adenylyl-transferase subunit of ATP sulfurylase, is upregulated by the bacilli inside its preferred host, the macrophage. This study demonstrates that cysD and cysNC orthologues exist in M. tuberculosis and constitute an operon whose expression is induced by sulfur limitation and repressed by the presence of cysteine, a major end-product of sulfur assimilation. The cysDNC genes are also induced upon exposure to oxidative stress, suggesting regulation of sulfur assimilation by M. tuberculosis in response to toxic oxidants. To ensure that the cysDNC operon encoded the activities predicted by its primary sequence, and to begin to characterize the products of the operon, they were expressed in Escherichia coli, purified to homogeneity, and tested for their catalytic activities. The CysD and CysNC proteins were shown to form a multifunctional enzyme complex that exhibits the three linked catalytic activities that constitute the sulfate activation pathway.