Mycobacterium tuberculosis nucleoside diphosphate kinase inactivates small GTPases leading to evasion of innate immunity.

Defining the mechanisms of Mycobacterium tuberculosis (Mtb) persistence in the host macrophage and identifying mycobacterial factors responsible for it are keys to better understand tuberculosis pathogenesis. The emerging picture from ongoing studies of macrophage deactivation by Mtb suggests that ingested bacilli secrete various virulence determinants that alter phagosome biogenesis, leading to arrest of Mtb vacuole interaction with late endosomes and lysosomes. While most studies focused on Mtb interference with various regulators of the endosomal compartment, little attention was paid to mechanisms by which Mtb neutralizes early macrophage responses such as the NADPH oxidase (NOX2) dependent oxidative burst. Here we applied an antisense strategy to knock down Mtb nucleoside diphosphate kinase (Ndk) and obtained a stable mutant (Mtb Ndk-AS) that displayed attenuated intracellular survival along with reduced persistence in the lungs of infected mice. At the molecular level, pull-down experiments showed that Ndk binds to and inactivates the small GTPase Rac1 in the macrophage. This resulted in the exclusion of the Rac1 binding partner p67(phox) from phagosomes containing Mtb or Ndk-coated latex beads. Exclusion of p67(phox) was associated with a defect of both NOX2 assembly and production of reactive oxygen species (ROS) in response to wild type Mtb. In contrast, Mtb Ndk-AS, which lost the capacity to disrupt Rac1-p67(phox) interaction, induced a strong ROS production. Given the established link between NOX2 activation and apoptosis, the proportion of Annexin V positive cells and levels of intracellular active caspase 3 were significantly higher in cells infected with Mtb Ndk-AS compared to wild type Mtb. Thus, knock down of Ndk converted Mtb into a pro-apoptotic mutant strain that has a phenotype of increased susceptibility to intracellular killing and reduced virulence in vivo. Taken together, our in vitro and in vivo data revealed that Ndk contributes significantly to Mtb virulence via attenuation of NADPH oxidase-mediated host innate immunity.

Mycobacterium tuberculosis employs Cpn60.2 as an adhesin that binds CD43 on the macrophage surface.

CD43 is a large sialylated glycoprotein found on the surface of haematopoietic cells and has been previously shown to be necessary for efficient macrophage binding and immunological responsiveness to Mycobacterium tuberculosis. Using capsular material from M. tuberculosis and recombinant CD43-Fc, we have employed affinity chromatography to show that Cpn60.2 (Hsp65, GroEL), and to a lesser extent DnaK (Hsp70), bind to CD43. Competitive inhibition using recombinant protein and polyclonal F(ab')(2) antibody-mediated epitope masking studies were used to evaluate M. tuberculosis binding to CD43(+/+) versus CD43(-/-) macrophages. Results showed that Cpn60.2, but not DnaK, acts as a CD43-dependent mycobacterial adhesin for macrophage binding. Assessment of the specific binding between Cpn60.2 and CD43 showed it to be saturable, with a comparatively weak affinity in the low micromolar range. We have also shown that the ability of Cpn60.2 to competitively inhibit M. tuberculosis binding to macrophages is shared by the Escherichia coli homologue, GroEL, but not by the mouse and human Hsp60 homologues. These findings add to a growing field of research that implicates molecular chaperones as having extracellular functions, including bacterial adherence to host cells. Thus, CD43 may act as a Pattern Recognition Receptor (PRR) for bacterial homologues of the 60 kDa molecular chaperone.

Mycobacterium tuberculosis Cpn60.2 and DnaK are located on the bacterial surface, where Cpn60.2 facilitates efficient bacterial association with macrophages.

Mycobacterium tuberculosis, the causative agent of tuberculosis, initially contacts host cells with elements of its outer cell wall, or capsule. We have shown that capsular material from the surface of M. tuberculosis competitively inhibits the nonopsonic binding of whole M. tuberculosis bacilli to macrophages in a dose-dependent manner that is not acting through a global inhibition of macrophage binding. We have further demonstrated that isolated M. tuberculosis capsular proteins mediate a major part of this inhibition. Two-dimensional polyacrylamide gel electrophoresis analysis of the capsular proteins showed the presence of a wide variety of protein species, including proportionately high levels of the Cpn60.2 (Hsp65, GroEL2) and DnaK (Hsp70) molecular chaperones. Both of these proteins were subsequently detected on the bacterial surface. To determine whether these molecular chaperones play a role in bacterial binding, recombinant Cpn60.2 and DnaK were tested for their ability to inhibit the association of M. tuberculosis bacilli with macrophages. We found that recombinant Cpn60.2 can inhibit approximately 57% of bacterial association with macrophages, while DnaK was not inhibitory at comparable concentrations. Additionally, when polyclonal F(ab')(2) fragments of anti-Cpn60.2 and anti-DnaK were used to mask the surface presentation of these molecular chaperones, a binding reduction of approximately 34% was seen for anti-Cpn60.2 F(ab')(2), while anti-DnaK F(ab')(2) did not significantly reduce bacterial association with macrophages. Thus, our findings suggest that while M. tuberculosis displays both surface-associated Cpn60.2 and DnaK, only Cpn60.2 demonstrates adhesin functionality with regard to macrophage interaction.

CD43 controls the intracellular growth of Mycobacterium tuberculosis through the induction of TNF-alpha-mediated apoptosis.

Establishment of Tuberculosis infection begins with the successful entry and survival of the pathogen within macrophages. We previously showed that macrophage CD43 is required for optimal uptake and growth inhibition of Mycobacterium tuberculosis both in vitro and in vivo. Here, we explore the mechanisms by which CD43 restricts mycobacterial growth in murine macrophages. We found that although M. tuberculosis grows more readily in resting CD43-/- macrophages, priming of cells with IFN-gamma returns the bacterial growth rate to that seen in CD43+/+ cells. To discern the mechanisms by which M. tuberculosis exhibits enhanced growth within resting CD43-/- macrophages, we assessed the induction of inflammatory mediators in response to infection. We found that absence of CD43 resulted in reduced production of TNF-alpha, IL-12 and IL-6 by M. tuberculosis-infected macrophages. We also found that infected resting, but not activated CD43-/- macrophages, showed decreased apoptosis and increased necrosis. Exogenous addition of the pro-inflammatory cytokine TNF-alpha restored control of M. tuberculosis growth and induction of apoptosis to CD43+/+ levels. We propose that CD43 is involved in the inflammatory response to M. tuberculosis and, through the induction of pro-inflammatory mediators, can regulate apoptosis to control intracellular growth of the bacterium.

Bacterial artificial chromosome fingerprint arrays for the differentiation of transcriptomic differences in mycobacteria.

Although microarray technology has become more widespread as a discovery tool for bacterial pathogenesis, it remains a method available only to laboratories with access to expensive equipment and costly analysis software. Mycobacterium tuberculosis, the causative agent for tuberculosis (TB), afflicts one-third of the global population, and kills between 2 and 3 million people per year. While the majority of cases of TB occur in developing areas of the world, facilities in these regions may not be able to support microarray analysis. Additionally, a major limitation of microarrays is that only genes on the array are being assayed. With acquired virulence and drug resistance in microbes, a method less dependent on a predetermined list of gene targets is advantageous. We present a method of expression analysis based on bacterial artificial chromosomes (BACs) that can be applied with standard laboratory equipment and free analysis software. This technique, bacterial artificial chromosome fingerprint arrays (BACFA), was developed and utilised to identify expression differences between intracellular strains of M. tuberculosis, one virulent (H37Rv) and one attenuated (H37Ra). Southern blots of restriction-enzyme digested BAC fragments were sequentially hybridised with strain-specific cDNA probes to generate expression profiles that were used to isolate expression differences in broth grown and intracellular bacteria. Repeat comparisons of intracellular profiles via BACFA identified genomic regions differentially expressed by the two strains. Quantitative real-time PCR was used to assess the genes located in these fragments in order to confirm or deny the differential regulation of genes. In total, we identified six genes that were differentially regulated between strains inside the host cell (pks2, aceE, Rv1571, and frdBCD). We report that BACFA is an effective technique in the expression analysis of bacteria and can be considered complementary to the high-throughput analysis offered by microarrays.

The value of orthodontics: Do parents' willingness to pay values reflect the IOTN?

INTRODUCTION: Given the limited evidence about the benefits of orthodontic treatment, many health care systems have rationed access to orthodontic care with the Index of Orthodontic Treatment Need (IOTN) being one tool used to attempt to allocate resources based on need. However, it is not clear whether patient and public valuations of different levels of need (as described by the IOTN) reflect the resource allocation decisions. The aim of this project was therefore to determine the values parents placed on correction of malocclusions at different IOTN levels using the willingness to pay (WTP) technique. METHOD: 401 parents of children attending hospital-based orthodontic clinics in the North of England were recruited to complete a questionnaire eliciting WTP for the correction of seven malocclusions with different IOTN scores. In addition demographic and orthodontic history characteristics were collected. Results were analysed with appropriate pairwise significance tests and regression. RESULTS: A significant difference in WTP was noted between all the possible pairs of malocclusions with the exception of overjets with moderate versus great need of treatments. At moderate levels, correction of crowding was valued less than overjet but this was reversed at great need levels. Very little of the variance in WTP was explained by the variables collected. When looking at factors affecting percentage difference between values for different pairs of malocclusions, in general, no factors predicted the magnitude of difference. CONCLUSION: Median valuations for correction of malocclusions vary significantly for different levels of need (as judged by IOTN), with increasing levels of need generating higher values. However, there was a limited effect of demographic or orthodontic characteristics on the magnitude of percentage difference in values for correcting malocclusions different levels of need.

An empirical review: Characteristics of plant microsatellite markers that confer higher levels of genetic variation.

During microsatellite marker development, researchers must choose from a pool of possible primer pairs to further test in their species of interest. In many cases, the goal is maximizing detectable levels of genetic variation. To guide researchers and determine which markers are associated with higher levels of genetic variation, we conducted a literature review based on 6782 genomic microsatellite markers published from 1997-2012. We examined relationships between heterozygosity (H e or H o) or allele number (A) with the following marker characteristics: repeat type, motif length, motif region, repeat frequency, and microsatellite size. Variation across taxonomic groups was also analyzed. There were significant differences between imperfect and perfect repeat types in A and H e. Dinucleotide motifs exhibited significantly higher A, H e, and H o than most other motifs. Repeat frequency and motif region were positively correlated with A, H e, and H o, but correlations with microsatellite size were minimal. Higher taxonomic groups were disproportionately represented in the literature and showed little consistency. In conclusion, researchers should carefully consider marker characteristics so they can be tailored to the desired application. If researchers aim to target high genetic variation, dinucleotide motif lengths with large repeat frequencies may be best.

Two-dimensional display and whole genome comparison of bacterial pathogen genomes of high G+C DNA content.

High-resolution comparison of bacterial genomes facilitates the identification of the genetic changes responsible for clinically relevant phenotypes. For this purpose we have established a method for the display and comparison of high G+C bacterial genomes in two dimensions. Here we describe the application of two-dimensional bacterial genomic display to resolve the genomes of Bordetella pertussis, Mycobacterium avium and Mycobacterium tuberculosis, and its utility in strain comparison and detection of insertion and substitution mutations.

Two-dimensional DNA displays for comparisons of bacterial genomes.

We have developed two whole genome-scanning techniques to aid in the discovery of polymorphisms as well as horizontally acquired genes in prokaryotic organisms. First, two-dimensional bacterial genomic display (2DBGD) was developed using restriction enzyme fragmentation to separate genomic DNA based on size, and then employing denaturing gradient gel electrophoresis (DGGE) in the second dimension to exploit differences in sequence composition. This technique was used to generate high-resolution displays that enable the direct comparison of > 800 genomic fragments simultaneously and can be adapted for the high-throughput comparison of bacterial genomes. 2DBGDs are capable of detecting acquired and altered DNA, however, only in very closely related strains. If used to compare more distantly related strains (e.g. different species within a genus) numerous small changes (i.e. small deletions and point mutations) unrelated to the interesting phenotype, would encumber the comparison of 2DBGDs. For this reason a second method, bacterial comparative genomic hybridization (BCGH), was developed to directly compare bacterial genomes to identify gain or loss of genomic DNA. BCGH relies on performing 2DBGD on a pooled sample of genomic DNA from 2 strains to be compared and subsequently hybridizing the resulting 2DBGD blot separately with DNA from each individual strain. Unique spots (hybridization signals) represent foreign DNA. The identification of novel DNA is easily achieved by excising the DNA from a dried gel followed by subsequent cloning and sequencing. 2DBGD and BCGH thus represent novel high resolution genome scanning techniques for directly identifying altered and/or acquired DNA.

Identifying protein kinase inhibitors using an assay based on inhibition of aerial hyphae formation in Streptomyces.

We have identified a strain of Streptomyces in which aerial hyphae formation appears to be especially sensitive to inhibition by protein kinase inhibitors. Using this assay, a number of bacterial cultures have been screened and novel inhibitors of eukaryotic protein kinases have been identified. Since M. tuberculosis possesses multiple eukaryotic-like protein kinase genes, we tested the active kinase inhibitors for the inhibition of mycobacterial growth and obtained several potent compounds. This identifies a new biochemical class of antimycobacterial agents.

Use of dental loupes among dental trainers and trainees in the UK.

INTRODUCTION: Magnification loupes are used in dentistry where increased visual performance is needed. The interest and use of loupes among dental practitioners and students appears to be growing. Despite this little quantitative data regarding the use of loupes in clinical practice and factors governing their purchase among dental trainers and trainees is available. OBJECTIVE: To determine the extent of use of loupes among dental trainers and trainees in the UK and identify what factors influence their choice of selection. METHOD: A proforma questionnaire was designed to collect the information required and was handed out dental trainers and trainees. RESULTS: Use of loupes is more common among the dental trainers (44%) than trainees (28%). Of the nonusers, increased interest among the trainees (83.6%) and trainers (50%) in purchasing loupes was noted. Price was found to be the most important factor during purchase. The majority of users opted for 2.5×; and a light source was a popular choice. Both users and nonusers in the study group had an affinity towards "through-the-lens" type. No significant variation in back/neck discomfort between the users and nonusers was found. CONCLUSION: Use of loupes among dental trainers and trainees remains low. Reduction in price and provision of information could increase the number of loupe users.

An efficient technique for primer development and application that integrates fluorescent labeling and multiplex PCR.

PREMISE OF THE STUDY: Development of genetic markers can be costly and time-consuming, especially when multiple primer pairs are fluorescently labeled. This step was streamlined by combining two techniques in the same PCR reaction: (1) custom-labeling of primers by the investigator and (2) multiplexing multiple primers together in the same reaction. • METHODS AND RESULTS: This technique was successfully used to develop microsatellite markers in several plant species. Microsatellites amplified with this multiplexing process were identical to those generated from PCR using individual primer pairs and with traditional methods using a priori labeled fluorescent primers. Tests of PCR cycling programs revealed that conditions recommended for the commercial kit generated stronger fragment peaks than the previously recommended cycling protocol. • CONCLUSIONS: This technique is an efficient and economical way to fluorescently label multiple microsatellite primers in the same reaction. It is also applicable to other markers used in PCR amplification of genetic material.

Contrasting transcriptional responses of a virulent and an attenuated strain of Mycobacterium tuberculosis infecting macrophages.

BACKGROUND: H37Rv and H37Ra are well-described laboratory strains of Mycobacterium tuberculosis derived from the same parental strain, H37, that show dramatically different pathogenic phenotypes. METHODOLOGY/PRINCIPAL FINDINGS: In this study, the transcriptomes of the two strains during axenic growth in broth and during intracellular growth within murine bone-marrow macrophages were compared by whole genome expression profiling. We identified and compared adaptations of either strain upon encountering an intracellular environment, and also contrasted the transcriptomes of the two strains while inside macrophages. In the former comparison, both strains induced genes that would facilitate intracellular survival including those involved in mycobactin synthesis and fatty acid metabolism. However, this response was stronger and more extensive for H37Rv than for H37Ra. This was manifested as the differential expression of a greater number of genes and an increased magnitude of expression for these genes in H37Rv. In comparing intracellular transcriptional signatures, fifty genes were found to be differentially expressed between the strains. Of these fifty, twelve were under control of the PhoPR regulon. Further differences between strains included genes whose products were members of the ESAT-6 family of proteins, or were associated with their secretion. CONCLUSIONS/SIGNIFICANCE: Along with the recent identification of single nucleotide polymorphisms in H37Ra when compared to H37Rv, our demonstration of differential expression of PhoP-regulated and ESX-1 region-related genes during macrophage infection further highlights the significance of these genes in the attenuation of H37Ra.

Adjusting to a new home: Mycobacterium tuberculosis gene expression in response to an intracellular lifestyle.

Mycobacterium tuberculosis remains the most significant single species of bacteria causing disease in mankind. The ability of M. tuberculosis to survive and replicate within host macrophages is a pivotal step in its pathogenesis. Understanding the microenvironments that M. tuberculosis encounters within the macrophage and the adaptations that the bacterium undergoes to facilitate its survival will lead to insights into possible therapeutic targets for improved treatment of tuberculosis. This is urgently needed with the emergence of multi- and extensively drug resistant strains of M. tuberculosis. Significant advances have been made in understanding the macrophage response on encountering M. tuberculosis. Complementary information is also accumulating regarding the counter responses of M. tuberculosis during the various stages of its interactions with the host. As such, a picture is emerging delineating the gene expression of intracellular M. tuberculosis at different stages of the interaction with macrophages.

Characterization of genes differentially expressed within macrophages by virulent and attenuated Mycobacterium tuberculosis identifies candidate genes involved in intracellular growth.

To identify genes involved in the intracellular survival of Mycobacterium tuberculosis we compared the transcriptomes of virulent (H37Rv) and attenuated (H37Ra) strains during their interaction with murine bone-marrow-derived macrophages. Expression profiling was accomplished via the bacterial artificial chromosome fingerprint array (BACFA) technique. Genes identified with BACFA, and confirmed via qPCR to be upregulated in the attenuated H37Ra at 168 h post-infection, were frdB, frdC and frdD. Genes upregulated in the virulent H37Rv were pks2, aceE and Rv1571. Further qPCR analysis of these genes at 4 and 96 h post-infection revealed that the frd operon (encoding the fumarate reductase enzyme complex) is expressed at higher levels in the virulent H37Rv at earlier time points while the expression of aceE and pks2 is higher in the virulent strain throughout the course of infection. Assessment of frd transcripts in oxygen-limited cultures of M. tuberculosis H37Ra and H37Rv showed that the attenuated strain displayed a lag in frdA and frdB expression at the onset of microaerophilic culture, when compared to microaerophilic cultures of H37Rv and aerated cultures of H37Ra. Lastly, treatment of intracellular bacteria with a putative inhibitor of fumarate reductase resulted in a significant reduction of bacterial growth.

Selection of transposon mutants of Mycobacterium tuberculosis with increased macrophage infectivity identifies fadD23 to be involved in sulfolipid production and association with macrophages.

Alterations to the composition or architecture of the mycobacterial cell envelope can affect the macrophage infectivity of the bacillus. To further characterize the mycobacterial gene products that modulate the interaction with host cells, we employed transposon mutagenesis and screened for mutants that demonstrated an enhanced binding affinity toward macrophages. After successive rounds of mutant selection and enrichment, a total of five mutants were isolated that harboured gene disruptions within loci involved in lipid synthetic pathways as well as genes coding for putative hypothetical proteins. One mutant in particular, with a disruption in the Rv3826 gene (fadD23), was repeatedly isolated during library screening. Analysis of the cell envelope constituents of the Tn : : fadD23 strain revealed a lack of sulfolipid production which was restored following complementation with the wild-type gene.

Absence of complement receptor 3 results in reduced binding and ingestion of Mycobacterium tuberculosis but has no significant effect on the induction of reactive oxygen and nitrogen intermediates or on the survival of the bacteria in resident and interferon-gamma activated macrophages.

The interaction of host macrophage (Mphi) and Mycobacterium tuberculosis (Mtb) is mediated by cell surface receptors and is important in establishing intracellular infection. Mphis can kill invading organisms via reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI). Using a Complement Receptor 3 (CR3) knockout mouse model we have examined whether the presence of CR3 affects the binding and uptake of viable Mtb by Mphis, the survival of the ingested bacteria and the induction of ROI and RNI during this interaction. We show that, although CR3 plays a role in the uptake of viable Mtb, the receptor plays no role in the subsequent survival of the bacteria. The finding holds true for resident Mphis and for interferon-gamma (IFN-gamma) activated Mphis, both in the absence and presence of serum opsonins. Activation of Mphi populations with IFN-gamma significantly inhibits the growth of Mtb in host Mphis and enhances the production of ROI and RNI. However, the presence of CR3 was not critical in any of these mechanisms. Furthermore, we demonstrate that the control of intracellular growth of Mtb in IFN-gamma activated Mphis is not mediated by a direct effect of RNI.

CD43 is required for optimal growth inhibition of Mycobacterium tuberculosis in macrophages and in mice.

We explored the role of macrophage (Mphi) CD43, a transmembrane glycoprotein, in the pathogenesis of Mycobacterium tuberculosis. Using gene-deleted mice (CD43-/-), we assessed the association of the bacterium with distinct populations of Mphi and found that CD43-/- Mphi bound less M. tuberculosis than CD43+/+ Mphi. Increased infective doses did not abrogate this difference. However, reduced association due to the absence of CD43 could be overcome by serum components. Mphi from heterozygote mice, which express 50% of wild-type CD43, bound more bacteria than CD43-/- but less than CD43+/+, proving that the gene dose of CD43 correlates with binding of M. tuberculosis. Furthermore, the reduced ability of CD43-/- Mphi to bind bacteria was restricted to mycobacterial species. We also found that the survival and replication of M. tuberculosis within Mphi was enhanced significantly in the absence of CD43, making this the first demonstration that the mechanism of mycobacterial entry influences its subsequent growth. Most importantly, we show here that the absence of CD43 in mice aerogenically infected with M. tuberculosis results in an increased bacterial load during both the acute and chronic stages of infection and more rapid development of granulomas, with greater lung involvement and distinctive cellularity.

Single mucosal, but not parenteral, immunization with recombinant adenoviral-based vaccine provides potent protection from pulmonary tuberculosis.

Bacillus Calmette-Guerin (BCG) vaccine has failed to control the global tuberculosis (TB) epidemic, and there is a lack of safe and effective mucosal vaccines capable of potent protection against pulmonary TB. A recombinant replication-deficient adenoviral-based vaccine expressing an immunogenic Mycobacterium tuberculosis Ag Ag85A (AdAg85A) was engineered and evaluated for its potential to be used as a respiratory mucosal TB vaccine in a murine model of pulmonary TB. A single intranasal, but not i.m., immunization with AdAg85A provided potent protection against airway Mycobacterium tuberculosis challenge at an improved level over that by cutaneous BCG vaccination. Systemic priming with an Ag85A DNA vaccine and mucosal boosting with AdAg85A conferred a further enhanced immune protection which was remarkably better than BCG vaccination. Such superior protection triggered by AdAg85 mucosal immunization was correlated with much greater retention of Ag-specific T cells, particularly CD4 T cells, in the lung and was shown to be mediated by both CD4 and CD8 T cells. Thus, adenoviral TB vaccine represents a promising novel vaccine platform capable of potent mucosal immune protection against TB. Our study also lends strong evidence that respiratory mucosal vaccination is critically advantageous over systemic routes of vaccination against TB.