Tuberculosis - Articles in Press

CXCL12 as a biological marker for the diagnosis of tuberculous pleurisy - Corrected Proof

2012-02-01

Summary: Although a chemokine CXCL12 is implicated in some infectious diseases, especially those in which T cell-mediated immunity plays critical roles, the relevance of CXCL12 to tuberculosis has never been elucidated. To determine the clinical efficacy of CXCL12 as a diagnostic marker for tuberculous (TB) pleurisy, we measured CXCL12 concentration in pleural fluid and serum from patients with various etiologies.Of 60 patients with pleural fluid, the median age of TB patients was 52 which was significantly lower than 71 of non-TB patients (P < 0.01). CXCL12 level in TB effusion (4456 ± 1013 pg/mL, n = 15) was significantly higher than non-TB effusion (2851 ± 1229 pg/mL, n = 45) (P < 0.01). On the other hand, serum CXCL12 level showed no significant differences among TB pleurisy, non-TB pleurisy, and normal healthy subjects. The sensitivity and specificity of CXCL12 in pleural fluid for the diagnosis of TB pleurisy was 60.0% and 93.2% (cut-off value = 4600 pg/mL), respectively. Area under the receiver operating characteristic (ROC) curve (AUC) for CXCL12 was 0.84. As the source of CXCL12, pleural mesothelium, endothelium of pulmonary vessels, bronchial epithelium, multinucleated giant epithelioid cells, and macrophages were positive for CXCL12 staining.Increased CXCL12 level in pleural fluid could be an informative diagnostic marker for differentiating TB pleurisy from other etiologies.

T cell responses to DosR and Rpf proteins in actively and latently infected individuals from Colombia - Corrected Proof

2012-01-09

Summary: Mycobacterium tuberculosis DosR regulon-encoded proteins elicit strong immune T-cell responses in individuals with latent tuberculosis (LTBI). Also, resuscitation (Rpf) proteins can induce such responses. However, variations in the immunogenicity of the DosR and Rpf proteins have been observed in European and African populations, and no data are published from other geographic areas. In Colombian LTBI and patients with recently diagnosed PTB, we therefore studied the immune response to DosR, Rpf, stress, and nominal antigens from Mtb, in 7-day stimulated cultures. Three DosR (Rv1737c, Rv2029c, Rv2628c) and 2 Rpf (Rv0867 and Rv2389c) antigens were recognized most prominently on the basis of the net IFNγ production (DosR) or the percentage of responding individuals (Rpf). Results show that the selected DosR antigens induced a higher proportion of CD4-T cells producing IFNγ from LTBI, compared to pulmonary TB patients (PTB), while there were no differences in the proportion of CD8-T cells. An increased frequency of CD4, but not CD8 T-cells with a CD45RO+CD27+ phenotype was observed in LTBI in response to Rv2029c, Rv0867c, and Rv2389c, compared to PTB. The levels of cytokines and chemokines in the supernatants of stimulated cells, showed that the DosR and Rpf antigens induced higher levels of IFNγ in cultures from LTBI compared to PTB, although the induced pattern of cytokines and chemokines was also antigen dependent. In summary, our results are consistent with the significant immunogenicity of Mtb DosR and Rpf antigens in LTBI individuals, and confirm and extend previously reported data from other TB affected human populations.

Identification of Rv0535 as methylthioadenosine phosphorylase from Mycobacterium tuberculosis - Corrected Proof

2012-01-06

Summary: 5′-methylthioadenosine (MTA) is a natural purine that is metabolized by methylthioadenosine phosphorylase (MTAP, E.C 2.4.2.28) in Eukarya and Archaea but generally not in bacteria. In this work, Rv0535, which has been annotated as a probable MTAP in Mycobacterium tuberculosis, was expressed in and purified from Escherichia coli BL21 (DE3). The purified protein displayed properties of a phosphorylase and MTA was the preferred substrate. Adenosine and S-adenosyl-l-homocysteine were poor substrates and no activity was detected with 5′-methylthioinosine, the other natural purines, or the natural pyrimidines. Kinetic analysis of M. tuberculosis MTAP showed that the Km value for MTA was 9 μM. Rv0535 was estimated as a 30 kDa protein on a denaturing SDS-PAGE gel, which agreed with the molecular mass predicted by its gene sequence. Using gel filtration chromatography, the native molecular mass of the enzyme was determined to be 60 ± 4 kDa, and thus indicated that M. tuberculosis MTAP is a dimer. Differences in active site between mycobacterial and human MTAPs were identified by homology modeling based on the crystal of the human enzyme. A complete structure–activity relationship analysis could identify differences in substrate specificity between the two enzymes to aid in the development of purine-based, anti-tuberculosis drugs.

Mycobacterium tuberculosis – Heterogeneity revealed through whole genome sequencing - Corrected Proof

2012-01-05

Summary: The emergence of whole genome sequencing (WGS) technologies as primary research tools has allowed for the detection of genetic diversity in Mycobacterium tuberculosis (Mtb) with unprecedented resolution. WGS has been used to address a broad range of topics, including the dynamics of evolution, transmission and treatment. Here, we have analyzed 55 publically available genomes to reconstruct the phylogeny of Mtb, and we have addressed complications that arise during the analysis of publically available WGS data. Additionally, we have reviewed the application of WGS to the study of Mtb and discuss those areas still to be addressed, moving from global (phylogeography), to local (transmission chains and circulating strain diversity), to the single patient (clonal heterogeneity) and to the bacterium itself (evolutionary studies). Finally, we discuss the current WGS approaches, their strengths and limitations.

Antibacterial activities of dendritic amphiphiles against nontuberculous mycobacteria - Corrected Proof

2012-01-03

Summary: The anti-mycobacterial activities of nine series of dicarboxyl and tricarboxyl dendritic amphiphiles with one alkyl, two alkyl, and cholestanyl tails against Mycobacterium abscessus, Mycobacterium avium, Mycobacterium chelonae, Mycobacterium marinum and Mycobacterium smegmatis have been measured. The dendritic amphiphiles overcame the limited aqueous solubility of natural long-chain fatty acids, alcohols, and amines to enable profiling the susceptibilities of the different mycobacterial species to the physicochemical properties of these amphiphiles. Several dendritic amphiphiles showed strong anti-mycobacterial activity with high critical micelle concentrations and low hemolytic activities thereby offering platforms for the development of antibiotics of higher activity against nontuberculous mycobacteria.

MTCID: A database of genetic polymorphisms in clinical isolates of Mycobacterium tuberculosis - Corrected Proof

Richa Bharti, Ram Das, Pragya Sharma, Kiran Katoch, Alok Bhattacharya — 2011-12-30

Summary: Tuberculosis (TB) is a major cause of morbidity and mortality throughout the world, particularly in developing countries. The response of the patients and treatment outcome depends, in addition to diagnosis, appropriate and timely treatment and host factors, on the virulence of Mycobacterium tuberculosis and genetic polymorphism prevalent in clinical isolates of the bacterium. A number of studies have been carried out to characterize clinical isolates of M. tuberculosis obtained from TB patients. However, the data is scattered in a large number of publications. Though attempts have been made to catalog the observed variations, there is no database that has been developed for cataloging, storing and dissemination of genetic polymorphism information. MTCID (M. tuberculosis clinical isolate genetic polymorphism database) is an attempt to provide a comprehensive repository to store, access and disseminate single nucleotide polymorphism (SNPs) and spoligotyping profiles of M. tuberculosis. It can be used to automatically upload the information available with a user that adds to the existing database at the backend. Besides it may also aid in maintaining clinical profiles of TB and treatment of patients. The database has ‘search’ features and is available at http://ccbb.jnu.ac.in/Tb.

Ultra-low dose of Mycobacterium tuberculosis aerosol creates partial infection in mice - Corrected Proof

2011-12-26

Summary: A murine low dose (LD) aerosol model is commonly used to test tuberculosis vaccines. Doses of 50–400 CFU (24h lung CFU) infect 100% of exposed mice. The LD model measures progression from infection to disease based on organ CFU at defined time points. To mimic natural exposure, we exposed mice to an ultra-low dose (ULD) aerosol. We estimated the presented dose by sampling the aerosol. Female C57BL/6 mice were exposed to Mycobacterium tuberculosis H37Rv aerosol at 1.0, 1.1, 1.6, 5.4, and 11 CFU presented dose, infecting 27%, 36%, 36%, 100%, and 95% of mice, respectively. These data are compatible with a stochastic infection event (Poisson distribution, weighted R2=0.97) or with a dose–response relationship (sigmoid distribution, weighted R2=0.97). Based on the later assumption, the ID50 was 1.6CFU presented dose (95% confidence interval, 1.2–2.1). We compared organ CFU after ULD and LD aerosols (5.4 vs. 395CFU presented dose). Lung burden was 30-fold lower in the ULD model at 4 weeks (3.4 vs. 4.8 logs, p<0.001) and 18 weeks (≤3.6 vs. 5.0 logs, p=0.01). Mice exposed to ULD aerosols as compared to LD aerosols had greater within-group CFU variability. Exposure to ULD aerosols leads to infection in a subset of mice, and to persistently low organ CFU. The ULD aerosol model may resemble human pulmonary tuberculosis more closely than the standard LD model, and may be used to identify host or bacterial factors that modulate the initial infection event.

Ability of Cricetomys rats to detect Mycobacterium tuberculosis and discriminate it from other microorganisms - Corrected Proof

2011-12-26

Summary: Trained African giant pouched rats (Cricetomys gambianus) have potential for diagnosis of tuberculosis (TB). These rats target volatile compounds of Mycobacterium tuberculosis (Mtb) that cause TB. Mtb and nontuberculous mycobacteria (NTM) species are related to Nocardia and Rhodococcus spp., which are also acid-fast bacilli and can be misdiagnosed as Mtb in smear microscopy. Diagnostic performance of C. gambianus on in vitro-cultured mycobacterial and related pulmonary microbes is unknown. This study reports on the response of TB detection rats to cultures of reference Mtb, clinical Mtb, NTM, Nocardia; Rhodococcus; Streptomyces; Bacillus; and yeasts. Trained rats significantly discriminated Mtb from other microbes (p < 0.008, Fisher’s exact test). Detection of Mtb cultures was age-related, with exponential and early stationary phase detected more frequently than early log phase and late stationary phase (p < 0.001, Fisher’s test) (sensitivity = 83.33%, specificity = 94.4%, accuracy = 94%). The detection of naturally TB-infected sputum exceeded that of negative sputum mixed with Mtb, indicating that C. gambianus are conditioned to detect odours of TB-positive sputum than spiked sputum. Although further studies on volatiles from detectable growth phases of Mtb are vital for identification of Mtb-specific volatiles detected by rats, our study underline the potential of C. gambianus for TB diagnosis.

The ins and outs of Mycobacterium tuberculosis protein export - Corrected Proof

Lauren S. Ligon, Jennifer D. Hayden, Miriam Braunstein — 2011-12-23

Summary: Mycobacterium tuberculosis is an important pathogen that infects approximately one-third of the world’s population and kills almost two million people annually. An important aspect of M. tuberculosis physiology and pathogenesis is its ability to export proteins into and across the thick mycobacterial cell envelope, where they are ideally positioned to interact with the host. In addition to the specific proteins that are exported by M. tuberculosis, the systems through which these proteins are exported represent potential targets for future drug development. M. tuberculosis possesses two well-known and conserved export systems: the housekeeping Sec pathway and the Tat pathway. In addition, M. tuberculosis possesses specialized export systems including the accessory SecA2 pathway and five ESX pathways. Here we review the current understanding of each of these export systems, with a focus on M. tuberculosis, and discuss the contribution of each system to disease and physiology.

Stimulation of the Mycobacterium tuberculosis transcription elongation by MtbMfd - Corrected Proof

Swayam Prabha, Arnab China, Desirazu N. Rao, Valakunja Nagaraja — 2011-11-30

Summary: The movement of RNA Polymerase (RNAP) along the template during transcription elongation is assisted by a diverse set of proteins with distinct roles. Transcription coupled DNA repair is carried out by Mfd, which dissociates blocked transcription elongation complexes at non-pairing lesions and mediates the repair via recruitment of DNA repair proteins. Mycobacterium tuberculosis Mfd (MtbMfd) influenced the stalled and backtracked RNAP by more than one way. It displaced stalled RNAP blocked by NTP starvation on T7A1 promoter based template in ATP or dATP-dependent manner. However, it did not affect initiating RNAP but released transcript after elongation complex disruption. An ATPase mutant of the protein (MfdD778A) failed to displace RNAP and release transcript whereas a C-terminal deletion mutant (MfdΔC) showed comparable activity. MtbMfd could displace stalled RNAPs from both mycobacterial and Escherichia coli elongation complexes. Arrested or backtracked elongation complexes were restored to the forward position by the activity of the factor in presence of NTPs resulting in productive elongation. MtbMfd also increased the rate of transcription on various GC rich templates possibly by its anti-backtracking activity. Our results indicate that the MtbMfd assists RNAP to dissociate and release the transcript or resume transcription under favorable conditions.

Characteristics of Mycobacterium smegmatis J15cs strain lipids - Corrected Proof

2011-11-07

Summary: Mycobacterium smegmatis is a rapidly growing, non-pathogenic mycobacterium, and M. smegmatis strain mc2155 in particular has been used as a tool for molecular analysis of mycobacteria because of its high rate of transformation. We examined another strain, M. smegmatis J15cs, which has the advantage of surviving for six days in murine macrophages. The J15cs strain produces a rough dry colony, and we hypothesized that the long survival of the J15cs strain was correlated with its cell wall components. Therefore, the lipid compositions of these two strains were compared. The subclasses and carbon species of the mycolic acids were very similar, and the major glycolipids and phospholipids were expressed in both strains. However, apolar glycopeptidolipids were deleted only in the J15cs strain. The presence of apolar glycopeptidolipids gives the cell wall a different structure. Moreover, the apolar glycopeptidolipids were recognized by macrophages via toll-like receptor 2, but not 4. We concluded that the absence of apolar glycopeptidolipids is a definitive feature of the J15cs strain, and affects its morphology and survival in host cells.

Inside or outside the phagosome? The controversy of the intracellular localization of Mycobacterium tuberculosis - Corrected Proof

Amanda Welin, Maria Lerm — 2011-10-28

Summary: The localization of Mycobacterium tuberculosis (Mtb) inside the macrophage has been a matter of debate in recent years. Upon inhalation, the bacterium is taken up into macrophage phagosomes, which are manipulated by the bacterium. Subsequent translocation of the bacilli into the cytosol has been observed by several groups, while others fail to observe this phenomenon. Here, we review the available literature in favour of and against this idea, and scrutinize the existing data on how human macrophages control Mtb infection, relating this to the robustness of the host cell. We conclude that both phagosomal maturation inhibition and escape from the phagosome are part of the greater infection strategy of Mtb. The balance between the host cell and the infecting bacterium is an important factor in determining the outcome of infection as well as whether phagosomal escape occurs and can be captured.

Informatics resources for tuberculosis – Towards drug discovery - Corrected Proof

2011-09-23

Summary: Integration of biological data on gene sequence, genome annotation, gene expression, metabolic pathways, protein structure, drug target prioritization and selection, has resulted in several online bioinformatics databases and tools for Mycobacterium tuberculosis. Alongside there has been a growth in the list of cheminformatics databases for small molecules and tools to facilitate drug discovery. In spite of these efforts there is a noticeable lag in the drug discovery process which is an urgent need in the case of emerging and re-emerging infectious diseases. For example, more than 25 online databases are available freely for tuberculosis and yet these resources have not been exploited optimally. Informatics-centered drug discovery based on the integration and analysis of both bioinformatics and cheminformatics data could fill in the gap and help to accelerate the process of drug discovery. This article aims to review the current standing of developments in tuberculosis-bioinformatics and highlight areas where integration of existing resources could lead to acceleration of drug discovery against tuberculosis. Such an approach could be adapted for other diseases as well.

Mouse and guinea pig models for testing new tuberculosis vaccines - Corrected Proof

Ian M. Orme — 2005-01-17

Summary: In this brief review I will discuss some of the more interesting [and in some cases, confusing] aspects that have arisen from the current NIH-funded TB vaccine screening program at Colorado State University, how they affect our understanding of the vaccination process, and how this may influence the rational vaccine design in the near future.