Samantha L. Sampson

Evolution and expansion of the Mycobacterium tuberculosis PE and PPE multigene families and their association with the duplication of the ESAT-6 (esx) gene cluster regions.

BackgroundThe PE and PPE multigene families of Mycobacterium tuberculosis comprise about 10% of the coding potential of the genome. The function of the proteins encoded by these large gene families remains unknown, although they have been proposed to be involved in antigenic variation and disease pathogenesis. Interestingly, some members of the PE and PPE families are associated with the ESAT-6 (esx) gene cluster regions, which are regions of immunopathogenic importance, and encode a system dedicated to the secretion of members of the potent T-cell antigen ESAT-6 family. This study investigates the duplication characteristics of the PE and PPE gene families and their association with the ESAT-6 gene clusters, using a combination of phylogenetic analyses, DNA hybridization, and comparative genomics, in order to gain insight into their evolutionary history and distribution in the genus Mycobacterium.ResultsThe results showed that the expansion of the PE and PPE gene families is linked to the duplications of the ESAT-6 gene clusters, and that members situated in and associated with the clusters represent the most ancestral copies of the two gene families. Furthermore, the emergence of the repeat protein PGRS and MPTR subfamilies is a recent evolutionary event, occurring at defined branching points in the evolution of the genus Mycobacterium. These gene subfamilies are thus present in multiple copies only in the members of the M. tuberculosis complex and close relatives. The study provides a complete analysis of all the PE and PPE genes found in the sequenced genomes of members of the genus Mycobacterium such as M. smegmatis, M. avium paratuberculosis, M. leprae, M. ulcerans, and M. tuberculosis.ConclusionThis work provides insight into the evolutionary history for the PE and PPE gene families of the mycobacteria, linking the expansion of these families to the duplications of the ESAT-6 (esx) gene cluster regions, and showing that they are composed of subgroups with distinct evolutionary (and possibly functional) differences.

Mycobacterial PE/PPE Proteins at the Host-Pathogen Interface

The mycobacterial PE/PPE proteins have attracted much interest since their formal identification just over a decade ago. It has been widely speculated that these proteins may play a role in evasion of host immune responses, possibly via antigenic variation. Although a cohesive understanding of their function(s) has yet to be established, emerging data increasingly supports a role for the PE/PPE proteins at multiple levels of the infectious process. This paper will delineate salient features of the families revealed by comparative genomics, bioinformatic analyses and genome-wide screening approaches and will summarise existing knowledge of subcellular localization, secretion pathways, and protein structure. These characteristics will be considered in light of findings on innate and adaptive host responses to PE/PPE proteins, and we will review the increasing body of data on B and T cell recognition of these proteins. Finally, we will consider how current knowledge and future explorations may contribute to a more comprehensive understanding of these intriguing proteins and their involvement in host pathogen interactions. Ultimately this information could underpin future intervention strategies, for example, in the area of new and improved diagnostic tools and vaccine candidates.

Protection Elicited by a Double Leucine and Pantothenate Auxotroph of Mycobacterium tuberculosis in Guinea Pigs

ABSTRACT We developed a live, fully attenuated Mycobacterium tuberculosis vaccine candidate strain with two independent attenuating auxotrophic mutations in leucine and pantothenate biosynthesis. The ΔleuD ΔpanCD double auxotroph is fully attenuated in the SCID mouse model and highly immunogenic and protective in the extremely sensitive guinea pig tuberculosis model, reducing both bacterial burden and disease pathology.

Transmission of a Multidrug-Resistant Mycobacterium tuberculosis Strain Resembling “Strain W” among Noninstitutionalized, Human Immunodeficiency Virus—Seronegative Patients

Since 1990, several outbreaks of multidrug-resistant tuberculosis (MDR-TB) have been described among institutionalized patients infected with human immunodeficiency virus (HIV). We describe a community MDR-TB outbreak among HIV-seronegative patients in Cape Town, South Africa. Isolates were characterized by restriction fragment length polymorphism (RFLP) analysis and dot-blot hybridization analysis of mutations conferring resistance for isoniazid, rifampin, streptomycin, and ethambutol. All isolates were identical on RFLP analysis. In 2 patients, RFLP analysis showed exogenous reinfection during or after treatment for drug-susceptible TB. Mutation analysis confirmed the genotypic identity of the isolates. The infecting strain was genotypically related to strain W, which is responsible for the majority of MDR-TB outbreaks in New York City. Transmission of MDR-TB is thus not limited to HIV-seropositive patients in an institutional setting but occurs within a community.

Drying a tuberculosis vaccine without freezing

With the increasing incidence of tuberculosis and drug resistant disease in developing countries due to HIV/AIDS, there is a need for vaccines that are more effective than the present bacillus Calmette–Guérin (BCG) vaccine. We demonstrate that BCG vaccine can be dried without traditional freezing and maintained with remarkable refrigerated and room-temperature stability for months through spray drying. Studies with a model Mycobacterium (Mycobacterium smegmatis) revealed that by removing salts and cryoprotectant (e.g., glycerol) from bacterial suspensions, the significant osmotic pressures that are normally produced on bacterial membranes through droplet drying can be reduced sufficiently to minimize loss of viability on drying by up to 2 orders of magnitude. By placing the bacteria in a matrix of leucine, high-yield, free-flowing, “vial-fillable” powders of bacteria (including M. smegmatis and M. bovis BCG) can be produced. These powders show relatively minor losses of activity after maintenance at 4°C and 25°C up to and beyond 4 months. Comparisons with lyophilized material prepared both with the same formulation and with a commercial formulation reveal that the spray-dried BCG has better overall viability on drying.

DNA fingerprinting and molecular epidemiology of tuberculosis: use and interpretation in an epidemic setting.

Tuberculosis (TB) is still a major cause of morbidity and mortality. It is clear that control requires more than simple availability of antibiotics. In order to gain insight into the disease, DNA fingerprinting has been applied to the study of bacterial population structure. This technology has been used to quantitate various components of the disease in a high‐incidence community, viz. recent transmission (RT) and reactivation (RA) and to monitor these over time as a tool to quantitate changes in the epidemic. In our high‐incidence community, we find unexpectedly high strain diversity, lower than predicted RT, and that reactivation disease dominates. This technology can be used to examine and challenge traditional dogmas. Quantitative measure of RT varies over time, using a two‐year sliding window for estimation as a useful period. The results show that the “epidemic” consists of subepidemics characterized by strain families that wax and wane in the community of TB patients. The technology is shown to be a useful and quantitative tool to assess disease status and can therefore be used to monitor intervention strategies and refine and monitor results of new control measures.

Cell Electrospinning: An In Vitro and In Vivo Study

Cell electrospinning and aerodynamically assisted bio-threading are novel bioplatforms for directly forming large quantities of cell-laden scaffolds for creating living sheets and vessels in three-dimensions. The functional biological architectures generated will be useful in both the laboratory and the clinic.

Phosphoproteomics analysis of a clinical Mycobacterium tuberculosis Beijing isolate: expanding the mycobacterial phosphoproteome catalog

Reversible protein phosphorylation, regulated by protein kinases and phosphatases, mediates a switch between protein activity and cellular pathways that contribute to a large number of cellular processes. The Mycobacterium tuberculosis genome encodes 11 Serine/Threonine kinases (STPKs) which show close homology to eukaryotic kinases. This study aimed to elucidate the phosphoproteomic landscape of a clinical isolate of M. tuberculosis. We performed a high throughput mass spectrometric analysis of proteins extracted from an early-logarithmic phase culture. Whole cell lysate proteins were processed using the filter-aided sample preparation method, followed by phosphopeptide enrichment of tryptic peptides by strong cation exchange (SCX) and Titanium dioxide (TiO2) chromatography. The MaxQuant quantitative proteomics software package was used for protein identification. Our analysis identified 414 serine/threonine/tyrosine phosphorylated sites, with a distribution of S/T/Y sites; 38% on serine, 59% on threonine and 3% on tyrosine; present on 303 unique peptides mapping to 214 M. tuberculosis proteins. Only 45 of the S/T/Y phosphorylated proteins identified in our study had been previously described in the laboratory strain H37Rv, confirming previous reports. The remaining 169 phosphorylated proteins were newly identified in this clinical M. tuberculosis Beijing strain. We identified 5 novel tyrosine phosphorylated proteins. These findings not only expand upon our current understanding of the protein phosphorylation network in clinical M. tuberculosis but the data set also further extends and complements previous knowledge regarding phosphorylated peptides and phosphorylation sites in M. tuberculosis.

Rapid in vivo assessment of drug efficacy against Mycobacterium tuberculosis using an improved firefly luciferase

Objectives In vivo experimentation is costly and time-consuming, and presents a major bottleneck in anti-tuberculosis drug development. Conventional methods rely on the enumeration of bacterial colonies, and it can take up to 4 weeks for Mycobacterium tuberculosis to grow on agar plates. Light produced by recombinant bacteria expressing luciferase enzymes can be used as a marker of bacterial load, and disease progression can be easily followed non-invasively in live animals by using the appropriate imaging equipment. The objective of this work was to develop a bioluminescence-based mouse model of tuberculosis to assess antibiotic efficacy against M. tuberculosis in vivo. Methods We used an M. tuberculosis strain carrying a red-shifted derivative of the firefly luciferase gene (FFlucRT) to infect mice, and monitored disease progression in living animals by bioluminescence imaging before and after treatment with the frontline anti-tuberculosis drug isoniazid. The resulting images were analysed and the bioluminescence was correlated with bacterial counts. Results Using bioluminescence imaging we detected as few as 1.7 × 103 and 7.5 × 104 reporter bacteria ex vivo and in vivo, respectively, in the lungs of mice. A good correlation was found between bioluminescence and bacterial load in both cases. Furthermore, a marked reduction in luminescence was observed in living mice given isoniazid treatment. Conclusions We have shown that an improved bioluminescent strain of M. tuberculosis can be visualized by non-invasive imaging in live mice during an acute, progressive infection and that this technique can be used to rapidly visualize and quantify the effect of antibiotic treatment. We believe that the model presented here will be of great benefit in early drug discovery as an easy and rapid way to identify active compounds in vivo.

A global perspective on pyrazinamide resistance: systematic review and meta-analysis

Background Pyrazinamide (PZA) is crucial for tuberculosis (TB) treatment, given its unique ability to eradicate persister bacilli. The worldwide burden of PZA resistance remains poorly described. Methods Systematic PubMed, Science Direct and Scopus searches for articles reporting phenotypic (liquid culture drug susceptibility testing or pyrazinamidase activity assays) and/or genotypic (polymerase chain reaction or DNA sequencing) PZA resistance. Global and regional summary estimates were obtained from random-effects meta-analysis, stratified by presence or risk of multidrug resistant TB (MDR-TB). Regional summary estimates were combined with regional WHO TB incidence estimates to determine the annual burden of PZA resistance. Information on single nucleotide polymorphisms (SNPs) in the pncA gene was aggregated to obtain a global summary. Results Pooled PZA resistance prevalence estimate was 16.2% (95% CI 11.2-21.2) among all TB cases, 41.3% (29.0-53.7) among patients at high MDR-TB risk, and 60.5% (52.3-68.6) among MDR-TB cases. The estimated global burden is 1.4 million new PZA resistant TB cases annually, about 270,000 in MDR-TB patients. Among 1,815 phenotypically resistant isolates, 608 unique SNPs occurred at 397 distinct positions throughout the pncA gene. Interpretation PZA resistance is ubiquitous, with an estimated one in six incident TB cases and more than half of all MDR-TB cases resistant to PZA globally. The diversity of SNPs across the pncA gene complicates the development of rapid molecular diagnostics. These findings caution against relying on PZA in current and future TB drug regimens, especially in MDR-TB patients.