Vasan K. Sambandamurthy

Specialized transduction: an efficient method for generating marked and unmarked targeted gene disruptions in Mycobacterium tuberculosis, M. bovis BCG and M. smegmatis

The authors have developed a simple and highly efficient system for generating allelic exchanges in both fast- and slow-growing mycobacteria. In this procedure a gene of interest, disrupted by a selectable marker, is cloned into a conditionally replicating (temperature-sensitive) shuttle phasmid to generate a specialized transducing mycobacteriophage. The temperature-sensitive mutations in the mycobacteriophage genome permit replication at the permissive temperature of 30 degrees C but prevent replication at the non-permissive temperature of 37 degrees C. Transduction at a non-permissive temperature results in highly efficient delivery of the recombination substrate to virtually all cells in the recipient population. The deletion mutations in the targeted genes are marked with antibiotic-resistance genes that are flanked by gammadelta-res (resolvase recognition target) sites. The transductants which have undergone a homologous recombination event can be conveniently selected on antibiotic-containing media. To demonstrate the utility of this genetic system seven different targeted gene disruptions were generated in three substrains of Mycobacterium bovis BCG, three strains of Mycobacterium tuberculosis, and Mycobacterium smegmatis. Mutants in the lysA, nadBC, panC, panCD, leuCD, Rv3291c and Rv0867c genes or operons were isolated as antibiotic-resistant (and in some cases auxotrophic) transductants. Using a plasmid encoding the gammadelta-resolvase (tnpR), the resistance genes could be removed, generating unmarked deletion mutations. It is concluded from the high frequency of allelic exchange events observed in this study that specialized transduction is a very efficient technique for genetic manipulation of mycobacteria and is a method of choice for constructing isogenic strains of M. tuberculosis, BCG or M. smegmatis which differ by defined mutations.

Survival perspectives from the world's most successful pathogen, Mycobacterium tuberculosis

Studying defined mutants of Mycobacterium tuberculosis in the mouse model of infection has led to the discovery of attenuated mutants that fall into several phenotypic classes. These mutants are categorized by their growth characteristics compared with those of wild-type M. tuberculosis, and include severe growth in vivo mutants, growth in vivo mutants, persistence mutants, pathology mutants and dissemination mutants. Here, examples of each of these mutant phenotypes are described and classified accordingly. Defining the importance of mycobacterial gene products responsible for in vivo growth, persistence and the induction of immunopathology will lead to a greater understanding of the host-pathogen interaction and potentially to new antimycobacterial treatment options.

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.

Characterization of the protective T-cell response generated in CD4-deficient mice by a live attenuated Mycobacterium tuberculosis vaccine

The global epidemic of tuberculosis, fuelled by acquired immune‐deficiency syndrome, necessitates the development of a safe and effective vaccine. We have constructed a ΔRD1ΔpanCD mutant of Mycobacterium tuberculosis (mc26030) that undergoes limited replication and is severely attenuated in immunocompromised mice, yet induces significant protection against tuberculosis in wild‐type mice and even in mice that completely lack CD4+ T cells as a result of targeted disruption of their CD4 genes (CD4–/– mice). Ex vivo studies of T cells from mc26030‐immunized mice showed that these immune cells responded to protein antigens of M. tuberculosis in a major histocompatibility complex (MHC) class II‐restricted manner. Antibody depletion experiments showed that antituberculosis protective responses in the lung were not diminished by removal of CD8+, T‐cell receptor γδ (TCR‐γδ+) and NK1.1+ T cells from vaccinated CD4–/– mice before challenge, implying that the observed recall and immune effector functions resulting from vaccination of CD4–/– mice with mc26030 were attributable to a population of CD4– CD8– (double‐negative) TCR‐αβ+, TCR‐γδ–, NK1.1– T cells. Transfer of highly enriched double‐negative TCR‐αβ+ T cells from mc26030‐immunized CD4–/– mice into naive CD4–/– mice resulted in significant protection against an aerosol tuberculosis challenge. Enriched pulmonary double‐negative T cells transcribed significantly more interferon‐γ and interleukin‐2 mRNA than double‐negative T cells from naive mice after a tuberculous challenge. These results confirmed previous findings on the potential for a subset of MHC class II‐restricted T cells to develop and function without expression of CD4 and suggest novel vaccination strategies to assist in the control of tuberculosis in human immunodeficiency virus‐infected humans who have chronic depletion of their CD4+ T cells.

Einstein Contained Aerosol Pulmonizer (ECAP): Improved Biosafety for Multi-Drug Resistant (MDR) and Extensively Drug Resistant (XDR) Mycobacterium tuberculosis Aerosol Infection Studies

A new apparatus enhances the biosafety of containment (biosafety level 3 [BSL-3]) and provides experimental reproducibility for aerosol infection experiments with MDR and XDR Mycobacterium tuberculosis. The methods are generally applicable to the study of air-borne pathogens.