Mycobacterial Mutants with Defective Control of Phagosomal Acidification

Abstract

The pathogenesis of mycobacterial infection is associated with an ability to interfere with maturation of the phagosomal compartment after ingestion by macrophages. Identification of the mycobacterial components that contribute to this phenomenon will allow rational design of novel approaches to the treatment and prevention of tuberculosis. Microarray-based screening of a transposon library was used to identify mutations that influence the fate of Mycobacterium bovis bacille Calmette-Guérin (BCG) following uptake by macrophages. A screen based on bacterial survival during a 3-d infection highlighted genes previously implicated in growth of Mycobacterium tuberculosis in macrophages and in mice, together with a number of other virulence genes including a locus encoding virulence-associated membrane proteins and a series of transporter molecules. A second screen based on separation of acidified and non-acidified phagosomes by flow cytometry identified genes involved in mycobacterial control of early acidification. This included the KefB potassium/proton antiport. Mutants unable to control early acidification were significantly attenuated for growth during 6-d infections of macrophages. Early acidification of the phagosome is associated with reduced survival of BCG in macrophages. A strong correlation exists between genes required for intracellular survival of BCG and those required for growth of M. tuberculosis in mice. In contrast, very little correlation exists between genes required for intracellular survival of BCG and those that are up-regulated during intracellular adaptation of M. tuberculosis. This study has identified targets for interventions to promote immune clearance of tuberculosis infection. The screening technologies demonstrated in this study will be useful to the study of pathogenesis in many other intracellular microorganisms.

Synopsis

The pathogenesis of Mycobacterium tuberculosis relies on an ability to survive inside host macrophages. Macrophages kill most other bacteria by engulfment into an intracellular compartment called a phagosome, which quickly matures to an acidic, hydrolytic organelle, resulting in bacterial death. M. tuberculosis and the related vaccine strain M. bovis bacille Calmette-Guérin (BCG) possess the ability to stop phagosome maturation and thus avoid its microbicidal properties. In this study, the researchers screened a library of mutant BCG bacteria to identify the bacterial genes responsible for preventing phagosome acidification. The predicted products of these genes span many different functional groups, but tend to be associated with the outside of the cell or secreted to the extracellular milieu. The researchers also demonstrated that mutant mycobacteria whose phagosomes acidify are unable to replicate in macrophages. This study identifies targets for new vaccines against tuberculosis.