Granulomas are organized host immune structures composed of tightly interposed macrophages and other cells that form in response to a variety of persistent stimuli, both infectious and noninfectious. The tuberculous granuloma is essential for host containment of mycobacterial infection, although it does not always eradicate it. Therefore, it is considered a host-beneficial, if incompletely efficacious, immune response. The Mycobacterium RD1 locus encodes a specialized secretion system that promotes mycobacterial virulence by an unknown mechanism. Using transparent zebrafish embryos to monitor the infection process in real time, we found that RD1-deficient bacteria fail to elicit efficient granuloma formation despite their ability to grow inside of infected macrophages. We showed that macrophages infected with virulent mycobacteria produce an RD1-dependent signal that directs macrophages to aggregate into granulomas. This Mycobacterium-induced macrophage aggregation in turn is tightly linked to intercellular bacterial dissemination and increased bacterial numbers. Thus, mycobacteria co-opt host granulomas for their virulence.

In tuberculosis, infecting mycobacteria are phagocytosed by macrophages, which then migrate into deeper tissue and recruit additional cells to form the granulomas that eventually contain infection. Mycobacteria are exquisitely adapted macrophage pathogens, and observations in the mouse model of tuberculosis have suggested that mycobacterial growth is not inhibited in macrophages until adaptive immunity is induced. Using the optically transparent and genetically tractable zebrafish embryo-Mycobacterium marinum model of tuberculosis, we have directly examined early infection in the presence and absence of macrophages. The absence of macrophages led rapidly to higher bacterial burdens, suggesting that macrophages control infection early and are not an optimal growth niche. However, we show that macrophages play a critical role in tissue dissemination of mycobacteria. We propose that residence within macrophages represents an evolutionary trade-off for pathogenic mycobacteria that slows their early growth but provides a mechanism for tissue dissemination.

The Mycobacterium tuberculosis exported repetitive protein (Erp) is a virulence determinant required for growth in cultured macrophages and in vivo. To better understand the role of Erp in Mycobacterium pathogenesis, we generated a mutation in the erp homologue of Mycobacterium marinum, a close genetic relative of M. tuberculosis. erp-deficient M. marinum was growth attenuated in cultured macrophage monolayers and during chronic granulomatous infection of leopard frogs, suggesting that Erp function is similarly required for the virulence of both M. tuberculosis and M. marinum. To pinpoint the step in infection at which Erp is required, we utilized a zebrafish embryo infection model that allows M. marinum infections to be visualized in real-time, comparing the erp-deficient strain to a ⌬RD1 mutant whose stage of attenuation was previously characterized in zebrafish embryos. A detailed microscopic examination of infected embryos revealed that bacteria lacking Erp were compromised very early in infection, failing to grow and/or survive upon phagocytosis by host macrophages. In contrast, ⌬RD1 mutant bacteria grow normally in macrophages but fail to induce host macrophage aggregation and subsequent cell-to-cell spread. Consistent with these in vivo findings, erpdeficient but not RD1-deficient bacteria exhibited permeability defects in vitro, which may be responsible for their specific failure to survive in host macrophages.Mycobacteria, typified by Mycobacterium tuberculosis, are intracellular pathogens that cause persistent infections despite vigorous host responses (13), and much of the work aimed at understanding mycobacterial pathogenesis has focused on the interactions between mycobacteria and the immune system. The infectious process can be viewed as a series of sequential steps, in which the bacteria interface with the immune system in multiple complex ways. Infection begins with the recruitment of macrophages to the site of infection and phagocytosis of infecting organisms. These infected macrophages then migrate to deeper tissues, and additional monocytes are recruited to the infected cell. As bacterial growth ensues, macrophages aggregate and differentiate into the characteristic epithelioid macrophages that constitute granulomas. Later, as adaptive immunity develops, other immune cells such as lymphocytes are recruited to the granulomas. The bacterium-host interactions that orchestrate this complex program are not well understood, and the bacterial determinants that permit both early growth and long-term persistence are now being identified.The last decade has seen significant advancements in the genetic manipulation of mycobacteria to define and characterize mycobacterial virulence factors (13,24,32,41). Mutations that reduce virulence have been identified, and the majority of these affect the structure and/or the function of the highly complex cell wall and outer lipid layer. These include mutations in genes involved in cell wall lipid synthesis, composition, or transport (8,9,11,14,19,20,22,23,28); the ...