Mycobacterium marinum Erp Is a Virulence Determinant Required for Cell Wall Integrity and Intracellular Survival

Abstract

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 ...

Mycobacterium abscessus cording prevents phagocytosis and promotes abscess formation

“…First, the time-course of death induced by M. abscessus is more rapid than with M. marinum, with up to 50% of embryos dying 5-7 dpi and 100% within 2 wk post infection (19,24,38). Second, despite the apparent lack of the ESX-1 secretion system, M. abscessus induces granuloma formation.…”

Immunity and Immunopathology in the Tuberculous Granuloma

“…Derp M. marinum displayed an intracellular growth defect in individual zebrafish larval macrophages, thus was compromised at an earlier step than DRD1 (Fig. 1) (Cosma et al 2006). Despite having no intracellular growth defect, DRD1 was as attenuated as Derp for overall growth in the animals.…”

Zebrafish development and regeneration: new tools for biomedical research

“…The latter genes are located in the well-known RD1 region, which is the distinctive mutation in the tuberculosis vaccine strain BCG. Injection of this mutant in zebrafish embryos resulted in aberrant granuloma formation in zebrafish embryos (Cosma et al, 2006;Gao et al, 2006;Volkman et al, 2004). This finding inspired us to do a forward genetic screen to identify M. marinum mutants impaired in granuloma formation in zebrafish embryos.…”

“…To further establish the zebrafish tuberculosis model, it was shown that M. marinum mutants of important M. tuberculosis virulence factors, such as the cell surface protein Erp and the secreted ESAT-6 and CFP-10 proteins, are attenuated in the zebrafish model (Cosma et al, 2006;Gao et al, 2006;Volkman et al, 2004). The latter genes are located in the well-known RD1 region, which is the distinctive mutation in the tuberculosis vaccine strain BCG.…”