Characterization of avirulent mutant Legionella pneumophila that survive but do not multiply within human monocytes.

Abstract

Legionella pneumophila, the causative agent of Legionnaires' disease, is a Gram-negative bacterium and a facultative intracellular parasite that multiplies in human monocytes and alveolar macrophages. In this paper, mutants of L. pneumophila avirulent for human monocytes were obtained and extensively characterized. The mutants were obtained by serial passage of wild-type L. pneumophila on suboptimal artificial medium. None of 44 such mutant clones were capable of multiplying in monocytes or exerting a cytopathic effect on monocyte monolayers. Under the same conditions, wild-type L. pneumophila multiplied 2.5-4.5 logs, and destroyed the monocyte monolayers. The basis for the avirulent phenotype was an inability of the mutants to multiply intracellularly. Both mutant and wild-type bacteria bound to and were ingested by monocytes, and both entered by coiling phagocytosis. Thereafter, their intracellular destinies diverged. The wild-type formed a distinctive ribosome-lined replicative phagosome, inhibited phagosome-lysosome fusion, and multiplied intracellularly. The mutant did not form the distinctive phagosome nor inhibit phagosome-lysosome fusion. The mutant survived intracellularly but did not replicate in the phagolysosome. In all other respects studied, the mutant and wild-type bacteria were similar. They had similar ultrastructure and colony morphology; both formed colonies of compact and diffuse type. They had similar structural and secretory protein profiles and LPS profile by PAGE. Both the mutant and wild-type bacteria were completely resistant to human complement in the presence or absence of high titer anti-L. pneumophila antibody. The mutant L. pneumophila have tremendous potential for enhancing our understanding of the intracellular biology of L. pneumophila and other parasites that follow a similar pathway through the mononuclear phagocyte. Such mutants also show promise for enhancing our understanding of immunity to L. pneumophila, and they may serve as prototypes in the development of safe and effective vaccines against intracellular pathogens.

A live avirulent mutant Legionella pneumophila vaccine induces protective immunity against lethal aerosol challenge.

“…Like the wild-type, the mutant enters monocytes by coiling phagocytosis (13,14). Thereafter, however, the mutant follows an intracellular pathway that differs from the wild-type.…”

“…Another study from this laboratory described an avirulent mutant form of L. pneumophila that survives but does not multiply in human monocytes (13). Like the wild-type, the mutant enters monocytes by coiling phagocytosis (13,14).…”

“…Whereas the wild-type phagosome interacts sequentially with host cell smooth vesicles, mitochondria, and ribosomes, a process culminating in the formation of a ribosome-lined replicative phagosome, the mutant phagosome does not interact with these host cell organelles (13). Moreover, whereas the wild-type phagosome does not fuse with lysosomes, the mutant phagosome does fuse with lysosomes so that the mutant comes to reside in a phagolysosome (13). In all other aspects examined the mutant resembles the wild-type.…”

Preventing bacterial DNA release and absent in melanoma 2 inflammasome activation by a Legionella effector functioning in membrane trafficking

“…However, the Dot/Icm system is critical for establishment of the LCV. The LCV serves as an intracellular niche, where L. pneumophila can efficiently replicate, as has been observed in human U937 monocytes (8).…”

Phagocytosis of Legionella pneumophila

“…Both kinds of strains enter phagocytes by coiling phagocytosis ; thereafter, their intracellular destinies diverge. The virulent organisms form a distinctive ribosome-lined replicative phagosome and inhibit phagosome fusion; the avirulent strain neither forms a distinctive phagosome nor inhibits phagosome-lysosome fusion.339 66 When PMNL were challenged with avirulent strains of L. pneumophila, they produced more chemiluminescence and superoxide anion than those challenged by virulent strains.67 This phenomenon may be due either to active suppression of PMNL responses by virulent strains or to varying degrees of opsonisation. A cytotoxin of low mol.…”