Analysis of Virulence Factors of Legionella pneumophila

Abstract

SummaryLegionella pneumophila, the causative agent of Legionnaires' disease is a facultative intracellular bacterium, which in the course of human infection multiplies in lung macrophages predominantly manifesting as pneumonia. The natural habitat of Legionella is found in sweet water reservoirs and man-made water systems. Virulent L. pneumophila spontaneously convert to an avirulent status at a high frequency. Genetic approaches have led to the identification of various L. pneumophila genes. The mip (macrophage infectivity potentiator) determinant remains at present the sole established virulence factor. The Mip protein exhibits activity of a peptidyl prolyl cis trans isomerase (PPiase), an enzyme which is able to bind the immunosuppressant FK506 and is involved in protein folding. The recently cloned major outer membrane protein (MOMP) could play a role in the uptake of legionellae by macrophages. Cellular models are useful in studying the intracellular replication of legionellae in eukaryotic cells. Human celllines and protozoan models are appropriate for this purpose. By using U 937 macrophage-like cells and Acanthamoeba castellanii as hosts, we could discriminate virulent and avirulent L. pneumophila variants since only the virulent strain was capable of intracellular growth at 37 oc. By using these systems we further demonstrated that a hemolytic factor cloned and characterized in our laboratory, legiolysin (lly), had no influence on the intracellular growth of L. pneumophila.

Discovery of virulence genes of Legionella pneumophila by using signature tagged mutagenesis in a guinea pig pneumonia model

“…We have demonstrated that use of the STM method identified a broad range of potential L. pneumophila virulence genes, far in excess of those discovered to date by using screening of transposon mutants in M cell lines or targeted mutagenesis (6)(7)(8)(27)(28)(29)(30). The key to obtaining the broad spectrum of mutants appears to be the use of an animal pneumonia model for screening the mutants, rather than M assays.…”

Isolation and Amino Acid Sequence of a New 22-kDa FKBP-like Peptidyl-prolyl cis/trans-Isomerase of Escherichia coli

“…Blots were destained by shaking in 50 mM Tris buffer, pH 7.5, containing 500 mM NaCl and 1% Tween 20. A polyclonal antiserum, specific to the Mip-protein (FKBP25mem from L. pneumophila Philadelphia I), was obtained from rabbit (28).…”

Detection of Legionella pneumophila by Real-Time PCR for the mip Gene

“…The genes that encode the 5S and 16S ribosomal subunits have been shown to contain signature sequences that are useful for the identification of L. pneumophila (8,14,15,17,24,28) and a variety of other organisms. More recently, target sequences on these genes have been used in conjunction with real-time PCR for the detection of the Legionella genus, as well as the species L. pneumophila (14,27).The macrophage infectivity potentiator gene, which encodes a 24-kDa protein virulence factor that facilitates the entry of legionellae into amoebae and macrophages, has sufficient sequence variability between the Legionella species to also afford the specific detection of L. pneumophila by PCR (2,6,7,12,13,14,16,21,22,23,25). Although two groups have described real-time PCR assays for the detection of L. pneumophila via detection of the mip gene in water samples, to date only one group has evaluated a real-time PCR for this genetic target for the detection of L. pneumophila in clinical specimens (1,14,28).…”

“…The macrophage infectivity potentiator gene, which encodes a 24-kDa protein virulence factor that facilitates the entry of legionellae into amoebae and macrophages, has sufficient sequence variability between the Legionella species to also afford the specific detection of L. pneumophila by PCR (2,6,7,12,13,14,16,21,22,23,25). Although two groups have described real-time PCR assays for the detection of L. pneumophila via detection of the mip gene in water samples, to date only one group has evaluated a real-time PCR for this genetic target for the detection of L. pneumophila in clinical specimens (1,14,28).…”