Demonstration of the intracellular production of tissue-destructive protease by Legionella pneumophila multiplying within guinea-pig and human alveolar macrophages

Abstract

The major extracellular enzyme of Legionellapneumophila, a metalloprotease, has been proposed as a pathogenic factor in Legionnaires' disease due to its cytotoxic, tissue-destructive, and phagocyte-inhibitory properties. The relevance of these activities depends on the production of the protease during infection, i.e. by L. pneumophila multiplying intracellularly. In this study, L. pneumophila was demonstrated to produce protease in guinea-pig and human alveolar macrophages infected in vitro. After 24 h infection, approximately 0.1 to 0.2 pg of protease per lo6 bacteria was measured by E L S A in culture supernatants and lysates of the infected cells, whereas no protease could be detected immediately after infection. Immunogold labelling using anti-protease antibody showed the enzyme to be located within phagosomes and distributed throughout the macrophages. Recent observations have shown that this protease could modify host defence mechanisms through inhibition of bacterial killing by neutrophils and monocytes. The intracellular production of the enzyme in infected macrophages demonstrated here further supports a role for the protease in the pathogenesis of Legionnaires' disease.

The Type II Secretion System ofLegionella pneumophilaElaborates Two Aminopeptidases, as Well as a Metalloprotease That Contributes to Differential Infection among Protozoan Hosts

“…On the other hand, it is possible that ProA/MspA degrades host factors that are designed to control bacterial growth or cleaves other secreted Legionella proteins that directly promote intracellular infection. In infected macrophages, the protease is seen in both the Legionella phagosome and the adjacent host cytoplasm (59), supporting the possibility of it having multiple intracellular targets. Clearly, the protease's greater role in Hartmannella hosts should facilitate future investigations into the protein's intracellular role.…”

“…Thus, proA/mspA represents an L. pneumophila gene that clearly shows differential importance among protozoan models. Although a need for ProA is only evident from infections of H. vermiformis, previous studies showed that proA and its protein product are expressed during infection of A. castellanii and macrophages (51,59). Thus, we hypothesize that in some hosts, such as acanthamoebae and macrophages, ProA/MspA is dispensable such that other proteases can, if necessary, fulfill its role, but that in other hosts, such as H. vermiformis, the need for ProA/MspA is greater and/or cannot be complemented by alternate proteases.…”

The Type II Secretion System of Legionella pneumophila Dampens the MyD88 and Toll-Like Receptor 2 Signaling Pathway in Infected Human Macrophages

“…Although TLR5 and TLR9 also signal through MyD88, the fact that we see a dampening of only the TLR2 pathway could be because there is more stimulation of TLR2 than of TLR5 and TLR9 during L. pneumophila infection. Compatible with the possibility of an effector directly targeting the host receptor or adaptor, there is already an example of a T2S-dependent substrate occurring in the cytosol of infected macrophages (86), although this particular effector, the ProA metalloprotease, is not required for the dampening of cytokine gene transcription (16). Thus, we posit the existence of a novel T2S-dependent effector that impedes the functioning of the TLR2 pathway.…”

Legionella pneumophila Type II Secretion Dampens the Cytokine Response of Infected Macrophages and Epithelia

“…On the other hand, ProA might be secreted by legionellae that (temporarily) reside extracellularly after lysis of the infected host cell. Finally, since ProA is expressed in infected macrophages and can be seen to some extent in the host cytoplasm (91), the effector may degrade the cytokine while both reside in the intracellular space.…”