Regulation, Integrase-Dependent Excision, and Horizontal Transfer of Genomic Islands in Legionella pneumophila

Legionella pneumophila is a Gram-negative freshwater agent which multiplies in specialized nutrient-rich vacuoles of amoebae. When replicating in human alveolar macrophages, Legionella can cause Legionnaires' disease. Recently, we identified a new type of conjugation/type IVA secretion system (T4ASS) in L. pneumophila Corby (named trb-tra). Analogous versions of trb-tra are localized on the genomic islands Trb-1 and Trb-2. Both can exist as an episomal circular form, and Trb-1 can be transferred horizontally to other Legionella strains by conjugation. In our current work, we discovered the importance of a site-specific integrase (Int-1, lpc2818) for the excision and conjugation process of Trb-1. Furthermore, we identified the genes lvrRABC (lpc2813 to lpc2816) to be involved in the regulation of Trb-1 excision. In addition, we demonstrated for the first time that a Legionella genomic island (LGI) of L. pneumophila Corby (LpcGI-2) encodes a functional type IV secretion system. The island can be transferred horizontally by conjugation and is integrated site specifically into the genome of the transconjugants. LpcGI-2 generates three different episomal forms. The predominant episomal form, form A, is generated integrase dependently (Lpc1833) and transferred by conjugation in a pilT-dependent manner. Therefore, the genomic islands Trb-1 and LpcGI-2 should be classified as integrative and conjugative elements (ICEs). Coculture studies of L. pneumophila wild-type and mutant strains revealed that the int-1 and lvrRABC genes (located on Trb-1) as well as lpc1833 and pilT (located on LpcGI-2) do not influence the in vivo fitness of L. pneumophila in Acanthamoeba castellanii

FliA expression analysis and influence of the regulatory proteins RpoN, FleQ and FliA on virulence and in vivo fitness in Legionella pneumophila

In Legionella pneumophila, the regulation of the flagellum and the expression of virulence traits are linked. FleQ, RpoN and FliA are the major regulators of the flagellar regulon. We demonstrated here that all three regulatory proteins mentioned (FleQ, RpoN and FliA) are necessary for full in vivo fitness of L. pneumophila strains Corby and Paris. In this study, we clarified the role of FleQ for fliA expression from the level of mRNA toward protein translation. FleQ enhanced fliA expression, but FleQ and RpoN were not necessary for basal expression. In addition, we identified the initiation site of fliA in L. pneumophila and found a putative σ(70) promoter element localized upstream. The initiation site was not influenced in the ΔfleQ or ΔrpoN mutant strain. We demonstrated that there is no significant difference in the regulation of fliA between strains Corby and Paris, but the FleQ-dependent induction of fliA transcription in the exponential phase is stronger in strain Paris than in strain Corby. In addition, we showed for the first time the presence of a straight hook at the pole of the non-flagellated ΔfliA and ΔfliD mutant strains by electron microscopy, indicating the presence of an intact basal body in these strains

European Resuscitation Council Guidelines for Resuscitation 2015. Section 1. Executive summary

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A Legionella pneumophila amylase is essential for intracellular replication in human macrophages and amoebae

Abstract Legionella pneumophila invades protozoa with an “accidental” ability to cause pneumonia upon transmission to humans. To support its nutrition during intracellular residence, L. pneumophila relies on host amino acids as the main source of carbon and energy to feed the TCA cycle. Despite the apparent lack of a requirement for glucose for L. pneumophila growth in vitro and intracellularly, the organism contains multiple amylases, which hydrolyze polysaccharides into glucose monomers. Here we describe one predicted putative amylase, LamB, which is uniquely present only in L. pneumophila and L. steigerwaltii among the ~60 species of Legionella. Our data show that LamB has a strong amylase activity, which is abolished upon substitutions of amino acids that are conserved in the catalytic pocket of amylases. Loss of LamB or expression of catalytically-inactive variants of LamB results in a severe growth defect of L. pneumophila in Acanthamoeba polyphaga and human monocytes-derived macrophages. Importantly, the lamB null mutant is severely attenuated in intra-pulmonary proliferation in the mouse model and is defective in dissemination to the liver and spleen. Our data show an essential role for LamB in intracellular replication of L. pneumophila in amoeba and human macrophages and in virulence in vivo