Molecular evolution of key genes for type II secretion in Legionella pneumophila Joana Costa, 1,2 Ana Filipa d’Avó, 1 Milton S. da Costa 1,2 and António Veríssimo 1,2 * 1 Centro de Neurociências e Biologia Celular, Universidade de Coimbra, 3004-517 Coimbra, Portugal. 2 Department of Life Sciences, University of Coimbra, Apartado 3046, 3001-401 Coimbra, Portugal. Summary Given the role of type II protein secretion system (T2S) in the ecology and pathogenesis of Legionella pneumophila, it is possible that this system is a target for adaptive evolution. The population genetic struc- ture of L. pneumophila was inferred from the partial sequences of rpoB and from the complete sequence of three T2S structural components (lspD, lspE and pilD) and from two T2S effectors critical for intracel- lular infection of protozoa (proA and srnA) of 37 strains isolated from natural and man-made environ- ments and disease-related from worldwide sources. A phylogenetic analysis was obtained for the concat- enated alignment and for each individual locus. Seven main groups were identified containing the same L. pneumophila strains, suggesting an ancient divergence for each cluster and indicating that the operating selective pressures have equally affected the evolution of the five genes. Although linkage dis- equilibrium analysis indicate a clonal nature for popu- lation structure in this sample, our results indicate that recombination is a common phenomenon among T2S-related genes on this species, as 24 of the 37 L. pneumophila isolates contained at least one locus in which recombination was identified. Furthermore, neutral selection acting on the analysed T2S-related genes emerged as a clear result, namely on T2S effec- tors, ProA and SrnA, indicating that they are probably implicated in conserved virulence mechanisms through legionellae hosts. Introduction Legionella pneumophila is a ubiquitous bacterium in natural and water distribution systems known for its ability to cause pneumonia in humans. The bacterium’s survival and spread depends on the ability to replicate inside phagocytic cells. In humans, L. pneumophila reaches the lungs after inhalation of contaminated aerosol droplets where is phagocytosed by alveolar macrophages, which are the major site for bacterial replication (Fields, 2008; Moliner et al., 2010; Newton et al., 2010). Legionella pneumophila has a rather exceptional number and wide variety of secretion systems for efficient and rapid deliverance of effector molecules into the phagocytotic host cells, underlining the importance of protein secretion for this pathogen (De Buck et al., 2007). Legionella pneumophila type II secretion system (T2S) was first identified based on the presence of PilD (Liles et al., 1998), a homologue of the Pseudomonas prepilin peptidase, which has the ability to process pilin and the so-called pseudopilins that are implicated both in the bio- genesis of type IV pili and a functional type II secretion system (Lory and Strom, 1997; Ayers et al., 2010). T2S is a multistep process where proteins intent for secretion are first translocated across the inner membrane by the Sec or Tat pathway, and upon delivery into the periplasm, where the unfolded proteins then assume their tertiary conformation. Finally, the proteins are translocated across the outer membrane by a multiprotein complex, the T2S apparatus (Cianciotto, 2009). T2S is present in many but not all Gram-negative bacteria, indicating its role as an important yet specialized secretion system (Cianciotto, 2005). It was showed that the system is operative in both pathogens and non-pathogens playing important roles in pathogenesis and/or contributing to bacterial fitness in different ecological niches (Cianciotto, 2005; Evans et al., 2008). T2S is critical for L. pneumophila intracellular infec- tion of protozoa cells and promotes the intracellular infec- tion of lung epithelial cells, dampens the cytokine output from infected macrophages and epithelia, and limits the levels of cytokine transcripts in infected macrophages (Cianciotto, 2009; McCoy-Simandle et al., 2011). More- over, at least 25 proteins have been shown to be T2S substrates (DebRoy et al., 2006; Cianciotto, 2009; Pearce and Cianciotto, 2009; Stewart et al., 2009). Although func- tional redundancy is prevalent among T2S substrates, a metalloprotease – ProA, and a ribonuclease – SrnA, were shown to be required for the optimal infection of amoeba (DebRoy et al., 2006; Rossier et al., 2008; 2009). Double mutants lacking both ProA and SrnA exhibited an Received 25 July, 2011; revised 9 September, 2011; accepted 23 October, 2011. *For correspondence. E-mail averiss@ci.uc.pt; Tel. (+351) 239824024; Fax (+351) 239826798. Environmental Microbiology (2012) 14(8), 2017–2033 doi:10.1111/j.1462-2920.2011.02646.x © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd