Structure, Dynamics and Cellular Insight Into Novel Substrates of the Legionella pneumophila Type II Secretion System.

Legionella pneumophila is a Gram-negative bacterium that is able to replicate within a broad range of aquatic protozoan hosts. L. pneumophila is also an opportunistic human pathogen that can infect macrophages and epithelia in the lung and lead to Legionnaires' disease. The type II secretion system is a key virulence factor of L. pneumophila and is used to promote bacterial growth at low temperatures, regulate biofilm formation, modulate host responses to infection, facilitate bacterial penetration of mucin gels and is necessary for intracellular growth during the initial stages of infection. The L. pneumophila type II secretion system exports at least 25 substrates out of the bacterium and several of these, including NttA to NttG, contain unique amino acid sequences that are generally not observed outside of the Legionella genus. NttA, NttC, and NttD are required for infection of several amoebal species but it is unclear what influence other novel substrates have within their host. In this study, we show that NttE is required for optimal infection of Acanthamoeba castellanii and Vermamoeba vermiformis amoeba and is essential for the typical colony morphology of L. pneumophila. In addition, we report the atomic structures of NttA, NttC, and NttE and through a combined biophysical and biochemical hypothesis driven approach we propose novel functions for these substrates during infection. This work lays the foundation for future studies into the mechanistic understanding of novel type II substrate functions and how these relate to L. pneumophila ecology and disease.

Structure and functional analysis of the Legionella pneumophila chitinase ChiA reveals a novel mechanism of metal-dependent mucin degradation.

Chitinases are important enzymes that contribute to the generation of carbon and nitrogen from chitin, a long chain polymer of N-acetylglucosamine that is abundant in insects, fungi, invertebrates and fish. Although mammals do not produce chitin, chitinases have been identified in bacteria that are key virulence factors in severe respiratory, gastrointestinal and urinary diseases. However, it is unclear how these enzymes are able to carry out this dual function. Legionella pneumophila is the causative agent of Legionnaires' disease, an often-fatal pneumonia and its chitinase ChiA is essential for the survival of L. pneumophila in the lung. Here we report the first atomic resolution insight into the pathogenic mechanism of a bacterial chitinase. We derive an experimental model of intact ChiA and show how its N-terminal region targets ChiA to the bacterial surface after its secretion. We provide the first evidence that L. pneumophila can bind mucins on its surface, but this is not dependent on ChiA. This demonstrates that additional peripheral mucin binding proteins are also expressed in L. pneumophila. We also show that the ChiA C-terminal chitinase domain has novel Zn2+-dependent peptidase activity against mammalian mucin-like proteins, namely MUC5AC and the C1-esterase inhibitor, and that ChiA promotes bacterial penetration of mucin gels. Our findings suggest that ChiA can facilitate passage of L. pneumophila through the alveolar mucosa, can modulate the host complement system and that ChiA may be a promising target for vaccine development.

Type II Secretion-Dependent Aminopeptidase LapA and Acyltransferase PlaC Are Redundant for Nutrient Acquisition during Legionella pneumophila Intracellular Infection of Amoebas.

Legionella pneumophila genes encoding LapA, LapB, and PlaC were identified as the most highly upregulated type II secretion (T2S) genes during infection of Acanthamoeba castellanii, although these genes had been considered dispensable on the basis of the behavior of mutants lacking either lapA and lapB or plaC A plaC mutant showed even higher levels of lapA and lapB transcripts, and a lapA lapB mutant showed heightening of plaC mRNA levels, suggesting that the role of the LapA/B aminopeptidase is compensatory with respect to that of the PlaC acyltransferase. Hence, we made double mutants and found that lapA plaC mutants have an ~50-fold defect during infection of A. castellanii These data revealed, for the first time, the importance of LapA in any sort of infection; thus, we purified LapA and defined its crystal structure, activation by another T2S-dependent protease (ProA), and broad substrate specificity. When the amoebal infection medium was supplemented with amino acids, the defect of the lapA plaC mutant was reversed, implying that LapA generates amino acids for nutrition. Since the LapA and PlaC data did not fully explain the role of T2S in infection, we identified, via proteomic analysis, a novel secreted protein (NttD) that promotes infection of A. castellanii A lapA plaC nttD mutant displayed an even greater (100-fold) defect, demonstrating that the LapA, PlaC, and NttD data explain, to a significant degree, the importance of T2S. LapA-, PlaC-, and NttD-like proteins had distinct distribution patterns within and outside the Legionella genus. LapA was notable for having as its closest homologue an A. castellanii protein.IMPORTANCE Transmission of L. pneumophila to humans is facilitated by its ability to grow in Acanthamoeba species. We previously documented that type II secretion (T2S) promotes L. pneumophila infection of A. castellanii Utilizing transcriptional analysis and proteomics, double and triple mutants, and crystal structures, we defined three secreted substrates/effectors that largely clarify the role of T2S during infection of A. castellanii Particularly interesting are the unique functional overlap between an acyltransferase (PlaC) and aminopeptidase (LapA), the broad substrate specificity and eukaryotic-protein-like character of LapA, and the novelty of NttD. Linking LapA to amino acid acquisition, we defined, for the first time, the importance of secreted aminopeptidases in intracellular infection. Bioinformatic investigation, not previously applied to T2S, revealed that effectors originate from diverse sources and distribute within the Legionella genus in unique ways. The results of this study represent a major advance in understanding Legionella ecology and pathogenesis, bacterial secretion, and the evolution of intracellular parasitism.