Phospholipase PlaB of Legionella pneumophila Represents a Novel Lipase Family

Abstract

Legionella pneumophila possesses several phospholipases capable of host cell manipulation and lung damage. Recently, we discovered that the major cell-associated hemolytic phospholipase A (PlaB) shares no homology to described phospholipases and is dispensable for intracellular replication in vitro. Nevertheless, here we show that PlaB is the major lipolytic activity in L. pneumophila cell infections and that PlaB utilizes a typical catalytic triad of Ser-Asp-His for effective hydrolysis of phospholipid substrates. Crucial residues were found to be located within the N-terminal half of the protein, and amino acids embedding these active sites were unique for PlaB and homologs. We further showed that catalytic activity toward phosphatidylcholine but not phosphatidylglycerol is directly linked to hemolytic potential of PlaB. Although the function of the prolonged PlaB C terminus remains to be elucidated, it is essential for lipolysis, since the removal of 15 amino acids already abolishes enzyme activity. Additionally, we determined that PlaB preferentially hydrolyzes long-chain fatty acid substrates containing 12 or more carbon atoms. Since phospholipases play an important role as bacterial virulence factors, we examined cell-associated enzymatic activities among L. pneumophila clinical isolates and non-pneumophila species. All tested clinical isolates showed comparable activities, whereas of the non-pneumophila species, only Legionella gormanii and Legionella spiritensis possessed lipolytic activities similar to those of L. pneumophila and comprised plaB-like genes. Interestingly, phosphatidylcholine-specific phospholipase A activity and hemolytic potential were more pronounced in L. pneumophila. Therefore, hydrolysis of the eukaryotic membrane constituent phosphatidylcholine triggered by PlaB could be an important virulence tool for Legionella pathogenicity.

Nutrient salvaging and metabolism by the intracellular pathogen Legionella pneumophila

“…Further strengthening this notion is the fact that L. pneumophila secretes a number of phospholipase enzymes, some of which have been determined to be virulence factors that act by destroying host membranes (Flieger et al, 2000, 2002, 2004; Bender et al, 2009; Kuhle and Flieger, 2013). …”

Bacterial Sphingomyelinases and Phospholipases as Virulence Factors

“…Surface-associated phospholipase A 2 s. The surface-associated phospholipase A 2 group includes the PlaB produced by different Legionella pneumophila strains as well as homologues, largely still uncharacterized, encoded in the genomes of other Legionella species, Pseudomonas aeruginosa, and other water-associated bacteria (80).…”

“…Amino acid residues Ser85, Asp203, and His251 are required for catalysis and likely constitute the catalytic triad, whereas Ser129 is important for PC substrate specificity (80). A Ser129 mutant displaying an ϳ90% reduction in its enzymatic activity against PC, but almost unchanged activity against PG, was not hemolytic to human erythrocytes (80).…”

Peer Review #3 of "A proposed update for the classification and description of bacterial lipolytic enzymes (v0.1)"

“…Jaeger and Eggert (2002) redefined these families by splitting family I.5 into two new families, family I.5 and family I.6, and moving the previous members of family I.6 into family I.7 (Jaeger & Eggert, 2002). In the 20 years since this bacterial lipolytic enzyme classification system was published, more than twice as many 'novel' families have been proposed (Handrick et al, 2001;Ewis, Abdelal & Lu, 2004;Lee et al, 2010Lee et al, , 2006Levisson, van der Oost & Kengen, 2007;Bender et al, 2009;Kim et al, 2009;Hu et al, 2010;Rao et al, 2011;Jeon et al, 2011;Bassegoda, Pastor & Diaz, 2012;Charbonneau & Beauregard, 2013;Zarafeta et al, 2016;Castilla et al, 2017;Parapouli et al, 2018). A recent update to this system expanded the system to include some (11 of 30) of the recently published novel lipases within 19 families and 8 true lipase sub-families, however many lipolytic proteins remain unclassified (Kovacic et al, 2019).…”

“…Examples of this are family X (Levisson, van der Oost & Kengen, 2007;Bassegoda, Pastor & Diaz, 2012) and family XV (Charbonneau & Beauregard, 2013;Zarafeta et al, 2016), both of which have been coined twice. Rao et al (2013) attempted to incorporate previously published families into their analysis, however their literature search did not include multiple families (Chu et al, 2008;Bender et al, 2009;Hu et al, 2010;Lee et al, 2010;Jeon et al, 2011;Nacke et al, 2011;Bassegoda, Pastor & Diaz, 2012). Ferrer et al (2015) also attempted to integrate additional families into the Arpigny and Jaeger (1999) classification system.…”