bdhA-patD Operon as a Virulence Determinant, Revealed by a Novel Large-Scale Approach for Identification of Legionella pneumophila Mutants Defective for Amoeba Infection

Abstract

Legionella pneumophila, the causative agent of Legionnaires' disease, is an intracellular parasite of eukaryotic cells. In the environment, it colonizes amoebae. After being inhaled into the human lung, the bacteria infect and damage alveolar cells in a way that is mechanistically similar to the amoeba infection. Several L. pneumophila traits, among those the Dot/Icm type IVB protein secretion machinery, are essential for exploiting host cells. In our search for novel Legionella virulence factors, we developed an agar plate assay, designated the scatter screen, which allowed screening for mutants deficient in infecting Acanthamoeba castellanii amoebae. Likewise, an L. pneumophila clone bank consisting of 23,000 transposon mutants was investigated here, and 19 different established Legionella virulence genes, for example, dot/icm genes, were identified. Importantly, 70 novel virulence-associated genes were found. One of those is L. pneumophila bdhA, coding for a protein with homology to established 3-hydroxybutyrate dehydrogenases involved in poly-3-hydroxybutyrate metabolism. Our study revealed that bdhA is cotranscribed with patD, encoding a patatin-like protein of L. pneumophila showing phospholipase A and lysophospholipase A activities. In addition to strongly reduced lipolytic activities and increased poly-3-hydroxybutyrate levels, the L. pneumophila bdhA-patD mutant showed a severe replication defect in amoebae and U937 macrophages. Our data suggest that the operon is involved in poly-3-hydroxybutyrate utilization and phospholipolysis and show that the bdhA-patD operon is a virulence determinant of L. pneumophila. In summary, the screen for amoeba-sensitive Legionella clones efficiently isolated mutants that do not grow in amoebae and, in the case of the bdhA-patD mutant, also human cells.

Legionella oakridgensis ATCC 33761 genome sequence and phenotypic characterization reveals its replication capacity in amoebae

“…lenticulata for a Scatter Screen (Aurass et al, 2009) This is in good agreement with the finding of all components of a functional Dot/Icm system within the genome sequence (see below). Interestingly, both dotD mutant strains investigated…”

“…The EZ-Tn5™ <KAN-2>Tnp Transposome™ Kit (Epicentre, Madison, WI, USA) was used for generation of a L. oakridgensis ATCC 33761 Tn5 mutant collection according to manufacturer´s instructions and as described previously (Aurass et al, 2009). Here, 5 µl of the transposon-transposase complexes were introduced in 80 µl competent L. oakridgensis ATCC 33761 and after 6 h of incubation with shaking at 220 rpm and 37 °C, 100 µl of the suspension were plated on BCYE plates containing kanamycin (12.5 µg/ml).…”

“…Here, 5 µl of the transposon-transposase complexes were introduced in 80 µl competent L. oakridgensis ATCC 33761 and after 6 h of incubation with shaking at 220 rpm and 37 °C, 100 µl of the suspension were plated on BCYE plates containing kanamycin (12.5 µg/ml). Received clones were analyzed in a Scatter Screen as described previously (Aurass et al, 2009 (Staden et al, 1998). To solve problems with misassembled regions caused by repetitive sequences and to close remaining sequence gaps of the genome sequence of L. oakridgensis ATCC 33761, PCR and primer walking were used.…”

Growth-related Metabolism of the Carbon Storage Poly-3-hydroxybutyrate in Legionella pneumophila

“…1). Interestingly, the inactivation of PHB degradation by deletion of lpp1788 in L. pneumophila Paris or bdhA in L. pneumophila Philadelphia-1 (lpp2264 homolog) led to a similar double-fold increased amount of PHB in the respective bacteria (48).…”

“…1). An earlier study reported that a bdhA-patD mutant strain of L. pneumophila Philadelphia-1 exhibits a 2-fold increased amount of PHB when compared with the WT strain (48). BdhA is a 3-hydroxybutyrate dehydrogenase, and the authors hypothesized that this enzyme is involved in the degradation of PHB.…”