The Legionella pneumophila PilT Homologue DotB Exhibits ATPase Activity That Is Critical for Intracellular Growth

Abstract

The ability of Legionella pneumophila to grow and cause disease in the host is completely dependent on a type IV secretion system known as the Dot/Icm complex. This membrane-spanning apparatus translocates effector molecules into host cells in a process that is poorly understood but that is known to require the putative ATPase DotB. One possible role for DotB is suggested by its similarity to the PilT family of proteins, which mediate pilus retraction. To better understand the molecular behavior of DotB, we have purified the protein and shown that it forms stable homohexameric rings and hydrolyzes ATP with a specific activity of 6.4 nmol of ATP/min/mg of protein. ATPase activity is critical to the function of DotB, as alteration of the conserved Walker box lysine residue resulted in a mutant protein, DotB K162Q, which failed to bind or hydrolyze ATP and which could not complement a ⌬dotB strain for intracellular growth in macrophages. Consistent with the ability of DotB to interact with itself, the dotBK162Q allele exhibited transdominance over wild-type dotB, providing the first example of such a mutation in L. pneumophila. Finally, the DotB K162Q mutant protein had a significantly enhanced membrane localization in L. pneumophila compared to wild-type DotB, suggesting a relationship between nucleotide binding and membrane association. These results are consistent with a model in which DotB cycles between the cytoplasm and the Dot/Icm complex at the membrane, where it hydrolyzes nucleotides to provide energy to the complex.Legionella pneumophila, an intracellular bacterial pathogen, causes Legionnaires' disease by replicating inside alveolar macrophages (13). This ability is dependent on a set of genes called dot or icm, which encode a specialized type IV secretion system (32, 37). The type IV secretion system family is composed of both classical plasmid transfer systems and a related protein export apparatus used by a wide range of pathogens to export toxins (reviewed in reference 4). For L. pneumophila, the Dot/Icm complex is believed to translocate numerous effector molecules into host cells in order to modify the endocytic pathway. Thus far, two translocated proteins, LidA and RalF, have been identified (6,19). RalF is an ADP-ribosylating factor that functions to recruit the macrophage protein ARF1 to L. pneumophila-containing phagosomes, while the function of LidA remains unknown. Because neither of these is absolutely required for L. pneumophila intracellular growth, additional Dot/Icm-secreted proteins are believed to exist (6).The Dot/Icm apparatus is a macromolecular complex that comprises a large number of membrane proteins and several proteins predicted to have cytoplasmic localization. One such cytoplasmic protein, DotB, contains conserved nucleotide binding domains present in many ATPases, as well as domains specific to ATPases found in bacterial type II and type IV secretion systems (23,27,39). Though DotB is known to be a critical component of the Dot/Icm complex (37, 38), its molecular function i...

The mating pair formation system of conjugative plasmids—A versatile secretion machinery for transfer of proteins and DNA

“…More data supporting this assumption have been reported: upon lipid binding, VirB11 is seen to undergo conformational changes (Krause et al, 2000b) and the same eVect is also observed for nucleotide binding (Savvides et al, 2003). Moreover, a functional nucleotide binding site is required for localization of VirB11 to the membrane (Sexton et al, 2004). This may be the reason why nucleotide binding motif (Walker A) mutations of VirB11 fail to transfer the T-DNA substrate to the channel components VirB6 and VirB8, as detected by TrIP analysis in A. tumefaciens (Atmakuri et al, 2004).…”

“…Along with VirB4 and the CP (VirD4), VirB11 is thought to energize the type IV secretion machinery either for complex assembly, pilus production and/or substrate secretion. The NTPase activity of several VirB11-like proteins has been determined in vitro (Christie et al, 1989;Krause et al, 2000b;Rivas et al, 1997;Sexton et al, 2004), revealing a rather weak activity similar to the one observed for chaperons like DnaK (Zylicz et al, 1983). VirB11 of A. tumefaciens has been reported to additionally possess an autophosphorylation activity (Christie et al, 1989), however, such an activity was not detected for any other member of the family of VirB11-like proteins.…”

Plasticity, dynamics, and inhibition of emerging tetracycline resistance enzymes

“…pneumophila 52 , bearing either the vector pJB1625 53 or the Tet(56) complementing clone pJB7207. Minimum inhibitory concentrations were determined according to Clinical and Laboratory Standards Institute (CLSI) procedures with the following modifications.…”

Regulation of Hypercompetence in Legionella pneumophila

“…Plasmid pJB955 contains a 3.2-kb HincII/EcoRI fragment of DNA adjacent to the dot/icm region II cloned into the HincII/EcoRI sites of pBluescript II KS ϩ . Natural competence reporter constructs pJB957, pJB964, and pJB1389 are pJB955 derivatives with Kan r , Cm r , or Gent r cassettes The cloning vector pJB1653, used to create proQ and comR complementing clones, is a Gent r derivative of the RSF1010 cloning vector pJB908 (43). First, the pJB908 polylinker HindIII site was replaced with a NotI site, creating plasmid pJB1301.…”