RESUMO
Legionella pneumophila is the causative agent of a severe pneumonia called Legionnaires' disease. A single strain of L. pneumophila encodes a repertoire of over 300 different effector proteins that are delivered into host cells by the Dot/Icm type IV secretion system during infection. The large number of L. pneumophila effectors has been a limiting factor in assessing the importance of individual effectors for virulence. Here, a transposon insertion sequencing technology called INSeq was used to analyze replication of a pool of effector mutants in parallel both in a mouse model of infection and in cultured host cells. Loss-of-function mutations in genes encoding effector proteins resulted in host-specific or broad virulence phenotypes. Screen results were validated for several effector mutants displaying different virulence phenotypes using genetic complementation studies and infection assays. Specifically, loss-of-function mutations in the gene encoding LegC4 resulted in enhanced L. pneumophila in the lungs of infected mice but not within cultured host cells, which indicates LegC4 augments bacterial clearance by the host immune system. The effector proteins RavY and Lpg2505 were important for efficient replication within both mammalian and protozoan hosts. Further analysis of Lpg2505 revealed that this protein functions as a metaeffector that counteracts host cytotoxicity displayed by the effector protein SidI. Thus, this study identified a large cohort of effectors that contribute to L. pneumophila virulence positively or negatively and has demonstrated regulation of effector protein activities by cognate metaeffectors as being critical for host pathogenesis.
Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Interações Hospedeiro-Patógeno/imunologia , Legionella pneumophila/patogenicidade , Doença dos Legionários/imunologia , Animais , Proteínas de Bactérias/imunologia , Citotoxicidade Imunológica , Modelos Animais de Doenças , Feminino , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Legionella pneumophila/genética , Legionella pneumophila/imunologia , Doença dos Legionários/microbiologia , Mutação com Perda de Função , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese Insercional , Fenótipo , Virulência/genéticaRESUMO
The segregation, or partition, of bacterial plasmids is driven by the action of plasmid-encoded partition ATPases, which work to position plasmids inside the cell. The most common type of partition ATPase, generally called ParA, is represented by the P1 plasmid ParA protein. ParA interacts with P1 ParB (the site-specific DNA binding protein that recognizes the parS partition site), and interacts with the bacterial chromosome via an ATP-dependent nonspecific DNA binding activity. ParA also regulates expression of the par genes by acting as a transcriptional repressor. ParA requires ATP for multiple steps and in different ways during the partition process. Here, we analyze the properties of mutations in P1 ParA that are altered in a key lysine in the Walker A motif of the ATP binding site. Four different residues at this position (Lys, Glu, Gln, Arg) result in four different phenotypes in vivo. We focus particularly on the arginine substitution (K122R) because it results in a worse-than-null and dominant-negative phenotype called ParPD. We show that ParAK122R binds and hydrolyzes ATP, although the latter activity is reduced compared with wild-type. ParAK122R interacts with ParB, but the consequences of the interaction are damaged. The ability of ParB to stimulate the ATPase activity of ParA in vitro and its repressor activity in vivo is defective. The K122R mutation specifically damages the disassembly of ParA-ParB-DNA partition complexes, which we believe explains the ParPD phenotype in vivo.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Plasmídeos/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Trifosfato de Adenosina/química , Substituição de Aminoácidos , Domínio Catalítico , DNA Primase/química , DNA Bacteriano/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Hidrólise , Fenótipo , Plasmídeos/genética , Regiões Promotoras Genéticas , Ligação ProteicaRESUMO
Localization of the P1 plasmid requires two proteins, ParA and ParB, which act on the plasmid partition site, parS. ParB is a site-specific DNA-binding protein and ParA is a Walker-type ATPase with non-specific DNA-binding activity. In vivo ParA binds the bacterial nucleoid and forms dynamic patterns that are governed by the ParB-parS partition complex on the plasmid. How these interactions drive plasmid movement and localization is not well understood. Here we have identified a large protein-DNA complex in vitro that requires ParA, ParB and ATP, and have characterized its assembly by sucrose gradient sedimentation and light scattering assays. ATP binding and hydrolysis mediated the assembly and disassembly of this complex, while ADP antagonized complex formation. The complex was not dependent on, but was stabilized by, parS. The properties indicate that ParA and ParB are binding and bridging multiple DNA molecules to create a large meshwork of protein-DNA molecules that involves both specific and non-specific DNA. We propose that this complex represents a dynamic adaptor complex between the plasmid and nucleoid, and further, that this interaction drives the redistribution of partition proteins and the plasmid over the nucleoid during partition.