Regulation of the Legionella mip-promotor during infection of human monocytes.
ABSTRACT The opportunistic pathogen Legionella pneumophila, the etiologic agent of Legionnaires disease, is able to invade and multiply intracellularly in human macrophages. This process is controlled by several bacterial virulence factors. As recently demonstrated, one of these virulence factors, the macrophage infectivity potentiator (Mip) protein, is important for invasion and proper intracellular establishment of L. pneumophila in macrophages and protozoa. Knockout mutants devoid of a functional mip-gene enter host cells much less effectively but intracellular replication is not affected. Using a P(mip)-green fluorescent protein reporter construct in L. pneumophila substrain Corby, P(mip) was recently shown to be constitutively active in replicating bacteria. A stringent regulation during the infection process could not be observed, neither in intracellular nor in BYE broth-grown bacteria. For enhanced temporal and quantitative resolution, we examined the activity of mip on RNA level in order to detect short transient regulatory events. Our results show that P(mip) of L. pneumophila is temporarily repressed directly after invasion of the monocytic human cell line MonoMac 6 and regains activity after 24 h of intracellular replication.
Article: Legionella pneumophila Mip, a surface-exposed peptidylproline cis-trans-isomerase, promotes the presence of phospholipase C-like activity in culture supernatants.[show abstract] [hide abstract]
ABSTRACT: The type II secretion system of Legionella pneumophila promotes pathogenesis. Among the Legionella type II-dependent exoenzymes is a p-nitrophenol phosphorylcholine (p-NPPC) hydrolase whose activity is only partially explained by the PlcA phospholipase C. In a screen to identify other factors that promote secreted hydrolase activity, we isolated a mip mutant. L. pneumophila Mip is a surface-exposed, FK506-binding protein that is needed for optimal infection and has peptidylproline cis-trans-isomerase (PPIase) activity. Since the molecular target of Mip was undefined, we investigated a possible relationship between Mip and the secreted p-NPPC hydrolase activity. In the mip mutant there was a 40 to 70% reduction in secreted activity that was successfully complemented by providing mip on a plasmid. A similar phenotype was observed when we examined four other independently derived mip mutants, and in all cases the defect was complemented by reintroduction of mip. Thus, mip promotes the presence of a p-NPPC hydrolase activity in culture supernatants. We also found that the C terminus of Mip is required for this effect. When supernatants were examined by anion-exchange chromatography, the p-NPPC hydrolase activity associated with Mip proved to be type II dependent but distinct from PlcA. This conclusion was supported by the phenotype of a newly constructed mip plcA double mutant. Thus, Mip promotes the elaboration of a new type II exoprotein. These data provide both the first evidence for a target for Mip and the first indication that a surface PPIase is involved in the secretion or activation of proteins beyond the outer membrane.Infection and Immunity 10/2006; 74(9):5152-60. · 4.16 Impact Factor
Article: The Neisseria meningitidis macrophage infectivity potentiator protein induces cross-strain serum bactericidal activity and is a potential serogroup B vaccine candidate.[show abstract] [hide abstract]
ABSTRACT: A gene encoding a 29-kDa protein from Neisseria meningitidis serogroup B strain MC58 with homology to the macrophage infectivity potentiator (MIP) protein of Legionella pneumophila was cloned and expressed in Escherichia coli, and the purified soluble recombinant protein (rMIP) was used for immunization studies. Analysis of the predicted amino acid sequences of MIP from 13 well-characterized meningococcal strains, isolated from carriers or patients and differing in serogroup, serotype, and subtype, showed that the protein was highly conserved (98 to 100%), with only three distinct sequence types (designated I, II, and III) found. Western blotting showed that the MIP protein was expressed at similar levels by all of these strains. Immunization of mice with type I MC58 rMIP in detergent micelles and liposomes containing monophosphoryl lipid A (MPLA) induced high levels of surface-reactive antibodies with serum bactericidal activity (SBA) titers of 1/1,024 against the homologous strain. Bactericidal antibodies were also induced with the protein in saline alone and liposomes alone (titers, 1/128) but not following adsorption to Al(OH)(3). Significantly, antisera raised against type I rMIP administered in saline or liposomes killed strains of heterologous sequence types II and III with similar SBA titers (1/128 to 1/256). Taken together, these findings suggest that rMIP can provide cross-strain protection against meningococci and should be considered a potential antigen for inclusion in new vaccines against meningococcal infection.Infection and immunity 06/2011; 79(9):3784-91. · 4.21 Impact Factor
Article: Genetic evidence that Legionella pneumophila RpoS modulates expression of the transmission phenotype in both the exponential phase and the stationary phase.[show abstract] [hide abstract]
ABSTRACT: The opportunistic pathogen Legionella pneumophila alternates between two states: replication within phagocytes and transmission between host amoebae or macrophages. In broth cultures that model this life cycle, during the replication period, CsrA inhibits expression of transmission traits. When nutrients become limiting, the alarmone (p)ppGpp accumulates and the sigma factors RpoS and FliA and the positive activators LetA/S and LetE promote differentiation to the transmissible form. Here we show that when cells enter the postexponential growth phase, RpoS increases expression of the transmission genes fliA, flaA, and mip, factors L. pneumophila needs to establish a new replication niche. In contrast, in exponential (E)-phase cells whose (p)ppGpp levels are low, rpoS inhibits expression of transmission traits, on the basis of three separate observations. First, rpoS RNA levels peak in the E phase, suggestive of a role for RpoS during replication. Second, in multiple copies, rpoS decreases the amounts of csrA, letE, fliA, and flaA transcripts and inhibits the transmission traits of motility, infectivity, and cytotoxicity. Third, rpoS blocks expression of cytotoxicity and motility by E-phase bacteria that have been induced to express the LetA activator ectopically. The data are discussed in the context of a model in which the alarmone (p)ppGpp enables RpoS to outcompete other sigma factors for binding to RNA polymerase to promote transcription of transmission genes, while LetA/S acts in parallel to relieve CsrA posttranscriptional repression of the transmission regulon. By coupling transcriptional and posttranscriptional control pathways, intracellular L. pneumophila could respond to stress by rapidly differentiating to a transmissible form.Infection and Immunity 06/2004; 72(5):2468-76. · 4.16 Impact Factor