Intracellular multiplication of Legionella pneumophila: human pathogen or accidental tourist?
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Animals↗
Biomedical subjects
L A Wiater
Publications and source records attributed to L A Wiater.
Early events in phagosome establishment are required for intracellular survival of Legionella pneumophila.
During infection, the Legionnaires' disease bacterium, Legionella pneumophila, survives and multiplies within a specialized phagosome that is near neutral pH and does not fuse with host lysosomes. In order to understand the molecular basis of this organism's ability to control its intracellular fate, we have isolated and characterized a group of transposon-generated mutants which were unable to kill macrophages and were subsequently found to be defective in intracellular multiplication. These mutations define a set of 20 genes (19 icm [for intracellular multiplication] genes and dotA [for defect in organelle trafficking]). In this report, we describe a quantitative assay for phagosome-lysosome fusion (PLF) and its use to measure the levels of PLF in cells that have been infected with either wild-type L. pneumophila or one of several mutants defective in different icm genes or dotA. By using quantitative confocal fluorescence microscopy, PLF could be scored on a per-bacterium basis by determining the extent to which fluorescein-labeled L. pneumophila colocalized with host lysosomes prelabeled with rhodamine-dextran. Remarkably, mutations in the six genes that were studied resulted in maximal levels of PLF as quickly as 30 min following infection. These results indicate that several, and possibly all, of the icm and dotA gene products act at an early step during phagosome establishment to determine whether L. pneumophila-containing phagosomes will fuse with lysosomes. Although not ruled out, subsequent activity of these gene products may not be necessary for successful intracellular replication.
HL-60 Cells↗
Escherichia coli F plasmid transfers to and replicates within Legionella pneumophila: an alternative to using an RP4-based system for gene delivery.
Derivatives of the self-transmissible F plasmid of Escherichia coli can be introduced into Legionella pneumophila by conjugation and maintained within only upon selection. In L. pneumophila. F-based replicons seem to exist as extrachromosomal elements since they were readily lost when F-containing L. pneumophila was grown on nonselective medium. The F-based plasmids were not self-transmissible in L. pneumophila. The mating defect may be due to an inability to form the F pilus since F-containing strains of L. pneumophila could neither be infected with the pilus-specific phage M13 nor transduced with f1-packaged ColE1 replicons. Currently, the most commonly used transfer system for introducing genetic information into L. pneumophila employs E. coli donors with a chromosomally integrated copy of RP4::Mu to mobilize plasmids bearing the RK2 origin of transfer (oriT). Use of this system to deliver TnphoA for mutagenesis of the L. pneumophila chromosome led to transconjugants that all contained cryptic DNA alterations that involved the plasmid RP4 and phage Mu. No TnphoA transposition was observed in L. pneumophila. The fact that F-mediated conjugation can be used to efficiently transfer plasmids containing the oriT of F to L. pneumophila provides an important alternative to the RP4-based plasmid transfer system and may avoid DNA anomalies in transconjugants that impede genetic analysis. Furthermore, our results demonstrate the promiscuous nature of the F conjugal transfer and replication systems.
Bacteriophage mu↗
Mutagenesis of Legionella pneumophila using Tn903 dlllacZ: identification of a growth-phase-regulated pigmentation gene.
Study of the molecular basis for Legionella pneumophila pathogenicity would be facilitated with an efficient mutagen that can not only mark genomic mutations, but can also be used to reflect gene expression during macrophage infection. A derivative of Tn903, Tn903dlllacZ, is shown to transpose with high efficiency in L. pneumophila. Tn903dlllacZ encodes resistance to kanamycin (KmR) and carries a 5' truncated 'lacZ gene that can form translational fusions to L. pneumophila genes upon transposition. The cis-acting Tn903 transposase is supplied outside Tn903dlllacZ, and hence chromosomally integrated copies are stable. KmR LacZ+ insertion mutants of L. pneumophila were isolated and shown by DNA hybridization to carry a single Tn903dlllacZ inserted within their chromosomes at various locations. One particular KmR LacZ+ mutant, AB1156, does not produce the brown pigment (Pig-) characteristic of Legionella species. Tn903dlllacZ is responsible for this phenotype since reintroduction of the transposon-linked mutation into a wild-type background results in a Pig- phenotype. L. pneumophila pigment production is normally observed in stationary-phase growth of cells in culture, and beta-galactosidase activity measured from the pig::lacZ fusion increased during the logarithmic-phase growth and peaked at the onset of stationary phase. Interestingly, pig::lacZ expression also increased during macrophage infection. The pigment itself, however, does not appear to be required for L. pneumophila to grow within or kill host macrophages.
Alleles↗
Identification of Legionella pneumophila genes required for growth within and killing of human macrophages.
Legionella pneumophila was mutagenized with Tn903dIIlacZ, and a collection of mutants was screened for defects in macrophage killing (Mak-). Of 4,564 independently derived mutants, 55 (1.2%) showed a reduced or complete lack in the ability to kill HL-60-derived human macrophages. Forty-nine of the Mak- mutants could be assigned to one of 16 DNA hybridization groups. Only one group (9 of the 10 members) could be complemented for macrophage killing by a DNA fragment containing icm and dot, two recently described L. pneumophila loci that are required for macrophage killing. Phenotypic analysis showed that none of the mutants were any more sensitive than the wild type to human serum, oxidants, iron chelators, or lipophilic reagents nor did they require additional nutrients for growth. The only obvious difference between the Mak-mutants and wild-type L. pneumophila was that almost all of the Mak- mutants were resistant to NaCl. The effects of LiCl paralleled the effects of NaCl but were less pronounced. Resistance to salt and the inability to kill human macrophages are linked since both phenotypes appeared when Tn903dIIlacZ mutations from two Mak- strains were transferred to wild-type backgrounds. However, salt sensitivity is not a requisite for killing macrophages since a group of Mak- mutants containing a plasmid that restored macrophage killing remained resistant to NaCl. Mak- mutants from groups I through IX associated with HL-60 cells similarly to wild-type L. pneumophila. However, like the intracellular-multiplication-defective (icm) mutant 25D, the Mak- mutants were unable to multiply within macrophages. Thus, the ability of L. pneumophila to kill macrophages seems to be determined by many genetic loci, almost all of which are associated with sensitivity to NaCl.
Bacterial Adhesion↗
The m gamma delta-1 element, a small gamma delta (Tn1000) derivative useful for plasmid mutagenesis, allele replacement and DNA sequencing.
Transposon gamma delta (Tn1000), a 6-kb member of the Tn3 family, is widely used for plasmid mutagenesis. A 1.8-kb derivative of gamma delta was constructed that contains the kan gene from Tn5 and the resolution (res) site from gamma delta cloned between 40-bp inverted repeats of gamma delta's delta (delta) end. This element, named m gamma delta-1, lacks the genes encoding transposase and resolvase, and therefore depends on its host to supply transposition and resolution functions. Thus, in strains lacking gamma delta, m gamma delta-1 will not transpose. The m gamma delta-1 element is shown to be useful for mutagenesis of plasmids, DNA sequencing, and allele replacement (in Streptomyces avermitilis).
Alleles↗
Gamma delta transposase. Purification and analysis of its interaction with a transposon end.
gamma delta, a member of the Tn3 family of prokaryotic transposons, encodes a transposase that binds to the 35-base pair (bp) terminal inverted repeats (IRs) which define the transposing DNA segment. The gamma delta transposase has been overexpressed, identified by molecular weight determination and by immunoblotting, and purified to homogeneity. Production of soluble transposase required the presence of Mg2+ prior to cell lysis. Fractions from a Sephacryl S-300 column contained levels of IR-binding activity that parallel the concentration of transposase, indicating that transposase alone is sufficient for binding to the ends of gamma delta. Hydroxyl radical footprinting indicated that transposase binds to one face of the DNA helix. The protected region extends across the IR and up to 17 bp into the flanking DNA. Integration host factor (IHF), which binds adjacent to transposase, also protects one face of the DNA helix and is shifted about 70 degrees around the helical axis from the transposase protection. Analysis of transposase-DNA complexes by electrophoresis on nondenaturing gels indicated that three complexes, two within the gel and one trapped at the well, result from specific interactions with the IR. The complex in the well and one complex in the gel were analyzed by methylation interference experiments. The results indicate that transposase interacts with specific base pairs between positions 10 and 37 of the IR, a region encompassing three consecutive major and minor grooves. Methylated bases at the very end of the transposon (positions 1-9) and in the flanking DNA did not inhibit transposase binding. Thus, although transposase seems to be in intimate contact throughout the IR of gamma delta and 17 bp of flanking DNA, specific base pair recognition needed for binding appears to be determined by the inner three-quarters of the IR.
Binding Sites↗
Integration host factor increases the transpositional immunity conferred by gamma delta ends.
The ends of the bacterial transposon gamma delta contain adjacent binding sites for gamma delta transposase and integration host factor (IHF). IHF+ and IHF- strains were used in conjunction with gamma delta transposon ends containing or lacking the site for IHF binding to determine the role that IHF plays in various gamma delta-mediated transposition events. IHF was not essential for the transposition of gamma delta and seemed to decrease its frequency of transposition about threefold. IHF played no role in determining the distribution of gamma delta inserts into a target replicon, nor did it significantly alter the frequency of simple transpositions. The only clear role discerned for IHF and the terminal IHF-binding sites was in transposition immunity. IHF stimulated the immunity of those plasmids that contain an end of gamma delta, provided the end included the terminal IHF-binding site. For both ends, the degree of stimulation of immunity was similar to the stimulation of binding of transposase by IHF.
Bacterial Proteins↗
Uncoupling of transpositional immunity from gamma delta transposition by a mutation at the end of gamma delta.
The transposon gamma delta, in common with other members of the Tn3 family, confers transpositional immunity, a phenomenon by which plasmids containing a single transposon end show reduced activity as targets for further insertion by the same element. We found that a copy of a mutant delta end, in which the two terminal base pairs (5' GG) were substituted with cytosines, conferred the same degree of immunity as the unaltered delta end. However, a transposon analog with the mutant delta end as its termini could not transpose. These results suggest that the binding of transposase to a site on a target replicon is sufficient to confer immunity and that immunity does not involve subsequent DNA transactions at the bound target site, analogous to the catalytic processes that occur at the transposon ends during transposition.
Base Composition↗
Gamma delta transposase and integration host factor bind cooperatively at both ends of gamma delta.
gamma delta, a prokaryotic transposon, encodes a transposase that is essential for its transposition. We show here, by DNase I protection experiments, that purified gamma delta transposase binds at the transposon's inverted repeats (IRs). Immediately adjacent to each transposase binding site (and within gamma delta DNA) we have identified a binding site for an additional protein factor, the Escherichia coli-encoded integration host factor (IHF). The binding of transposase and IHF to these adjacent sites is mutually cooperative. An IHF binding-site was also found in the original target DNA, just outside one of the ends of gamma delta. The affinity of IHF for this flanking site is reduced by transposase. These results demonstrate that gamma delta transposase binds at the IRs of gamma delta, and suggest that IHF may be involved in forming a transposase-DNA complex and/or influencing the target site selection during the transposition of gamma delta.
Adenosine Triphosphate↗
Passive transport of K+ and Na+ in human red blood cells: sulfhydryl binding agents and furosemide.
Passive transport pathways for K+ and Na+ were studied in fresh human red blood cells (pretreated with ouabain) by measuring unidirectional influxes. The effects of the sulfhydryl binding agents N-ethylmaleimide (NEM) and p-chloromercuribenzene sulfonate (p-CMBS) and the loop diuretic furosemide were studied. Influxes were measured at equimolar K+ and Na+ concentrations (50 mM) with both ions present and also in K+-free or Na+-free media. Some experiments were carried out in Cl--free media (with NO-3 as the substitute). NEM stimulated K+ influx twofold; the stimulation required Cl- but not Na+. NEM inhibited Na+ influx 20%. Furosemide inhibited both K+ and Na+ influxes. All of furosemide-inhibitable Na+ influx required the presence of K+. However 30% of furosemide-inhibitable K+ influx did not require Na+. All of furosemide-inhibitable K+ influx required Cl-. The ratio of Na+-dependent K+ influx to K+-dependent Na+ influx was 3:1. p-CMBS stimulated both Na+ and K+ influxes. K+ influx in p-CMBS cells required neither Na+ nor Cl-. Likewise p-CMBS-promoted Na+ influx did not require K+. These various results are consistent with two Cl--dependent pathways for K+ transport, one requiring Na+ [perhaps (Na + K + Cl) cotransport] and one independent of Na+ [perhaps (K + Cl) cotransport]. The pathways promoted by p-CMBS are probably independent of the apparent cotransport systems.
4-Chloromercuribenzenesulfonate↗