The Legionella pneumophila response regulator LqsR promotes host cell interactions as an element of the virulence regulatory network controlled by RpoS and LetA

Legionella pneumophila is an opportunistic human pathogen that replicates within environmental amoebae including Acanthamoeba castellanii and Dictyostelium discoideum. The Icm/Dot type IV secretion system promotes phagocytosis and intracellular replication of L. pneumophila in an endoplasmic reticulum-derived 'Legionella-containing vacuole' (LCV). L. pneumophila adopts a biphasic life cycle consisting of a replicative growth phase and a transmissive (stationary) phase, the latter of which is characterized by the preferential expression of genes required for motility and virulence. A bioinformatic analysis of the L. pneumophila genome revealed a gene cluster homologous to the Vibrio cholerae cqsAS genes, encoding a putative quorum sensing autoinducer synthase (lqsA) and a sensor kinase (lqsS), which flank a novel response regulator (lqsR). We report here that an L. pneumophila lqsR deletion mutant grew in broth with the same rate as wild-type bacteria, but entered the replicative growth phase earlier. Overexpression of lqsR led to an elongated morphology of the bacteria. The lqsR mutant strain was found to be more salt-resistant and impaired for intracellular growth in A. castellanii, D. discoideum and macrophages, formation of the ER-derived LCV and toxicity. Moreover, L. pneumophila lacking LqsR, as well as strains lacking the stationary sigma factor RpoS or the two-component response regulator LetA, were phagocytosed less efficiently by A. castellanii, D. discoideum or macrophages. The expression of lqsR was dependent on RpoS and, to a lesser extent, also on LetA. DNA microarray experiments revealed that lqsR regulates the expression of genes involved in virulence, motility and cell division, consistent with a role for LqsR in the transition from the replicative to the transmissive (virulent) phase. Our findings indicate that LqsR is a novel pleiotropic regulator involved in RpoS- and LetA-controlled interactions of L. pneumophila with phagocytes.     

The global regulatory proteins LetA and RpoS control phospholipase A, lysophospholipase A, acyltransferase, and other hydrolytic activities of Legionella pneumophila JR32

Legionella pneumophila possesses a variety of secreted and cell-associated hydrolytic activities that could be involved in pathogenesis. The activities include phospholipase A, lysophospholipase A, glycerophospholipid:cholesterol acyltransferase, lipase, protease, phosphatase, RNase, and p-nitrophenylphosphorylcholine (p-NPPC) hydrolase. Up to now, there have been no data available on the regulation of the enzymes in L. pneumophila and no data at all concerning the regulation of bacterial phospholipases A. Therefore, we used L. pneumophila mutants in the genes coding for the global regulatory proteins RpoS and LetA to investigate the dependency of hydrolytic activities on a global regulatory network proposed to control important virulence traits in L. pneumophila. Our results show that both L. pneumophila rpoS and letA mutants exhibit on the one hand a dramatic reduction of secreted phospholipase A and glycerophospholipid:cholesterol acyltransferase activities, while on the other hand secreted lysophospholipase A and lipase activities were significantly increased during late logarithmic growth phase. The cell-associated phospholipase A, lysophospholipase A, and p-NPPC hydrolase activities, as well as the secreted protease, phosphatase, and p-NPPC hydrolase activities were significantly decreased in both of the mutant strains. Only cell-associated phosphatase activity was slightly increased. In contrast, RNase activity was not affected. The expression of plaC, coding for a secreted acyltransferase, phospholipase A, and lysophospholipase A, was found to be regulated by LetA and RpoS. In conclusion, our results show that RpoS and LetA affect phospholipase A, lysophospholipase A, acyltransferase, and other hydrolytic activities of L. pneumophila in a similar way, thereby corroborating the existence of the LetA/RpoS regulation cascade.     

A two-component regulator induces the transmission phenotype of stationary-phase Legionella pneumophila

Pathogenic Legionella pneumophila evolved as a parasite of aquatic amoebae. To persist in the environment, the microbe must be proficient at both replication and transmission. In laboratory cultures, as nutrients become scarce a stringent response-like pathway coordinates exit from the exponential growth phase with induction of traits correlated with virulence, including motility. A screen for mutants that express the flagellin gene poorly identified five activators of virulence: LetA/LetS, a two-component regulator homologous to GacA/GacS of Pseudomonas and SirA/BarA of Salmonella; the stationary-phase sigma factor RpoS; the flagellar sigma factor FliA; and a new locus, letE. Unlike wild type, post-exponential-phase letA and letS mutants were not motile, cytotoxic, sodium sensitive or proficient at infecting macrophages. L. pneumophila also required fliA to become motile, cytotoxic and to infect macrophages efficiently and letE to express sodium sensitivity and maximal motility and cytotoxicity. When induced to express RelA, all of the strains exited the exponential phase, but only wild type converted to the fully virulent form. In contrast, intracellular replication was independent of letA, letS, letE or fliA. Together, the data indicate that, as the nutrient supply wanes, ppGpp triggers a regulatory cascade mediated by LetA/ LetS, RpoS, FliA and letE that coordinates differentiation of replicating L. pneumophila to a transmissible form.     

The Legionella pneumophila rpoS Gene Is Required for Growth within Acanthamoeba castellanii

To investigate regulatory networks in Legionella pneumophila, the gene encoding the homolog of the Escherichia coli stress and stationary-phase sigma factor RpoS was identified by complementation of an E. coli rpoS mutation. An open reading frame that is approximately 60% identical to the E. coli rpoS gene was identified. Western blot analysis showed that the level of L. pneumophila RpoS increased in stationary phase. An insertion mutation was constructed in the rpoS gene on the chromosome of L. pneumophila, and the ability of this mutant strain to survive various stress conditions was assayed and compared with results for the wild-type strain. Both the mutant and wild-type strains were more resistant to stress when in stationary phase than when in the logarithmic phase of growth. This finding indicates that L. pneumophila RpoS is not required for a stationary-phase-dependent resistance to stress. Although the mutant strain was able to kill HL-60- and THP-1-derived macrophages, it could not replicate within a protozoan host, Acanthamoeba castellanii. These data suggest that L. pneumophila possesses a growth phase-dependent resistance to stress that is independent of RpoS control and that RpoS likely regulates genes that enable it to survive in the environment within protozoa. Our data indicate that the role of rpoS in L. pneumophila is very different from what has previously been reported for E. coli rpoS.     

Identification of CpxR as a positive regulator of icm and dot virulence genes of Legionella pneumophila

To date, 24 Legionella pneumophila genes (icm and dot genes) have been shown to be required for intercellular growth and host cell killing. A previous report indicated that the regulation of these genes is complicated and probably involves several regulatory proteins. In this study, a genetic screen performed in Escherichia coli identified the CpxR response regulator as an activator of the L. pneumophila icmR gene. Construction of an L. pneumophila cpxR insertion mutant showed that the expression of icmR is regulated by CpxR. In addition, a conserved CpxR binding site (GTAAA) was identified in the icmR regulatory region and L. pneumophila His-tagged CpxR protein was shown to bind to the icmR regulatory region using a mobility shift assay. Besides its dramatic effect on the icmR level of expression, the CpxR regulator was also found to affect the expression of the icmV-dotA and icmW-icmX operons, but to a lesser extent. The role of CpxA, the cognate sensor kinase of CpxR, was also examined and its effect on the icmR level of expression was found to be less pronounced than the effect of CpxR. The RpoE sigma factor, which was shown to coregulate genes together with CpxR, was examined as well, but it did not influence icm and dot gene expression. In addition, when the cpxR mutant strain, in which the expression of the icmR gene was dramatically reduced, and the cpxA and rpoE mutant strains were examined for their ability to grow inside Acanthamoeba castellanii and HL-60-derived human macrophages, no intracellular growth defect was observed. This study presents the first evidence for a direct regulator (CpxR) of an icm-dot virulence gene (icmR). The CpxR regulator together with other regulatory factors probably concerts with the expression of icm and dot genes to result in successful infection.     

Temperature-dependent parasitic relationship between Legionella pneumophila and a free-living amoeba (Acanthamoeba castellanii)

We analyzed the effects of temperature on the interaction of Legionella pneumophila with Acanthamoeba castellanii. At <20 degrees C, overexpression of type 1 metacaspase, a stimulator of A. castellanii encystation, was associated with a reduced number of bacteria within amoeba. At low temperatures, A. castellanii seems to eliminate L. pneumophila by encystation and digestion.     

Legionella pneumophila CsrA is a pivotal repressor of transmission traits and activator of replication

Legionella pneumophila can replicate inside amoebae and also alveolar macrophages to cause Legionnaires' Disease in susceptible hosts. When nutrients become limiting, a stringent-like response coordinates the differentiation of L. pneumophila to a transmissive form, a process mediated by the two-component system LetA/S and the sigma factors RpoS and FliA. Here we demonstrate that the broadly conserved RNA binding protein CsrA is a global repressor of L. pneumophila transmission phenotypes and an essential activator of intracellular replication. By analysing csrA expression and the phenotypes of csrA single and double mutants and a strain that expresses csrA constitutively, we demonstrate that, during replication in broth, CsrA represses every post-exponential phase phenotype examined, including cell shape shortening, motility, pigmentation, stress resistance, sodium sensitivity, cytotoxicity and efficient macrophage infection. At the transition to the post-exponential phase, LetA/S relieves CsrA repression to induce transmission phenotypes by both FliA-dependent and -independent pathways. For L. pneumophila to avoid lysosomal degradation in macrophages, CsrA repression must be relieved by LetA/S before phagocytosis; conversely, before intracellular bacteria can replicate, CsrA repression must be restored. The reciprocal regulation of replication and transmission exemplified by CsrA likely enhances the fitness of microbes faced with fluctuating environments.     

The response regulator PmrA is a major regulator of the icm/dot type IV secretion system in Legionella pneumophila and Coxiella burnetii

Legionella pneumophila and Coxiella burnetii have been shown to utilize the icm/dot type IV secretion system for pathogenesis and recently a large number of icm/dot-translocated substrates were identified in L. pneumophila. Bioinformatic analysis has revealed that 13 of the genes encoding for L. pneumophila-translocated substrates and five of the C. burnetii icm/dot genes, contain a conserved regulatory element that resembles the target sequence of the PmrA response regulator. Experimental analysis which included the construction of a L. pneumophila pmrA deletion mutant, intracellular growth analysis, comparison of gene expression between L. pneumophila wild type and the pmrA mutant, construction of mutations in the PmrA conserved regulatory element, controlled expression studies as well as mobility shift assays, demonstrated the direct relation between the PmrA regulator and the expression of L. pneumophila icm/dot-translocated substrates and several C. burnetii icm/dot genes. Furthermore, genomic analysis identified 35 L. pneumophila and 68 C. burnetii unique genes that contain the PmrA regulatory element and few of these genes from L. pneumophila were found to be new icm/dot-translocated substrates. Our results establish the PmrA regulator as a fundamental regulator of the icm/dot type IV secretion system in these two bacteria.     

Multiplication of different Legionella species in Mono Mac 6 cells and in Acanthamoeba castellanii.

Survival and distribution of legionellae in the environment are assumed to be associated with their multiplication in amoebae, whereas the ability to multiply in macrophages is usually regarded to correspond to pathogenicity. Since most investigations focused on Legionella pneumophila serogroup 1, we examined the intracellular multiplication of different Legionella species in Mono Mac 6 cells, which express phenotypic and functional features of mature monocytes, and in Acanthamoeba castellanii, an environmental host of Legionella spp. According to the bacterial doubling time in Mono Mac 6 cells and in A. castellanii, seven clusters of legionellae could be defined which could be split further with regard to finer differences. L. longbeachae serogroup 1, L. jordanis, and L. anisa were not able to multiply in either A. castellanii or Mono Mac 6 cells and are members of the first cluster. L. dumoffi did not multiply in Mono Mac 6 cells but showed a delayed multiplication in A. castellanii 72 h after infection and is the only member of the second cluster. L. steigerwaltii, L. gormanii, L. pneumophila serogroup 6 ATCC 33215, L. bozemanii, and L. micdadei showed a stable bacterial count in Mono Mac 6 cells after infection but a decreasing count in amoebae. They can be regarded as members of the third cluster. As the only member of the fourth cluster, L. oakridgensis was able to multiply slight in Mono Mac 6 cells but was killed within amoebae. A strain of L. pneumophila serogroup 1 Philadelphia obtained after 30 passages on SMH agar and a strain of L. pneumophila serogroup 1 Philadelphia obtained after intraperitoneal growth in guinea pigs are members of the fifth cluster, which showed multiplication in Mono Mac 6 cells but a decrease of bacterial counts in A. castellanii. The sixth cluster is characterized by intracellular multiplication in both host cell systems and consists of several strains of L. pneumophila serogroup 1 Philadelphia, a strain of L. pneumophila serogroup 2, and a fresh clinical isolate of L. pneumophila serogroup 6. Members of the seventh cluster are a strain of agar-adapted L. pneumophila serogroup 1 Bellingham and a strain of L. pneumophila serogroup 1 Bellingham which was passaged fewer than three times on BCYE alpha agar after inoculation and intraperitoneal growth in guinea pigs. In comparison to members of the sixth cluster, both strains showed a slightly enhanced multiplication in Mono Mac 6 cells but a reduced multiplication in amoebae. From our investigations, we could demonstrate a correlation between prevalence of a given Legionella species and their intracellular multiplication in Mono Mac 6 cells. Multiplication of members of the genus Legionella in A. castellanii seems to be dependent on mechanisms different from those in monocytes.     

Characterization of a Legionella pneumophila relA Insertion Mutant and Roles of RelA and RpoS in Virulence Gene Expression

To investigate the involvement of RelA in the regulation of Legionella pneumophila virulence, a deletion substitution was constructed in the relA gene. The relA knockout resulted in an undetectable level of ppGpp in the cells during the stationary phase, but the original level was restored when the relA gene product was supplied on a plasmid. The effect of the relA mutation was examined with two systems that are known to be expressed during the stationary phase in L. pneumophila. Pigment production was found to be dependent on the relA gene product, and only one-half as much pigment was produced by the relA mutant as by the wild-type strain. Flagellum gene expression was also found to be dependent on the relA gene product, as determined with a flaA::lacZ fusion. However, the relA gene product was found to be dispensable for intracellular growth both in HL-60-derived human macrophages and in the protozoan host Acanthamoeba castellanii. To determine the involvement of the relA gene product in expression of L. pneumophila genes required for intracellular growth (icm/dot genes), nine icm::lacZ fusions were constructed, and expression of these fusions in the wild-type strain was compared with their expression in relA mutant strains. Expression of only one of the icm::lacZ fusions was moderately reduced in the relA mutant strain. Expression of the nine icm::lacZ fusions was also examined in a strain containing an insertion in the gene that codes for the stationary-phase sigma factor RpoS, and similar results were obtained. We concluded that RelA is dispensable for intracellular growth of L. pneumophila in the two hosts examined and that both RelA and RpoS play minor roles in L. pneumophila icm/dot gene expression.     

Icm/Dot-dependent upregulation of phagocytosis by Legionella pneumophila

Legionella pneumophila is the causative agent of Legionnaires' disease, a severe pneumonia. Dependent on the icm/dot loci, L. pneumophila survives and replicates in macrophages and amoebae within a specialized phagosome that does not fuse with lysosomes. Here, we report that phagocytosis of wild-type L. pneumophila is more efficient than uptake of icm/dot mutants. Compared with the wild-type strain JR32, about 10 times fewer icm/dot mutant bacteria were recovered from HL-60 macrophages in a gentamicin protection assay. The defect in phagocytosis of the mutants could be complemented by supplying the corresponding genes on a plasmid. Using fluorescence microscopy and green fluorescent protein (GFP)-expressing strains, 10-20 times fewer icm/dot mutant bacteria were found to be internalized by HL-60 cells and human monocyte-derived macrophages (HMMPhi). Compared with icm/dot mutants, wild-type L. pneumophila infected two to three times more macrophages and yielded a population of highly infected host cells (15-70 bacteria per macrophage) that was not observed with icm/dot mutant strains. Wild-type and icmT mutant bacteria were found to adhere similarly and compete for binding to HMMPhi. In addition, wild-type L. pneumophila was also phagocytosed more efficiently by Acanthamoeba castellanii, indicating that the process is independent of adherence receptor(s). Wild-type L. pneumophila enhanced phagocytosis of an icmT mutant strain in a synchronous co-infection, suggesting that increased phagocytosis results from (a) secreted effector(s) acting in trans.     

An oxaloacetate decarboxylase homologue protein influences the intracellular survival of Legionella pneumophila

Abstract Legionella pneumophila is a facultative intracellular parasite which is able to survive in various eukaryotic cells. We characterised a Tn5-mutant of the L. pneumophila Corby strain and were able to identify the insertion site of the transposon. It is localised within an open reading frame which shows high homology to the α-subunit of the oxaloacetate decarboxylase (OadA) of Klebsiella pneumoniae . The OadA homologous protein of L. pneumophila was detected in the wild-type strain by Western blotting. Since the intracellular multiplication of the oad A⁻ mutant strain is reduced in guinea pig alveolar macrophages and human monocytes, it is concluded that the oad A gene product has an effect on the intracellular survival of L. pneumophila .     

Studies on the uptake and intracellular replication of Legionella pneumophila in protozoa and in macrophage-like cells

Abstract Legionella pneumophila strains isolated from different sources were tested for their host range in the protists Acanthamoeba castellanii, Hartmannella vermiformis and Entamoeba histolytica . It has been shown that A. castellanii and H. vermiformis but not E. histolytica support the intracellular replication of L. pneumophila . Furthermore it could be demonstrated that in vivo virulence in the guinea pig and the intracellular growth in U937 cells coincides with the capability to replicate intracellularly in A. castellanii at 37°C. The infectivity of L. pneumophila that had sustained a 48 hours nutrient deprivation was not significantly different from that of legionellae grown to log-phase on BCYE plates. In contrast the nutrient limitation on A. castellanii increased the amount of intracellular legionellae at the beginning of infection. An initial opsonin independent attachement stage of legionellae to U937 cells was demonstrated by scanning electron microscopy. In contrast, L. pneumophila's capability of stable or long term attachmennt to A. castellanii was shown to be inefficient.     

Control of cell division by sex factor F in Escherichia coli. II. Identification of genes for inhibitor protein and trigger protein on the 42.84-43.6 F segment

The genetic structure of the 42.84-43.6 F (BamHI-PstI) segment of the F plasmid, which contains all the F DNA sequences necessary for coupling cell division of F+ bacteria with plasmid DNA replication, was analyzed by isolating a series of amber mutants. Two cistrons were found in this region and they were designated letA and letD (an abbreviation for lethal mutation). The letA and letD cistrons were mapped on the 42.84-43.35 F (BamHI- XmaI ) segment and the 43.07-43.6 F (HincII-PstI) segment, respectively, and are presumed to correspond to the first (43.04-43.26 F) and second (43.26-43.57 F) open reading frames, respectively, which were found in this region by nucleotide sequencing. The letD gene product acts to inhibit cell division of the host bacteria and to induce prophages in lysogenic bacteria, whereas the letA gene product acts to suppress the activity of the letD gene product. Taking into consideration the fact that the 42.84-43.6 F segment carries all the F plasmid genes necessary for coupling cell division with plasmid DNA replication, and that the expression of the genes is likely to be controlled by plasmid DNA replication, we constructed the following hypothesis. Before completion of plasmid DNA replication, LetD protein acts to prevent cell division of the host bacteria. When plasmid DNA replication is completed, synthesis of LetA protein (and also LetD protein) takes place and the LetA protein synthesized acts to suppress the activity of LetD protein and make the cell ready for cell division. Actual cell division will take place when replication of both chromosomal and plasmid DNA is completed and the termination protein of the chromosome and the LetA protein of F plasmid are both synthesized. When cell division takes place LetA protein is consumed, and as a result LetD protein becomes active and prevents cell division until the next round of DNA replication is completed.     

Free-living freshwater amoebae differ in their susceptibility to the pathogenic bacterium Legionella pneumophila

Legionella pneumophila is known as a facultative intracellular parasite of free-living soil and freshwater amoebae, of which several species have been shown to support the growth of the pathogenic bacteria. We report for the first time the behaviour of two strains (c2c and Z503) of the amoeba Willaertia magna towards different strains of L. pneumophila serogroup 1 and compared it with Acanthamoeba castellanii and Hartmannella vermiformis , known to be L. pneumophila permissive. In contrast to the results seen with other amoebae, W. magna c2c inhibited the growth of one strain of Legionella ( L. pneumophila , Paris), but not of others belonging to the same serogroup ( L. pneumophila , Philadelphia and L. pneumophila , Lens). Also, the different L. pneumophila inhibited cell growth and induced cell death in A. castellanii, H. vermiformis and W. magna Z503 within 3–4 days while W. magna c2c strain remained unaffected even up to 7 days. Electron microscopy demonstrated that the formation of numerous replicative phagosomes observed within Acanthamoeba and Hartmannella is rarely seen in W. magna c2c cocultured with L. pneumophila . Moreover, the morphological differences were observed between L. pneumophila cultured either with Willaertia or other amoebae. These observations show that amoebae are not all equally permissive to L. pneumophila and highlight W. magna c2c as particularly resistant towards some strains of this bacterium.