The Legionella pneumophila prp locus; required during infection of macrophages and amoebae

Microbial Pathogenesis 1999; 27: 369–376

Article available online at http://www.idealibrary.com on

Article No. mpat.1999.0311

MICROBIAL PATHOGENESIS

The Legionella pneumophila prp locus; required during infection of macrophages and amoebae Barbara J. Stone, Adam Brier & Yousef Abu Kwaik∗ Department of Microbiology and Immunology, University of Kentucky Chandler Medical Center, Lexington, Kentucky 40536-0084, U.S.A. (Received March 23, 1999; accepted in revised form June 16, 1999)

Transposon mutagenesis was performed using mTn10phoA to identify Legionella pneumophila genes that are expressed under certain in vitro conditions, and are required for intracellular replication. Of the 1653 PhoA fusions examined, 19 PhoA+ fusion mutants were isolated and screened for differential expression of fusion proteins after growth at 30 or 37°C, in the presence of low iron, or increased magnesium concentrations. The mutants were examined for their cytopathogenicity and intracellular replication within U937 macrophage-like cells and the protozoan Hartmannella vermiformis. One of the mutants generated, BS10, was defective in its multiplication within U937 macrophage-like cells and H. vermiformis. The defect in BS10 was complemented with a cosmid clone containing the wild type locus. The open reading frame interrupted by the insertion was homologous to prpD of Salmonella typhimurium and mmgE of Bacillus subtilis.  1999 Academic Press

Key words: intracellular, pmi, iron, PhoA, prpD.

Introduction Legionella pneumophila is the causative agent of Legionnaires’ disease. Infection with L. pneumophila is a leading cause of atypical pneumonias in immunocompetent adults with communityacquired pneumonia (see [1, 2] for recent reviews). An estimate of 25 000 cases of pneumonia are caused by L. pneumophila each year in the United States [1, 3]. Documented cases of fatal Legionnaires’ disease in cattle have also been ∗ Author for correspondence. 0882–4010/99/120369+08 $30.00/0

reported [4]. The hallmark of Legionnaires’ disease is the intracellular bacterial replication within macrophages [1, 2]. In addition, it is most likely that intracellular replication within type I and II alveolar epithelial cells contribute to the pathogenesis of Legionnaires’ disease [5–7]. In the environment, L. pneumophila invades and replicates within protozoa, and this host– parasite interaction is central to bacterial ecology and pathogenesis [2, 3]. Initial bacterial attachment to host cells is mediated by several adhesins including the type IV CAP pilus [8–10], the HSP60 [11], and the major outer membrane protein opsonized by  1999 Academic Press

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complement [12]. Uptake by mammalian cells is mediated by the complement receptor and also by noncomplement-mediated mechanisms [12–14]. Uptake by the protozoan H. vermiformis is mediated by the Gal/GalNAc lectin receptor [3, 15, 16]. Following entry into the host cell, L. pneumophila replicates within a phagosome that is blocked from maturation through the endosomal–lysosomal degradation pathway [17, 18], and is surrounded by the rough endoplasmic reticulum [19, 20] (see [2, 3] for recent reviews). Therefore, the phagosome has been designated as endosomal maturation-blocked (EMB) phagosome [2, 3]. Intracellular bacterial replications is associated with cytopathogenicity of the host cell. We have recently provided a biphasic model by which L. pneumophila kills mammalian cells [21]. Apoptosis is induced during early stages of the infection [21, 22] followed by rapid and independent induction of necrosis, which seems to be mediated by temporal expression of the pore-forming toxin upon termination of replication [23, 24]. It has been shown that within the unique intracellular niche, L. pneumophila undergoes dramatic phenotypic modulations during the intracellular infection [23, 25–30]. Several approaches to characterize the bacterial genes induced during the intracellular infection have identified several genes induced by L. pneumophila, including Hsp60 [27, 30], ppa [25], and gspA [31]. Promoter fusion studies have shown that gspA is a stress-induced gene that is also induced throughout the intracellular infection period, indicating constitutive exposure of intracellular L. pneumophila to stress stimuli [32]. Differential display-PCR has been used to identify eml, a locus induced during early stages of infection in macrophages [33]. The intracellular signals that trigger the alterations in bacterial gene expression are still to be identified. Many mutants of L. pneumophila that are defective for intracellular replication have previously been identified. Mutants defective in iron acquisition and assimilation and also defective in the intracellular infection have been isolated, and one of the defective loci encodes for aerobactin synthetase [34, 35]. The dot/icm loci, which are thought to be involved in the assembly of a type IV-like secretion apparatus, are required for intracellular replication within macrophages [36, 37]. Mutants that are defective within both macrophages and protozoa have been identified, and designated as protozoa and

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macrophage-infectivity loci (pmi) [38]. Interestingly, the defect in three of the pmi mutants is due to insertions within dotA and icmwxyz, clearly indicating that the dot/icm secretion apparatus is essential for infectivity of both macrophages and protozoa [38]. These data have been recently substantiated by showing that another 13 icm genes that are completely required for infectivity of macrophages are also required for infectivity of protozoa [39]. In addition, mutants that are specifically defective in intracellular replication within macrophages but exhibit a wild type phenotype within protozoa have been isolated, and designated as macrophage-specific infectivity loci (mil) [40]. Three phoA fusion mutants that are defective in protozoa but exhibit a wild type phenotype within macrophages have also been isolated [41]. In this study, we attempted an alternative approach to identify bacterial genes responsive to environmental signals that may mimic the phagosomal microenvironment. Our approach was to focus on bacterial proteins that are secreted or localized to the outer membrane. These genes are candidates for encoding bacterial factors that interact with the host cell, and are required for intracellular survival and replication.

Results and Discussion Isolation of PhoA+ mutants Since understanding of the environmental signals preceding and during intracellular replication is limited, mutants with differential expression of fusion proteins after growth under various conditions were isolated and subsequently examined for defects in infectivity of macrophages and amoebae. We utilized phoA fusion to identify bacterial factors that are secreted or are outer membrane proteins. Of 1653 kanR sucS colonies isolated, 19 were phoA+ under at least one of the conditions screened (Table 1). The phoA+ isolates were screened three times to confirm their phenotypes. One phoA+ fusion was selectively expressed at 37°C but not at 30°C and one was selectively expressed at 30°C but not at 37°C. Fourteen fusions were induced under low iron conditions, and five were induced by 50 mM Mg2+. Some fusions were induced by more than one environmental condition tested. Studies of two of the mutants (BS12 and BS19) were not pursued as the mutants exhibited reduced growth on BCYE plates.

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37°C

30°C

Low Iron

50 mM MgSO4

AA100 BS1 BS2 BS3 BS4 BS5 BS6 BS7 BS8b BS9 BS10 BS11 BS12 BS13b BS14b BS15b BS16b BS17 BS18 BS19

− − − + + + + + − − − + − − − − − + + +

− − − + + + + + − − − + + − − − − + + −

− + + + + + + + − + + + + − − − − + + +

− − − + + + − + − − − + − − − − − +/− +/− −

a

Phenotypes indicated are representative after three screenings; + and − indicate PhoA+ or PhoA− under the respective condition, respectively; − indicates inconclusive results. b Mutant exhibiting a PhoA+ phenotype during the initial screen which was not reproduced during subsequent testing.

The rest of the mutants were indistinguishable from the parent strain, when compared for growth on BCYE agar plates.

Cytopathogenicity of the mutants to U937 macrophage-like cells U937 macrophage-like cells were infected at an moi of 0.1 and cytopathogenicity was determined at 72 h post-infection using Alamar Blue. Of the 17 phoA+ isolates tested, seven exhibited decreased cytopathogenicity to U937 macrophage-like cells, compared to the wildtype strain AA100 (Fig. 1). Reduction in the ability of the seven mutants to kill macrophages was less than 50%. Therefore, the seven mutants exhibited defects in cytopathogenicity to U937 macrophage-like cells. We have recently proposed a model to show that L. pneumophilamediated cytopathogenicity is manifested in two phases. These two phases are mediated by two

100 75 50 25 0

AA100 BS1 BS2 BS3 BS4 BS5 BS6 BS7 BS8 BS9 BS10 BS11 BS13 BS14 BS15 BS16 BS17 BS18 Uninfected

Strain

125 Macrophage survival (%)

Table 1. L. pneumophila strain phoA+ phenotypesa under various culture conditions

Figure 1. Cytopathogenicity of L. pneumophila phoA+ mutants and the wild type strain AA100 to U937 macrophage-like cells. Viability of macrophages was determined at 72 h post-infection. Error bars represent standard deviations.

independent processes that include both apoptosis during early stages of the infection followed by necrosis during late stages of the infection [21]. It is most likely that the reduced cytopathogenicity of these mutants is due to the lower number of intracellular bacteria that did not become cytotoxic because the bacteria did not reach the post-exponential phase of growth [23]. However, we cannot exclude that some of them may be defective in induction of apoptosis.

Intracellular replication of the mutants within U937 macrophage-like cells and amoebae Although seven phoA+ mutants were defective for cytopathogenicity, only one of the mutants, BS10, was defective when compared to AA100 for survival and intracellular replication within U937 macrophage-like cells (Fig. 2 and data not shown). This mutant was selected because it showed a marked defect in intracellular replication. Although U937 macrophage-like cells were infected with equal numbers of bacteria of AA100 and BS10, there was a 20 to 50-fold reduction in the number of intracellular BS10 when examined at the first time point following the gentamicin treatment to kill extracellular bacteria (2 h post-inoculation). This early decrease in the number of BS10 indicated that either BS10 was taken into U937 macrophagelike cells less efficiently than AA100 or that BS10

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8

*

6

*

7

*

*

*

*

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Log cfu/ml

Log cfu/ml

6

4

4

*

* 3

2

*

5

3

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20 30 Time (h)

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Figure 2. Intracellular replication of BS10 within macrophage-like cells. Equal numbers of bacteria were added to U937 macrophages for 1 h followed by washing off the extracellular bacteria and gentamicin treatment for 1 h to kill extracellular bacteria. The initial time point (t=0) represent the first time point immediately after gentamicin treatment. The intracellular bacteria were enumerated at the time points indicated after gentamicin treatment. (∗), AA100; (Β), BS10; (Χ), BS10 pCC7. Error bars represent standard deviations.

bacteria were partially killed just after entry into U937 macrophage-like cells, or possibly, both. BS10 was also partially defective for intracellular replication, as compared to AA1000. At 48 h post-infection, the number of intracellular BS10 was at least a 100-fold less than that of the parental strain AA100. Intracellular replication was also determined for BS10 in the protozoan H. vermiformis. Although equal numbers of AA100 and BS10 (104 cfu/ml) were added to H. vermiformis cultures, BS10 was defective for survival at early time points of the infection, similar to that of macrophages. This most likely represents intracellular killing, since viability of the bacteria was not altered in the medium alone (data not shown). At 24 h post-infection, the number of intracellular BS10 was approximately a 100-fold less than that of the parental strain AA100. BS10 was subsequently able to reach the same intracellular replication endpoint as AA100 by day 7 after infection (Fig. 3). Interestingly, the intracellular growth of BS10 within amoebae was closer to the wild type strain, in contrast to the marked

2

0

1

2

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4 5 Time (day)

6

7

8

Figure 3. Intracellular replication of BS10 within H. vermiformis compared to the wild type strain AA100. Equal numbers of bacteria were added to triplicate cultures of H. vermiformis and bacteria were enumerated at the time points indicated. (∗), AA100; (Β), BS10; (Χ), BS10 pCC7. Error bars represent standard deviations.

defect in macrophages. Interestingly, the phenotype of the BS10 mutant is similar to many other mutants of L. pneumophila, including the mip mutant [42, 43]. The phenotype of BS10 is distinct from the three phoA insertion mutants that have been recently reported, and are defective only in H. vermiformis but not in U937 macrophages [41].

Complementation of the defective locus in BS10 To identify the defective locus in BS10, chromosomal DNA from BS10 was restriction-digested with EcoRI, and a 6 kb fragment containing the kan resistance marker was cloned into pBC SK+ to probe the cosmid library of strain AA100 [8]. The wild type locus mutated in BS10 was isolated on the cosmid pCC7, which was confirmed by Southern hybridizations (data not shown). The pCC7 cosmid contains approximately 35 kb of DNA (data not shown). Southern hybridization and restriction digestion analyses indicated that pCC7 contained the whole defective locus in BS10 and several kbs of DNA upstream and downstream of it (data not shown). When introduced into BS10, cosmid pCC7 restored cytopathogenicity of BS10 to

The prp locus of Legionella pneumophila

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125

Macrophage survival (%)

100

75

50

Uninfected

BS10 pCC7

BS10

0

AA100

25

Figure 4. Restoration of cytopathogenicity to BS10 by pCC7. Bacteria were added to macrophages and viability of the host cell was determined at 72 h postinfection. Error bars represent standard deviations.

macrophages (Fig. 4). The cosmid also restored the early defect in intracellular survival of BS10 to the wild type levels during infections of H. vermiformis (Fig. 3). Additionally, pCC7 partially restored the defects for uptake/early survival and intracellular replication during infection of U937 macrophage-like cells (Fig. 2). These data indicated that pCC7 complemented the Tn10phoA insertion mutation in BS10.

Characterization of the defective locus in BS10 The DNA sequence flanking the insertion mutation in BS10 was determined from pBJ118 (GenBank accession number AF157018), and aligned to published sequences in genetic databases. The predicted amino acid sequence derived from the open reading frame disrupted by the transposon insertion contained 68% identity and 79% similarity to the first 246 (of 483) amino acids of prpD from Salmonella typhimurium and Escherichia coli and 65% identity and 76% similarity to the first 238 (of 472) amino acids of mmgE from Bacillus subtilis [44]. Therefore, based upon sequence homology, the L. pneumophila locus has been designated prpD. The transposon insertion was in the opposite orientation of transcription of

the disrupted open reading frame of prpD. Therefore, the observed iron-regulated PhoA fusion protein produced was not an indication of environmental responsive expression from prpD. The reason for the iron-regulated phenotype of BS10 and its relationship to the prp locus is not known at this time. In S. typhimurium, the prp locus is an operon comprised of four genes with the genetic organization prpBCDE, and is involved in proprionate catabolism [45]. prpB encodes for carboxyphosphoenolpyruvate phosphomutase, prpC encodes for citrate synthase, prpD encodes for a protein of unknown function, and prpE encodes for acetyl coenzyme A synthase [45]. Interestingly, the complete open reading frame upstream of prpD in L. pneumophila also had homology to a lage number of citrate synthase genes including prpC of S. typhimurium (52% identity and 75% similarity). Therefore, this L. pneumophila gene was designated as prpC. The prpC and prpD genes in L. pneumophila appeared to be separated by 32 nucleotides, consistent with the genetic organization in S. typhimurium. In conclusion, the prp locus of L. pneumophila is required for cytopathogenicity to U937 macrophage-like cells, intracellular survival and replication within macrophages and amoebae. As BS10, the prpD mutant, contains a transposon insertion, the possibility of polar affects on other prp genes within the prp operon, and downstream of prpD, is possible. However, homology of the prpCD of L. pneumophila to that of S. typhimurium and E. coli suggests a role for the prp locus of L. pneumophila in a biosynthetic pathway required during infection of U937 macrophage-like cells and amoebae. Interestingly, this biosynthetic pathway was not required for growth in vitro.

Materials and Methods Transposon mutagenesis L. pneumophila strain AA100 [27] was mutagenized by the transposon mTn10phoA to generate phoA fusions in genes encoding secreted or membrane products. Plasmid pMM237 [46] was electroporated into L. pneumophila wild-type strain AA100 and mutants were selected on buffered charcoal yeast extract agar (BCYE) [47] containing 10 g/ml kanamycin (kan) and 5% sucrose, as we described previously [38]. Mutant

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strains were grown on BCYE containing 20 g/ ml kan after the original selection.

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were removed at the indicated time points, and the bacteria were enumerated following growth on agar plates.

Selection of mutants Genetic manipulations Kanamycin resistant colonies wre replica-plated onto BCYE plates for growth under various conditions. Activity of the PhoA gene fusion was compared for growth at 37°C, 30°C, in the presence of 50 mM MgSO4, and in the presence of low levels of iron (addition of ferric pyrophosphate was omitted from BCYE). Kanamycin resistant mutants were screened for PhoA activity on agar plates containing 5-bromo-4chloro-3-indolyl phosphate buffered to pH 11.0, as previously described [48].

Cytopathogenicity assays The human U937 macrophage-like cell line was maintained and differentiated into macrophagelike cells by phorbol 12-myristate 13-acetate (Sigma, St. Louis, MO, U.S.A.), as we previously described [38]. Infections were performed, in triplicate, in 96 well plates at an moi of 0.1 at 37°C. Cytopathogenicity (loss of cell viability) of the host cell was determined at 72 h postinfection using the Alamar blue (AccuMed, Westlake, OH, U.S.A.) assay, and expressed as we described previously [21].

Intracellular replication within U937 macrophage-like cells and H. vermiformis U937 macrophage-like cells were infected with the mutant strains as we described previously [38]. Briefly, bacteria were added, in triplicate, to monolayers at an moi of 10 for 1 h then treated with gentamicin for 1 h to kill extracellular bacteria, which were subsequently washed off. The initial time point (t=0) represents the first time point immediately after gentamicin treatment, and represents intracellular bacteria that were enumerated following hypotonic lysis of the host cell. Infections were allowed to proceed until the indicated time point prior to hypotonic lysis of macrophages and enumeration of bacteria following growth on agar plates. Maintenance and infection of H. vermiformis by L. pneumophila was performed as we previously described [49]. Briefly, infections were performed in triplicate at an moi of 0.1. Aliquots

Plasmid and cosmid DNA preparations were performed using the QIAGEN plasmid Kit according to manufacturer’s recommendations (QIAGEN Inc, Chatsworth, CA, U.S.A.). Transformations were carried out by electroporation using a Bio Rad Gene Pulser as recommended (Bio Rad, Hercules, CA, U.S.A.). Purification of DNA fragments from agarose gels for Southern hybridization was carried out using a QIAEX kit, according to manufacturer’s recommendations (QIAGEN Inc, Chatsworth, CA, U.S.A.). Transfer of DNA from agarose gels onto membranes, flouresceine labeling of DNA probes, hybridizations, and detection were performed as described before [31]. To identify the locus mutated in BS10, chromosomal DNA from BS10 was restriction-digested with EcoRI (Promega, Madison, WI, U.S.A.) and a 6 kb fragment containing the kan resistance marker was cloned into pBC SK+ (Stratagene, La Jolla, CA, U.S.A.) to generate pBJ118. The 6 kb fragment was used to probe, by in situ colony hybridization, the cosmid library constructed from AA100 chromosomal DNA, as we previously described [8]. The wild type locus mutated in BS10 was isolated on the cosmid pCC7 and introduced into BS10 by triparental conjugation, as we described previously [33]. DNA sequence analysis was performed, as described previously [8].

Acknowledgements We thank N. C. Engleberg and M. McClain for the pmm 237 plasmid. B. J. S. was supported by National Research Service Award CA09509 and YA is supported by Public Health Service Award R29AI38410.

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