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Infect Immun. 2008 July; 76(7): 3075–3085.
Published online 2008 April 21. doi:  10.1128/IAI.00209-08
PMCID: PMC2446704

Significant Role for ladC in Initiation of Legionella pneumophila Infection[down-pointing small open triangle]

Abstract

Previously, we identified ladC in a cohort of genes that were present in Legionella pneumophila but absent in other Legionella species. Here we constructed a ladC mutant of L. pneumophila and assessed its ability to replicate in mammalian cell lines and Acanthamoeba castellanii. The ladC mutant was recovered in significantly lower numbers than wild-type L. pneumophila at early time points, which was reversed upon transcomplementation with ladC but not ladCN430A/R434A, encoding a putative catalytically inactive derivative of the protein. In fact, complementation of ladC::Km with ladCN430A/R434A resulted in a severe replication defect within human and amoeba cell models of infection, which did not follow a typical dominant negative phenotype. Using differential immunofluorescence staining to distinguish adherent from intracellular bacteria, we found that the ladC mutant exhibited a 10-fold reduction in adherence to THP-1 macrophages but no difference in uptake by THP-1 cells. When tested in vivo in A/J mice, the competitive index of the ladC mutant dropped fivefold over 72 h, indicating a significant attenuation compared to wild-type L. pneumophila. Although localization of LadC to the bacterial inner membrane suggested that the protein may be involved in signaling pathways that regulate virulence gene expression, microarray analysis indicated that ladC does not influence the transcriptional profile of L. pneumophila in vitro or during A. castellanii infection. Although the mechanism by which LadC modulates the initial interaction between the bacterium and host cell remains unclear, we have established that LadC plays an important role in L. pneumophila infection.

Legionella pneumophila is a widespread gram-negative aquatic bacterium that parasitizes a range of protozoan hosts (43). This close relationship with a eukaryotic host has equipped L. pneumophila with a genetic repertoire that can result in disease when the organism is inhaled by humans. Once introduced into the lungs, L. pneumophila is phagocytosed by alveolar macrophages but is not degraded via phagosome-lysosome fusion events (19). Instead, the L. pneumophila-containing vacuole (LCV) rapidly recruits host proteins involved in organelle trafficking as well as rough endoplasmic reticulum-derived vesicles to generate a protected intracellular niche in which the bacteria replicate (19, 21). Subsequent host cell egress, immunopathology, and lung tissue damage can lead to the potentially fatal form of infectious pneumonia termed Legionnaires' disease.

Central to establishment of the intracellular replicative niche and to L. pneumophila virulence is a type IV secretion system termed the Dot/Icm system (2, 46). Many proteins that are translocated into the host cell via this secretion apparatus have been identified, although mutation of most substrates does not result in altered intracellular trafficking and replication (11, 29, 35, 51). This may be due to a high level of functional redundancy and/or host cell-specific substrates. However, Dot/Icm substrates that contribute to the ability of L. pneumophila to manipulate host cell trafficking events have been identified. These include RalF, a guanine nucleotide exchange factor for ARF GTPases; SdhA, which has been shown to prevent host cell death; and DrrA/SidM, which is a bifunctional protein that acts as a guanine nucleotide exchange factor for Rab1 and also as a displacement factor for the GDP dissociation inhibitor that maintains inactive Rab1 in the host cell cytosol (20, 25, 30, 31, 34, 35). DrrA/SidM therefore plays a central role in the recruitment and activation of Rab1 on the LCV membrane (20, 30).

Other determinants also enhance the virulence of L. pneumophila; these include a type II secretion system (Lsp), a family of Sel1 repeat proteins, the macrophage infectivity potentiator Mip, and a secreted nucleotide triphosphate diphosphohydrolase, Lpg1905 (Lpp1880/Lpl1869), that is similar to human CD39 (8, 9, 37, 42, 45). Our recent genomic comparison between L. pneumophila and Legionella micdadei identified a range of potential virulence determinants, including the Sel1 repeat protein LpnE, which is important for host cell entry and early trafficking events (37, 38), and LadC, a putative adenylate cyclase. Previous work has suggested that LadC is involved in the pathogenesis of L. pneumophila, as ladC is one of a cohort of genes that is not active during in vitro growth but is induced during infection of macrophages (40). Adenylate cyclases are universal enzymes that convert ATP to cyclic AMP (cAMP). Despite regulating a vast range of functions, signaling via cAMP involves three fundamental aspects: (i) activation of an adenylate cyclase by a specific signal to produce cAMP, (ii) propagation of the signal by cAMP binding proteins, and (iii) hydrolysis of cAMP by phosphodiesterases that regulate the signaling event (1). Cyclic nucleotide signaling is crucial to the virulence of many bacterial pathogens either through direct interference with cAMP levels within host cells, as mediated by the Bordetella pertussis toxin CyaA (24), ExoY of Pseudomonas aeruginosa (53), the edema factor of Bacillus anthracis (26), and cholera toxin of Vibrio cholerae (13), or by acting as a signaling molecule within the bacterium. The latter is true for Pseudomonas, Yersinia, and Vibrio spp., where cAMP, produced by adenylate cyclases in response to an external signal such as host cell contact, acts as a cofactor for cAMP regulatory proteins (CRPs) (17, 22, 39, 51). The first CRP identified was the catabolite gene activator protein (CAP) of Escherichia coli (15, 57). The CAP homodimer complex, when bound to cAMP, directly binds DNA to activate transcription at more than 100 promoters (5). Homologues of CAP are found in many pathogens, so the cAMP-CRP complex is considered to be a master regulator of virulence that modulates the transcription of many essential virulence components such as type III secretion systems, flagella, pili, hemolysin, and proteases (17, 22, 39, 51).

Given that LadC was present only in strains of L. pneumophila and not in other, less pathogenic, species of Legionella (37), we hypothesized that this protein may play an important role in L. pneumophila virulence. In this study we investigated the contribution of ladC to the ability of L. pneumophila to replicate in macrophages, epithelial cells, Acanthamoeba castellanii, and the lungs of A/J mice.

MATERIALS AND METHODS

Bacterial strains and culture conditions.

The bacterial strains used in this study are shown in Table Table1.1. L. pneumophila was grown on buffered charcoal-yeast extract (BCYE) agar or in ACES [N-(2-acetamido)-2-aminoethanesulfonic acid]-buffered yeast extract broth at 37°C (36). E. coli strains were cultured aerobically in Luria broth (LB) or on LB agar. When required, antibiotics were used at the following final concentrations: ampicillin at 100 μg/ml, kanamycin at 100 μg/ml for E. coli and 25 μg/ml for L. pneumophila, and chloramphenicol at 12.5 μg/ml for E. coli and 6 μg/ml for L. pneumophila.

TABLE 1.
Bacterial strains and plasmids used in this study

DNA cloning, purification, and sequence analysis.

DNA-modifying enzymes were used in accordance with the manufacturer's recommendations (Promega, WI). PCR products and restriction digests were purified using the Perfectprep gel cleanup kit (Eppendorf, Hamburg, Germany). Plasmids used and produced in this study are listed in Table Table1.1. Plasmid DNA, purified using the QIAprep spin miniprep kit (Qiagen, Hilden, Germany), was prepared for nucleotide sequencing using a Prism Ready Reaction Dye Deoxy Terminator cycle sequencing kit (Applied Biosystems, CA). Automated DNA sequencing was performed with an Applied Biosystems 3730 DNA analyzer. Subsequent DNA sequences were analyzed using Sequencher 3.1.1 software (Gene Codes Corp., MI) and BLAST programs. L. pneumophila genome sequences were accessed via http://genolist.pasteur.fr/LegioList/index.html and http://genome3.cpmc.columbia.edu/~legion/. Bioinformatic analysis of putative adenylate cyclases of L. pneumophila was performed with the Simple Modular Architecture Research Tool (http://smart.embl-heidelberg.de/).

Disruption and transcomplementation of ladC in L. pneumophila 130b.

An insertional mutation in ladC was created via homologous recombination. A 1,360-bp fragment of ladC was amplified by PCR using an annealing temperature of 44°C and the oligonucleotide primers ladCF (5′-CGGATCCTGGCCTACTTCTCATTGT-3′) and ladCR (5′-CCATCGATGAAATGCCGTATGATCTG-3′). The resulting product was cloned into the BamHI and ClaI sites of pCR-Script, and a kanamycin resistance gene from Tn5 was introduced into the native EcoRI site of ladC at nucleotide position 748. The construct was introduced into L. pneumophila 130b via natural transformation, as described previously (37). Kanamycin-resistant clones were assessed by PCR analysis and ampicillin sensitivity to detect replacement of ladC with ladC::Km and the loss of pCR-Script. L. pneumophila 130b ladC::Km was transcomplemented by the introduction of ladC or ladCN430A/R434A on the expression vector pMMB2002 (42). Full-length ladC, with an additional 100 bp upstream, was amplified using 5′-GCTCTAGACATCGTTCTGGAGATTGG-3′ and ladCR, with a PstI recognition sequence replacing the ClaI restriction site, and cloned into pGEM-T Easy (Promega, WI), creating pGEMLadC. The XbaI/PstI fragment was transferred to pMMB2002 to create both pLadC and pLadCN430A/R434A. XbaI/PstI flanking PCR products were also amplified for ladC1-296 and the CycC domain of LadC, ladC297-483, and cloned into pMMB2002. The ladC1-296 construct was amplified using 5′-GCTCTAGACATCGTTCTGGAGATTGG-3′ and 5′-TTATACTTGCGGTAACTCACC-3′, and the CycC domain was amplified from pGEMLadC and pGEMLadCN430A/R434A using 5′-AAGGAATTATAAATGTTTAGCGAGAGACGTAAAG-3′ (the ribosome binding site is underlined and the start codon shown in bold) and ladCR. The resulting PCR products were cloned into the XbaI/PstI sites of pMMB2002 to produce pLadC1-296, pCYC, and pCYCN430A/R434A. These constructs were introduced into L. pneumophila 130b by electroporation.

Site-directed mutagenesis of ladC.

Site-directed mutagenesis of ladC was performed using the QuikChange II site-directed mutagenesis kit (Stratagene, CA) following the manufacturer's instructions. Double-stranded pGEMLadC was used as templates for the complementary pair of mutagenesis oligonucleotides incorporating two amino acid substitutions (N430A and R434A), 5′-GGCGATACTGTCGCTTTGGCGTCAGCAATAGAAAATGC-3′. Underlined codons represent changes to alanine, with specific nucleotide changes shown in bold. As two amino acid changes were introduced simultaneously, 18 cycles of the mutagenesis PCR were performed.

Tissue culture conditions and intracellular infection by L. pneumophila.

A human monocytic cell line, THP-1, and a type II alveolar epithelial cell line, A549, were maintained in RPMI 1640 with 10% fetal bovine serum in 5% CO2 at 37°C. These cells were prepared for infection with stationary-phase L. pneumophila as previously described (37). THP-1 cells were infected at a multiplicity of infection (MOI) of 5 and A549 cells at an MOI of 100.

Coculture of A. castellanii and L. pneumophila.

A. castellanii ATCC 50739 was cultured in PYG 712 medium at 20°C for 72 h prior to harvesting for L. pneumophila infection (4). A. castellanii cells were washed once with A.c. buffer (0.1% trisodium citrate, 0.4 mM CaCl2, 2.5 mM KH2PO4, 4 mM MgSO4, 2.5 mM Na2HPO4, 0.05 mM ferric pyrophosphate) and seeded into 24-well tissue culture trays (Sarstedt, Leicestershire, United Kingdom) at a density of 105 cells/well. Stationary-phase L. pneumophila was added at an MOI of 0.01 and incubated at 37°C. At specific time points, entire coculture wells were collected and plated onto BCYE agar for viable counts. Infections for transcription analysis were performed in 75-cm2 tissue culture flasks (Sarstedt) at an MOI of 50. After a 1-h invasion period, the A. castellanii cells were washed twice with A.c. buffer, and the infection period commenced at 37°C.

Quantitative examination of cell-associated bacteria.

Immunofluorescence was employed to closely examine the initial stages of L. pneumophila infection of THP-1 cells. A total of 2.5 × 105 cells were chemically differentiated with phorbol 12-myristate 13-acetate onto 12-mm glass coverslips (Menzel-Glaser, Braunschweig, Germany) in 24-well tissue culture trays (Sarstedt) for 72 h before being infected with stationary-phase L. pneumophila. Following 15-min and 1-h incubations at 37°C, cells were washed six times with phosphate-buffered saline (PBS) to remove unattached L. pneumophila before being fixed with 4% (wt/vol) paraformaldehyde (pH 7.4) for 1 h. Samples were then stained with rabbit anti-L. pneumophila antibodies (Biodesign, ME), washed with PBS, and then exposed to anti-rabbit immunoglobulin G Alexafluor 594 (Invitrogen, CA). This resulted in specific labeling of cell-associated extracellular bacteria before permeabilization of the THP-1 cells with PBS containing 10% fetal bovine serum and 0.05% saponin for 1 h. Intracellular bacteria were then labeled as described above, using anti-rabbit immunoglobulin G Alexafluor 488 to distinguish between extracellular bacteria, which stained red/green, and intracellular bacteria, which stained green only. All antibodies were diluted in PBS with 10% fetal bovine serum, used at a dilution of 1:50 (Legionella) or 1:200 (secondary antibodies), and incubated with the cells for 1 h at 37°C. Coverslips were mounted in DAKO fluorescent mounting medium (DAKO Corporation, Carpinteria, CA) and stored at 4°C in the dark. Slides were examined under a 100× objective using an Olympus BX51 microscope (Olympus, Tokyo, Japan). Intracellular and extracellular bacteria were quantified blind, and at least 100 fields of view or 100 bacteria were counted for each of three coverslips per strain for three independent infections.

Pulmonary infection of A/J mice with L. pneumophila.

The comparative virulence of L. pneumophila 130b and the ladC::Km derivative within A/J mice was examined via competition assays, as described previously (42). Briefly, 6- to 8-week-old female A/J mice (Jackson Laboratory, ME) were anesthetized and inoculated intratracheally with approximately 105 CFU of each L. pneumophila strain under investigation. At 24 and 72 h following inoculation, mice were sacrificed and their lung tissue isolated. Tissue was homogenized, and complete host cell lysis was achieved by incubation in 0.1% saponin for 15 min at 37°C. Serial dilutions of the homogenate were plated onto both plain and antibiotic-selective BCYE agar to determine the number of viable bacteria and the ratio of wild-type to mutant bacteria colonizing the lung. Competition infections were also performed between L. pneumophila 130b and the complemented ladC mutant.

Production of recombinant LadC and antibody preparation.

The oligonucleotide 5′-AACTGCAGGAGGATATCCAGAAATTTAA-3′ was used in conjunction with ladCR, using an annealing temperature of 46°C, to produce a 1,300-bp DNA fragment which was cloned into the PstI site of the expression vector pRSET C. Expression of recombinant LadC, lacking the first 55 amino acids containing a hydrophobic secretion signal and predicted transmembrane domain, was induced in E. coli BL21(DE3), using 1 mM isopropyl β-d-thiogalactoside (IPTG). Induced E. coli BL21(DE3) cells were lysed using a French pressure cell (ThermoSpectronic, NY) and the insoluble pellet collected via centrifugation. Recombinant LadC was partially purified from inclusion bodies by two washes in 0.1% Triton X-100 in PBS followed by two washes in 0.1% deoxycholate in PBS and then resuspension in PBS. Polyclonal antibodies to LadC were produced by immunization of a rabbit with 500 μg of recombinant LadC suspended in incomplete Freund's adjuvant on days 0, 28, and 49 before exsanguination on day 66 (Chemicon International Inc., CA). Immune serum was absorbed against L. pneumophila 130b ladC::Km and used for immunoblotting, diluted 1:500 in 0.05% (vol/vol) Tween in Tris-buffered saline.

Fractionation of L. pneumophila proteins.

Fifty-milliliter stationary-phase cultures of L. pneumophila 130b ladC::Km(pLadC) were induced with 1 mM IPTG and harvested for fractionation by centrifugation. Following washing in cold PBS, the bacterial pellet was resuspended in 10 ml of 50 mM Tris (pH 7.0) and lysed with 1-mm glass beads in a FastPrep machine for five bursts of 30 s. Intact bacteria and glass beads were removed via centrifugation (10 min, 10,000 × g, 4°C), and the lysed cells were centrifuged using a TLA 100.3 rotor (1 h, 50,000 × g, 4°C). The supernatant from this spin was designated the cytoplasmic protein fraction. The cellular membrane pellet was washed once with 50 mM Tris (pH 7.0) before being gently resuspended in 1 ml of 50 mM Tris (pH 7.0). Ten percent (vol/vol) Triton X-100 was slowly added to this suspension to a final concentration of 1% before being incubated for 30 min on ice and then centrifuged (1 h, 50,000 × g, 4°C). The supernatant was collected and constituted the Triton X-100 soluble fraction. The pellet, containing Triton X-100-insoluble protein, was directly resuspended in sample buffer. Equivalent volumes of L. pneumophila 130b ladC::Km(pLadC) protein fractions and trichloroacetic acid-precipitated culture supernatants were probed, via Western blotting, for the presence of LadC and DotA, using monoclonal anti-DotA antibodies (kindly provided by C. R. Roy).

ladC-phoA gene fusions and alkaline phosphatase assays.

E. coli phoA lacking the start codon and signal sequence was amplified from XL1-Blue genomic DNA using phoAF (5′-CAGGGCGATATTACTGC-3′) and phoAR (5′-GCTCTGGGGCTGAAATAA-3′) with a PCR annealing temperature of 48°C. This 1,368-bp DNA fragment was cloned into the PstI/HindIII sites of pMMB2002 to produce pPhoA. Both full-length ladC and truncated ladC, amplified with ladCF and 5′-GAGTGGACGTAGACGCC-3′ (designed to end after amino acid 184 before the second predicted transmembrane domain), were cloned into the XbaI/PstI sites to create in-frame ladC-phoA fusions on pLadC:PhoA and pLadC1-184-PhoA. These constructs were introduced into L. pneumophila via electroporation. Alkaline phosphatase assays were performed as described previously (44). Briefly, overnight cultures of L. pneumophila 130b carrying pLadC-PhoA or pLadC1-184-PhoA were diluted to an optical density at 600 nm (OD600) of 0.2 and allowed to grow for 6 h before the addition of 1 mM IPTG to induce expression of the PhoA fusion proteins. Following a 1-h induction period, three tubes of 1-ml aliquots of the bacterial culture were pelleted and resuspended in 1 ml of 0.1 M CAPS [3-(cyclohexlamino)-1-propanesulfonic acid] (pH 11.0). An additional aliquot of culture was retained as a whole-cell extract for immunoblotting with rabbit anti-PhoA (Millipore, MA). The CAPS suspensions were diluted 10-fold, and 0.1 ml of 0.4% Sigma 104, dissolved in 0.1 M CAPS (pH 11.0), was added. The reaction mixture was incubated at 37°C until a yellow color change was observed, and 0.1 ml of 1 M K2HPO4 was added to terminate the reaction. Bacteria were pelleted by centrifugation, and the OD420 of the supernatant was measured. Alkaline phosphatase units were calculated using the formula (1,000 × OD420)/(minutes of reaction × culture OD600). The alkaline phosphatase units for the three separate tubes were averaged to provide a value for an individual culture. This experiment was performed with three independent cultures of each strain.

DNA-DNA hybridization.

A microarray containing 70-mer oligonucleotides representing all open reading frames (ORFs) of the L. pneumophila strain Paris genome was utilized to examine genes present in L. pneumophila strain 130b. Two micrograms of genomic DNA was extracted with phenol-chloroform, digested with RsaI, and used as a template for incorporation of Cy5 and Cy3-dUTP (Perkin-Elmer, MA) by a randomly primed polymerization reaction. Labeling reactions were performed at 37°C for 3 h. Labeled DNA was purified using Qiaquick minicolumns (Qiagen), and the integration of cyanine was measured by OD at a range of wavelengths. Hybridizations were performed with 250 pmol of Cy3- and Cy5-labeled genomic DNA within Telechem hybridization chambers following the manufacturer's recommendations (Corning, NY). A dye-swap microarray was also carried out. Slides were scanned on a GenePix 4000A scanner (Axon Instruments, CA) and analyzed using Genepix Pro 4.0 software (Molecular Devices, CA). The presence or absence of genes was determined using the GACK software (http://falkow.stanford.edu/whatwedo/software/software.html). For normalization and differential analysis, the R software (http://www.R-project.org) was used.

RNA isolation and labeling and transcriptional analysis of the ladC mutant.

RNA was extracted from L. pneumophila in coculture with A. castellanii (in vivo) and from in vitro cultures as described previously (4). In vivo RNA isolation involved an additional 30-second FastPrep spin prior to bacterial cell lysis to minimize A. castellanii RNA contamination. RNA was reverse transcribed and indirectly labeled with Cy3 or Cy5 as described by the manufacturer (Amersham Biosciences, NJ). A 70-mer microarray containing all L. pneumophila Paris ORFs and ORFs specific to both strains Lens and Philadelphia was utilized to examine transcription differences between L. pneumophila 130b and the ladC insertional mutant as described previously (4). Hybridizations were performed as described above, with 250 pmol of labeled cDNA. Biological replicates and dye-swap experiments were performed. Array scanning and analysis was achieved as described for the DNA-DNA hybridizations.

RESULTS

Bioinformatic analysis of the adenylate cyclases of L. pneumophila.

Class III adenylate cyclases are considered the universal group of adenylate cyclases, since they are found in both prokaryotes and eukaryotes. Within this enzyme class there is large variability in topology and physiological roles, although the catalytic site remains highly conserved, as does the mechanism of purine binding (55). The most conserved residues, essential for adenylate cyclase activity, are an asparagine/arginine pair, separated by 3 amino acids, required to stabilize the transition state of the enzyme (28, 54). Searching of the L. pneumophila genome sequences revealed the presence of five putative class III adenylate cyclases (Fig. (Fig.1).1). All five possess the conserved CycC activity domain containing the catalytic requirements of an adenylate cyclase. However, lpp0730 lacks the essential arginine residue and a lysine is present in its place, suggesting that this enzyme is inactive. Other domains found within the putative adenylate cyclases of L. pneumophila are also commonly associated with this group of enzymes. HAMP domains (so designated for their presence in histidine kinases, adenylate cyclases, methyl binding proteins, and phosphatases), present in ladC and lpp1277, comprise a conserved region of approximately 50 amino acids that forms an α-helical region with the ability to regulate activity (27). The CHASE2 (defined as cyclases/histidine kinases associated sensory extracellular) domain, conserved in lpp1704, is an extracellular sensory domain that in Myxococcus xanthus participates in signal transduction during osmotic stress (23, 56). Finally, the GAF region, found in lpp1446, is one of the largest families of small-molecule binding domains that act in both signaling and sensory roles (32). The GAF (cyclic GMP adenylyl cyclase FhlA) domain was first recognized in cGMP-specific cyclic nucleotide phosphodiesterases and adenylate cyclases for their ability to bind cGMP and cAMP, respectively.

FIG. 1.
Putative adenylate cyclases of L. pneumophila. A schematic representation of the domain structure of the five predicted adenylate cyclases of L. pneumophila (lpp1131/lpl1135/lpg1130, lpp0730/lpl0710/lpg0674, lpp1277/lpl1276/lpg1322, lpp1446/lpl1538/lpg1490, ...

DNA-DNA hybridization L. pneumophila 130b on Paris array.

ladC is present in the sequenced strains of L. pneumophila serogroup 1 (lpp1131, lpl1135, and lpg1130) and, according to low-stringency Southern hybridizations, 22 other L. pneumophila isolates of various serogroups (37). To complete the adenylate cyclase survey of L. pneumophila 130b and appraise the shared gene content of this common research strain with the sequenced strains Philadelphia, Lens, and Paris, we performed DNA-DNA hybridization with a Paris-specific microarray to examine L. pneumophila genes that were absent in strain 130b. Using an estimated probability of presence of 100%, GACK software determined that 264 genes on this array were absent in strain 130b whereas 2,675 genes were shared by these two strains (data not shown). The remaining 147 genes required further investigation to determine their presence in strain 130b, which was not performed here. Gene variation among bacterial strains of the same species does not usually represent such a high proportion of the genome, although the variation seen here is not dissimilar to that observed among the three sequenced L. pneumophila genomes (6, 7). There are 280 genes present in strain Lens and absent in strain Paris and 428 genes present in strain Paris but absent in strain Lens (6). While the Paris-specific plasmid was shown to be absent in strain 130b, two of the plasmid-carried genes, plpp0064 and plpp0069, were present. Interestingly this hybridization showed that 4 of the 20 ankyrin domain-containing proteins of strain Paris (lpp0202, lpp1100, lpp2058 and lpp2065) are absent in 130b. This may indicate an area of functional redundancy among the L. pneumophila genomes, as these genes are also absent in strain Philadelphia and only lpp2058 and lpp2065 have homologues within strain Lens. Importantly, the DNA-DNA hybridization confirmed that all five putative adenylate cyclases of L. pneumophila were also present in strain 130b.

ladC contributes to initial infection of THP-1 and A549 cells and A. castellanii.

To investigate the putative role of ladC in host-pathogen interactions, we inactivated ladC by insertion of a kanamycin resistance cassette. In addition, two transcomplemented derivatives of the ladC mutant were constructed by introducing pMMB2002 carrying either native ladC or ladCN430A/R434A, the latter carrying the amino acid changes N430A and R434A to abrogate predicted adenylate cyclase activity. At 24, 48, and 72 h after infection, the ladC mutant was recovered in numbers similar to those for L. pneumophila 130b from A549 and THP-1 cells (Fig. 2A and B). However, at 3 h after infection, the ladC::Km mutant was recovered in significantly lower numbers than L. pneumophila 130b from both cell types. In A549 cells this represented a ~5-fold reduction in bacterial numbers and in THP-1 cells a ~2-fold reduction (Fig. 2A, B, D, and E). By analyzing the ratio of bacteria present at each time point to the inoculum, we could establish that the intracellular growth rates of 130b and the ladC mutant were the same after this early delay (Fig. 2D and E). Importantly, the observed early defect was complemented by the introduction of ladC but not ladCN430A/R434A (Fig. 2A and B). Indeed, the introduction of ladCN430A/R434A led to a pronounced intracellular growth defect in THP-1 cells but, interestingly, not in A549 cells (Fig. 2D and E). Incubation of these L. pneumophila derivatives in conditioned tissue culture medium alone did not alter the viability or recovery of any of these strains (data not shown), confirming that the observed difference is a direct result of bacterium-host cell interactions.

FIG. 2.
(A to C) Replication of L. pneumophila 130b and the ladC::Km, ladC::Km(pLadC), and ladC::Km(pLadCN430A/R434A) mutants within the alveolar epithelial cell line A549 (A), the macrophage cell-line THP-1 (B), and A. castellanii (C). Results are expressed ...

We also tested the ability of the ladC mutant to replicate in A. castellanii. The number of bacteria per well was determined at 2, 6, 12, 24, 48, and 72 h postinoculation (Fig. (Fig.2C).2C). Again the ladC mutant was recovered in lower numbers at early time points and achieved wild-type numbers of bacteria only at 72 h after infection (Fig. 2C and F). At 6 h postinfection this represented a 7.5-fold reduction compared to wild-type L. pneumophila. Transcomplementation of the ladC mutant with native ladC restored the ability of L. pneumophila to infect A. castellanii; however, transcomplementation with ladCN430A/R434A resulted in the same replication defect observed in THP-1 cells (Fig. 2C and F).

Examination of the replication defect of the ladC::Km pLadCN430A/R434A mutant.

pLadC and pLadCN430A/R434A were introduced into wild-type L. pneumophila to examine whether the replication defect observed for the ladC::Km mutant carrying pLadCN430A/R434A was a true dominant negative effect. Interestingly, these complementation constructs had no effect on the ability of L. pneumophila to replicate within A. castellanii (Fig. 3A and B), and even after 72 h these strains were recovered at levels similar to those for L. pneumophila carrying pMMB2002 alone. This was in contrast to the ladC::Km mutant carrying pLadCN430A/R434A, which was recovered in significantly lower numbers at 72 h after infection (Fig. (Fig.3B)3B) (P = 0.0046, unpaired two-tailed t test). Truncated forms of LadC, namely, LadC1-296, which lacks the C-terminal catalytic domain but possesses the HAMP domain, as well as just the catalytically active (CYC) and inactive (CYCN430A/R434A) CycC domains, were cloned into pMMB2002 and introduced into both wild-type L. pneumophila and the ladC::Km mutant. All strains were then examined for a replication defect. As observed for full-length LadC derivates, the presence of LadC1-296, CYC, or CYCN430A/R434A had no effect on the replication of wild-type L. pneumophila in A. castellanii. Indeed only full-length LadCN430A/R434A, in the ladC::Km background induced a replication defect (Fig. (Fig.3B).3B). The results also indicated that only full-length native LadC encoded on pLadC was able to rescue the early defect of the ladC mutant at 6 h after infection, suggesting that the catalytic domain alone, which was represented by pCYC, was not sufficient to perform the role of full-length LadC during infection.

FIG. 3.
Replication of L. pneumophila 130b and the ladC::Km mutant carrying ladC derivatives on pMMB2002, expressed as the ratio of recovered bacteria to the inoculum at 6 h (A) and 72 h (B) postinfection, from A. castellanii. Results are expressed as the mean ...

Early interactions between the ladC::Km mutant and host cells.

To examine the initial interactions between the ladC mutant and THP-1 cells more closely and determine if the defect we observed related to bacterial adherence or uptake into THP-1 cells, we used differential immunofluorescence staining to distinguish between extracellular adherent bacteria and intracellular bacteria. Following 15-min and 1-h infection periods, antibodies specific for L. pneumophila lipopolysaccharide (LPS) were used to stain bacteria before and after permeabilization of THP-1 cells to allow distinction between extracellular and intracellular bacteria using different secondary antibodies and fluorophores. We determined the proportion of bacteria that were intracellular from total cell-associated bacteria (representing uptake) as well as the proportion of cell-associated bacteria from the total inoculum (representing adherence) for at least 100 fields of view (Fig. (Fig.4).4). Although the percentage of the inoculum that was cell associated increased with infection time, there was no significant difference in uptake of the wild-type and ladC mutant strains (Fig. 4A and C). However, the ladC mutant was impaired for adherence compared to wild-type L. pneumophila (Fig. 4B and D) (P = 0.000006, unpaired two-tailed t test). The ability of the ladC mutant to adhere to THP-1 cells was significantly improved by complementation with pLadC, although not to wild-type levels, but complementation with pLadCN430A/R434A did not result in any rescue of the adherence defect of the ladC mutant (Fig. 4B and D). Together these results indicated that LadC activity was important for the initial adherence of L. pneumophila to THP-1 cells. Given that L. pneumophila expressing the CYC domain alone did not rescue the ladC mutant defect in A. castellanii (Fig. (Fig.3A),3A), we assumed that expression of CYC alone would also not restore adherence to the ladC mutant, and so this strain was not tested with THP-1 cells.

FIG. 4.
Quantification of differential fluorescence staining to determine the ratio of intracellular and cell-associated bacteria at 15 min (A and B) and 1 h (C and D) after infection of THP-1 cells with L. pneumophila 130b and the ladC::Km, ladC::Km(pLadC), ...

ladC contributes to L. pneumophila virulence in A/J mice.

A/J mice provide a well-established small-animal model to investigate L. pneumophila lung colonization. The in vitro adherence defect of the ladC mutant suggested that LadC might have an effect on the ability of L. pneumophila to colonize the lung. Competition experiments were performed to examine the relative fitness of the ladC mutant compared to the wild-type L. pneumophila 130b. Bacteria were isolated from lung tissue by host cell lysis with saponin, a detergent that does not lyse bacterial cells. Trial studies were performed to ensure that the ladC::Km mutation did not alter the viability of L. pneumophila cells incubated in 0.1% saponin solution (data not shown). Quantification of bacteria at 24 h and 72 h after infection allowed the competitive index (CI) to be calculated from the ratio of test strain CFU to reference strain CFU recovered from animals divided by the ratio of test strain CFU to reference strain CFU in the inoculum. A test strain with a CI of <0.5 was considered to be attenuated, whereas a CI of ≥1 indicated that the test strain colonized at least as well as the reference strain (3). Three independent competition experiments demonstrated that the ladC mutant was less virulent than wild-type L. pneumophila 130b and that colonization of the lung by the ladC mutant was compromised, particularly 72 h after infection (Fig. 5A, B, and C). Although we observed much variability in the CIs obtained in these experiments, the mutant was consistently attenuated compared to wild-type L. pneumophila 130b, showing a ~5-fold drop in CI from 24 h to 72 h in all experiments. In contrast, two independent infections using the ladC mutant complemented with native ladC encoded on pLadC in competition with wild-type L. pneumophila 130b demonstrated no significant differences in colonization between the two strains, indicating that the virulence defect of the ladC mutant was due to loss of ladC (Fig. 5D and E).

FIG. 5.
CI values from mixed infections of A/J mice. Mice were inoculated with approximately 105 CFU of each strain under investigation and were sacrificed at 24 or 72 h after infection to examine the bacterial content of their lungs. (A, B, and C) Three independent ...

LadC is present within the L. pneumophila inner membrane.

To understand whether LadC contributed to virulence by direct interaction with the host or whether it may act as an internal bacterial signaling protein, we determined the localization of LadC within L. pneumophila. Rabbit polyclonal antibodies to recombinant LadC lacking the N-terminal region that contains both a predicted signal sequence and transmembrane domain were created. The resulting monospecific antibodies were used to examine bacterial localization of LadC in stationary-phase cultures of L. pneumophila 130b and L. pneumophila ladC::Km(pLadC). LadC could not be detected in wild-type L. pneumophila grown in vitro, which is consistent with previous work showing that expression occurs only during in vivo growth (data not shown) (40). By inducing expression of LadC from pLadC in the mutant background, we detected a reactive band of the predicted size (54.7 kDa), which was present in the Triton X-100-soluble fractions of L. pneumophila ladC::Km(pLadC) yet absent in the fractionated ladC::Km strain (Fig. (Fig.6A).6A). LadC was not detected in the cytoplasmic protein fraction, the Triton X-100-insoluble fraction, or the secreted protein fractions, which is consistent with the prediction that LadC is an inner membrane protein (44, 50). Immunoblotting was also performed on the same fractions using monoclonal antibodies to DotA to serve as a control for inner membrane protein localization (44). DotA was visualized within the Triton X-100-soluble inner membrane fraction and to a lesser extent within the insoluble outer membrane fraction, which may indicate that Triton X-100 solubilization was incomplete, although some association of DotA with the Triton X-100-insoluble fraction has been shown previously (44). Processed DotA was also strongly detected within the trichloroacetic acid-precipitated culture supernatant, as described previously (36).

FIG. 6.
Localization and topology of LadC in the bacterial inner membrane. (A) Immunoblot analysis of fractions from the ladC::Km(pLadC) mutant with anti-LadC and anti-DotA antibodies. Bacteria were grown to stationary phase and induced with IPTG, and proteins ...

To confirm the localization of LadC in the bacterial inner membrane, signal sequence-deficient E. coli phoA was fused to the C termini of both full-length ladC and truncated ladC1-184, the latter encoding only one of the two predicted transmembrane domains. These constructs, pLadC-PhoA and pLadC1-184-PhoA, were introduced into L. pneumophila 130b and assessed for alkaline phosphatase activity (Fig. 6B and C). The presence of alkaline phosphatase activity would indicate the translocation of active PhoA to the periplasmic side of the inner membrane (44). While both PhoA fusion proteins were expressed equally well (Fig. (Fig.6B),6B), only LadC1-184-PhoA exhibited alkaline phosphatase activity (Fig. (Fig.6C).6C). This indicated that the region of LadC between the two predicted transmembrane domains was located within the periplasmic space and that the second transmembrane domain served to localize the catalytic region of LadC to the cytoplasmic face of the inner membrane.

In vitro and in vivo transcriptional comparison between L. pneumophila 130b and the ladC::Km mutant.

Since transcomplementation of ladC::Km with ladCN430A/R434A did not restore the ability of the ladC mutant to initiate infection in THP-1 and A549 cells or A. castellanii, we inferred that the contribution of LadC to virulence depended on adenylate cyclase activity of LadC. As adenylate cyclases are known to contribute to transcriptional regulation in a range of human pathogens through the cAMP-CRP complex and as the localization and topology of LadC within the bacterial inner membrane suggested that the protein may be involved in signal transduction, the role of LadC in transcriptional regulation was analyzed.

A microarray containing gene-specific 70-mer oligonucleotides covering the entire Paris genome (3,823 genes) as well as 302 Lens-specific and 285 Philadelphia-specific genes was utilized to examine global changes in transcription. The transcription profiles of broth cultures of stationary-phase L. pneumophila 130b and the ladC::Km derivative were compared and showed no statistical differences in the transcription of any ORFs (data not shown). Indeed, the level of ladC transcription in L. pneumophila 130b was extremely low, as expected from previous work reporting that the ladC promoter is inactive in vitro (data not shown) (40). To compare in vivo transcription profiles, L. pneumophila RNA was extracted from infected A. castellanii cultures following a 14-h infection at an MOI of 50. This infection period consistently rendered a large proportion of A. castellanii cells infected with motile L. pneumophila, which corresponds to the virulent, transmissive phase (4). In addition, ladC is upregulated more than twofold in the transmissive phase in vivo (4). Unfortunately, microarray analysis, including dye swaps, of three independent infections showed no consistent or significant transcriptional differences between wild-type L. pneumophila 130b and the ladC::Km mutant (data not shown). These results suggested that the contribution of LadC to virulence was not mediated through the transcriptional regulation of virulence determinants.

DISCUSSION

Crucial to the ecology and subsequent disease-causing capability of L. pneumophila is its unique relationship with protozoa. The ability of L. pneumophila to replicate within amoebae allows the pathogen to persist in aquatic environments and also replicate in mammalian cells. Many processes important for subsequent environmental persistence are also principal virulence processes. Despite a high number of strain-specific genes among different isolates of L. pneumophila serogroup 1 (6), all strains share the core genes required for their unique intracellular life cycle.

In this study we established that the gene encoding a putative L. pneumophila-specific adenylate cyclase, LadC, is among a cohort of genes important for initial infection of epithelial cells, macrophages, and A. castellanii. The differences in bacterial numbers recovered from cells infected with wild-type L. pneumophila or the ladC mutant were most obvious at 3 h after infection. When we examined the initial interaction between THP-1 macrophages and derivatives of L. pneumophila, we found that the ladC mutant adhered in smaller numbers to the host cell than wild-type L. pneumophila and that this was already evident at 15 min after infection. Despite the reduction in total numbers of the ladC mutant interacting with host cells, the proportion of cell-associated ladC mutant bacteria internalized by THP-1 cells was equivalent to that of the wild-type strain. Consistent with the absence of a defect in bacterial uptake for the ladC mutant, there was no difference in trafficking of the LCV between wild-type L. pneumophila and the ladC mutant, as measured by acquisition of the late endosomal marker LAMP-1 (data not shown). The adherence defect of the ladC mutant in vitro also conferred a colonization defect in vivo upon infection of A/J mice. This confirmed that ladC makes an important contribution to the virulence of L. pneumophila in a respiratory infection model, presumably by influencing initial contact between the bacterium and host cell.

Amino acid sequence analysis predicted that the putative LadC protein was capable of converting ATP to cAMP. cAMP is one of the most ubiquitous signaling molecules in both prokaryotes and eukaryotes and allows an organism to respond rapidly to a variety of stimuli. In prokaryotes, cAMP traditionally mediates biological change via regulation of transcription through its interaction with CRPs. However, here microarray studies demonstrated that inactivation of ladC had no influence on L. pneumophila transcription either in vitro or during infection of A. castellanii. This was surprising, as we demonstrated using fractionation and immunoblotting as well as PhoA fusions that LadC localized to the bacterial inner membrane. Given that our microarray studies suggested that the predicted product of ladC was not involved in the regulation of transcription, LadC may aid virulence by modulating protein-protein interactions, signal transduction, and protein activity. cAMP is also able to bind to and activate a number of proteins not involved in transcriptional regulation and signal transduction. This is not a well-studied mechanism of cAMP function in bacteria; however, there are many examples of posttranslational modifications of protein function in eukaryotes by cAMP. Classical examples of this include the activation of protein kinase A leading to protein phosphorylation, regulation of ion channels, and, more recently, activation of the Ras-like GTPase Rap1 (14, 49).

The behavior of the ladC mutant complemented with ladCN430A/R434A is another intriguing aspect of this study. Clearly this putative catalytically inactive form of ladC was unable to complement the attenuated ladC mutant, suggesting that production of cAMP is crucial for LadC function in virulence. However, this strain demonstrated a severe replication defect in THP-1 cells and A. castellanii, and interestingly, this attenuation was not due to a true dominant negative phenotype, as wild-type L. pneumophila carrying ladCN430A/R434A did not show diminished replication in A castellanii. In addition, this phenotype was observed only for full-length ladCN430A/R434A, as complementation of ladC::Km with pLadC1-296, pCYC, and pCYCN430A/R434A did not confer a similar replication defect. The presence of an inactive enzyme may have resulted in aberrant signaling and/or interfered with the balance of ATP and cAMP and in so doing disrupted the function and signaling roles of the four other putative adenylate cyclases present in L. pneumophila. However, at this stage the reason for the increased attenuation of the ladC mutant complemented with ladCN430A/R434A is unknown.

Analysis of the L. pneumophila genome revealed no close CRP homologues; however, there are five genes encoding predicted proteins with conserved cAMP binding domains. Two of these, lpp2063 and lpp2777 (also termed legN, delineating homology to eukaryotic motifs [12]), encode putative proteins with conserved cyclic nucleotide monophosphate binding domains and may act similarly to traditional prokaryotic CRPs. There are another three genes encoding putative proteins that contain a cyclic nucleotide monophosphate binding domain, which may indicate other roles for cAMP in L. pneumophila. These are lpp0611, a putative flavin adenine dinucleotide-dependent oxidoreductase; lpp3069, a putative sulfate transporter; and lpp1482. lpp1482 contains a conserved CaaX amino terminal protease domain, where “a” represents aliphilic amino acids. CaaX proteases play an important role in eukaryotes, cleaving the aaX amino acids from C-terminal farnesylated CaaX motifs (52). This modification is one step within CaaX processing that renders proteins, such as Rab GTPases, hydrophobic at their C termini, allowing membrane association (18, 52). Of these, lpp2063, lpp2777, lpp3069, and lpp1482 are also upregulated two- to fourfold in the transmissive phase of L. pneumophila growth, similar to the case for ladC (4).

Several similarities can be drawn between the intracellular pathogens L. pneumophila and Mycobacterium tuberculosis in relation to their adenylate cyclase repertoire and their ability to replicate inside mammalian cells and prevent phagolysosome fusion (10). M. tuberculosis strain H37Rv encodes 16 class III cyclases, 4 of which have conserved HAMP domains, similar to the case for LadC (47). Many of the M. tuberculosis adenylate cyclases have undergone biochemical characterization, although their role in virulence remains unclear (47). It is known that cAMP can influence transcription within M. tuberculosis, and 10 putative CRPs have been identified from the genome sequence (41). However, the wide array of both adenylate cyclases and putative cAMP binding proteins suggests more diverse action of cAMP in M. tuberculosis than in L. pneumophila (47, 48). In this study, recombinant full-length LadC and recombinant protein encompassing just the CycC domain of LadC were analyzed repeatedly for adenylate cyclase activity under a variety of conditions in vitro (data not shown). However, these attempts at the biochemical characterization of LadC were unsuccessful, and we concluded that we were unable to mimic the biological conditions where LadC is active.

This study has demonstrated that the putative adenylate cyclase LadC is involved in the ability of L. pneumophila to infect a broad range of hosts, specifically through initiating an interaction between the bacterium and host cell. The mechanism by which this is achieved remains unclear; however, it appears that under the conditions examined here, it is not accomplished via the traditional role of cAMP as a second messenger to regulate bacterial transcription. We also did not detect any obvious differences in LPS staining or in protein secretion and outer membrane protein profile between wild-type L. pneumophila and the ladC mutant (data not shown), although a sensitive mass spectrometry approach may yet identify variations in LPS profile or protein abundance that could account for the adherence defect of the ladC mutant. Above all, further investigation of the five conserved adenylate cyclases of L. pneumophila and the various roles of cAMP during host cell infection will help to clarify this complex signaling system and its intrinsic link to pathogenesis.

Acknowledgments

We are indebted to Craig Roy for his kind gift of anti-DotA antibodies.

H.J.N. was the recipient of an Australian Postgraduate Award, a Victoria Fellowship, and an AFAST-FEAST Fellowship, which assisted this work. F.M.S. was the recipient of a Monash Postgraduate Award. H.B. is supported by a German Academy of Natural Scientists Leopoldina Fellowship, and C.A.-W. holds a Bavarian Research Foundation Fellowship. This research was supported by NHMRC grant 284214 awarded to E.L.H., NIH grant AI43987 awarded to N.P.C., and AFSSET project number ARCL-2005-002 awarded to C.B.

Notes

Editor: J. L. Flynn

Footnotes

[down-pointing small open triangle]Published ahead of print on 21 April 2008.

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