Dept of Veterinary and Biomedical Sciences, University of Nebraska, Lincoln, 203 VBS, Fair and East Campus Loop, Lincoln, NE 68583, USA1
Author for correspondence: Jeffrey D. Cirillo. Tel: +1 402 472 8587. Fax: +1 402 472 9690. e-mail: jcirillo1{at}unl.edu
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ABSTRACT |
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Keywords: phagocytosis, cytotoxicity, Sel-1, invasion, RTX
Abbreviations: Enh, enhanced entry (phenotype); RTX, structural toxin
The GenBank accession numbers for the enh1 and enh2 loci reported in this paper are AF057703 and AF057704, respectively.
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INTRODUCTION |
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Several mechanisms of L. pneumophila adherence to host cells have been demonstrated, including opsonization with complement (Husmann & Johnson, 1992 ; Marra et al., 1990
; Payne & Horwitz, 1987
) and specific antibodies (Horwitz & Silverstein, 1981
; Husmann & Johnson, 1992
; Nash et al., 1984
). In support of mechanisms involving bacterial opsonization, both Fc (Husmann & Johnson, 1992
) and complement receptors (Marra et al., 1990
; Payne & Horwitz, 1987
) have been shown to mediate adherence. Complement-mediated adherence has been shown to result in phagocytosis by monocytes (Payne & Horwitz, 1987
) and is thought to involve the L. pneumophila major outer-membrane protein (Bellinger-Kawahara & Horwitz, 1990
; Krinos et al., 1999
). However, no specific L. pneumophila mutants have been constructed in this gene, which prevents elucidation of its exact role in entry. It has been suggested that non-opsonic adherence mechanisms may also exist (Gibson et al., 1994
; Rodgers & Gibson, 1993
). Potential host-cell receptors for non-opsonic adherence have been suggested (Harb et al., 1998
; Venkataraman et al., 1997
) and a type IV pilus that can mediate this type of adherence has been described (Stone & Abu Kwaik, 1998
). However, in the absence of this pilus L. pneumophila adheres at 53% of the wild-type levels, suggesting the presence of multiple adherence mechanisms.
In the current study, we designed a strategy to identify additional bacterial factors involved in entry of L. pneumophila into host cells. This strategy is based on the observation that entry by L. pneumophila is enhanced after growth in the environmental host Acanthamoeba castellanii, compared to growth on laboratory media (Cirillo et al., 1994 ). This observation suggests that the genes involved in entry into host cells by L. pneumophila are down-regulated when grown on laboratory media. Using chemical mutagenesis and a positive-selection strategy, we isolated mutants that display enhanced entry into host cells when grown on standard laboratory media (BCYE agar). Screens for the presence of a dominant mutation in one of these mutants allowed the identification, sequencing and characterization of three loci that have the ability to confer an enhanced entry (Enh) phenotype. Identification of these genes may help to clarify the unusual mechanism of entry utilized by L. pneumophila. In addition, the strategy used to isolate these genes should be generally applicable to the identification of other regulated virulence determinants from L. pneumophila and a wide range of bacterial pathogens.
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METHODS |
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The vector pJDC15 was constructed on the backbone of pTLP4 (Moffat et al., 1994 ; Stibitz et al., 1986
). The streptomycin sensitivity gene and cos site were removed by digestion with SphI and replaced with the SphI fragment containing the kanamycin-resistance gene from pYUB8 (McAdam et al., 1995
). The resulting plasmid, designated pJDC1, was then cut with PvuII to remove the chloramphenicol-resistance gene and ColE1 origin of replication and replace them with the R6K origin of replication (Kolter et al., 1978
) carried on a SmaI fragment from pBDJ121 (Jones & Falkow, 1994
). The promoterless streptomycin-sensitive rpsL allele was inserted into the BamHI and XbaI sites of the resulting plasmid, pJDC6, by PCR from pTLP4 with the addition of flanking BglII and XbaI sites creating plasmid pJDC7. The promoterless chloramphenicol-resistance gene was inserted into the XbaI and SacI sites of pJDC7 by PCR from pTLP4 with the addition of SacI and BamHI sites on the 5' end and a XbaI site on the 3' end. The resulting vector, pJDC8, was digested with SacI/BamHI and ligated to the L5 mycobacteriophage promoter produced by PCR from pYUB215 (Cirillo et al., 1991
) with the addition of flanking 5' SacI and 3' BamHI sites. The sacB gene (Blomfield et al., 1991
) was cloned into the resulting plasmid, pJDC13, by ligation of the sacB-containing EcoRV fragment of pCVD442 (Mobley et al., 1993
) into the EcoRV site, producing pJDC15. pJDC15 is a suicide plasmid in any bacterial strain that does not contain the Pir protein in trans and carries kanamycin and chloramphenicol resistance, and streptomycin and sucrose sensitivity as well as an origin of transfer for conjugation. These characteristics make pJDC15 an ideal vector for allelic exchange in a number of bacterial species.
Cell culture.
HEp-2 cells (ATCC CCL23), established from a human epidermoid carcinoma, were grown in RPMI 1640 plus 5% heat-inactivated foetal calf serum (Gibco). THP-1 cells (ATCC TIB202), a human monocytic cell line, were grown in RPMI 1640 plus 10% heat-inactivated foetal calf serum.
Phenotypic characterization of strains.
Assays to determine the intracellular growth rate of each strain were carried out essentially as described previously (Cirillo et al., 1999 ). The THP-1 cells used for viability assays were seeded into 24-well tissue culture dishes at 1·5x106 cells per well in RPMI 1640 plus 10% serum, 5 µg LPS ml-1 (Difco; E. coli 0127:B8) and 40 U human
-interferon ml-1 (Boehringer Mannheim). The cells were incubated for 2 d with this medium and fresh medium added for an additional 24 h to allow activation of the monocytes prior to entry. Bacteria were added to the cells at a m.o.i. of 10100 and incubated at 37 °C for 5 min, washed three times with warm PBS, and suspended in fresh medium with human
-interferon and LPS before lysis of different sample wells with water every 2 h for 24 h. Dilutions of the resulting lysates were plated to determine c.f.u. at each time point.
Growth rate in laboratory media was assessed essentially as described by Byrne & Swanson (1998) . Bacteria were inoculated from BCYE agar into 25 ml BYE broth (Edelstein, 1981
) at a concentration of 105 c.f.u. ml-1 and grown at 37 °C with agitation. Optical density readings were taken at 600 nm and dilutions plated on BCYE agar every 2 h for 24 h.
The presence of pili and flagella was assessed by transmission electron microscopy of negatively stained specimens essentially as described by Chandler et al. (1980) and Rodgers et al. (1980)
. The bacteria were suspended at 106 c.f.u. ml-1 in PBS and a drop of this suspension applied to Formvar carbon coated copper grids. The samples were then stained with 1% uranyl acetate and examined by transmission electron microscopy.
The ultrastructural morphology of the bacteria was examined as described previously (Cirillo et al., 1994 ). Samples were fixed in 2% glutaraldehyde and 1% OsO4 for 2 h and stained with 0·5% uranyl acetate overnight at 4 °C. Samples were then embedded and thin sections examined by transmission electron microscopy.
Motility was assessed using microscopy, essentially as described by Wei & Bauer (1998) . Bacteria were grown in BYE to stationary phase and diluted to 104 c.f.u. ml-1 in sterile deionized water prior to examination with a Nikon TE300 inverted microscope with differential-interference-contrast optics. Digital video captures were obtained from at least three independent fields from three independent suspensions and the number of bacteria quantitated using NIH Image 1.61 on selected frames. Repeated examination of the captured video allowed quantitation of the number of motile bacteria in each field. The total number of bacteria per field used was always greater than 50, resulting in quantitation of the percentage of motile bacteria from greater than 450 bacteria per strain.
Entry and adherence assays.
Entry assays were carried out essentially as described previously (Cirillo et al., 1994 ). HEp-2 cells were seeded in 24-well tissue culture dishes (Falcon) at a concentration of 2·5x105 cells per well and allowed to adhere overnight at 37 °C. The bacteria to be assayed were suspended and diluted in the same medium as the cells that were to be infected. After adding the bacteria at an m.o.i. of 100 they were allowed to interact with cells for various times, though all data shown are for 90 min. The cells were then washed with PBS and incubated in the appropriate culture medium plus 100 µg gentamicin ml-1 for 2 h. After antibiotic treatment, the cells were washed with PBS, then with water and lysed by incubation for 10 min in 1 ml water followed by vigorous pipetting. In the case of THP-1 cells, the bacteria were allowed to interact with cells for various times, though all data shown are for 30 min and the assays were carried out in suspension. This requires that the cells be pelleted by centrifugation at 100 g for 1 min before each change of solution. After lysis, the number of intracellular bacteria was determined by plating for c.f.u. on BCYE (L. pneumophila) or LB (E. coli) agar. Entry levels were determined by calculating the percentage of the inoculum that became gentamicin-resistant over the course of the assay [i.e. % entry=100x(c.f.u. gentamicin-resistant/c.f.u. inoculum)]. To correct for variation in levels of uptake between experiments, entry is reported relative to AA100 (i.e. relative entry= % entry test strain/% entry AA100). Adhesion was tested in a similar manner to that for entry, except that bacteria were added to the cells, mixed and immediately washed three times to remove non-adherent bacteria prior to lysis.
Chemical and transposon mutagenesis.
AA100 was mutagenized at a concentration of 1x108 c.f.u. ml-1 with 30 µg EMS ml-1 for 1 h at 37 °C in minimal A buffer (Miller, 1972 ). This method of EMS treatment resulted in a level of 1·25% survival of L. pneumophila. The pool of mutagenized bacteria was then washed twice in minimal A buffer and grown overnight on BCYE agar. The pool was suspended in RPMI 1640 and aliquots (106 c.f.u.) were used to isolate enhanced entry mutants.
Cosmids carrying L. pneumophila genomic fragments were mutagenized with a chloramphenicol-resistant mini-Tn10 transposon (Alexeyev & Shokolenko, 1995 ) carried on pKV32 (Visick & Ruby, 1997
). The plasmid pKV32 has an R6K
-origin of replication, which prohibits its replication in strains that do not carry the
protein in trans (Miller & Mekalanos, 1988
). Mutagenesis of the cosmids 1A3, 2A4 and 2A6 with this transposon was carried out by transformation of the XL-1 Blue strain containing each of these cosmids with pKV32. The resulting transformants were then allowed to recover in SOC medium (Dower et al., 1988
) with 2 mM IPTG for 1 h at 37 °C to induce transposition (Alexeyev & Shokolenko, 1995
). The culture was then incubated for an additional 4 h in the presence of kanamycin and IPTG. Aliquots were plated on LB agar plates with kanamycin and chloramphenicol. The 100200 colonies that arose from transposition events were pooled, plasmid DNA isolated by alkaline lysis (Sambrook et al., 1989
) and transformed into XL-1 Blue. This procedure allows isolation specifically of those transposition events into the cosmid. Individual colonies were then screened for the presence of unique insertions into the cosmid by restriction analysis. Unique transposon insertions were then electroporated into AA100 using the same method described for E. coli (Dower et al., 1988
) and tested for the ability to confer enhanced entry.
Selection for enhanced-entry mutants.
Mutants that displayed enhanced entry were isolated through the use of a modified HEp-2 cell entry assay. This selective entry assay was accomplished by growing EMS-mutagenized L. pneumophila strain AA100 on BCYE agar at 37 °C for 5 d and then pooling the resulting colonies in RPMI 1640. A standard entry assay into HEp-2 cells was carried out with this suspension as described above, except that the bacteria were only allowed to interact with the HEp-2 cells for 5 min. The bacteria that entered during this assay were grown on BCYE agar for 5 d and individual colonies suspended in PBS with 50% (v/v) glycerol and kept frozen at -70 °C until use. To test the entry phenotype of individual clones, they were grown on BCYE agar for 3 d and compared to AA100 in a standard entry assay. Those clones that displayed greater than a twofold increase in entry over AA100 were considered to have an Enh phenotype.
Library construction and screening for dominant mutations.
Contiguous and non-contiguous L. pneumophila strain AA100 and C3 genomic DNA libraries were constructed in the cosmid vector pYUB289. pYUB289 was constructed by first producing a PacI cassette in SuperCos 1 (Stratagene) as described previously for pYUB328 (Balasubramanian et al., 1996 ). The resulting cassette and adjacent cos site were moved to pACYC177 by digestion with NheI/AatII, isolation of the appropriate DNA fragments and ligation. The resulting cosmid carries a single cos site, the PacI cassette, kanamycin resistance, ampicillin resistance and the low copy-number origin of replication p15A. To construct the Legionella genomic libraries, total genomic DNA was isolated as described for E. coli (Silhavy et al., 1984
), digested partially with Sau3AI to produce fragments of approximately 20 kbp in length for non-contiguous libraries or 50 kbp for contiguous libraries. For non-contiguous libraries, these fragments were ligated to BamHI-cut and dephosphorylated pYUB289. For contiguous libraries, these fragments were dephosphorylated and ligated to BamHI-cut pYUB289. The resulting ligations were in vitro packaged with Gigapack II Gold (Stratagene) packaging mix and used to infect
2819 for in vivo packaging (Jacobs et al., 1986
). Over 10000 kanamycin-resistant
2819 colonies were pooled for in vivo packaging, producing a lysate that had a titre of greater than 109 cosmid-containing phages ml-1. This lysate was used to infect XL-1 Blue and plated on LB agar plates containing kanamycin such that approximately 20000 colonies were produced on ten plates. The resulting colonies were pooled in two separate pools of approximately 10000 colonies each and plasmid prepared from them.
To screen for dominant mutations, the two resulting plasmid preparations from the non-contiguous C3 library were electroporated independently into AA100 and dilutions plated on BCYE agar with kanamycin. Approximately 10000 kanamycin-resistant AA100 colonies from each transformation were pooled and clones that conferred the enhanced-entry phenotype isolated in the same manner as the original isolation of EMS-mutagenized enhanced-entry mutants. Individual colonies were isolated following the selective entry assay and their ability to enter HEp-2 cells compared to wild-type AA100. Those clones that entered HEp-2 cells at two-fold higher frequencies than AA100 were considered to confer the Enh phenotype. For each of the transposon insertions, the ability to confer the Enh phenotype was tested in a similar manner except that the purified plasmids carrying insertions were directly transformed into AA100 and tested for the Enh phenotype.
To ensure that the cosmid carried by each clone was responsible for the enhanced entry rather than the acquisition of a spontaneous mutation in the host bacteria, each cosmid that conferred the Enh phenotype was transferred from AA100 into XL-1 Blue, purified, retransformed into AA100 and retested in the entry assay. Cosmids were transferred into E. coli by the technique of direct electroporation.
Isolation and subcloning of contiguous enh loci.
To demonstrate that the Enh phenotype was due to the loci indicated by transposon mutagenesis and not the result of scrambled genes, we identified and subcloned each of the loci involved from the contiguous genomic libraries of C3 and AA100. A 609 bp region beginning 33 bp upstream of the putative translational start for rtxA was used as a probe in colony hybridizations to isolate the contiguous cosmid clones that contain the enh1 locus. A 438 bp region beginning 590 bp upstream of the putative translational stop for enhC was used as a probe in colony hybridizations to isolate the contiguous cosmid clones that contain the enh2 locus. Restriction and Southern analyses confirmed that the enh1 and enh2 loci on these cosmids were on 5265 bp EcoRI and 5263 bp SwaI fragments, respectively, that were the same size in C3 and AA100 chromosomal DNA digests (data not shown). One of each of the cosmids isolated in this manner containing enh1 or enh2 was digested with either EcoRI or SwaI, respectively, and the appropriate size fragment was purified and ligated into EcoRI or ScaI digested pYUB289. The resulting cosmids, pJDC19, pJDC20, pJDC23 and pJDC24, are listed in Table 1. The presence of the complete contiguous loci on each of these subclones was confirmed by restriction and sequence analysis (data not shown).
Construction and complementation of rtxA and
enhC mutations.
In-frame deletions were constructed in the rtxA and enhC genes by overlapping PCR in a similar manner to that described previously (Fang et al., 1999 ; Horton et al., 1989
; Sandhu et al., 1992
). An in-frame deletion was constructed in the rtxA gene, producing the amino-terminal 6 aa and the carboxy-terminal 124 aa of RtxA. This construct does not contain domains thought to be required for RTX (structural toxin) activity (Boehm et al., 1990
; Welch, 1991
). An in-frame deletion was constructed in the enhC gene, producing the amino-terminal 12 aa and the carboxy-terminal 158 aa of EnhC. The resulting mutations were then cloned into the NotI site of pJDC15, transformed into AA100 and plated on kanamycin to select for single-crossover events resulting from integration of the plasmids by homologous recombination. These recombinants were then plated on BCYE plus 5% sucrose plates to select for the presence of the desired mutations in the chromosome and loss of pJDC15. The presence of the appropriate
rtxA and
enhC mutations in the L. pneumophila chromosome were confirmed by Southern analysis and extended PCR (data not shown). The resulting
rtxA and
enhC mutants were designated
lp24 and
lp30, respectively. Complementation of these mutations was performed by transformation with the appropriate plasmids pJDC20 (
rtxA) and pJDC24 (
enhC) that contain only the complete wild-type enh locus and promoter region. Each of the resulting mutants and complementing strains were then compared to wild-type in entry assays.
Southern analysis and colony hybridization.
Probes were labelled by nick translation or PCR with digoxigenin using the methods suggested by the manufacturer of the Genius System (Boehringer Mannheim). Membranes were prepared by the suggested methods for both colony hybridization and Southern analysis. Hybridization and washes were carried out at high stringency.
DNA sequence analysis.
DNA sequence analysis was carried out initially using a forward primer TrnF (5'-CCACTAGTTCTAGAGCGGCC-3') from the mini-Tn10 transposon (Alexeyev & Shokolenko, 1995 ). The sequence was continued by primer walking directly on the cosmid of interest. All regions were sequenced completely in both directions using Big Dye Terminator (Applied Biosystems) cycle sequencing and subsequent analysis on an ABI 310 automated sequencing apparatus (Applied Biosystems). Sequence analysis and assembly was carried out using Gene Construction Kit 2 (Textco) and comparison with known sequences using BLAST (Altschul et al., 1997
). Analysis of putative signal sequences was done using SignalP (Nielsen et al., 1997
), potential protein profiles using ProfileScan (Lüthy et al., 1994
; Thompson et al., 1994
), and secondary structure and multiple sequence alignments using Lasergene (DNASTAR) software.
Statistical analyses.
All in vitro experiments were carried out in triplicate and repeated at least twice. Significance of the results was analysed using ANOVA. Values of P<0·05 were considered significant.
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RESULTS |
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A cosmid library of C3 total genomic DNA was constructed and transferred into wild-type AA100 to allow isolation of the genes responsible for the C3 Enh phenotype. Due to the possibility that the C3 Enh phenotype was due to multiple mutations, we utilized a non-contiguous genomic library for these experiments. A low copy-number cosmid vector was chosen for these studies to reduce the possibility of copy-number effects, such as toxicity of L. pneumophila gene products in E. coli, on the comprehensiveness of the library. Recombinant cosmids that conferred the Enh phenotype were isolated using selective-entry assays. A total of 19 cosmids were isolated from two independent pools of 20000 AA100 transformants with the C3 genomic library. The ability of each of these recombinant clones to enter HEp-2 cells was compared to wild-type in standard entry assays (Fig. 2a). Seven cosmid-containing clones consistently displayed the Enh phenotype, two from the first pool (containing cosmids 1A3 and 1A7) and five from the second pool (containing cosmids 2A3, 2A4, 2A6, 2A7 and 2A9). A restriction map of each of these cosmids was constructed using BamHI (data not shown). All of these cosmids had unique restriction maps excluding 2A4 and 2A6, which were identical. Cosmid 1A3 consistently displayed the highest relative entry of those tested. On the basis of these data, cosmids 1A3, 2A4 and 2A6 were chosen for further analysis.
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Identification of enhanced-entry (enh) loci
Detailed physical maps of cosmids 1A3, 2A4 and 2A6 were constructed using BamHI, NheI and XhoI (data not shown). Cosmids 2A4 and 2A6 have identical physical maps with these three enzymes. However, cosmid 1A3 has no apparent regions of overlap with cosmids 2A4 and 2A6. Southern analysis was carried out with AA100 chromosomal DNA and purified 1A3, 2A4 and 2A6 cosmid DNA digested with BamHI, using 1A3 and 2A6 separately as probes (data not shown). No overlapping regions were identified by Southern analysis for 1A3 with the 2A4 and 2A6 cosmids. However, 2A6 hybridized with all restriction fragments of 2A4, indicating that these cosmids are identical. Thus, only cosmids 1A3 and 2A6 were analysed further.
Transposon mutagenesis of cosmids 1A3 and 2A6 was carried out in XL-1 Blue using mini-Tn10. Greater than 100 transposon insertions in each cosmid were isolated and partially mapped with restriction endonucleases to determine the location of each transposon insertion (data not shown). At least 26 unique transposon insertions per cosmid were tested for their ability to confer the Enh phenotype of the original cosmid in HEp-2 cells (Fig. 3). The location and phenotype of each transposon insertion is shown in Fig. 4
. Two different loci, designated enh1 (~4 kbp) and enh2 (~5 kbp), on cosmid 1A3 were involved in the ability of this cosmid to confer the Enh phenotype. In the case of cosmid 2A6 one locus, designated enh3 (~4 kbp), was required. Since only cosmid 1A3 confers a phenotype similar to the C3 mutant strain, all further studies were carried out on the enh1 and enh2 loci from this cosmid. To confirm that each locus, separate from adjoining fragments, was sufficient to confer the Enh phenotype and this phenotype was due to the presence of contiguous chromosomal fragments rather than scrambled genes, we identified and subcloned these loci from our contiguous C3 and AA100 genomic libraries.
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Characterization of the rtxA and enhABC genes
Analysis of the sequence of the rtxA gene revealed the presence of several distinctive characteristics (Fig. 7). Standard translational start (ATG) and stop (TAA) sequences were present, allowing the translation of a protein of 1208 aa. However, a somewhat unusual ribosome-binding site (AAGTAG) was found upstream of the translational start. Eight repeats of the RTX consensus sequence in two separate regions of the protein (at aa 519 and 1060) were found within the L. pneumophila rtxA gene (Fig. 7a
). The first of these regions has five 9 aa RTX repeats, whereas the second region has only three. The 9 aa repeats were nearly identical for all copies present in the L. pneumophila rtxA gene. When the carboxy-terminal regions of the RTX repeats were aligned, additional sequence similarity was observed in the 40 aa carboxy-terminal of the repeats. An alignment of the two L. pneumophila RTX repeat regions with similar RTX regions from other species is shown in Fig. 7b
. The positions of glycine, aspartate and phenylalanine residues continue to be highly conserved in this region.
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Mutations in the rtxA and enhC genes affect entry
To clearly demonstrate that the genes identified are involved in entry of L. pneumophila, we constructed in-frame deletions in rtxA and enhC and attempted to complement them with a plasmid containing the appropriate enh locus. The ability of the resulting strains to enter epithelial cells and monocytes was then compared to the wild-type L. pneumophila strain AA100 (Fig. 9). Each of the mutations significantly (P<0·006) affects the ability of L. pneumophila to enter both cell types. This reduced-entry phenotype can be complemented by expression of the appropriate enh locus from a low-copy-number plasmid. However, no complementation was observed in the mutants containing the plasmid vector without the appropriate enh locus (data not shown). These results indicate that the enh1 and enh2 loci are important for entry of L. pneumophila into monocytes.
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DISCUSSION |
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The enh1 locus contains two genes, rtxA and arpB. The rtxA gene product has eight RTX repeats, which are thought to be involved in the Ca2+-binding activity that is required for toxin activity and host-cell attachment (Boehm et al., 1990 ; Welch, 1991
). The involvement of this domain in binding to host cells has been observed with RTX toxins including the Actinobacillus actinomycetemcomitans leukotoxin (Lally et al., 1997
), E. coli haemolysin (Lally et al., 1997
) and B. pertussis cytotoxin (Glaser et al., 1989
). An observation that supports a role for the rtxA gene in adherence and entry is that RTX toxins use ß2 integrins as receptors on target cells (Lally et al., 1997
). Previous studies in L. pneumophila have demonstrated a role for complement receptors, which are ß2 integrins, in both adherence and entry by L. pneumophila (Marra et al., 1990
; Payne & Horwitz, 1987
). These data combined with the presence of an RTX in L. pneumophila suggest that further investigation of direct interactions of L. pneumophila with complement receptors is warranted.
The enh2 locus contains three genes, enhA, enhB and enhC, that have putative Gram-negative signal sequences. The observation that enhC affects the ability of L. pneumophila to enter host cells suggests the presence of a novel mechanism of entry that has not been observed in other bacterial systems. The presence of unusual mechanisms of entry in L. pneumophila is not surprising considering that entry has been shown to occur by coiling phagocytosis (Horwitz, 1984 ), which has not been observed at significant levels in other bacterial species with the exception of spirochaetes (Rittig et al., 1992
, 1998
). The role of enhC in entry may be through a direct interaction with a target-cell receptor or through regulation of the available receptors on the host cell, similar to Sel-1 (Grant & Greenwald, 1996
). Unfortunately, the receptor for Sel-1 on target cells is not yet known, which would facilitate analysis of its interaction with these cells. Studies are ongoing to differentiate between the possible role of enhC in entry as well as the roles of the two other genes present at this locus.
The current study has laid a foundation for further examination of the entry mechanisms utilized by L. pneumophila. The strategy used to isolate the enh1 and enh2 loci should be broadly applicable to the isolation of virulence determinants from bacterial pathogens. This is not the first time that gene dosage effects have been used to control the regulation of genes of interest (Aiba et al., 1982 ; Blanc-Potard et al., 1999
; OSullivan et al., 1996
). However, this is the first time that these effects have been utilized to directly select for genes of interest, particularly virulence determinants. One caveat of this strategy may be that the operator regions present on the fragments present in multiple copies could saturate global regulators, resulting in down- or up-regulation of unlinked loci in the same regulon. This potential problem was not observed in the current study, since only transposon insertions in coding regions affected the Enh phenotype conferred by these plasmids. One might expect that overexpression of genes under growth conditions where they are not normally expressed would be detrimental to the bacteria. Thus, the use of the low copy-number p15A origin of replication is likely to have contributed to the success of the current study by producing only moderately enhanced levels of expression. Application of this strategy to the analysis of the molecular determinants of L. pneumophila entry has provided insight into the potential mechanisms involved and should lead to a better understanding of the pathogenesis of Legionnaires disease.
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ACKNOWLEDGEMENTS |
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Received 28 January 1999;
accepted 14 February 2000.