Laboratory of Bacteriology, Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
Correspondence
Elke Lammertyn
Elke.Lammertyn{at}rega.kuleuven.ac.be
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ABSTRACT |
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These two authors contributed equally to the work described in this paper and to the writing of the paper.
The GenBank accession number for the sequence of the L. pneumophila lepB gene is AJ608705.
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INTRODUCTION |
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Type I SPases belong to a novel group of serine proteases and have been classified into the evolutionary clan of serine proteases SF, which utilize a Ser-Lys or Ser-His catalytic dyad rather than the more common Ser-His-Asp triad mechanism (Black, 1993; Paetzel et al., 1998
). Type I SPases can be irreversibily inhibited by certain penem compounds which are responsible for the acylation of the serine residue in the active site (Black & Bruton, 1998
; Paetzel et al., 1998
). SPase activity was found to be essential for cell viability, defining type I SPases as potential targets for the development of novel antibacterial agents.
We report here on the cloning and transcriptional analysis of the L. pneumophila lepB gene encoding the SPase I. In addition, functional activity of the encoded protein was demonstrated by complementation analysis in an E. coli SPase mutant. Finally, the effect of a penem derivative on growth of L. pneumophila and an E. coli temperature-sensitive SPase mutant expressing L. pneumophila lepB was evaluated.
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METHODS |
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DNA manipulations.
All DNA manipulations, including purification, restriction digestion, ligation, agarose gel electrophoresis, PCR amplification and transformation of E. coli cells, were performed using standard techniques (Sambrook et al., 1989). Restriction endonucleases and other DNA-modifying enzymes were purchased from Roche Diagnostics and Invitrogen-Life Technologies. Oligonucleotides (Table 1
) were obtained from Eurogentec. Nucleotide sequence analysis was performed on ALFexpress (Amersham Biosciences) using Cy5-labelled oligonucleotides. Genomic DNA of Legionella strains was extracted with the Wizard Genomic DNA Purification Kit (Promega).
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Southern blot hybridization.
Genomic DNA from L. pneumophila was digested with specific restriction enzymes (EcoRV, EcoRI and HindIII) and separated by agarose gel electrophoresis. DNA fragments were subsequently transferred to Hybond-N membranes (Amersham Biosciences) using a vacuum blot device (VacuGene, Amersham Biosciences). A lepB-specific DNA probe was generated by labelling the PCR fragment obtained with primers LspF and LspR with digoxigenin (DIG) according to the instructions of the manufacturer (Roche Diagnostics). The UV cross-linked DNA fragments were hybridized with the lepB-specific probe as described previously (Engler-Blum et al., 1993). Hybridization signals were detected using 0·25 mM CDP-Star (Roche Diagnostics) according to the method of Hoeltke et al. (1995)
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RNA isolation and RT-PCR experiments.
Total RNA was isolated from L. pneumophila cultures grown to exponential and stationary phase using the RNeasy mini kit (Qiagen). For this purpose, the cells from 3 ml of culture were collected by centrifugation, the RNAs were fixed using the RNA Protect kit (Qiagen) and the samples frozen at 20 °C until immediately prior to use. To investigate whether the L. pneumophila lepB gene was also expressed when the cells were grown intracellularly, total RNA was isolated from L. pneumophila grown within A. castellanii. For these experiments, A. castellanii, adherently grown in a 24-well plate, was infected with L. pneumophila cells at an m.o.i. of 1. Infection was allowed to proceed for 2 h at 37 °C and then gentamicin (100 µg ml1) was added for 1 h to kill the bacteria present in the extracellular environment; this represents the initial time point (0 h). At 24 h and 48 h post-infection, culture supernatants from 12 wells were collected and the bacteria were released from the amoebae by hypotonic lysis with ice-cold distilled water. After mixing the released bacteria with those present in the cell culture supernatant, bacteria were collected and RNA was isolated as described above. As L. pneumophila does not replicate in cell culture medium, the bacteria present in the samples result from intracellular growth and subsequent host cell lysis. After isolation, a control for the quality of the RNA samples was carried out by 1·3 % agarose-formaldehyde gel electrophoresis followed by ethidium bromide staining. RT-PCR experiments were carried out using the various primers given in Table 1 (see also Fig. 3
) by means of the Access RT-PCR kit (Promega). Initial synthesis of the cDNA strand and subsequent amplification were carried out starting from 0·5 µg total RNA using the following cycling conditions: 45 min at 48 °C, 3 min at 95 °C, 40 cycles of (30 s at 95 °C, 45 s at 50 °C, 2 min at 72 °C), 5 min at 72 °C. The same reactions were also performed on RNA treated with RNase to check for the possible presence of traces of DNA in the samples.
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Computer-aided analysis.
Nucleotide and amino acid sequences were analysed using Vector NTI Suite (Informax) and NCBI BLAST search analysis (Altschul et al., 1997). Amino acid sequence alignment was done using the Vector NTI Suite software and topology prediction was performed using HMMTOP (Tusnady & Simon, 1998
).
Complementation analysis of the L. pneumophila lepB gene.
The complementation assay protocol was similar to those described previously (Inada et al., 1989; Cregg et al., 1996
; Chu et al., 2002
). E. coli strain IT89 (temperature-sensitive lepB mutant) was transformed with either pEXLepLpn, pBCNLepLpn or control plasmids pEX50 or pBCN. Single transformants were grown overnight in LB medium at 27 °C. After dilution to an OD540 of 0·02, cultures were grown at the non-permissive temperature 42 °C in the absence of IPTG and the OD540 was followed as a function of time.
In addition, growth of E. coli IT89(pEXLepLpn) at 42 °C was monitored in the presence of 1 mM or 2 mM IPTG to stimulate expression of the L. pneumophila lepB gene.
In vivo analysis of the inhibitory effect of (5S,6S)-6-[(R)-acetoxyethyl]-penem-3-carboxylate on LepB.
Overnight cultures of E. coli IT89(pEXLepLpn) and L. pneumophila were diluted to an OD540 of 0·02 and 0·2, respectively (in triplicate). To one of the cultures 100 µM (5S,6S)-6-[(R)-acetoxyethyl]-penem-3-carboxylate (dissolved in DMSO) was added. As a control, an equal amount of DMSO was added to one of the cultures to investigate the effect of this solvent on both bacterial strains. No DMSO or inhibitor was added to the third culture. The cultures were then incubated at 42 °C [E. coli IT89(pEXLepLpn)] or 37 °C (L. pneumophila). Growth of the different cultures was compared during exponential growth [OD540 0·5 for E. coli IT89(pEXLepLpn) without DMSO or inhibitor added; OD540 1·5 for L. pneumophila without DMSO or inhibitor] and post-exponential growth [OD540 0·8 for E. coli IT89(pEXLepLpn) without DMSO or inhibitor; OD540 4·0 for L. pneumophila without DMSO or inhibitor].
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RESULTS AND DISCUSSION |
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Transcriptional analysis of the L. pneumophila lepB gene
In the first instance, the organization of the genes in the neighbourhood of the lepB gene was analysed (Fig. 3a). The lepB gene is situated in the middle of two other putative genes that are transcribed in the same direction. Upstream of lepB, a gene encoding a homologue of GTP-binding LepA proteins was found, while downstream of lepB, a gene encoding a ribonuclease III (Rnc) homologue is present. The intergenic region between the lepA and lepB gene is 72 bp, while that between the SPase gene and the putative rnc gene is 390 bp. The predicted ORFs located further upstream and downstream are transcribed in the opposite direction and encode proteins with homology to lytic murein transglycolases and an ATP-binding protein of an ABC transporter, respectively.
To investigate whether L. pneumophila expresses the lepB gene and whether the three unidirectionally described genes constitute an operon, RT-PCR experiments on total RNA were carried out (Fig. 3b). Total RNA was isolated either from L. pneumophila grown to exponential and stationary phase in liquid culture, or from L. pneumophila grown intracellularly in A. castellanii. RT-PCR with primers LspF and LspR, both binding to the lepB gene, clearly showed the presence of a 0·8 kb transcript in the case of L. pneumophila grown in liquid culture, as well as in L. pneumophila cells arising from infection of, and intracellular multiplication within, A. castellanii. Similar results were obtained with RNA isolated from exponential- or stationary-phase cultures (data not shown). RT-PCR on RNase-treated samples, included to detect traces of DNA in the samples, did not result in a band on agarose gel, indicating the absence of DNA and hence confirming a specific RNA-dependent amplification. In addition, the presence of a transcript of 1·7 kb comprising the three genes could be demonstrated, indicating that the three genes are cotranscribed. The lower intensity of the 1·7 kb fragment compared to that of the smaller fragments could be an indication of a post-transcriptional cleavage of the longer message but might also be the consequence of a lower stability of the mRNA, or even of a less efficient PCR amplification. Based on these results and taking into account that the more upstream located gene is transcribed in the opposite direction, we assume the presence of a promoter upstream of the lepA gene. Based on database analysis, the lepAlepBrnc cluster, with highly variable intergenic distances, can also be found in the genomes of other Gram-negative human pathogens, such as enteropathogenic E. coli, Vibrio cholerae, Yersinia pestis, Haemophilus influenzae, Shigella flexneri and Salmonella typhimurium genomes. For most of the pathogens, however, information on transcriptional organization is not available. In contrast to L. pneumophila, where lepB and rnc are part of the same operon (lepAlepBrnc), for E. coli it was shown that the lepB gene is situated in an operon structure together with the upstream lepA gene (March & Inouye, 1985
) and this lep operon is followed immediately by the rnc operon. Recently, it was shown that in the case of the obligate intracellular pathogen Rickettsia rickettsii, the lepB gene is cotranscribed with the secF gene (encoding a membrane protein involved in protein translocation across the inner membrane), the nuoF gene (encoding a putative NADH dehydrogenase I chain F), and the rnc gene in the secFnuoFlepBrnc cluster (Rahman et al., 2003
). In Rhodobacter capsulatus, no lepA is present but the lepB gene forms an operon with the downstream genes, rnc and era (encoding a GTP-binding protein) (Rauhut et al., 1996
). From these examples it is clear that, although homologous genes are often present in the lep region, the transcriptional organization can vary considerably between different organisms. A tripartite operon structure consisting of lepAlepBrnc as observed in L. pneumophila is unique so far.
Expression and functional analysis of the L. pneumophila lepB gene in E. coli
Assessment of in vivo activity of L. pneumophila LepB depended on complementation of the temperature-sensitive LepB mutant of E. coli IT89 (Inada et al., 1989). E. coli IT89 contains an amber mutation in the lepB gene at codon 39, which gives a temperature-sensitive phenotype (Cregg et al., 1996
). The strain shows normal growth at 27 °C, but growth is dramatically affected at the non-permissive temperature (42 °C). This temperature-sensitive mutation can be complemented by a plasmid carrying a functional lepB gene. This assay has already been used to demonstrate type I SPase activity for SPase genes of Salmonella typhimurium (van Dijl et al., 1990
), Bradyrhizobium japonicum (Bairl & Müller, 1998
; Müller et al., 1995
), Staphylococcus aureus (Cregg et al., 1996
), Streptococcus pneumoniae (Zhang et al., 1997
), Streptomyces lividans (Parro et al., 1999
), Bacillus amyloliquefaciens (Chu et al., 2002
), and Rickettsia rickettsii and Rickettsia typhi (Rahman et al., 2003
).
The L. pneumophila lepB gene was cloned on a low-copy-number (pEX50) and a high-copy-number (pBCN) plasmid to evaluate the effect of overexpression of L. pneumophila LepB on its complementation capacity. The pBCN plasmid has a 10 times higher copy number than pEX50. Growth of E. coli IT89 harbouring pEXLepLpn, pBCNLepLpn and the respective control plasmids pEX50 and pBCN at 42 °C was monitored in the absence of IPTG. Fig. 4(a) clearly shows that L. pneumophila LepB supported growth at the non-permissive temperature, demonstrating that L. pneumophila LepB correctly inserts into the E. coli inner membrane and that it can process all E. coli proteins necessary for cell viability. The growth rate of E. coli IT89(pBCNLepLpn) was significantly retarded compared to that of E. coli IT89(pEXLepLpn). This observation, together with the finding that addition of 1 mM and 2 mM IPTG to E. coli IT89(pEXLepLpn) resulted in a decrease of growth rate, suggests that overexpression of the L. pneumophila lepB gene results in lethality.
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Effect of a penem derivative on the activity of L. pneumophila LepB
To evaluate L. pneumophila LepB as a target for novel antibiotics, growth of E. coli IT89(pEXLepLpn) and L. pneumophila was monitored in the presence and absence of (5S,6S)-6-[(R)-acetoxyethyl]-penem-3-carboxylate, a specific SPase inhibitor (Allsop et al., 1995; Black & Bruton 1998
; Paetzel et al., 1998
). As a control, the effect of adding DMSO, used to dissolve the penem compound, was included. Fig. 5
shows that addition of 100 µM (5S,6S)-6-[(R)-acetoxyethyl]-penem-3-carboxylate resulted in a clear reduction (1418 %) of the growth of E. coli IT89(pEXLepLpn) cells at 42 °C compared to the control with only DMSO added. A clearly retarded growth (1825 %) was also observed when L. pneumophila was grown in BYE medium containing 100 µM of the penem derivative. In this case, no effect due to the addition of DMSO was observed. For both strains, reduction of the growth was observed for exponentially-growing cells as well as for stationary-phase cultures. The antibacterial activity of the penem derivative is limited, most likely because of the difficulty in penetrating the outer membrane to reach its target (Black & Bruton, 1998
). When present in a similar concentration (100 µM), this compound was reported to cause a 31 % reduction of the growth of E. coli TOP10(pMB2) (Barbosa et al., 2002
).
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In summary, we have cloned and characterized the functional type I signal peptidase gene of the human pathogen L. pneumophila. Further experiments will reveal the extent to which this key enzyme is important in the secretion of established virulence factors and to what degree it may be useful, from a therapeutic point of view, as a target for novel antibiotic compounds.
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ACKNOWLEDGEMENTS |
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Received 9 December 2003;
revised 22 January 2004;
accepted 26 January 2004.
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