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1 Department of Microbiology and Immunology, University of British Columbia, Vancouver, BC V5Z 4H4, Canada
2 Departments of Microbiology and Immunology and Paediatrics, University of British Columbia; Division of Infectious and Immunological Diseases, British Columbia's Children's Hospital, Vancouver, BC V5Z 4H4, Canada
Correspondence
Richard W. Stokes
rstokes{at}interchange.ubc.ca
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Whilst the currently available antimicrobials are often effective at eradicating the infection, there are issues with patient drug compliance and the emergence of drug-resistant M. tuberculosis (Espinal, 2003; Ormerod, 2005). In addition, the rising incidence of HIV infection, particularly in parts of Africa, has further increased the mortality associated with tuberculosis (Nunn et al., 2005). At the onset of infection in the lung, pathogenic mycobacteria gain access to the alveoli, where the bacteria encounter the resident macrophages. While much is known with regard to macrophage receptors that mediate bacterial recognition and subsequent phagocytosis (El-Etr & Cirillo, 2001; Ernst, 1998), the bacterial surface components that participate in the ligand–receptor interactions remain largely uncharacterized. A thorough understanding of the molecular mechanisms employed by pathogenic mycobacteria when faced with host cells could potentially identify targets for antimicrobial design and lead to the development of novel therapies.
Previously, we reported that disruption of the M. tuberculosis surface capsule following sonication significantly increases the level of bacterial association with murine and human macrophages (Stokes et al., 2004). A similar phenotype was also reported following deletion of the pks3–pks4 locus of M. tuberculosis, which reduced the retention of the surface capsule due to a deficiency in mycosanoic and mycolipenic acid synthesis that resulted in the absence of diacyltrehaloses and polyacyltrehaloses in the cell envelope (Dubey et al., 2002; Rousseau et al., 2003). Increased bacterial recognition by macrophages has also been demonstrated in non-tuberculous mycobacteria lacking surface glycopeptidolipids (Etienne et al., 2002). Together these findings suggest that modifications to the mycobacterial cell wall can potentially lead to the exposure of bacterial ligands that are readily recognized by macrophages. Interestingly, a recent study of M. tuberculosis clinical isolates reported that strains associated with extra-respiratory sites of infection correlated with an increased level of macrophage infectivity, suggesting that the initial interactions with the host macrophage directly affect bacterial pathogenesis (Garcia de Viedma et al., 2005).
The focus of the present study was to identify and characterize mycobacterial factors that participate in the initial binding interaction with host macrophages. A transposon library of M. tuberculosis was screened for mutants that showed an increased level of macrophage association as compared to the parental wild-type. Our expectation was that this screening strategy would identify mycobacterial genes that contribute, in some manner, to the biogenesis of the cell wall. A total of five mutants were selected and the sites of transposon disruptions were localized to loci encoding enzymes involved in lipid biosynthesis as well as hypothetical proteins. One mutant containing a transposon disruption within the fadD23 gene was repeatedly isolated from the library. Lipid analysis of the fadD23 mutant indicated a deficiency in the production of cell wall sulfolipids. Genetic complementation of the transposon mutant with the wild-type gene led to restoration of the wild-type phenotype for both macrophage binding and sulfolipid production.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). For the preparation of lipid extracts, mycobacteria were cultured in glycerol/alanine/salts (GAS) medium (Takayama et al., 1975). Escherichia coli strains were grown in Luria–Bertani (LB) broth or on LB-agar plates. When required, hygromycin and kanamycin were used at concentrations of 50 µg ml–1 and 25 µg ml–1 respectively.
Transposon mutagenesis.
A transposon library was generated using the pHAE87 vector and Tn5370 as described previously (Bardarov et al., 1997). Briefly, a 100 ml culture of M. tuberculosis was grown to OD600 0.8–1.0. The cells were washed twice and resuspended in a final volume of 10 ml phage adsorption buffer (10 mM Tris/HCl, pH 7.6, 100 mM NaCl, 10 mM MgSO4, 2 mM CaCl2). The bacteria were infected with the phage lysate at an m.o.i. of 10 : 1 (phage particles:bacteria) for 4 h at 39 °C. The infected cell suspension was centrifuged at 16 000 g for 5 min to recover the bacteria before plating onto selective media. After 4–6 weeks of growth, hygromycin-resistant colonies were scraped from the agar plates and cultured in 7H9 in separate pools each containing roughly 500 mutants. After 7–10 days of growth, the cultures were aliquoted and stored at –80 °C. A total of 20 library pools were used in the screening experiments.
Southern blot analysis on a total of 38 randomly selected mutants confirmed that significant diversity in mutant genotypes was present in the transposon library (data not shown). Briefly, genomic DNA from the 38 mutants was digested with PvuII, which does not cut within the transposon, and transferred onto nylon membrane as described elsewhere (Sambrook et al., 1989). The blots were hybridized with a DIG-labelled DNA fragment derived from the hygromycin-resistance cassette according to the directions of the manufacturer (Roche Applied Science). The insertion patterns and limitations of Tn5370 transposon have been previously characterized (McAdam et al., 2002). Thus, while the library may not be representative of the whole genome, it is likely that insertions are present in a significant number of the genes.
Infection of THP-1 cells.
The human macrophage-like cell line THP-1 (ATCC TIB202) was cultured in RPMI 1640 (Gibco) supplemented with 10 % heat-inactivated fetal calf serum (Gibco), 10 mM L-glutamine (Gibco) and 10 mM sodium pyruvate (Gibco) and maintained in an atmosphere of 5 % CO2 at 37 °C as described previously (Stokes & Doxsee, 1999). For infection assays, THP-1 cells were seeded onto acid-washed glass coverslips (2x105 cells per coverslip) or into T-75 cm2 flasks (5x106 cells) and differentiated in the presence of 100 nM phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich) over 3 days. Prior to infection, the monolayers were washed with binding medium to remove any residual PMA.
Single-cell suspensions of mycobacteria for use during infection assays were prepared using two separate techniques, essentially as described previously (Stokes et al., 2004). Briefly, the bacteria were pelleted by centrifugation (16 000 g for 5 min) and resuspended in 500 µl binding medium (138 mM NaCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, 2.7 mM KCl, 0.6 mM CaCl2, 1 mM MgCl2 and 5.5 mM D-glucose; Smith & Iden, 1981). The bacterial clumps were then dispersed by 10 repeated passages through a 25-gauge needle attached to a 1 ml syringe or by three 30 s pulses of bath sonication using a VC50T 50 W microcup horn (Sonics & Materials) as indicated for each experiment (Stokes et al., 2004). The volume of the cell suspension was then adjusted using binding medium to achieve the desired bacterial concentration.
The differentiated THP-1 cells were infected with mycobacteria for two purposes: (i) to screen the transposon library and (ii) to test the macrophage binding affinities for the mutant clones selected for further study. For procedure (i), THP-1 cells cultured in T-75 cm2 flasks and on coverslips were infected with the library pools (syringed suspensions) at an m.o.i. of 100 : 1 (bacteria : macrophages) for 4 h. Non-adherent mycobacteria were removed by vigorously washing the monolayer three times with binding medium (10 ml for flasks, 500 µl for coverslips), a method that we have previously shown to be effective for the removal of non-adherent bacteria (Stokes et al., 1986). The infected coverslips were fixed (10 min in 10 % formaldehyde in ethanol) and mounted onto microscope slides followed by staining with Kinyoun's Carbol Fuchsin and malachite green. The level of bacterial binding for each mutant pool was determined by microscopic count. The infected monolayers in the T-75 cm2 flasks were used to enrich for mutants with enhanced host-cell-binding capabilities; the THP-1 cells were dislodged using a cell scraper, resuspended in PBS containing 0.1 % Tween-80 (PBS-Tween) and briefly sonicated to lyse the macrophages. The macrophage-associated bacteria were recovered and cultured for 7–10 days in 7H9 before being used as the inoculum in a subsequent round of selection. After six to eight rounds of enrichment, the library pools that displayed an increased level of macrophage association (as determined by microscopic count) were plated onto selective agar plates and individual mutant clones were assessed for macrophage infectivity.
For procedure (ii), syringed and sonicated suspensions of mycobacteria were prepared as described above and used to infect THP-1 monolayers on coverslips at an m.o.i. of 100 : 1 (bacteria : macrophages) for 3 h. The monolayers were fixed and stained as described above. The percentage of infected macrophages was determined by counting 100 macrophages for each coverslip.
Identification of the transposon insertion site.
Genomic DNA from the selected transposon mutants was extracted (Belisle & Sonnenberg, 1998), restriction digested with PvuII and analysed by Southern hybridization using a labelled fragment from the hygromycin-resistance cassette. The presence of a single hybridizing DNA fragment on the Southern blot verified that a single copy of the transposon had incorporated into the chromosome. The precise location of the transposon insertion was determined by sequencing the amplicons generated by ligation-mediated inverse PCR (Cox et al., 1999). Briefly, 2.5 µg DNA was digested with RsaI. The restriction digest was extracted once with phenol/chloroform/isoamyl alcohol (25 : 24 : 1) and precipitated with ethanol. An aliquot of the digested DNA (
200 ng) was ligated overnight at 16 °C, followed by ethanol precipitation. The DNA sequences adjacent to the transposon were amplified using primers corresponding to the left (O84L-F 5'-GTCATCCGGCTCATCACCAG-3' and O84L-R 5'-AACTGGCGCAGTTCCTCTGG-3') and right (O84R-F 5'-ATACACGCGCACCGGTTCTAGC-3 and O84R-R 5'-CACGGCGAACCGCTGGTG-3') ends of the transposon. Sequence comparisons were done using BLAST 2.0 at www.ncbi.nlm.nih.gov/. The transposon insertion within the fadD23 gene was also confirmed by PCR using primers that flank the site of transposon integration; the FADD-L 5'-TCTGACAACATCCGCGATAA-3' primer corresponds to nucleotides 348–368 and the FADD-R 5'-GTCACTTCGAAGGCGAAGTT-3' primer matches nucleotides 807–827 of the fadD23 gene.
Complementation of the fadD23 transposon mutant.
The complementation construct was generated by amplifying the fadD23 gene along with 455 bp of upstream sequence from M. tuberculosis chromosomal DNA using Pfu DNA polymerase (Stratagene) with the following primers (FADJLL 5'-CGTAGAACTCGTCGCAATCC-3' and FADJLR 5'-GTGGATCCGACCGTAAGACC-3'). The amplicon was blunt-end ligated into the mycobacterial integration vector pMV306, which had been digested with EcoRV (Stover et al., 1991). The resulting plasmid construct was sequenced and transformed into the mutant by electroporation as described elsewhere (Parish & Stoker, 1998). Transformed bacteria were recovered using kanamycin selection and the presence of the wild-type fadD23 gene in the complemented mutant was confirmed by PCR using the FADD-L and FADD-R primers.
Lipid analysis.
Apolar and polar lipids were extracted from bacterial cultures that were pelleted by centrifugation, autoclaved (121 °C, 60 min) and subsequently lyophilized as described previously (Besra, 1998). Lipids were separated by two-dimensional thin-layer chromatography (2D-TLC) on Silica Gel 60 plates (EM Science) using five different solvent systems (A–E) for mycobacterial lipid analysis that have been described elsewhere (Besra, 1998; Dobson et al., 1985). Sulfolipids were localized using solvent system D, which uses chloroform/methanol/water (100 : 14 : 0.8) for the first-dimension solvent system and chloroform/acetone/methanol/water (50 : 60 : 2.5 : 3) for the second dimension. Lipids were visualized by spraying TLC plates with either 5 % ethanolic phosphomolybdic acid or 2.5 % 
-naphthol/10 % sulfuric acid followed by charring. Sulfolipid 1 (SL-1) was also detected by staining TLC plates with cresyl violet as described elsewhere (Soto et al., 2000). Briefly, the lipids were resolved on TLC plates using solvent system D and the plates were immersed in a solution of 0.02 % cresyl violet in 1 % aqueous acetic acid for 10 min followed by briefly destaining with 1 % aqueous acetic acid (data not shown).
Statistical analysis.
The results shown are the mean±SEM. Statistically significant differences between groups were determined with the two-tailed, unpaired Student's t test or analysis of variance. P values <0.05 were considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). A biological screening assay consisting of iterative rounds of macrophage infection was used to select for transposon mutants with an enhanced ability to bind macrophages. As outlined in Fig. 1, 20 pools from a transposon library (each containing 500 mutants) of M. tuberculosis were used to infect THP-1 cell monolayers. Transposon mutants that preferentially associated with host cells were recovered from the monolayer and used as the initial inoculum in a subsequent macrophage infection assay. We hypothesized that the desired phenotype would become enriched within the output mutant population after successive rounds of selection. Between the sixth and eighth round of successive macrophage infection assays, three of the library pools emerged that consistently bound the cell monolayers more readily than the syringed M. tuberculosis control (Fig. 2). The three pools were plated onto 7H10 agar and 40 mutants from each pool were isolated at random. Southern blotting was used to identify siblings within each pool (data not shown) and the level of macrophage association was determined for each of the 40 mutants from the three pools (a total of 120 mutants). The site of transposon insertion was identified using inverse PCR and DNA sequencing of mutants within the pools with the highest levels of macrophage association (Table 1). None of the mutants appeared to have any alterations in colony morphology or growth rate in broth when compared to the wild-type (data not shown).
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, all of the mutants identified from the selection procedure exhibited a binding phenotype that more closely mimicked that of the sonicated preparation of wild-type M. tuberculosis. These increases in macrophage infectivity suggested that the transposon insertions led to an altered bacterial surface and increased recognition by the host macrophages. To confirm that the increases in bacterial binding were not merely a side effect of the mutagenesis procedure, we included a transposon mutant (mutant 1) from library pool A as a negative control. As shown in Fig. 3, the mutant 1 strain did not exhibit any increase in macrophage association and was indistinguishable from a syringed preparation of wild-type M. tuberculosis, suggesting that the phenotypes of the mutants identified from the screening process could not be simply attributed to the transposon construct. It is also important to note that the transposon mutants that were identified from the screening procedure did not exhibit any increased bacterial clumping when cultured in vitro and that the syringe-dispersal treatment used prior to macrophage infection was sufficient for the disruption of small clumps of mycobacteria, as previously described (Stokes et al., 2004)
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. The fadD23 mutant displayed an increased binding affinity to THP-1 cells as compared to wild-type (P<0.05, ANOVA). Complementation of the mutation with the fadD23 gene in trans resulted in binding levels that were intermediate between the wild-type and the mutant, and not significantly different from either (P>0.05, ANOVA), indicating that partial restoration of the binding phenotype was achieved following complementation (Fig. 4b). The close proximity of the fadD23 gene to other loci that have been linked to sulfolipid biosynthesis prompted us to investigate whether the biosynthesis of cell wall glycolipids was affected in the fadD23 transposon mutant. The apolar and polar lipid fractions were extracted from whole-cell pellets of the parental wild-type, the mutant and the complemented strain for analysis by TLC. As shown in Fig. 5, comparison of the apolar lipid fractions revealed a lack of sulfolipid production in the absence of fadD23. Confirmation that the lipid fraction was indeed sulfolipid was obtained by staining the TLC plates with cresyl violet (Soto et al., 2000) (data not shown). Importantly, sulfolipid production could be restored following complementation of the mutant with the wild-type fadD23 gene.
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DISCUSSION |
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). We hypothesized that this observation could be used as a library screening tool to identify mutants that had incurred cell wall alterations as a result of mutagenesis. In this investigation, we screened a transposon library of M. tuberculosis using successive rounds of infection assays with THP-1 cell monolayers. Five mutants were identified from the screen that exhibited an increased binding affinity for host cells as compared to the parental wild-type, and at least three of these disrupted genes (Rv3335c, pks6 and Rv1115) have been previously linked to mycobacterial pathogenesis.
The pks6 gene encodes a predicted polyketide synthase that was initially identified by signature-tagged mutagenesis and mutants in this gene were found to be impaired for growth in the lungs of Balb/c mice (Camacho et al., 1999). Separate studies have also suggested that Pks6 is involved in countering host defences that involve interferon-
(Hisert et al., 2004). Although the precise biological function of Pks6 is unknown, a recent report suggests its involvement in the synthesis of polar lipids within the M. tuberculosis cell envelope (Waddell et al., 2005). The Rv1115 and Rv3335c genes encode hypothetical proteins for which there is little functional information, although these loci have been previously identified from separate transposon libraries; a transposon mutant in Rv3335c has been previously shown to be defective for growth within the spleens of C57/BL6 mice using the transposon-site hybridization (TRASH) system (Sassetti et al., 2003) and disruption of the Rv1115 gene in Mycobacterium bovis BCG leads to increased accumulation of the mutant within acidified phagosomes following macrophage infection (Stewart et al., 2005). The Rv1505c gene encodes a predicted protein with a conserved domain that is associated with enzymes belonging to the hexapeptide repeat superfamily of acetyltransferases (Raetz & Roderick, 1995; Vuorio et al., 1994; Wenzel et al., 2005). It is important to note that confirmation of the phenotypes for the Rv3335c, pks6, Rv1115 and Rv1505c mutants will require genetic complementation to rule out the possibility of polar effects. In particular, the ORFs downstream from the Rv1505c and Rv1115 genes may be affected as a result of the transposon insertions. The Rv1505c gene appears to reside within an operon that encompasses Rv1505c–Rv1503c. The Rv1503c and Rv1504c genes, which are immediately downstream of Rv1505c, belong to the DegT/DnrJ/EryC1/StrS aminotransferase family in the protein families (Pfam) database (Bateman et al., 2004). Interestingly, the Rv1503–Rv1504 proteins share a high degree of similarity with the WecE protein (59 % identity, 71 % similarity) of E. coli and Salmonella enterica. Within these enteric pathogens, WecE is a sugar aminotransferase that functions in the synthesis of the carbohydrate component of the enterobacterial common antigen, a glycolipid located on the outer leaflet of the Gram-negative outer membrane. In the case of the Rv1115 transposon mutant, the downstream locus (Rv1116) is located just over 100 bp downstream and encodes a hypothetical protein of unknown function. Polar effects from the transposon are less likely to be an issue with the pks6 and Rv3335c mutants since the downstream loci are not cotranscribed in the same orientation.
The fadD23 transposon mutant was chosen for further study as this mutation was repeatedly identified within the output population of mutants that remained after the selection assays. The fadD23 gene is located upstream of two loci that have been confirmed to be involved in sulfolipid biosynthesis. The Pks2 polyketide synthase catalyses the formation of the sulfolipid acyl chains (Sirakova et al., 2001) while the MmpL8 protein mediates the transport of mature sulfolipid or its precursors from the cytosol into the cell envelope (Converse et al., 2003; Domenech et al., 2004). The fadD23 gene probably encodes a fatty-acyl CoA ligase that would function in concert with the Pks2 enzyme to generate the glycolipid acyl chains (Trivedi et al., 2004). The fadD23 gene is flanked on the other side by the Rv3827c gene, which encodes a putative transposase. It is unlikely that polar effects play a role in the phenotype of the fadD23 mutant given that the transcription of the Rv3827c gene is in the opposite orientation relative to fadD23 and that mutant complementation was sufficient to restore the wild-type phenotype. Thus from the data presented in this report, we propose that the FadD23 protein should now also be included in the list of SL-1 biosynthetic enzymes. Comparative TLC analysis of the apolar lipid fractions from the parental wild-type and the fadD23 transposon mutant confirmed the absence of mature SL-1 on the cell surface of the mutant strain. In addition, SL-1 production could be restored by complementation of the mutant with the fadD23 gene. We found that disruption of the fadD23 gene resulted in an increased level of bacterial association with macrophages which was restored to wild-type levels following complementation.
The correlation of SL-1 loss with increased binding of M. tuberculosis to macrophages could reflect alterations in the organization of the cell envelope leading to the exposure of previously masked bacterial ligands. Alternatively, the block in sulfolipid production seen in the mutant may lead to an upregulation in the synthesis of other cell wall lipids. Recently, Jain et al. (2007) reported that PDIM mutants of M. tuberculosis synthesized an increased abundance of sulfolipid due to the increased availability of methylmalonyl-CoA precursors. While the authors did not identify a reciprocal increase in PDIM synthesis in SL-1 deficient mutants (Jain et al., 2007), there remains the possibility that the transposon mutation in fadD23 may cause a disturbance in the total distribution of cell wall lipids. Finally, the absence of SL-1 may also affect the overall surface charge of the bacillus, thereby increasing the level of cell–cell interactions. In support of this possibility, we have previously shown a correlation between a negatively charged mycobacterial surface and enhanced macrophage association (Stokes et al., 2004).
There are other examples in the literature where loss of cell surface lipids has resulted in improved association of mycobacteria and host cells. Mycobacterium smegmatis mutants lacking cell wall glycopeptidolipids (GPL) are more rapidly internalized by macrophages than are wild-type bacteria (Etienne et al., 2002), perhaps due to the inhibitory effects of GPL on M. smegmatis binding (Villeneuve et al., 2003). In addition, inactivation of the pks3–pks4 locus in M. tuberculosis coincides with an increase in macrophage binding due to loss of diacyltrehaloses and polyacyltrehaloses (Rousseau et al., 2003). Collectively, these and our earlier findings (Stokes et al., 2004) suggest that the interaction of mycobacteria with macrophages is limited by the presence of an intact outer cell envelope, disruption of which leads to enhanced binding.
In summary, transposon mutants were identified that were more apt to associate with phagocytic cells, implying that these strains had sustained some cell surface alterations responsible for their increased uptake. Lipid analysis of the fadD23 transposon mutant confirmed the involvement of fadD23 in the synthesis of cell wall sulfolipid. However, it is important to note that the collection of mutants identified in this study does not represent an exhaustive list of all the mycobacterial genes involved in binding to host macrophages. In particular, the use of large pools of mutants as part of the screening process does not favour the selection of mutant clones that have reduced rates of in vitro replication. There is also the possibility that neighbouring bacteria may complement the phenotypes of other mutants within the same pool (Sassetti & Rubin, 2003). Further investigation will focus on the cell wall compositions of the remaining mutants and will determine whether the alterations in the cell envelope correlate with changes in bacterial virulence.
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ACKNOWLEDGEMENTS |
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Edited by: S. V. Gordon
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Received 6 March 2007;
revised 18 May 2007;
accepted 4 June 2007.
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