| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Department of Microbiology and Immunology, University of North Carolina School of Medicine, Chapel Hill, NC 27599-7290, USA
2 Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA
Correspondence
Miriam Braunstein
Miriam_Braunstein{at}med.unc.edu
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). Mycobacterium tuberculosis, the causative agent of this disease, is an intracellular pathogen and the ability of this bacterium to survive and grow in macrophages is essential to its virulence. Multiple processes are likely employed by M. tuberculosis to avoid destruction in macrophages. These include residing in a phagosome that fails to mature into an acidified phagolysosome and resisting reactive radicals (as reviewed by Russell, 2007 and Zahrt & Deretic, 2002). As in other bacterial pathogens, M. tuberculosis proteins exported beyond the cytoplasm to the bacterial cell envelope (composed of the cytoplasmic membrane and cell wall) or secreted into the environment are ideally positioned to interact with host-cell components and promote survival in macrophages. Consequently, exported and secreted proteins make good candidates for virulence factors, drug targets for disease intervention, and vaccine antigens.
Mycobacteria possess two conserved pathways for exporting proteins: the general secretion (Sec) pathway and the twin-arginine translocation (Tat) pathway (Braunstein et al., 2001; Kurtz & Braunstein, 2005; McDonough et al., 2005; Owens et al., 2002; Posey et al., 2006; Saint-Joanis et al., 2006). These systems recognize precursor proteins synthesized with amino-terminal signal sequences and transport them across the cytoplasmic membrane (DeLisa et al., 2003; Mori & Ito, 2001). The proteins exported by these pathways can remain associated with the cell envelope or be further secreted by the bacterium. The signal sequences of Sec and Tat substrates share a similar domain structure; however, Tat substrates are distinguished by the presence of the twin-arginine motif, R-R-x-
- (=uncharged residue). The two pathways also differ in their mode of transport. Sec substrates are translocated across the cytoplasmic membrane in an unfolded state, whereas Tat substrates are translocated in a folded conformation. M. tuberculosis also has at least two specialized protein export pathways: the SecA2-dependent system and the ESX-1 (ESAT-6) system (Braunstein et al., 2003; Guinn et al., 2004; Hsu et al., 2003; Pym et al., 2003; Stanley et al., 2003). Interestingly, both pathways appear capable of secreting specific subsets of proteins that lack conventional Sec or Tat signal sequences.
In M. tuberculosis, proteomic and genetic methods have been used to experimentally identify proteins exported beyond the cytoplasm (reviewed by Kurtz & Braunstein, 2005). The genetic methods rely on reporter enzymes that are fused to M. tuberculosis protein sequences and report on the subcellular location of the fusion proteins (Braunstein et al., 2000; Chubb et al., 1998; Downing et al., 1999; Lim et al., 1995; Wiker et al., 2000). Surrogate hosts such as non-pathogenic Mycobacterium smegmatis or Escherichia coli have been used in most of these studies, often because endogenous enzyme activities in M. tuberculosis precluded their use directly in the pathogen. The use of surrogate hosts is a problem for identifying proteins that are only exported by pathogenic M. tuberculosis.
β-Lactamase is an export reporter that was not initially employed directly in M. tuberculosis because of endogenous β-lactam resistance. β-Lactamase catalyses the hydrolysis of β-lactams, a class of antibiotic that targets cell-wall biosynthetic enzymes located outside of the cytoplasmic membrane. Therefore, β-lactamase must be exported beyond the cytoplasm to protect the bacterium from the drug. For this reason, when fused to another protein, it can be used as an export reporter with β-lactam resistance as a powerful indicator of export. We recently reported that a 
blaC mutant of M. tuberculosis, lacking the chromosomally encoded β-lactamase BlaC, is β-lactam sensitive (Flores et al., 2005). Further, we showed that BlaC is a native Tat substrate and that a truncated 'BlaC lacking a signal sequence can function as a reporter of Tat-dependent export directly in a blaC mutant of M. tuberculosis (McDonough et al., 2005). This was shown by fusing a Tat signal sequence to 'BlaC and demonstrating that the resulting hybrid protein confers resistance to the β-lactam antibiotic carbenicillin in the blaC background. Interestingly, the 'BlaC reporter works with Tat- but not Sec-exported proteins. Here we expanded the β-lactamase tools that can be used directly in M. tuberculosis by demonstrating that the TEM-1 β-lactamase (BlaTEM-1), originally identified in a clinical isolate of E. coli (Datta & Kontomichalou, 1965), functions as an export reporter in the blaC mutant of M. tuberculosis. The 'BlaTEM-1 reporter has the significant advantage of being compatible with both Sec and Tat signal sequences.
The proteomic and genetic approaches used in previous work for identifying exported proteins of M. tuberculosis are limited by their reliance on in vitro-grown bacteria. Consequently, a potentially interesting collection of proteins only exported or secreted while M. tuberculosis are inside host cells are missed. In this paper, we demonstrate that β-lactamase reporters have the novel capability of identifying M. tuberculosis proteins that are exported during intracellular growth in β-lactam-treated THP-1 macrophage-like cells. The system we describe will be of significant value for identifying the most interesting category of exported M. tuberculosis proteins – those exported during growth in the host environment.
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
was grown in Luria–Bertani medium (Fisher) supplemented with the following concentrations of antibiotics as required: carbenicillin, 100 µg ml–1; kanamycin, 40 µg ml–1. M. tuberculosis strains H37Rv (wild-type), PM638 (blaC, H37Rv) (Flores et al., 2005) and all derivative strains were cultured in Middlebrook 7H9 medium or on Middlebrook 7H10 agar medium (Difco; BD Biosciences) supplemented with 10 % ADS [0.5 % BSA, fraction V (Roche); 0.2 % glucose (dextrose); and 0.85 % NaCl], 0.5 % glycerol and 0.05 % Tween 80 (Fisher). Antibiotics for mycobacteria were used at the following concentrations: carbenicillin, 50 µg ml–1; kanamycin, 20 µg ml–1. 7H10 plates supplemented with carbenicillin lacked Tween, as the combination of Tween and carbenicillin appeared detrimental to growth of fusion-expressing strains.
Construction of 'blaTEM-1 fusion plasmids
Plasmids used in this study are listed in Table 1. All subcloned PCR products were sequenced and determined to be error free. Sequence encoding the mature domain (lacking the N-terminal signal sequence) of E. coli BlaTEM-1 was amplified from pUC19 plasmid DNA (Invitrogen) using the following primers: TEMbla1 (5'-AGATCTCACCCAGAAACGCTGGTGAAAG-3') and TEMbla2 (5'-GTTACCAATGCTTAATCAGTGAGGCACC-3'). The resulting PCR product was cloned into the pCC1 vector (Epicentre) to generate pJM114. The 'blaTEM-1 reporter was subcloned as a BglII–BamHI fragment into each of the multi-copy vectors described below.
|
ss, 'blaTEM-1.
(ii) ssplcB-'blaTEM-1.
The 'blaTEM-1 fragment was subcloned into BamHI-cut pMB222. The resulting plasmid, pJES101, contains an in-frame fusion of DNA encoding the signal sequence of PlcB/Rv2350c (ssplcB) to 'blaTEM-1 under the control of the hsp60 promoter.
(iii) ssmpt63-'blaTEM-1.
The 'blaTEM-1 fragment was subcloned into BamHI-cut pMB227. The resulting plasmid, pJES103, contains an in-frame fusion of ssmpt63 (Rv1926c) to 'blaTEM-1 under the control of the hsp60 promoter.
(iv) ssmpt83-'blaTEM-1.
DNA encoding the signal sequence and the first 31 amino acids of the mature M. tuberculosis Mpt83 (Rv2873) protein along with the native mpt83 promoter (Juarez et al., 2001) was amplified from M. tuberculosis genomic DNA using the following primers: mpt83HindIIIF (5'-CAAGCTTCGTCGGATCCGTGGTAGGGGATGTC-3') and mpt83HindIIIR (5'-CAAGCTTCGGGGTCAGCCATTGCCGCCGTGG-3') and cloned into the pCR2.1 vector (Invitrogen) to generate pJES125. A HindIII fragment from pJES125, carrying ssmpt83 and upstream genomic sequence was cloned into HindIII-cut pJES128 (Table 1
). The resulting plasmid, pJES129, contains an in-frame fusion of ssmpt83 to 'blaTEM-1 under the control of the native mpt83 promoter (Pmpt83).
Protein quantification by immunoblotting.
Whole-cell lysates of M. tuberculosis strains were prepared as described previously (Braunstein et al., 2001) with the following modifications. M. tuberculosis cultures were grown in 5 ml volumes to mid-exponential phase. The cultures were washed twice and resuspended in PBS containing 0.02 % Tween 80. An equal volume of 10 % formalin was added to the washed cultures, which were then incubated at room temperature for 1 h with frequent mixing by inversion. The formalin-fixation step was necessary to kill M. tuberculosis before further processing. Bacteria were then harvested by centrifugation at 3000 r.p.m., washed once in PBS 0.02 % Tween to remove residual formalin, and bead-beaten lysates were then obtained from each sample. Protein concentration for each lysate was measured using a bicinchoninic acid protein quantification kit (Pierce). Lysates were boiled for 10 min, subjected to SDS-PAGE and immunoblots were performed using standard conditions. Primary antibodies specific for BlaTEM-1 were used at a concentration of 1 : 5000 (QED Biosciences), and horseradish peroxidase-conjugated anti-mouse secondary antibodies were used at a concentration of 1 : 20 000. Bands were visualized using Western Lightning Chemiluminescent Reagent Plus (Perkin Elmer) and quantified using ImageJ Image Processing and Analysis software (http://rsb.info.nih.gov/ij/). Whole-cell lysates with the highest level of expression were diluted to enable direct comparison of all hybridization signals on a single blot. The comparative quantification was determined by measuring pixel density of an equal area for each blotted lysate in duplicate. Signal intensity per µg of whole-cell lysate loaded was determined and is reported as the amount relative to protein detected in the 'BlaTEM-1-expressing strain.
Macrophage infections.
THP-1 cells were maintained in RPMI (Gibco)/10 % heat-inactivated fetal calf serum (FCS) at 37 °C and 5 % CO2. To prepare THP-1 monolayers for infection, cells were spun down at 300 g, washed once in RPMI, then resuspended in RPMI/10 % FCS at a concentration of 1x106 cells ml–1. Cells were seeded into eight-well tissue culture slides at 2x105 cells per well and treated with phorbol myristate acetate (PMA) at a final concentration of 50 ng ml–1 for 48 h.
M. tuberculosis was grown to mid-exponential phase (OD600 0.5–1.0). Immediately prior to infection, the bacterial culture was pelleted, washed once in PBS containing 0.05 % Tween 80 (PBS-Tw), and resuspended in an equal volume of PBS-Tw. The culture was then briefly sonicated to break up clumps of bacteria, diluted in RPMI/10 % FCS medium and added to the THP-1 monolayer at m.o.i.=0.1.
THP-1 monolayers were infected with M. tuberculosis strains for 4 h at 37 °C and 5 % CO2. Overlying medium was then removed, the monolayers were washed three times with RPMI to remove non-cell-associated bacteria, and triplicate wells were lysed and plated to determine uptake (day 0 time point). The infected monolayers were then overlaid with RPMI/10 % FCS, or RPMI/10 % FCS supplemented with carbenicillin, and maintained at 37 °C and 5 % CO2. At 3 days post-infection, the overlying medium was replenished with RPMI/10 % FCS medium or medium supplemented with carbenicillin, as appropriate. On days 1, 3 and 5 post-infection, triplicate wells for each infection were washed to remove antibiotic and lysed with 0.05 % SDS. The resulting lysates were diluted and plated on 7H10 agar to enumerate intracellular bacteria during the course of infection. On day 0 and day 5 of the infection, cell lysates were also plated on 7H10 agar supplemented with 50 µg carbenicillin ml–1. This demonstrated that selection of spontaneous β-lactam-resistant mutants did not occur during the course of infection. To determine the appropriate carbenicillin concentration necessary to kill intracellular bacteria, THP-1 infection experiments were performed with a range of antibiotic concentrations (see Fig. 4b). Carbenicillin at 1 mg ml–1 was determined to be the lowest concentration of antibiotic that caused optimal killing of sensitive intracellular M. tuberculosis and was used in subsequent experiments.
|
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
blaC mutant of M. tuberculosis or blaS mutant of M. smegmatis (McDonough et al., 2005). Since the Sec pathway is likely responsible for the majority of protein export in M. tuberculosis, we were interested in utilizing a β-lactamase reporter that additionally works with Sec-exported proteins. For this reason, we tested the E. coli TEM-1 β-lactamase (BlaTEM-1), which has been used in other bacteria to report on proteins exported by Sec, Tat, type II and type III secretion systems (Broome-Smith et al., 1990; Charpentier & Oswald, 2004; Sauvonnet & Pugsley, 1996; Stanley et al., 2002).
A series of multi-copy kanamycin-marked 'blaTEM-1 plasmids were constructed and electroporated into the blaC mutant of M. tuberculosis (Fig. 1). The resulting kanamycin-resistant strains were tested for the ability to grow in the presence of 50 µg ml–1 of the β-lactam carbenicillin. When the truncated 'blaTEM-1 reporter without a signal sequence was expressed in the blaC mutant of M. tuberculosis, the strain remained carbenicillin-sensitive. In fact, no colonies of the strain expressing the truncated 'BlaTEM-1 grew on agar containing carbenicillin even after extended incubation (Figs 1 and 2). However, expression of a hybrid protein composed of a Sec signal sequence from Mpt63, a well-established secreted protein of M. tuberculosis (Horwitz et al., 1995; Manca et al., 1997), fused to 'BlaTEM-1 (ssMpt63-'BlaTEM-1) protected the blaC mutant from carbenicillin, as was evident by the ability of this strain to grow on carbenicillin agar plates (Figs 1 and 2) . We similarly tested a fusion protein in which the Sec signal sequence of a proven cell-wall-associated lipoprotein, Mpt83 (Hewinson et al., 1996), was fused to 'BlaTEM-1. This construct also conferred β-lactam resistance to blaC M. tuberculosis (Fig. 1). Of note, the ssMpt83-'BlaTEM-1 fusion protein included the first 31 amino acids of the mature Mpt83 protein as well as the native mpt83 promoter, which is reported to be active at very low levels in vitro (Hewinson et al., 1996; Said-Salim et al., 2006).
|
|
; Raynaud et al., 2002). PlcB has a predicted Tat signal sequence, and the ssPlcB-'BlaTEM-1 fusion also allowed blaC M. tuberculosis to grow in the presence of carbenicillin (Fig. 1).
To determine whether the ssPlcB-'BlaTEM-1 fusion was exported by the Tat pathway, it was tested in blaS M. smegmatis and in a tatA blaS M. smegmatis double mutant (McDonough et al., 2005) in two independent experiments. When the ssPlcB-'BlaTEM-1 fusion protein was expressed in blaS M. smegmatis, 92 % of colonies were carbenicillin resistant. However, when the same construct was expressed in the tatA blaS mutant, only an average 7 % of colonies were carbenicillin resistant, indicating that the Tat pathway functions in the export of this fusion protein. To show that a functional Tat pathway was not required for export of the Sec signal sequence 'BlaTEM-1 fusion, we similarly evaluated export of ssMpt63-'BlaTEM-1. When expressed in blaS and the tatA blaS mutants, ssMpt63-'BlaTEM-1 conferred carbenicillin resistance to 90 % and 95 % of colonies, respectively. This indicated, as expected, no role for the Tat pathway in exporting a Sec signal sequence 'BlaTEM-1 fusion.
In each example where a M. tuberculosis signal sequence (Sec or Tat) was fused to 'BlaTEM-1, blaC M. tuberculosis was protected from β-lactam attack. To demonstrate that the inability of the 'BlaTEM-1 reporter lacking a signal sequence to protect against carbenicillin was due to lack of export, as opposed to lack of expression, whole-cell extracts of 'BlaTEM-1 expression strains were prepared and assayed for cell-associated β-lactamase. To test for enzyme activity, we used the chromogenic β-lactam nitrocefin, which turns red following cleavage by β-lactamase (O'Callaghan et al., 1972). During a 15 min incubation the nitrocefin was hydrolysed by all strains expressing 'BlaTEM-1 constructs, while blaC M. tuberculosis demonstrated no activity, similar to PBS alone (data not shown). Importantly, β-lactamase activity was detected with the truncated 'BlaTEM-1 reporter lacking a signal sequence. In fact, the lysate from the 'BlaTEM-1 strain converted nitrocefin to the red product almost instantaneously and faster than any other strain tested. We similarly detected β-lactamase activity in whole-cell lysates of blaC M. tuberculosis expressing the 'BlaC reporter lacking its native signal sequence.
We also compared the level of each 'BlaTEM-1 fusion protein present in whole-cell lysates from the respective M. tuberculosis strains by immunoblots with antibodies specific for BlaTEM-1. This revealed a wide variation in the amount of 'BlaTEM-1 protein produced by the different strains (Fig. 3). The non-exported 'BlaTEM-1 expressed off the hsp60 promoter (Phsp60) was the most abundant protein detected. Phsp60 is considered a relatively strong promoter and is, therefore, present on many mycobacterial shuttle vectors (Stover et al., 1991). In comparison, the Phsp60-driven ssPlcB-'BlaTEM-1 and ssMpt63-'BlaTEM-1 were expressed at lower levels (59 % and 0.9 % of the level of the non-exported 'BlaTEM-1 construct, respectively). Since mpt83 is expressed at relatively low levels in vitro, we expected the ssMpt83-'BlaTEM-1 fusion to be weakly expressed (Hewinson et al., 1996; Said-Salim et al., 2006; Schnappinger et al., 2003). In fact, it was nearly undetectable by immunoblotting, present at only 0.4 % of the amount of non-exported 'BlaTEM-1 construct. The bands detected on the immunoblot are in general agreement with the predicted molecular mass of the expressed proteins. 'BlaTEM-1, lacking a signal sequence, has a predicted size of 28 kDa. Since whole-cell lysates were analysed in these experiments it is possible to see processed protein and/or uncleaved cytosolic precursor, which may explain the larger-sized ssPlcB-'BlaTEM-1 product. The signal sequences of PlcB and Mpt63 would add approximately 3 and 4 kDa, while the Mpt83 signal sequence and fused portion of the mature protein would add approximately 11 kDa, if left intact.
|
blaC M. tuberculosis 'BlaTEM-1 must be exported to confer protection against β-lactam antibiotics, that β-lactam resistance can be used to report on export, and that this reporter can be exported by Sec or Tat signal sequences and is compatible with different levels of expression.
The blaC mutant of M. tuberculosis is sensitive to β-lactams during intracellular growth in human THP-1 cells
β-Lactam antibiotics can be used for clinical treatment of intracellular pathogens such as Listeria monocytogenes (Safdar & Armstrong, 2003), and have been shown to reduce the population of phagocytosed Staphylococcus aureus (Barcia-Macay et al., 2006). This indicates that β-lactams can enter macrophages and inhibit intracellular growth of some bacteria. The blaC mutant of M. tuberculosis is sensitive to β-lactams in vitro, and we set out to test if this mutation also makes M. tuberculosis susceptible to β-lactams during growth in host cells.
Intracellular growth of the blaC mutant was not previously evaluated; therefore, we first tested the ability of this mutant to grow within human monocytic THP-1 cells. THP-1 cells were infected at a m.o.i. of 0.1 with either the blaC mutant or the virulent parental H37Rv strain. After a 4 h period of infection, the THP-1 monolayer was washed to remove non-cell-associated bacilli and fresh medium was added back. Growth over 5 days was assessed by plating of infected host-cell lysates for viable bacilli. The blaC mutant showed no difference in intracellular growth when compared to H37Rv (Fig. 4a). Of note, we confirmed that M. tuberculosis does not grow in the THP-1 culture medium as previously reported (Zhang et al., 1998).
To determine if the blaC mutant was sensitive to β-lactams during intracellular growth, THP-1 cells were infected with blaC M. tuberculosis and, following the washes to remove extracellular bacilli, medium containing different concentrations of carbenicillin was added to the infected monolayers. After 5 days incubation, the infected monolayers were washed to remove carbenicillin and lysed to plate for viable bacilli. In the absence of carbenicillin, the blaC mutant grew in THP-1 cells as previously seen. However, as the concentration of carbenicillin during the intracellular growth period increased, growth of the mutant diminished. At carbenicillin concentrations of 
0.8 mg ml–1, substantial killing of the mutant was observed (Fig. 4b). These results indicated that the blaC mutant is sensitive to β-lactam antibiotics during intracellular growth, and it suggested that the β-lactamase reporters could be used to study protein export during intracellular growth. Additional experiments showed that a carbenicillin concentration of 1 mg ml–1 was sufficient to achieve significant killing of the blaC mutant of M. tuberculosis in THP-1 cells, and this concentration was used in all subsequent experiments.
Export of β-lactamase protects intracellular blaC M. tuberculosis from β-lactam antibiotics
A reporter system that works with intracellularly growing M. tuberculosis would be of great value for identifying exported proteins that are expressed and exported only during infection. Having shown that the blaC mutant was sensitive to β-lactams during intracellular growth, we tested if β-lactamase could be used to report on protein export by M. tuberculosis growing in host cells. We tested fusion proteins expressing the 'BlaC and 'BlaTEM-1 reporters for the ability to protect the blaC mutant in β-lactam-treated THP-1 cells. In each experiment we compared an exported fusion protein to the truncated reporter alone. To test the 'BlaC reporter, which works with Tat-exported proteins only, THP-1 cells were infected with the M. tuberculosis blaC mutant expressing ssPlcB-'BlaC or 'BlaC only. Medium with or without 1 mg carbenicillin ml–1 was added and the course of infection was monitored over 5 days. In the absence of carbenicillin, both strains grew in THP-1 cells during the course of the experiment. However, in the presence of carbenicillin, the strain expressing the truncated reporter alone did not grow and was reduced by 10-fold over 5 days while the strain expressing the exported ssPlcB-'BlaC fusion protein was protected from carbenicillin and grew normally (Fig. 5a).
|
blaC M. tuberculosis expressing either the exported ssMpt63-'BlaTEM-1 or the 'BlaTEM-1 reporter alone, only the strain expressing ssMpt63-'BlaTEM-1 fusion grew in THP-1 cells in the presence of carbenicillin. The non-exported 'BlaTEM-1 strain was sensitive to the β-lactam and was reduced in number by 10-fold (Fig. 5b). Similarly, blaC M. tuberculosis exporting ssMpt83-'BlaTEM-1 fusion was able to grow in carbenicillin-treated THP-1 cells, while the non-exported 'BlaTEM-1 construct did not confer resistance to the blaC mutant (Fig. 5c). These experiments demonstrated that both the Tat-specific 'BlaC reporter and the more permissive 'BlaTEM-1 reporter can report on protein export while M. tuberculosis is growing in β-lactam-treated host cells. The use of β-lactamase reporters with intracellular M. tuberculosis represents a powerful tool for the study and identification of proteins exported during growth in host cells.
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). In fact, there is a growing list of M. tuberculosis exported and secreted proteins shown to contribute to virulence or to development of a host immune response (Kurtz & Braunstein, 2005). Genetic reporters have proven to be powerful tools for identifying these extracytoplasmic proteins. The construction of a β-lactam-sensitive blaC mutant of M. tuberculosis opened the door for using β-lactamases as reporters of protein export directly in M. tuberculosis. The 'BlaC reporter can be used as a Tat-specific reporter, while the 'BlaTEM-1 reporter, as shown here, can work with Sec or Tat signal sequences. An advantage of β-lactamase reporters is that they can be used to select for exported fusion proteins, as opposed to more labour-intensive screening. In addition, we showed here for the first time that resistance to β-lactam antibiotics can be used to report on protein export during intracellular growth of bacteria. Even in more genetically tractable bacterial pathogens, the identification of proteins exported or secreted from within host cells is a challenge.
Because β-lactams target cell-wall-modifying enzymes, β-lactamases must be exported in order to protect against these drugs. This export requirement was previously exploited with fusion proteins expressed in E. coli and other bacteria grown in vitro (Broome-Smith et al., 1990; Lee & Hughes, 2006). Here we showed that BlaTEM-1 can also report on protein export directly in blaC M. tuberculosis. The three M. tuberculosis signal sequences tested in our study are from well-established secreted or cell-wall-associated proteins. Mpt63 (Rv1926c, 16 kDa protein) has a predicted Sec signal sequence and is one of the four most abundant M. tuberculosis proteins secreted into culture media during in vitro growth (Horwitz et al., 1995). Mpt83 (Rv2873) is a glycosylated lipoprotein (Hewinson et al., 1996; Sutcliffe & Harrington, 2004) that is exported to the cell wall of M. tuberculosis. Mpt83 has a predicted Sec signal sequence with a lipoprotein signal peptidase (LspA) cleavage site and the requisite conserved cysteine for lipid modification. PlcB (Rv2350c, phospholipase C) is a cell-wall-associated protein of M. tuberculosis shown to function in virulence (Johansen et al., 1996; Raynaud et al., 2002). Unlike Mpt63 and Mpt83, PlcB has a predicted Tat signal sequence including a twin-arginine motif (Dilks et al., 2003). Signal sequences from all three of these proteins were able to promote export of a fused 'BlaTEM-1 reporter on the basis of production of β-lactam resistance. Notably, the ssMpt83-'BlaTEM-1 fusion protein was expressed from the native mpt83 promoter and the fusion protein included the predicted signal sequence plus 31 amino acids of the mature Mpt83 protein. This demonstrated the ability of the reporter to work with different strength promoters and extended protein sequences. It is important to note that even though variable levels of fusion protein were detected in M. tuberculosis whole-cell lysates as determined by immunoblot, each exported fusion provided sufficient protection against 50 µg carbenicillin ml–1 while the most abundant 'BlaTEM-1 without an export signal did not confer β-lactam resistance.
Previously, we showed that the PlcB signal sequence is able to drive export of functional 'BlaC in a Tat- and RR-dependent manner (McDonough et al., 2005). In E. coli the 'BlaTEM-1 reporter works with both Sec and Tat signal sequences (Broome-Smith et al., 1990; Stanley et al., 2002). The Sec and Tat pathways appear essential in M. tuberculosis (Braunstein et al., 2001; Saint-Joanis et al., 2006; Sassetti et al., 2003). Therefore, to investigate the mode of export of the ssPlcB-'BlaTEM-1 fusion protein, it was tested in M. smegmatis blaS and in a M. smegmatis tatA blaS double mutant. A 93 % reduction in β-lactam-resistant colonies was observed in the M. smegmatis tatA blaS double mutant. Thus, the Tat pathway is involved in the export of ssPlcB-'BlaTEM-1, although other export pathways participate as well. The signal sequence of PlcB may be promiscuous in targeting the Tat or Sec pathway for export, depending on the folded or unfolded nature of a fused reporter element. Similar results were recently shown for some predicted Tat signal sequences in E. coli (Tullman-Ercek et al., 2007).
In addition to working with the Sec and Tat pathways, the 'BlaTEM-1 reporter has been used with type II and type III secretion systems of Gram-negative bacteria (Charpentier & Oswald, 2004; Sauvonnet & Pugsley, 1996). Since substrates of the type III secretion system lack conventional N-terminal signal sequences, it remains possible that the 'BlaTEM-1 reporter will also work with non-conventional exported proteins of M. tuberculosis.
An interesting category of exported proteins that has been largely overlooked are those proteins only expressed and/or exported during the course of infection. We hypothesize that these are proteins exclusively exported in the host environment, including virulence factors and protective antigens. Furthermore, only a small number of the exported M. tuberculosis proteins identified in vitro have ever been directly investigated during intracellular growth in host cells (Kurtz & Braunstein, 2005). For most of these studies, immunomicroscopy was used to localize the proteins in M. tuberculosis-infected macrophages, which required development of suitable antibodies. We reasoned that if β-lactam antibiotics can reach intracellular blaC M. tuberculosis, β-lactamase reporters should additionally work during intracellular growth. β-Lactam antibiotics do not normally accumulate in eukaryotic cells; however, antibiotics of this class freely diffuse in and out of host cells (Tulkens, 1991), and β-lactam antibiotics are used to treat some intracellular bacterial infections (Safdar & Armstrong, 2003). More specifically, β-lactams reach intracellular S. aureus and L. monocytogenes and prevent growth of these organisms in THP-1 cells (Barcia-Macay et al., 2006; Carryn et al., 2003). Here we showed that blaC M. tuberculosis in THP-1 cells was also susceptible to carbenicillin. Thus, BlaC is responsible for M. tuberculosis resistance to β-lactam antibiotics during intracellular growth, indicating that the chromosomal blaC is a key factor preventing the use of β-lactams to treat M. tuberculosis infection.
When the set of exported β-lactamase fusion proteins was tested for the ability to protect blaC M. tuberculosis from β-lactam treatment during intracellular growth, all exported fusions conferred resistance. In contrast, the truncated non-exported β-lactamase reporters were not protective. These experiments demonstrated the effectiveness of both 'BlaC and 'BlaTEM-1 reporters to identify M. tuberculosis sequences that drive export of each reporter during growth within host cells. Because the ssMpt83-'BlaTEM-1 fusion was expressed from the native promoter, our results indicate that Mpt83, a protein of unknown function, is expressed and exported during intracellular infection. This result is consistent with the reported induction of mpt83 in macrophages (Schnappinger et al., 2003).
Several approaches have described proteins exported by M. tuberculosis in vitro, but a different suite of proteins may be exported during infection of the host. The intracellular β-lactamase reporter system we describe represents a new genetic tool for studying protein export in M. tuberculosis. It can be used to directly test the intracellular export of a protein of interest. We also hope to use it in combination with multiple rounds of infection and selection of β-lactam-resistant clones from a M. tuberculosis fusion library. This should serve to identify the most interesting category of proteins, namely those that are exported during intracellular growth and missed by alternative methods.
|
ACKNOWLEDGEMENTS |
|---|
Edited by: W. Bitter
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Braunstein, M., Griffin, T. I., Kriakov, J. I., Friedman, S. T., Grindley, N. D. & Jacobs, W. R., Jr (2000). Identification of genes encoding exported Mycobacterium tuberculosis proteins using a Tn552'phoA in vitro transposition system. J Bacteriol 182, 2732–2740.
Braunstein, M., Brown, A. M., Kurtz, S. & Jacobs, W. R., Jr (2001). Two nonredundant SecA homologues function in mycobacteria. J Bacteriol 183, 6979–6990.
Braunstein, M., Espinosa, B. J., Chan, J., Belisle, J. T. & Jacobs, W. R., Jr (2003). SecA2 functions in the secretion of superoxide dismutase A and in the virulence of Mycobacterium tuberculosis. Mol Microbiol 48, 453–464.[CrossRef][Medline]
Broome-Smith, J. K., Tadayyon, M. & Zhang, Y. (1990). Beta-lactamase as a probe of membrane protein assembly and protein export. Mol Microbiol 4, 1637–1644.[Medline]
Carryn, S., Van Bambeke, F., Mingeot-Leclercq, M. P. & Tulkens, P. M. (2003). Activity of beta-lactams (ampicillin, meropenem), gentamicin, azithromycin and moxifloxacin against intracellular Listeria monocytogenes in a 24 h THP-1 human macrophage model. J Antimicrob Chemother 51, 1051–1052.
Charpentier, X. & Oswald, E. (2004). Identification of the secretion and translocation domain of the enteropathogenic and enterohemorrhagic Escherichia coli effector Cif, using TEM-1 beta-lactamase as a new fluorescence-based reporter. J Bacteriol 186, 5486–5495.
Chubb, A. J., Woodman, Z. L., da Silva Tatley, F. M., Hoffmann, H. J., Scholle, R. R. & Ehlers, M. R. (1998). Identification of Mycobacterium tuberculosis signal sequences that direct the export of a leaderless beta-lactamase gene product in Escherichia coli. Microbiology 144, 1619–1629.[Abstract]
Datta, N. & Kontomichalou, P. (1965). Penicillinase synthesis controlled by infectious R factors in Enterobacteriaceae. Nature 208, 239–241.[CrossRef][Medline]
DeLisa, M. P., Tullman, D. & Georgiou, G. (2003). Folding quality control in the export of proteins by the bacterial twin-arginine translocation pathway. Proc Natl Acad Sci U S A 100, 6115–6120.
Dilks, K., Rose, R. W., Hartmann, E. & Pohlschroder, M. (2003). Prokaryotic utilization of the twin-arginine translocation pathway: a genomic survey. J Bacteriol 185, 1478–1483.
Downing, K. J., McAdam, R. A. & Mizrahi, V. (1999). Staphylococcus aureus nuclease is a useful secretion reporter for mycobacteria. Gene 239, 293–299.[CrossRef][Medline]
Finlay, B. B. & Falkow, S. (1997). Common themes in microbial pathogenicity revisited. Microbiol Mol Biol Rev 61, 136–169.[Abstract]
Flores, A. R., Parsons, L. M. & Pavelka, M. S., Jr (2005). Genetic analysis of the beta-lactamases of Mycobacterium tuberculosis and Mycobacterium smegmatis and susceptibility to beta-lactam antibiotics. Microbiology 151, 521–532.
Guinn, K. M., Hickey, M. J., Mathur, S. K., Zakel, K. L., Grotzke, J. E., Lewinsohn, D. M., Smith, S. & Sherman, D. R. (2004). Individual RD1-region genes are required for export of ESAT-6/CFP-10 and for virulence of Mycobacterium tuberculosis. Mol Microbiol 51, 359–370.[CrossRef][Medline]
Hewinson, R. G., Michell, S. L., Russell, W. P., McAdam, R. A. & Jacobs, W. J. (1996). Molecular characterization of MPT83: a seroreactive antigen of Mycobacterium tuberculosis with homology to MPT70. Scand J Immunol 43, 490–499.[CrossRef][Medline]
Horwitz, M. A., Lee, B. W., Dillon, B. J. & Harth, G. (1995). Protective immunity against tuberculosis induced by vaccination with major extracellular proteins of Mycobacterium tuberculosis. Proc Natl Acad Sci U S A 92, 1530–1534.
Hsu, T., Hingley-Wilson, S. M., Chen, B., Chen, M., Dai, A. Z., Morin, P. M., Marks, C. B., Padiyar, J., Goulding, C. & other authors (2003). The primary mechanism of attenuation of bacillus Calmette-Guerin is a loss of secreted lytic function required for invasion of lung interstitial tissue. Proc Natl Acad Sci U S A 100, 12420–12425.
Johansen, K. A., Gill, R. E. & Vasil, M. L. (1996). Biochemical and molecular analysis of phospholipase C and phospholipase D activity in mycobacteria. Infect Immun 64, 3259–3266.[Abstract]
Juarez, M. D., Torres, A. & Espitia, C. (2001). Characterization of the Mycobacterium tuberculosis region containing the mpt83 and mpt70 genes. FEMS Microbiol Lett 203, 95–102.[Medline]
Kurtz, S. & Braunstein, M. (2005). Protein secretion and export in Mycobacterium tuberculosis. In Mycobacterium Molecular Biology, pp. 71–138. Edited by T. Parish. Norwich, UK: Horizon Bioscience.
Lee, H. J. & Hughes, K. T. (2006). Posttranscriptional control of the Salmonella enterica flagellar hook protein FlgE. J Bacteriol 188, 3308–3316.
Lim, E. M., Rauzier, J., Timm, J., Torrea, G., Murray, A., Gicquel, B. & Portnoi, D. (1995). Identification of Mycobacterium tuberculosis DNA sequences encoding exported proteins by using phoA gene fusions. J Bacteriol 177, 59–65.
Manca, C., Lyashchenko, K., Wiker, H. G., Usai, D., Colangeli, R. & Gennaro, M. L. (1997). Molecular cloning, purification, and serological characterization of MPT63, a novel antigen secreted by Mycobacterium tuberculosis. Infect Immun 65, 16–23.[Abstract]
McDonough, J. A., Hacker, K. E., Flores, A. R., Pavelka, M. S., Jr & Braunstein, M. (2005). The twin-arginine translocation pathway of Mycobacterium smegmatis is functional and required for the export of mycobacterial beta-lactamases. J Bacteriol 187, 7667–7679.
Mori, H. & Ito, K. (2001). The Sec protein-translocation pathway. Trends Microbiol 9, 494–500.[CrossRef][Medline]
O'Callaghan, C. H., Morris, A., Kirby, S. M. & Shingler, A. H. (1972). Novel method for detection of beta-lactamases by using a chromogenic cephalosporin substrate. Antimicrob Agents Chemother 1, 283–288.
Owens, M. U., Swords, W. E., Schmidt, M. G., King, C. H. & Quinn, F. D. (2002). Cloning, expression, and functional characterization of the Mycobacterium tuberculosis secA gene. FEMS Microbiol Lett 211, 133–141.[CrossRef][Medline]
Posey, J. E., Shinnick, T. M. & Quinn, F. D. (2006). Characterization of the twin-arginine translocase secretion system of Mycobacterium smegmatis. J Bacteriol 188, 1332–1340.
Pym, A. S., Brodin, P., Majlessi, L., Brosch, R., Demangel, C., Williams, A., Griffiths, K. E., Marchal, G., Leclerc, C. & Cole, S. T. (2003). Recombinant BCG exporting ESAT-6 confers enhanced protection against tuberculosis. Nat Med 9, 533–539.[CrossRef][Medline]
Raynaud, C., Guilhot, C., Rauzier, J., Bordat, Y., Pelicic, V., Manganelli, R., Smith, I., Gicquel, B. & Jackson, M. (2002). Phospholipases C are involved in the virulence of Mycobacterium tuberculosis. Mol Microbiol 45, 203–217.[CrossRef][Medline]
Russell, D. G. (2007). Who puts the tubercle in tuberculosis? Nat Rev Microbiol 5, 39–47.[CrossRef][Medline]
Safdar, A. & Armstrong, D. (2003). Antimicrobial activities against 84 Listeria monocytogenes isolates from patients with systemic listeriosis at a comprehensive cancer center (1955–1997). J Clin Microbiol 41, 483–485.
Said-Salim, B., Mostowy, S., Kristof, A. S. & Behr, M. A. (2006). Mutations in Mycobacterium tuberculosis Rv0444c, the gene encoding anti-SigK, explain high level expression of MPB70 and MPB83 in Mycobacterium bovis. Mol Microbiol 62, 1251–1263.[CrossRef][Medline]
Saint-Joanis, B., Demangel, C., Jackson, M., Brodin, P., Marsollier, L., Boshoff, H. & Cole, S. T. (2006). Inactivation of Rv2525c, a substrate of the twin arginine translocation (Tat) system of Mycobacterium tuberculosis, increases beta-lactam susceptibility and virulence. J Bacteriol 188, 6669–6679.
Sassetti, C. M., Boyd, D. H. & Rubin, E. J. (2003). Genes required for mycobacterial growth defined by high density mutagenesis. Mol Microbiol 48, 77–84.[CrossRef][Medline]
Sauvonnet, N. & Pugsley, A. P. (1996). Identification of two regions of Klebsiella oxytoca pullulanase that together are capable of promoting beta-lactamase secretion by the general secretory pathway. Mol Microbiol 22, 1–7.[Medline]
Schnappinger, D., Ehrt, S., Voskuil, M. I., Liu, Y., Mangan, J. A., Monahan, I. M., Dolganov, G., Efron, B., Butcher, P. D. & other authors (2003). Transcriptional adaptation of Mycobacterium tuberculosis within macrophages: insights into the phagosomal environment. J Exp Med 198, 693–704.
Stanley, N. R., Sargent, F., Buchanan, G., Shi, J., Stewart, V., Palmer, T. & Berks, B. C. (2002). Behaviour of topological marker proteins targeted to the Tat protein transport pathway. Mol Microbiol 43, 1005–1021.[CrossRef][Medline]
Stanley, S. A., Raghavan, S., Hwang, W. W. & Cox, J. S. (2003). Acute infection and macrophage subversion by Mycobacterium tuberculosis require a specialized secretion system. Proc Natl Acad Sci U S A 100, 13001–13006.
Stover, C. K., de la Cruz, V. F., Fuerst, T. R., Burlein, J. E., Benson, L. A., Bennett, L. T., Bansal, G. P., Young, J. F., Lee, M. H. & Hatfull, G. F. (1991). New use of BCG for recombinant vaccines. Nature 351, 456–460.[CrossRef][Medline]
Sutcliffe, I. C. & Harrington, D. J. (2004). Lipoproteins of Mycobacterium tuberculosis: an abundant and functionally diverse class of cell envelope components. FEMS Microbiol Rev 28, 645–659.[CrossRef][Medline]
Tulkens, P. M. (1991). Intracellular distribution and activity of antibiotics. Eur J Clin Microbiol Infect Dis 10, 100–106.[CrossRef][Medline]
Tullman-Ercek, D., Delisa, M. P., Kawarasaki, Y., Iranpour, P., Ribnicky, B., Palmer, T. & Georgiou, G. (2007). Export pathway selectivity of Escherichia coli twin-arginine translocation signal peptides. J Biol Chem 282, 8309–8316.
WHO (2007). WHO Information tuberculosis fact sheet. World Health Organization. http://www.who.int/mediacentre/factsheets/fs104/en/
Wiker, H. G., Wilson, M. A. & Schoolnik, G. K. (2000). Extracytoplasmic proteins of Mycobacterium tuberculosis – mature secreted proteins often start with aspartic acid and proline. Microbiology 146, 1525–1533.
Zahrt, T. C. & Deretic, V. (2002). Reactive nitrogen and oxygen intermediates and bacterial defenses: unusual adaptations in Mycobacterium tuberculosis. Antioxid Redox Signal 4, 141–159.[CrossRef][Medline]
Zhang, M., Gong, J., Lin, Y. & Barnes, P. F. (1998). Growth of virulent and avirulent Mycobacterium tuberculosis strains in human macrophages. Infect Immun 66, 794–799.
Received 28 March 2007;
revised 14 June 2007;
accepted 18 June 2007.
This article has been cited by other articles:
|
|
 |
|
  J. A. McDonough, J. R. McCann, E. M. Tekippe, J. S. Silverman, N. W. Rigel, and M. Braunstein Identification of Functional Tat Signal Sequences in Mycobacterium tuberculosis Proteins J. Bacteriol., October 1, 2008; 190(19): 6428 - 6438. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. M. Hou, N. G. D'Lima, N. W. Rigel, H. S. Gibbons, J. R. McCann, M. Braunstein, and C. M. Teschke ATPase Activity of Mycobacterium tuberculosis SecA1 and SecA2 Proteins and Its Importance for SecA2 Function in Macrophages J. Bacteriol., July 15, 2008; 190(14): 4880 - 4887. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||
