PPE38 Modulates the Innate Immune Response and Is Required for ...

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PPE38 Modulates the Innate Immune Response and Is Required for Mycobacterium marinum Virulence Dandan Dong,a Decheng Wang,a Ming Li,b Hui Wang,a Jia Yu,a* Chuan Wang,a Jun Liu,b and Qian Gaoa Key Laboratory of Medical Molecular Virology, Institute of Biomedical Sciences and Institute of Medical Microbiology, Shanghai Medical College, Fudan University, Shanghai, China,a and Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canadab

The proline-glutamic acid (PE) and proline-proline-glutamic acid (PPE) family proteins are prevalent in pathogenic mycobacteria and play a diverse role in mycobacterial pathogenesis. While some members have been studied, the function of most PE/PPE proteins remains unknown. In this study, we isolated a transposon-inactivated PPE38 mutant of Mycobacterium marinum and characterized its phenotype. We found that the PPE38 protein is associated with the cell wall and exposed on the cell surface. The inactivation of PPE38 altered the bacterial cell surface properties and led to deficiencies in cord formation, sliding motility, and biofilm formation. The PPE38 mutant was defective in phagocytosis by macrophages and exhibited reduced virulence in adult zebrafish. We also found that PPE38 is involved in the induction of proinflammatory cytokines in infected macrophages. Together, our results indicate that PPE38, a previously uncharacterized protein, plays a role in mycobacterial virulence, presumably by modulating the host innate immune response.

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ycobacterium tuberculosis is a highly successful human pathogen that latently infects one-third of the world’s population, causing 10 million new infections and 2 million deaths annually. One reason for the success of M. tuberculosis lies in its ability to evade host immune defense mechanisms and to create a niche within host cells, enabling the bacterium to persist for long periods. M. tuberculosis infects and survives within macrophages by evading macrophage killing mechanisms through a variety of strategies (reviewed in references 51 and 56). During persistent infection, M. tuberculosis is thought to reside within a granuloma, a cellular accumulation around the bacilli that is comprised mainly of macrophages, dendritic cells, T cells, B cells, and fibroblasts (reviewed in reference 31). Granulomas are generally thought to contain the infection by limiting bacterial growth and spread (21, 61). However, recent studies by Ramakrishnan and coworkers using zebrafish embryos demonstrated that the granuloma is a dynamic structure and that Mycobacterium marinum, an aquatic mycobacterium closely related to M. tuberculosis, uses the granuloma as a niche to recruit uninfected macrophages, raising the possibility that granulomas are exploited by the pathogen for expansion and dissemination in early infection (23, 25, 26). PE and PPE family proteins, named for the conserved N-terminal-domain-containing proline-glutamic acid (PE) motif or proline-proline-glutamine (PPE) motif, are unique to mycobacteria and particularly prevalent in pathogenic mycobacteria (35). They account for a significant fraction (⬃10%) of the coding capacity of pathogenic mycobacteria. There are 168 PE/PPE proteins in M. tuberculosis (22) and 281 in M. marinum (64). The relatively conserved N termini are approximately 110 and 180 amino acids in the PE and PPE families, respectively. The C-terminal domains of both the PE and PPE protein families are highly variable in both size and sequence and often contain repetitive DNA sequences that differ in copy numbers between genes. Large sequence polymorphisms in the C termini of PE/PPE proteins led to the hypothesis that they could provide a source of antigen variation and a mechanism for immune evasion (3, 14, 20, 22, 27, 47). Consistent with this view, a number of PE/PPE proteins are cell wall associated or secreted into the extracellular mi-

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lieu (1–4, 13, 28, 29, 33, 58), and at least 20 PE/PPE proteins are able to elicit T cell responses (reviewed in reference 57). Those studies suggest that PE/PPE proteins interact with host immune components and possibly subvert critical innate immune pathways, allowing bacterial survival. However, the direct role of PE/ PPE proteins in virulence has been demonstrated for only a few PE/PPE proteins due to their high degree of homology and redundancy, which makes it a significant challenge to generate targeted deletion mutants. Disruptions by a transposon of two PE_PGRS genes (PE_PGRS30 and PE_PGRS62) in M. marinum (54) or PPE25 in M. avium (46) attenuated the virulence of the respective organisms, including reduced replication in macrophages and animals. PPE46 (Rv3018c) was found previously to be associated with M. tuberculosis virulence in vivo by signature-tagged mutagenesis (17). A PE_PGRS33 transposon mutant of Mycobacterium bovis BCG exhibited reduced entry into macrophages, suggesting that PE_PGRS33 may promote macrophage uptake (15). The function of most members of these large families of proteins remains unknown, and whether there is a functional redundancy of certain members remains unclear. The PPE38 (Rv2352c) gene is localized in region of difference 7 (RvD7), which occurs in the M. tuberculosis H37Rv genome but is absent in M. bovis and M. bovis BCG (47). A recent genome analysis showed that in the ancestral M. tuberculosis complex (MTBC) member Mycobacterium canettii, the PPE38 region contains two

Received 20 April 2011 Returned for modification 16 August 2011 Accepted 18 October 2011 Published ahead of print 28 October 2011 Editor: J. L. Flynn Address correspondence to Jun Liu, [email protected], or Qian Gao, [email protected] * Present address: Institute of Genetics and Biostatistics, School of Life Sciences, Fudan University, Shanghai, China. Copyright © 2012, American Society for Microbiology. All Rights Reserved. doi:10.1128/IAI.05249-11

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FIG 1 Identification of the PPE38 mutant of M. marinum. (A) Genetic organization of the PPE38 locus in the genome of M. marinum and the transposon insertion sites (vertical triangles) in 05B1, 05D7, and 05F6. The PPE38-esxP_3-esxN_4 region is duplicated as PPE71-esxP_3-esxN_4 and is highlighted. (B) Colony morphology of M. marinum strains cultured on 7H10 medium. The 05B1 mutant exhibits a rough, granular, and dry colony without a translucent border, which is distinct from the WT. Mutant strains 05D7 and 05F6 showed a colony morphology similar to that of 05B1 (not shown). The transformation of plasmids harboring PPE38 of M. marinum or M. tuberculosis into the 05B1 mutant restored the colony morphology, whereas the transformation of the cloning vector did not.

identical PPE genes (PPE38 and PPE71), separated by 2 esat-6 (esx)-like genes, which derives from an esx-esx-PPE duplication (47). The PPE38 region is hypervariable among clinical isolates of M. tuberculosis, which contain 0 to 2 copies of the intact PPE38 or PPE71 gene due to frequent IS6110 integration, IS6110-associated recombination, and homologous recombination and gene conversion events between PPE38 and PPE71 (47). The function of PPE38 and the impact of polymorphisms in the PPE38 region in clinical isolates remain unknown. Outside the MTBC, PPE38 is found only in the genome of M. marinum, which contains intact PPE38 and PPE71 as well as four copies of the esat-6-like genes (47) (Fig. 1A). In this study, we identified three M. marinum mutants in which the PPE38 gene was disrupted by a transposon. We show that PPE38 is localized on the cell surface, plays a role in biofilm formation, and is required for the full virulence of M. marinum in zebrafish. Moreover, we show that PPE38 plays a role in modulating the innate immune response. These findings suggest that polymorphisms in the PPE38 region in clinical isolates of M. tuberculosis could potentially give rise to differences in the immune response, which may have an impact on the course of infection. MATERIALS AND METHODS Bacterial strains and growth. M. marinum strain M (ATCC BAA-535) was used as the wild-type (WT) strain for this study. Strains 05B1, 05D7, and 05F6 are three independent PPE38 mutants (MMAR_3661:: ␾MycoMar) identified from the transposon insertion library (see below). M. marinum strains were grown in Middlebrook 7H9 broth (Difco) supplemented with 10% oleic acid-albumin-dextrose-catalase (OADC), 0.5% glycerol, and 0.05% Tween 80 or on Middlebrook 7H10 agar supplemented with 10% OADC and 0.5% glycerol at 32°C. When necessary, the growth media were supplemented with the antibiotic kanamycin or hygromycin at 50 ␮g/ml.

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Escherichia coli strain DH5␣ ␭pir116 was used for the isolation of a transposon-containing plasmid (52). E. coli strain DH5␣ was used for molecular cloning and the propagation of plasmids. E. coli cells were grown in Luria-Bertani (LB) broth or on agar at 37°C supplemented with the following antibiotics as appropriate: ampicillin at 100 ␮g/ml and hygromycin at 200 ␮g/ml. Generation and screening of the M. marinum ␾MycoMar insertion library. The mariner-based transposon system was used to generate a transposon insertion mutant library of M. marinum as described previously (6, 60, 77). Kanamycin-resistant (i.e., transposon-containing) M. marinum colonies were patched onto Middlebrook 7H10 agar, and colonies with unusual morphologies were identified by visual inspection. Localization of the ␾MycoMar insertion. The method used to localize and identify the transposon-disrupted gene was described previously (6). Briefly, total chromosomal DNA of the transposon insertion mutant was cleaved with BamHI and then self-ligated with T4 DNA ligase and transformed into competent E. coli DH5␣ ␭pir116 cells. The MycoMar element contains an R6K origin and an aph gene such that recircularized fragments containing the transposon are able to replicate as kanamycinresistant plasmids. Plasmid DNA was isolated from Kmr E. coli transformants, and MycoMar-specific primers were used to determine the DNA sequence at the transposon/chromosomal junction. These DNA sequences were compared with the genome sequences of M. marinum and M. tuberculosis at the GenoList website (http://genodb.pasteur.fr/cgi-bin /WebObjects/GenoList). Molecular cloning. The intact PPE38 (MMAR_3661) gene of M. marinum was PCR amplified by using forward primer 5=-CGGGATCCAGG AGGGGTTGTGATGATTTTGGACTTTG-3= and reverse primer 5=-TAC CAAGCTTTTCAAAAGACATTCAGACCCGACCAA-3=, which contain a BamHI site and a HindIII site, respectively (underlined). The amplified PCR product was cloned into vector pSMT3LxEGFP (63), creating pMMppe38. The PPE38 (Rv2352c) gene of M. tuberculosis was cloned similarly by using forward PCR primer 5=-CGGGATCCTGGAGGGGTT GCGATGATTTTTGGATTTTTC-3= and reverse PCR primer 5=-GGACT AGTACCGCTTCGGTGCACTTCATTTC-3= to generate pRVppe38.

Infection and Immunity

PPE38 Is Required for M. marinum Virulence

These constructs were electroporated into mutant strain 05B1 of M. marinum, and transformants were selected on 7H10 agar containing 50 ␮g/ml of hygromycin. Constructs for the expression of FLAG-tagged PPE38 (at the C terminus) in M. marinum were made by using forward primer 5=-CG GGATCCAGGAGGGGTTGTGATGATTTTGGACTTTG-3= and reverse primer 5=-GCACTAGTTTATTTATCGTCATCGTCTTTGTAGTCTCC GACACCGATCCGCGGCACCA-3= for M. marinum PPE38 or forward primer 5=-CGGGATCCTGGAGGGGTTGCGATGATTTTTGGATTTTT C-3= and reverse primer 5=-CGACTAGTCTATTTATCGTCATCGTCTT TGTAGTCTCCGATCCCGACCCGCGGCACCA-3= for M. tuberculosis PPE38 (restriction enzyme sites are underlined). The PCR products were cloned into pSMT3LxEGFP to generate pMMppe38_FLAG and pRvppe38_FLAG, respectively. qRT-PCR. Determinations of the expression levels of PPE38 and PPE71 were accomplished by quantitative real-time PCR (qRT-PCR) using universal primers for both genes (forward primer 5=-CTGCTGACAT GCCCAAGATG-3= and reverse primer 5=-CAACCGAGCCTTACCCAA ATC-3=). Similarly, the expression levels of the esxN_4 and esxP_3 genes were determined by qRT-PCR using primers specific for each gene (esxN_4F [5=-GACGATCAATTACCAGTTCGGTGA-3=], esxN_4R [5=-G CCAACACATCACGCACAATC-3=], esxP_3F [5=-GTGACTACTCGGT TTATGACTGACC-3=], and esxP_3R [5=-CTCATCCTCGACCGTCTG C-3=]). Primers specific for sigA (forward primer 5=-GAAAAACCACCT GCTGGAAG-3= and reverse primer 5=-CGCGTAGGTGGAGAACTTG T-3=) were used as an endogenous control to normalize the amount of cDNA template added to each qRT-PCR sample. The cDNA used in these experiments was prepared from RNA samples obtained from three biological replicates. qRT-PCRs were carried out in duplicate by using a 7500 real-time PCR system (Applied Biosystems) and iQ SYBR green supermixture (Bio-Rad). Sliding motility assay. The sliding motility assay was described previously (55). Briefly, Middlebrook 7H9 base medium supplemented with 6% glycerol was solidified with 0.3% high-grade agarose (Promega). Plates were inoculated in their center with 5 ␮l of the cultures after adjusting the optical density at 600 nm (OD600) to 0.5. Spreading was evaluated after incubation for 6 days at 32°C in a humidified incubator. Biofilm formation assay. The biofilm formation of M. marinum strains was assayed by a method previously developed for M. tuberculosis biofilms (49). Briefly, polystyrene petri dishes (6 cm in diameter) containing 7 ml Sauton medium (without Tween 80) were inoculated with 100 ␮l of saturated planktonic M. marinum cultures and incubated without shaking at 32°C for 5 weeks. The dishes were wrapped with Parafilm during incubation. Localization of the PPE38 protein. Recombinant M. marinum strains harboring the pMMppe38_FLAG and pRvppe38_FLAG constructs were grown and subjected to cell fractionation by using a method described previously (19). Briefly, the cells were lysed by sonication, and cell debris and unlysed cells were removed by centrifugation at 3,000 ⫻ g for 5 min. The supernatant was subjected to ultracentrifugation at 27,000 ⫻ g for 30 min at 4°C. The pellet from this centrifugation step was considered the cell wall fraction, and the supernatant was considered the combined cell membrane and cytosol fractions. Equal amounts of protein (7 ␮g) from each fraction were subjected to Western blotting using monoclonal antibodies against FLAG (dilution, 1:2,000) and green fluorescent protein (GFP) (dilution, 1:2,000) (control) (Sigma) for analyses of PPE38 expression. Proteinase K and trypsin sensitivity assays. Proteinase K and trypsin sensitivity assays were performed as described previously (19). Briefly, cells of recombinant M. marinum harboring pMMppe38_FLAG and pRvppe38_FLAG were mixed with proteinase K or trypsin at a concentration of 100 ␮g/ml and incubated at 37°C for the indicated times. The reaction was stopped by the addition of 100 nM phenylmethylsulfonyl fluoride (PMSF) to the mixture. The samples were dissolved in SDS load-

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ing buffer and analyzed by Western blotting using anti-FLAG and antiGFP antibodies. Cell wall lipid analysis. Cell wall lipid compositions were analyzed by thin-layer chromatography (TLC) according to previously reported procedures (6, 16). Briefly, the apolar and polar lipids as well as mycolic acids were prepared from M. marinum cells (50 mg dry biomass), and these lipids were analyzed by two-dimensional thin-layer chromatography (2D-TLC) on silica gel 60 plates (EMD Chemicals Inc.). Apolar lipids were developed with petroleum ether-ethyl acetate (98:2, 3⫻) in the first dimension and petroleum ether-acetone (98:2) in the second dimension. Lipids were visualized by spraying plates with 5% phosphomolybdic acid, followed by the gentle charring of the plates. For the detection of phenolic glycolipids (PGLs), the apolar lipid extract was developed with chloroform-methanol (96:4, vol/vol) in the first direction and with toluene-acetone (80:20, vol/vol) in the second direction, followed by charring with ␣-naphthol. Polar lipids were separated with chloroformmethanol-water (60:30:6) in the first dimension and chloroform-acetic acid-methanol-water (40:25:3:6) in the second dimension and were detected by charring with ␣-naphthol. Mycolic acid methyl esters were developed in petroleum ether-diethyl ether (95:5, 4⫻) and charred with 5% phosphomolybdic acid. Macrophage infection. The infection of the murine macrophage cell line J774A.1 (ATCC TIB67) with M. marinum was performed as described previously (53, 55, 68). Briefly, J774 cells were maintained at 37°C in 5% CO2 in Dulbecco’s modified Eagle medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco). Cells were seeded into 24-well tissue culture plates (Corning) at a density of 5 ⫻ 104 cells per well in DMEM supplemented with 10% FBS 24 h before infection. The single-cell suspension of bacteria was prepared by subjecting cells to vortexing with glass beads, followed by low-speed centrifugation and passage through a 5-␮m syringe filter to remove bacterial aggregates. J774 cells were infected with M. marinum at a multiplicity of infection (MOI) of 0.5 (i.e., one bacterium for two macrophages). The dosages of bacteria were confirmed by plating and determining CFU in each experiment. The infection was allowed to proceed for 4 h at 32°C in 5% CO2, after which time the J774 cells were washed twice with sterile phosphate-buffered saline (PBS). Extracellular M. marinum bacteria were killed by incubating the tissue culture with 200 ␮g/ml gentamicin (Amersco) for 2 h. Cells were again washed twice with PBS and subsequently incubated at 32°C in 5% CO2 in fresh medium with 20 ␮g/ml gentamicin. On days 0 (4 h), 1, 3, and 4, the infected macrophage monolayers (three wells per strain) were washed twice with fresh medium and then lysed with 0.1 ml of 1% Triton X-100 (Sigma) to release intracellular mycobacteria. The number of intracellular mycobacteria was enumerated by plating appropriate dilutions onto Middlebrook 7H10 agar plates containing appropriate antibiotics. For experiments measuring the entry of M. marinum into macrophages, infection experiments were performed at an MOI of 25. Immediately following the infection (3 h at 32°C), macrophage cells were washed three times with RPMI 1640 to remove extracellular bacteria, and the CFU recovered from macrophages was enumerated by plating onto 7H10 agar. Zebrafish infection. Zebrafish infection with M. marinum was performed as previously described (77). Briefly, adult zebrafish were infected by intraperitoneal injection with 10 ␮l of a single-cell bacterial suspension in PBS prepared as described above with a dosage of 104 CFU bacteria per fish. The bacterial CFU was confirmed by plating the suspension onto 7H10 agar at the time of injection. For survival studies, 20 or 25 fish per group were injected with individual strains and the PBS negative control for each experiment. To assess the bacterial burdens at various time points postinfection, the livers of fish were obtained and homogenized. The homogenates were plated by serial dilution to determine CFU. Six to eight fish were used for each condition per time point. For histopathological studies, three fish were sacrificed at the indicated times. Fish were fixed for at least 96 h in 10% formalin and then dehydrated with ethanol. After paraffin embedding and sectioning, serial paraffin sections (5 ␮m) were prepared and subjected to hematoxylin and

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eosin (H&E) staining and modified Ziehl-Neelson staining (BA-409 TB stain kit; Baso, Zhuhai, China) according to the manufacturer’s instruction. Sections were examined under an Olympus BH2 microscope (Tokyo, Japan), and images were collected with a digital camera (TKC1481BEC; JVC, Tokyo, Japan). Cytokine assay. The THP-1 human monocytic cell line was maintained at 37°C in 5% CO2 in RPMI 1640 medium (Gibco) supplemented with 10% FBS. Prior to experimentation, THP-1 cells were differentiated into macrophages in 96-well plates (5 ⫻ 105 cells/well) with 200 nM phorbol myristate acetate (PMA) (Sigma) for 24 h, followed by incubation with fresh medium without PMA for 24 h. THP-1 cells were infected with mycobacteria at an MOI of 5 or 10 for 4 h at 32°C or 37°C in 5% CO2 in triplicate. The extracellular bacteria were removed by washing and incubation with 200 ␮g/ml gentamicin for 2 h. THP-1 cells were then maintained in RPMI 1640 supplemented with 10% FBS as well as 20 ␮g/ml of gentamicin for the indicated times. At 24 and 48 h postinfection, cell-free supernatants were collected, and cytokine levels were determined by an enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions (BD Biosciences).

RESULTS

A PPE38 mutant exhibits altered colony morphology. Three independent PPE38 mutants, 05B1, 05D7, and 05F6, were identified by the screening of a transposon mutation library of M. marinum for altered colony morphology followed by DNA sequencing. All three mutants exhibited rough, granular, and dry colonies, which were distinctive from the wild-type (WT) colony (see Fig. 1B for 05B1 as an example). Subsequent molecular cloning and DNA sequencing revealed that the three mutants each had a unique transposon insertion site within the coding region of MMAR_3661 (Fig. 1A), which showed 73% identity in amino acid sequence to PPE38 of M. tuberculosis. To confirm that the observed phenotype was a result of the PPE38 inactivation, the intact PPE38 gene from M. marinum was cloned into a shuttle vector, pSMT3LxEGFP (63), to generate plasmid pMMppe38. Recombinant strain 05B1 containing pMMppe38 restored the WT colony morphology (Fig. 1B). Moreover, the PPE38 gene of M. tuberculosis also complemented the morphological phenotype of the M. marinum mutant (Fig. 1B), indicating that these two proteins are indeed homologous to each other. The PPE38 mutant is defective in cord formation, sliding motility, and biofilm formation. The morphological phenotype of the PPE38 mutant prompted us to investigate several other phenotypes associated with alternations of cell surface properties. The PPE38 mutant grew equally as well as the WT strain in 7H9 broth medium; however, the mutant was highly aggregative and quickly precipitated in standing culture (Fig. 2A). Under microscopic observation, the PPE38 mutant did not form serpentine cords that are characteristic of pathogenic mycobacteria including M. marinum (Fig. 2B). The mutant also exhibited reduced sliding motility and was defective in biofilm formation compared to the WT strain (Fig. 2C and D). Importantly, all of these phenotypes were fully complemented by the intact PPE38 gene from M. marinum or M. tuberculosis (Fig. 2), indicating that the inactivation of PPE38 is responsible for the observed phenotypes of the mutant. Similar phenotypes, albeit to different extents, were observed previously for an M. marinum mutant defective in certain cell wall lipid components, such as lipooligosaccharides (55). Analysis of cell wall lipids, including the apolar lipids phthiocerol dimycocerosate, phenolic glycolipid, and triacylglycerol; the polar lipids phosphatidylinositol mannoside and lipooligosaccharide; as well

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as mycolic acids, did not reveal a difference between the PPE38 mutant and WT cells (data not shown). PPE38 is associated with cell wall and is surface exposed. A number of PE/PPE proteins have been found to be associated with the cell wall or secreted into the extracellular medium (3, 28, 29, 33, 58). We hypothesized that PPE38 could be localized in the cell wall, which would provide an explanation for the observed phenotypes associated with the alteration of cell surface properties. To test this hypothesis, we made constructs that express the FLAGtagged PPE38 protein of M. marinum or M. tuberculosis and transformed these constructs individually into PPE38 mutant strain 05B1. As a control and as a marker for cytoplasmic proteins, we transformed a construct, pSMT3LxEGFP, that expresses enhanced green fluorescent protein (EGFP) in the same cells. Recombinant M. marinum strains expressing FLAG-tagged PPE38 and GFP were subjected to cell fractionation experiments, followed by Western blot analysis. The majority of the PPE38 proteins were detected in the cell wall fraction (Fig. 3A), indicating that PPE38 is associated with the cell wall. As expected, GFP was detected only in the cytoplasm. To further confirm the cell surface association of PPE38, whole cells of recombinant M. marinum strains expressing FLAG-tagged PPE38 or GFP were subjected to proteinase K and trypsin digestion as described previously (19, 28). The PPE38 protein expressed in M. marinum cells was rapidly degraded by proteinase K and trypsin within 10 min of the reaction (Fig. 3B and C). In contrast, GFP, which localizes in the bacterial cytosol, was not digested at all. Taken together, these results demonstrate that PPE38 is localized in the cell wall and exposed on the cell surface. PPE38 is involved in the entry of M. marinum into macrophages. Macrophages are the primary target of pathogenic mycobacteria (56). As the first step to determine the role of PPE38 in mycobacterial virulence, we performed macrophage infection experiments and compared the intracellular replications of the WT and the mutant strains. J774 cells were infected with M. marinum strains at an MOI of 0.5 (1 bacterium for 2 macrophage cells) at 32°C for 4 h, followed by washing and gentamicin treatment to remove extracellular bacteria. The intracellular growth of bacteria was assayed by enumerating the CFU at different time points postinfection. Inside macrophages, PPE38 mutant strain 05B1 grew at a rate equivalent to that of the WT during the course of infection (Fig. 4A). The mutant appeared to enter macrophages less efficiently than the WT strain. To confirm this, the entry of macrophages was measured at an MOI of 25 to allow a more accurate estimate of entry. Immediately following the infection (3 h at 32°C), macrophage cells were washed three times to remove extracellular bacteria, and the CFU recovered from macrophages was enumerated. The result showed that the PPE38 mutant entered macrophages less efficiently than the WT strain (Fig. 4B); the number of CFU recovered from macrophages infected with the mutants was significantly lower (P ⬍ 0.01, determined by Student’s t test) than that recovered from macrophages infected with the WT strain (Fig. 4B). The deficiencies were restored in the complemented mutant strains (Fig. 4B). PPE38 is required for the full virulence of M. marinum in zebrafish. Zebrafish have been widely used as a laboratory model for studying M. marinum infection, which manifest both acute disseminated disease and chronic persistent infection (70). To assess the role of PPE38 in virulence, adult zebrafish (25 per group) were infected with WT M. marinum and three PPE38 mutant

Infection and Immunity

PPE38 Is Required for M. marinum Virulence

FIG 2 Phenotypes of the PPE38 mutant. (A) The PPE38 mutant is highly aggregative in 7H9 broth. (B) The PPE38 mutant is defective in cord formation. The M. marinum WT strain formed cords in liquid culture, whereas PPE38 mutant strain 05B1 did not. (C) The PPE38 mutant is defective in sliding motility. An example of sliding motility of M. marinum strains on 0.3% agar plates is shown at the left. The quantification of the sliding motility of M. marinum strains is shown at the right. The diameters of each halo were measured. Data are from three independent experiments (ⴱⴱ, P ⬍ 0.01, determined by Student’s t test). (D) The PPE38 mutant is defective in biofilm formation. The biofilm formed by M. marinum strains at the air-liquid interface after a 5-week incubation is shown. The PPE38 mutant formed atypical biofilms: cells clumped together to form floating islands rather than a single continuous layer across the surface. The complementation of mutant strain 05B1 with PPE38 of M. marinum or M. tuberculosis restored all the above-described phenotypes.

strains, 05B1, 05D7, and 05F6, and were monitored for survival. In three independent experiments, the median survival times of fish infected with WT M. marinum were 14.5, 14, and 12 days (Fig. 5), whereas the median survival times for fish infected with three independent PPE38 mutants, 05B1, 05D7, and 05F6, were 23, 23, and 21 days, respectively (Fig. 5). Fish infected with the WT M. marinum strain succumbed to disease significantly sooner than those infected with the mutant strains (P ⬍ 0.001, determined by log rank test). The complementation of the mutant (05B1) with

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the intact PPE38 gene of M. marinum restored virulence (Fig. 5A). The median survival time of fish infected with the complemented mutant strain (05B1 containing pMMppe38) was 16 days, which is significantly shorter than that of fish infected with 05B1 (P ⬍ 0.05, determined by log rank test). The complementation of the mutant with PPE38 of M. tuberculosis partially restored virulence, with a median survival time of 18 days (Fig. 5A) (P ⫽ 0.128 for 05B1 versus 05B1 containing pRvppe38). The suboptimal complementation of the virulence phenotype by M. tuberculosis PPE38 might

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FIG 3 PPE38 is associated with the cell wall and exposed on the cell surface. (A) Recombinant M. marinum strains expressing FLAG-tagged PPE38 from M. marinum or M. tuberculosis were subjected to fractionation experiments, and the presence of the PPE38 protein in different fractions was detected by Western blotting using anti-FLAG antibody. The expression of cytosolic GFP in each fraction was also detected by anti-GFP antibody as a control. Lanes 1 to 4, samples from a recombinant M. marinum strain expressing PPE38 of M. marinum; lanes 5 to 8, samples from recombinant M. marinum expressing PPE38 of M. tuberculosis; lane 9, cell lysate of M. marinum transformed with the control vector only. Lanes 1 and 5, whole-cell lysate; lanes 2 and 6, cell wall fraction; lanes 3 and 7, cytosolic fraction; lanes 4 and 8, cell debris. (B and C) Recombinant M. marinum strains expressing the FLAG-tagged PPE38 protein were digested by proteinase K (B) or trypsin (C) for the indicated times. The samples were analyzed for the abundance of PPE38 by Western blotting using anti-FLAG antibody. Cytosolic GFP was analyzed by Western blotting using anti-GFP antibody as a negative control.

have been caused by the subtle sequence difference between PPE38 of M. tuberculosis and PPE38 of M. marinum. Disease progression was also assayed by determining the bacterial burden in fish liver at different time points following infection. During early infection (day 1 postinfection), there were no differences in bacterial burdens in livers of fish infected with the WT and the mutant strains (Fig. 6A). However, at day 11 postinfection, fish infected with the mutant strains had significantly lower bacterial CFU [05B1, (1.51 ⫾ 1.31) ⫻ 105 CFU (mean ⫾ standard

FIG 5 The PPE38 mutant is attenuated in zebrafish. Shown are data for the survival of adult zebrafish during a 30-day infection. Fish were intraperitoneally infected with 104 CFU of the M. marinum WT or the PPE38 mutants: 05B1 and 05B1 complemented with PPE38 from M. tuberculosis or M. marinum (A), 05D7 (B), and 05F6 (C). PBS was injected as the negative control.

FIG 4 The PPE38 mutant is defective in macrophage phagocytosis. (A) Growth of M. marinum inside macrophages. Data were generated from three indepen-

dent experiments (ⴱ, P ⬍ 0.05, determined by Student’s t test). (B) Infection of macrophages by M. marinum at day 0 (4 h postinfection). An MOI of 25 was used for infection. Data were generated from four independent experiments (ⴱⴱ, P ⬍ 0.01, determined by Student’s t test).

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Infection and Immunity

PPE38 Is Required for M. marinum Virulence

FIG 6 Bacterial burden in infected zebrafish. (A) CFU of M. marinum in fish livers infected with the WT and mutant strain 05B1, 05D7, or 05F6 at day 1 and day 11 postinfection. Twenty-five fish were infected by each strain, and 6 to 8 fish were sacrificed to enumerate the CFU at each time point. An asterisk indicates a significant difference (P ⬍ 0.05, determined by one-way ANOVA) between the CFU of the WT strain and those of the mutant strains at day 11 postinfection. (B) CFU of M. marinum in fish livers infected with the WT, 05B1, 05B1 plus pMMppe38, or 05B1 plus pRvppe38 at days 1, 5, and 8 postinfection. A significant difference in mean bacterial burdens at day 8 postinfection was found between the mutant and the WT or between the mutant and the mutant complemented with PPE38 of M. marinum (ⴱ, P ⬍ 0.05, determined by Student’s t test).

deviation); 05D7, (1.59 ⫾ 0.89) ⫻ 105 CFU; 05F6, (0.89 ⫾ 0.85) ⫻ 105 CFU] than those infected with the WT strain [(3.15 ⫾ 2.32) ⫻ 105 CFU; P ⬍ 0.05 by one-way analysis of variance (ANOVA)] (Fig. 6A). A similar result was found in an independent experiment (Fig. 6B) that showed that at day 8 postinfection, there was a significantly lower number of bacteria recovered from fish infected with mutant strain 05B1 [(2.4 ⫾ 0.30) ⫻ 104 CFU; P ⬍ 0.05, determined by Student’s t test) than that recovered from fish infected with the WT [(6.2 ⫾ 0.60) ⫻ 104 CFU] or the mutant strain complemented with intact PPE38 from M. marinum [(5.3 ⫾ 0.33) ⫻ 104 CFU] (Fig. 6B). Fish infected with the mutant strain exhibited significantly less-severe pathology than fish infected with the WT strain. At day 15 postinfection, extensive tissue damage and dissemination of bacteria were detected in fish infected with WT M. marinum (Fig. 7A to H). A large number of bacterial cells were observed in the intestinal villus and peritoneal space. Granulomas with bacteria residing inside were detected at a low number. In contrast, in fish infected with PPE38 mutant strain 05B1, well-organized granulomas with bacteria inside were observed (Fig. 7I to P). Limited tissue damage was detected, and there was no dissemination of the bacterial cells in fish infected with the mutant strain. Together, these results suggest that PPE38 is required for the full virulence of M. marinum. PPE38 induces TNF and IL-6 production. To explore whether PPE38 contributes to the subversion of early immune responses,

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we investigated the response of the human monocytic cell line THP-1 to infection with WT, mutant, and complemented mutant strains. Macrophages infected with the PPE38 mutant secreted significantly smaller amounts of the proinflammatory cytokines tumor necrosis factor (TNF) and interleukin-6 (IL-6) than did the WT-infected cells (Fig. 8A and B). There was no difference in the levels of other inflammatory proteins, including IL-1␤, IL-8, and monocyte chemoattractant protein 1 (data not shown). The levels of IFN-␥, IL-12p40, and IL-12p70 in the supernatants of macrophages infected with all three M. marinum strains were negligible. Macrophages infected with the complemented strains elicited levels of TNF and IL-6 production equivalent to those of the WTinfected macrophages (Fig. 8A and B). To further confirm the above-described observations, we overexpressed PPE38 of M. marinum or M. tuberculosis in M. smegmatis, a nonpathogenic mycobacterium that does not contain a PPE38 ortholog, and repeated the macrophage infection experiments. At two different MOIs, macrophages infected with recombinant M. smegmatis overexpressing PPE38 induced higher levels of TNF and IL-6 secretion than did those infected with the control strain (Fig. 8C and D). Taken together, these results suggest that PPE38 plays a role in modulating the innate immune response. PPE38 but not PPE71 is highly expressed in M. marinum. The PPE38 region is duplicated in the M. marinum genome, which contains two copies of the PPE38 gene (PPE38 and PPE71, which share 96% identity in encoded amino acid sequences) and four copies of the esat-6-like genes (two copies of the esxP_3 and esxN_4 genes) (Fig. 1A). It is somewhat surprising that the disruption of PPE38 affects the virulence of the organism. To determine if both genes are expressed in M. marinum, we performed quantitative real-time PCR (qRT-PCR) to quantify the transcript levels of PPE38 and PPE71. Because the DNA sequences of these two genes are nearly identical, we employed PCR primers that amplify mRNAs of both genes and compared the transcript levels between the WT and the PPE38 mutant. The result showed that the combined transcript levels of PPE38 and PPE71 in the WT strain were about 100⫻ those in the PPE38 mutant (Fig. 9A), suggesting that PPE38 is highly expressed in the WT strain, whereas PPE71 is not. Since one copy of the esxP_3 and esxN_4 genes is immediately downstream of PPE38 (Fig. 1A), we tested whether the transposon disruption of PPE38 could cause a polar effect on the expression of these esat-6-like genes. Primers specific for esxP_3 or esxN_4 were designed, and their expression levels in the WT, the PPE38 mutant, and the complemented strain were determined by qRT-PCR. The expression levels of esxP_3 and esxN_4 in the mutant were about 6⫻ (5.58⫻ ⫾ 1.59⫻) and 13⫻ (12.62⫻ ⫾ 1.35⫻), respectively, lower than those in the WT (Fig. 9B and C). However, the complementation of the mutant with an intact PPE38 gene in trans did not restore the expression levels of esxP_3 and esxN_4. This result suggests that PPE38 and its downstream esxP_3 and esxN_4 genes may form an operon. Since the complementation of the PPE38 mutant with an intact PPE38 gene in trans restored all the phenotypes observed in our study, we conclude that the disruption of the PPE38 gene is directly responsible for these phenotypes. DISCUSSION

In this study, we describe the function of PPE38, a member of the PE/PPE family proteins that has not been studied previously. We show that PPE38 is associated with the cell wall and

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FIG 7 The PPE38 mutant caused less-severe pathology. Shown are histopathologies of zebrafish infected by WT M. marinum (A to H) and PPE38 mutant strain 05B1 (I to P). Three fish at day 15 postinfection were sacrificed and subjected to histological analysis with hematoxylin and eosin staining (A, B, C, D, I, J, K, and L) and Ziehl-Neelson staining (E, F, G, H, M, N, O, and P) of serial sections. Arrows indicate bacteria outside granulomas, while arrowheads show bacteria inside granulomas. Magnifications, ⫻64 for all sections.

exposed on the cell surface. The disruption of PPE38 altered the cell surface properties, which manifested in several phenotypes, including colony morphology, cord formation, sliding motility, and biofilm formation. Cording morphology was first described by Robert Koch, and previous studies revealed a correlation between cording and virulence (24, 42, 48). To date, a few genes involved in cord formation have been genetically identified, but they are all involved in cell wall lipid biosynthesis (11, 34, 36–38, 40, 50, 76). In our study, the cell wall lipid composition was not affected by the PPE38 mutation, indicating that cell wall-associated proteins such as PPE38 also play a role in cording. Furthermore, we showed that PPE38 is involved in the entry of bacteria into macrophages and plays a role in the induction of proinflammatory cytokine secretion. The inactivation of PPE38 leads to a reduction of the bacterial burden in infected animals, limited levels of tissue damage, and, consequently, reduced virulence. Contrary to the prediction that PPE38 may not be important for mycobacterial virulence (47), our findings support such a role for PPE38.

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Although the PPE38 region is duplicated in the M. marinum genome, which contains two copies of the PPE38 genes (PPE38 and PPE71), we found that only PPE38 was expressed under the conditions of our experiments, whereas PPE71 was not. Despite the high sequence identity at the coding regions of PPE38 and PPE71 (97% identity in DNA sequences), their promoter regions are dissimilar, and there is no significant sequence homology between DNA sequences 200 bp upstream of the coding regions of these two genes (data not shown). Together, these data suggest that PPE38 and PPE71 are differentially regulated. The majority (128/169) of the PE/PPE genes of M. tuberculosis were found to be differentially regulated under 15 different conditions tested (75), suggesting that there is likely a high degree of plasticity in the PE/PPE expression repertoire. As such, members of the PE/PPE family of proteins may play subtly different functional roles and are required at different stages of disease. PPE38 was identified as one of the most highly expressed proteins of M. tuberculosis after a 90-day infection of guinea pig, suggesting that PPE38 plays a potentially critical role in persistent infection (43). This is in accor-

Infection and Immunity

PPE38 Is Required for M. marinum Virulence

FIG 8 PPE38 induces proinflammatory cytokine secretion. (A and B) Secretion of TNF (A) or IL-6 (B) by macrophages infected with M. marinum. THP-1 macrophages were infected with WT, 05B1, and complemented mutant strains at an MOI of 10. Supernatants of infected cells were collected 24 and 48 h after infection, and levels of secreted TNF and IL-6 were measured by ELISA. Data were generated from three independent experiments. (C and D) Secretion of TNF (C) or IL-6 (D) by macrophages infected with recombinant M. smegmatis. THP-1 macrophages were infected with M. smegmatis expressing PPE38 (MS⫹pMMppe38 and MS⫹pRvppe38) or M. smegmatis harboring the vector alone (MS⫹pSMT3), at the indicated MOIs. Supernatants of infected cells were collected at different time points after infection, and levels of secreted TNF and IL-6 were measured by ELISA. Data were generated from three independent experiments. ⴱ, P ⬍ 0.05; ⴱⴱ, P ⬍ 0.01; ⴱⴱⴱ, P ⬍ 0.001 (determined by Student’s t test).

dance with our finding that PPE38 plays a role in the virulence of M. marinum. Our results suggest that PPE38 plays a role in virulence, presumably by modulating the host immune response. We found that the WT or the mutant strain complemented with the intact PPE38

gene induced higher levels of TNF and IL-6 secretion in macrophages. A similar finding for other PE/PPE family proteins was reported recently, in which the disruption of Rv0485, a transcription factor that positively regulates the expression of PE13 and PPE18 in M. tuberculosis, resulted in reduced levels of TNF and

FIG 9 (A) PPE38 is highly expressed in M. marinum. The combined mRNA levels of PPE38 and PPE71 in the WT and the PPE38 mutant strain are shown. qRT-PCR analysis was carried out on cDNA samples prepared from the WT and PPE38 mutant strain 05B1. Relative expression data (fold change) were obtained by using universal primers for the PPE38 and PPE71 genes, and sigA was used as a reference gene for normalization. (B and C) Expression of esxP_3 (B) and esxN_4 (C) in the WT, the PPE38 mutant, and the complemented mutant strain. qRT-PCR analysis was carried out on cDNA samples prepared from the three strains. Relative expression data (fold change) were obtained by using primers for the esxP_3 and esxN_4 genes, and sigA was used as a reference gene for normalization.

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IL-6 secretion in infected macrophages (39). PE_PGRS33 was found previously to interact with Toll-like receptor 2 (TLR-2) and induce TNF secretion in macrophages (9). TNF is a pleiotropic cytokine that plays an important role in the outcome of mycobacterial infections by controlling a range of cytokines and chemokines (30). In the mouse model of infection, TNF is required for the control of acute M. tuberculosis infection (32) and plays a critical role in granuloma formation (reviewed in references 30 and 72). IL-6 has been shown to have both pro- and antiinflammatory properties in the host response to M. tuberculosis and is important for the initial innate response to the pathogen (44, 62, 73). Excessive proinflammatory responses can lead to deleterious effects for the host. In active tuberculosis (TB), emerging evidence suggests that a high level of TNF could accelerate disease progression (5, 10, 12, 39, 69, 71). The production of proinflammatory cytokines, including TNF and IL-6, is present at the site of disease (8, 18, 45), and these proinflammatory cytokines are partially responsible for systemic manifestations such as cachexia and wasting in tuberculosis patients (59, 74). Consistent with this view, we observed more extensive tissue damage and bacterial dissemination in fish infected with WT M. marinum strains than in fish infected with the PPE38 mutant strain, which may correlates with higher levels of TNF and IL-6 production induced by the former in macrophages. At the same stage of infection, we observed fewer granulomas in fish infected with the WT strain than in fish infected with the mutant. The granuloma is currently viewed as a niche both for bacteria to grow and persist and as a host infection control mechanism to prevent the dissemination of infection (31). The balance of host immune responses in granulomas affects the outcome of the infection (7, 31). The excessive production of proinflammatory mediators and tissue damage may lead to the dissolution of granulomas and the dissemination of the infection, which is consistent with our observation. Although it remains to be determined whether PPE38 also interacts with TLR-2, thereby mediating the secretion of TNF and IL-6, emerging evidence suggests that selected members of the PE/PPE family may potentiate mycobacterial virulence by interfering with host immune responses. The highly homologous sequences in the conserved 5= regions of the PE/PPE genes, the lengthy stretches of GC-rich sequences, and the imperfect triplet repeats within associated genes make them hotspots for recombination events and other mutations, including the insertion of transposable elements (22, 47). It is not surprising that several PE/PPE genes, including PPE38, PE_PGRS33, PE_PGRS16, and PE_PGRS26, were found to exhibit extensive polymorphisms in clinical isolates of M. tuberculosis (41, 47, 66, 67). Consequently, it was speculated that these proteins represent a source of antigenic variation which allows the pathogen to evade antigen-specific host responses (22, 41, 66). A moderately significant association of large sequence variations in the PE_PGRS33 gene with noncavitary TB and case clustering was reported previously (65). It remains to be determined whether this is a true biological link and whether there are some associations of polymorphisms of other PE/PPE genes, including PPE38, with certain clinical and epidemiological characteristics of the disease. ACKNOWLEDGMENTS We thank E. J. Rubin, Harvard University, for providing the MycoMarT7 mariner transposon phage and D. B. Young, Imperial College London, for plasmid pSMT3.

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This work was supported by the Key Project of Chinese National Programs (2012ZX10003004-003), National Natural Science Foundation of China grant 30872259 (to Q.G.), and Canadian Institutes of Health Research (CIHR) grants CCI-85667, MOP-15107, and MOP-106559 (to J.L.).

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