In Vivo Expression of Streptococcus pyogenes Immunogenic Proteins during Tibial Foreign Body Infection Jeffrey A. Freiberg, Kevin S. McIver and Mark E. Shirtliff Infect. Immun. 2014, 82(9):3891. DOI: 10.1128/IAI.01831-14. Published Ahead of Print 7 July 2014.
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In Vivo Expression of Streptococcus pyogenes Immunogenic Proteins during Tibial Foreign Body Infection Jeffrey A. Freiberg,a Kevin S. McIver,b Mark E. Shirtliffc,d Graduate Program in Life Sciences, University of Maryland, Baltimore, Maryland, USAa; Department of Cell Biology and Molecular Genetics, Maryland Pathogen Research Institute, University of Maryland, College Park (UMCP), College Park, Maryland, USAb; Department of Microbial Pathogenesis, School of Dentistry, University of Maryland, Baltimore, Maryland, USAc; Department of Microbiology and Immunology, School of Medicine, University of Maryland, Baltimore, Maryland, USAd
S
treptococcus pyogenes (group A streptococcus [GAS]) is an important human pathogen that can cause a wide range of diseases in human hosts. Although it is most commonly responsible for self-limiting, superficial infections such as impetigo and pharyngitis, GAS can also cause more-invasive diseases such as necrotizing fasciitis, streptococcal toxic shock syndrome, or osteomyelitis (1). These invasive diseases are of great concern because they cause much higher morbidity and mortality (2). Altogether, GAS causes approximately 700 million infections worldwide each year and is responsible for over 500,000 deaths (3). Currently, there is no commercially available licensed vaccine against GAS (4). The development of such a vaccine would be a much-needed solution to invasive GAS infections, which are often identified too late for effective medical intervention (5). A vaccine would also be useful in preventing rheumatic heart disease, a postinfection complication commonly encountered in the developing world and a leading cause of heart disease globally (3). Although all known clinical strains of S. pyogenes are still susceptible to penicillins, a number of studies have shown a treatment failure rate of 20% to 40% when using antibiotics to treat GAS infections (6). Several hypotheses have been put forward as possible explanations for this failure of antibiotic treatment. One explanation that has been put forward is the ability of GAS to form biofilms (7). A biofilm can be classified as a sessile, microbially derived community where cells grow attached to a surface or a floating microbial conglomerate and secrete an extracellular matrix (8). When in the biofilm mode of growth, the majority of S. pyogenes strains display tolerance toward penicillin, making this antibiotic ineffective at microbial clearance (9). Since the ability of GAS to form a biofilm was first recognized over a decade ago, there has been only a modest appreciation of the role of GAS biofilms in
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an infection. Along with an initial study showing evidence of GAS forming a biofilm in a skin infection, GAS microcolonies indicative of in vivo biofilm formation have also been found in children who underwent tonsillectomies due to recurrent GAS infections (10, 11). A complex relationship between S. pyogenes and other respiratory tract streptococci has been demonstrated and suggests that the formation of a multispecies biofilm with the resident oral microbiota might be involved in asymptomatic GAS persistence (12). Also, it has been found that GAS survival in fomites is enhanced by growth as a biofilm, thereby providing important clinical implications in the transmission of GAS from infected hosts (13). Biofilms have also been implicated in increased in vivo survival and transformability of GAS, thereby contributing to genomic diversity (14). Several additional studies have found evidence of GAS biofilms in animal models of infection with M1 and M14 GAS strains (15– 18). In addition, cellular fibronectin, a component of the extracellular matrix, has been shown to enhance GAS biofilm formation (19). However, those previous studies focused mainly on superficial GAS infections and largely ignored the role of biofilms in an invasive S. pyogenes infection. For our study, we examined the role
Received 27 March 2014 Returned for modification 10 May 2014 Accepted 29 May 2014 Published ahead of print 7 July 2014 Editor: A. Camilli Address correspondence to Mark E. Shirtliff,
[email protected]. Copyright © 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/IAI.01831-14
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Group A streptococcus (GAS) is an important human pathogen that causes a number of diseases with a wide range of severities. While all known strains of GAS are still sensitive to penicillin, there have been reports of antibiotic treatment failure in as many as 20% to 40% of cases. Biofilm formation has been implicated as a possible cause for these failures. A biofilm is a microbially derived, sessile community where cells grow attached to a surface or as a bacterial conglomerate and surrounded by a complex extracellular matrix. While the ability of group A streptococcus to form biofilms in the laboratory has been shown, there is a lack of understanding of the role of GAS biofilms during an infection. We hypothesized that during infections, GAS exhibits a biofilm phenotype, complete with unique protein expression. To test this hypothesis, a rabbit model of GAS osteomyelitis was developed. A rabbit was inoculated with GAS using an infected indwelling device. Following the infection, blood and tissue samples were collected. Histological samples of the infected tibia were prepared, and the formation of a biofilm in vivo was visualized using peptide nucleic acid fluorescent in situ hybridization (PNA-FISH) and confocal microscopy. In addition, Western blotting with convalescent rabbit serum detected cell wall proteins expressed in vitro under biofilm and planktonic growth conditions. Immunogenic proteins were then identified using matrix-assisted laser desorption ionization–time of flight tandem mass spectrometry (MALDI-TOF/TOF MS). These identities, along with the in vivo results, support the hypothesis that GAS forms biofilms during an infection. This unique phenotype should be taken into consideration when designing a vaccine or any other treatment for group A streptococcus infections.
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MATERIALS AND METHODS Bacterial strain and growth conditions. The strain of GAS used in this study was 5448, an M1TI strain previously described (23). Unless indicated otherwise, GAS cells were grown in Todd-Hewitt broth supplemented with 0.2% yeast extract (THYB; BD Laboratories) at 37°C. Production of osteomyelitis. Strain 5448 was grown overnight in a mixture containing THYB and 10 mg/ml dextran beads (Cytodex microcarrier beads; Sigma) representing a foreign body. The overnight culture was spun down and washed with saline solution. Two New Zealand White female rabbits (Charles Rivers Laboratory), 8 weeks of age and weighing 1.5 to 2.0 kg, were used. The rabbits were anesthetized using an intramuscular injection of ketamine (Vetalar; Boehringer Ingelheim Vetmedica, Inc., St. Joseph, MO) (35 mg/kg of body weight), xylazine (AnaSed; Akorn, Decatur, IL) (10 mg/kg), and buprenorphine [Buprenex; Reckitt Benckiser Healthcare (UK) Ltd., Hull, England] (0.015 mg/kg). A bacterial infection was introduced following a previously described method (24). Briefly, an 18-gauge needle was inserted into the intramedullary cavity of the left tibial metaphysis. A 0.1-ml volume of a 5% (wt/vol) solution of sodium morrhuate (Eli Lilly, Indianapolis, IN), 0.1 ml of the GAS mixture (⬃2 ⫻ 107 CFU), and 0.2 ml of sterile saline solution were injected sequentially (25–29). Serum was collected prior to inoculation and at 28 days postinoculation. All procedures were performed per humane criteria set forth by the University of Maryland, Baltimore, Animal Care and Use Committee. Organ cultures. After 28 days, the rabbits were sacrificed by an intravenous injection of sodium pentobarbital. Both tibias (right and left) were removed, dissected free of all soft tissue, and processed for bacterial cultures and histology. The bones were split into small pieces, and a sample of the infected tibia was fixed in 4% paraformaldehyde (PFA) for further imaging. The remaining bone fragments were then pulverized and suspended in 3 ml of sterile 0.85% saline solution per gram of tissue. In addition, both kidneys and the spleen were removed and suspended in 3 ml of sterile 0.85% saline solution per gram of tissue, as well. All the organs were homogenized, and then serial 10-fold dilutions were spotted onto Todd-Hewitt agar plates supplemented with 0.2% yeast extract to determine the presence of GAS.
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Imaging of infected tissue. The PFA-fixed bone fragment was decalcified and embedded in paraffin at the University of Maryland Histology Core. Slides prepared from the paraffin-embedded sample were Gram stained and hematoxylin and eosin (H&E) stained, and peptide nucleic acid fluorescent in situ hybridization (PNA-FISH) was performed with a fluorescein isothiocyanate (FITC)-labeled Universal bacterial probe per the manufacturer’s instructions (AdvanDx, Woburn, MA). The slides were then examined with a Zeiss LSM 510 confocal scanning laser microscope (Carl Zeiss, Thornwood, NY) for green fluorescence using an FITC filter and a 63⫻ objective. Growth of GAS in vitro. Planktonic GAS cultures were grown as described above. Planktonic cultures were harvested by centrifugation for 10 min at 4,000 ⫻ g and 4°C. Planktonic cultures were harvested at time points corresponding to the early log phase, the late log phase, and the early stationary phase (4, 6, and 8 h after subculture, respectively). The pellets were resuspended in 1 ml of ice-cold protein preservation solution (PPS; 2.8 mM phenylmethylsulfonyl fluoride [PMSF], 50 mM Tris-Cl, 1 mM EDTA [pH 8.0], and 0.01% sodium azide). Samples were recentrifuged for 1 min at 13,000 rpm and 4°C. The resulting supernatant was discarded, and the cell pellets were frozen at ⫺20°C until protein extraction could be performed. In vitro GAS biofilm samples were obtained using a flow reactor system, allowing for reproducible biofilm growth as previously described (24), with the addition of a bubble trap (BioSurface Technologies Corporation, Bozeman, MT) upstream of the pump. Briefly, the reactor consisted of silicon tubing through which a 1:5 dilution of THYB broth flowed in a once-through fashion to a waste container under the control of a peristaltic pump. An overnight culture of GAS was diluted 1:100 into prewarmed THYB and allowed to grow at 37°C until the exponential phase was reached. A 10-ml volume of the exponential-phase bacterial culture was injected into each silicon tube through an injection port. The system was allowed to incubate without flow for 3 h, giving the bacteria time to adhere to the inner side of the tubing. The flow was then restored at a flow rate of 0.8 ml/min, providing a 7.5-min residence time. The entire flow reactor system was contained within a 37°C incubator during the entire time. Biofilm cultures were harvested at time points corresponding to earlystage biofilm, mid-stage biofilm, and late-stage biofilm (16 h, 6 days, and 10 days after restarting flow, respectively). Biofilms were harvested by squeezing the biofilm from the tubing into 10 ml ice-cold PPS. The tubing was then flushed with an additional 10 ml of PPS, and the entire collected sample was subjected to centrifugation for 10 min at 4,000 ⫻ g and 4°C. The pellets were resuspended and prepared for storage in the same manner as the planktonic samples described above. Purification of cell wall fraction. The cell wall protein fraction was isolated using a modified version of the previously described protocol for Streptococcus pyogenes cell wall extraction (30). Briefly, cell culture pellets were resuspended in 1 ml lysis buffer (50 mM ammonium acetate [pH 6.2], 10 mM CaCl2, 1 M dithiothreitol [DTT], and Roche Complete protease inhibitors). Equal numbers of cells from the samples, as determined by the optical density at 600 nm, were transferred to fresh tubes. The cells were pelleted and resuspended in lysis buffer containing 20% sucrose, 125 U of mutanolysin/ml, and 5 mg of lysozyme/ml. Cells were digested for 3 h at 37°C with constant rotation. Following digestion, the samples were centrifuged and the supernatant containing the cell wall fraction was saved for further analysis by 2DGE. Two-dimensional gel electrophoresis (2DGE). In order to conduct two-dimensional electrophoresis, the crude cell wall protein fraction was first extracted by adding a 1/10 volume of an ice-cold 1:10 mixture of trichloroacetic acid (Sigma, St. Louis, MO) and acetone. The resulting pellet was then resuspended in 0.2 ml saline solution and quantified using an Advanced protein assay (Cytoskeleton, Denver, CO). From the resuspended fraction, 0.1 mg of protein was further purified using a PerfectFocus kit (G-Biosciences, St. Louis, MO) following the manufacturer’s instructions. The resulting pellet was solubilized in rehydration buffer
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of GAS biofilms in osteomyelitis and foreign body infection. Osteomyelitis and infections of foreign bodies can result from primary seeding (e.g., perioperative contamination or trauma) or secondary seeding from a distal site of infection. In particular, GAS osteomyelitis has important clinical significance, especially in the pediatric population. Approximately 6% to 10% of all cases of pediatric osteomyelitis are due to GAS (20–22). In addition, GAS has also been isolated along with Staphylococcus aureus in cases of polymicrobial osteomyelitis (22). The difficulty in resolving GAS osteomyelitis by treatment with antibiotics suggests that biofilm formation has a potential role in osteomyelitis due to GAS. In this study, we demonstrated the presence of GAS biofilm in a rabbit model of tibial osteomyelitis and foreign body infection. This is, to our knowledge, the first GAS osteomyelitis model. In addition, we characterized differences between the biofilm and the planktonic growth phases in levels of cell wall protein expression for GAS. Using in vitro cultures and convalescent-phase serum from infected rabbits, we were able to identify GAS antigens expressed in vivo by the use of two-dimensional gel electrophoresis (2DGE) and immunoblotting followed by matrix-assisted laser desorption ionization–time of flight tandem mass spectrometry (MALDI-TOF/TOF MS). These proteins have potentially important implications for understanding the regulation of GAS biofilms in an infection and are also potential targets for vaccines and therapeutics to treat GAS infections.
In Vivo Expression of S. pyogenes Antigens
{0.1 mM urea, 25 M thiourea, 0.35 M DTT, 0.5% [wt/vol] CHAPS, i.e., 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate, and 1.6% Pharmalyte 3-10}. These samples were applied to (linear) Immobiline Dry Strips (GE Healthcare, Piscataway, NJ) (11 cm in length; pH 4 to 7). The isoelectric focusing, which separated the proteins based on their isoelectric point (pI) values, was performed using a Multiphor II system from Amersham per the manufacturer’s directions. Prior to analysis using the second dimension, the immobilized pH gradient (IPG) strips were equilibrated (per the manufacturer’s directions) and subsequently applied to SDS-PAGE gels. For the resolution of GAS protein extracts in the second dimension, a Bio-Rad Criterion Cell (Bio-Rad, Hercules, CA) was used. Proteins were separated on a 12.5% resolving gel and then either transferred to a polyvinylidene difluoride (PVDF) membrane for Western blotting or stained using Sybro Ruby protein gel stain (Lonza, Basel, Switzerland). Western blotting. Bacterial proteins that were transferred to a PVDF membrane following 2DGE were blocked in 5% nonfat dry milk–Trisbuffered saline–Tween 20 (pH 7.6) (TBS-T) (20 mM Tris, 137 mM NaCl, 0.1% Tween 20) overnight at 4°C. The membrane was treated with convalescent-phase sera (diluted 1:10) from one of the rabbits in TBS-T for 60 min and then washed two times for 20 min each time in TBS-T at 25°C. Goat anti-rabbit IgG horseradish peroxidase (HRP)-conjugated secondary antibody (Bio-Rad, Hercules, CA) was used at a dilution of 1:2,500 for 60 min in TBS-T, followed by two washes as described above. Immunogenic proteins were detected using SuperSignal chemiluminescent substrate (Pierce, Rockford, IL) as described by the manufacturer. The treated membranes were then imaged using a FluorChem 8900 imaging system and the associated imaging software (AlphaEaseFC). Western blotting was performed in triplicate for each combination of growth condition and time point. Images of the 2DGE gels and Western blots were compared to identify immunogenic protein spots. These spots were excised and identified using MALDI-TOF/TOF MS. MALDI-TOF/TOF MS. For identification of immunogenic proteins, an Applied Biosystems Voyager-DE STR MALDI-TOF mass spectrometer was used operated in the positive-ion mode with a cyano-4-hydroxycinnamic acid matrix for ionization. At least 100 laser shots per spectrum were averaged. Mass spectral peaks with a signal-to-noise ratio greater than 5:1 were deisotoped, and the resulting monoisotopic masses were subjected to protein identification using mass fingerprint analysis. The software used for protein identification was the Profound search engine
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using Genomic Solution Knexus software (ver 2004.03.15), and the database used was the latest NCBI nonredundant database obtained from NIH. Proteins with an expectation score of 1 ⫻ 10⫺3 or less were considered positive identities.
RESULTS
Characterization of the rabbit S. pyogenes osteomyelitis model. A novel rabbit model of tibial osteomyelitis and foreign body infection was developed in order to evaluate the architecture, phenotype, and proteomic character of GAS biofilms in vivo. In this model, the left tibiae of rabbits were inoculated with a mixture of dextran beads (which served as a deeply implanted foreign body representing an implanted medical device) and S. pyogenes. The rabbits were followed for 28 days. Rabbits developed manifestations of inflammation and pain, as demonstrated by non-weightbearing behavior using the infected limb over the course of the first week that persisted until the third week. At the end of 28 days, the rabbits were euthanized and the tibias, kidneys, and spleen were harvested. The infected left tibias showed significant inflammation and bone thickening, weighing greater than 2 times the uninfected tibia on average (11.91 g versus 5.42 g). A set of left and right tibias removed from the same rabbit postinfection is shown in Fig. 1. Bacterial colonies were recovered only from left tibias (Table 1), thereby demonstrating the production of chronic and localized osteomyelitis. Visualization of S. pyogenes biofilm. In addition to the gross
TABLE 1 Recovery of S. pyogenes M1T1 (5448) from tissue S. pyogenes M1T1 CFU/g Tissue
Rabbit 1
Rabbit 2
Left tibia Right tibia Left kidney Right kidney Spleen
3.60 ⫻ 105 0 0 0 0
3.73 ⫻ 105 0 0 0 0
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FIG 1 Left (infected) and right (uninfected) tibia from rabbit.
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FIG 2 Stained histologic sections from infected rabbit tibia. (A) H&E-stained dextran bead (asterisk) surrounded by inflammatory infiltrate. (B) Gram-stained
examination, one of the infected left tibias was examined histologically for the presence of a biofilm. Histological sections were prepared from the infected and uninfected tibia and stained using H&E stain (Fig. 2A) and Gram stain (Fig. 2B). Microscopic analysis of these sections confirmed the presence of a GAS infection as shown by inflammatory infiltrate surrounding the implants in H&E-stained samples as well as chains of cocci contained within the bone in Gram-stained sections. In addition, the presence of a biofilm was visualized in the infected tibial tissue using confocal microscopy. Following the staining of tissue samples with a peptide nucleic acid fluorescent in situ hybridization probe for the bacterial 16S rRNA, the presence of fluorescent S. pyogenes growing in dense microcolonies was readily identified (Fig. 3). Identification of immunogenic proteins. Due to the difficulty in recovering bacterium-specific protein from a mammalian host, an alternative approach was taken to examine protein expression. S. pyogenes was grown in the laboratory and harvested under both planktonic and biofilm growth conditions. Cells were harvested at multiple time points to account for temporal variations in protein production. In order to target proteins that are likely to be accessible to the immune system, the cell wall protein fraction was preferentially isolated. Cell wall protein extracted from the harvested cells was separated by 2DGE and subjected to Western blotting using serum obtained from infected rabbits. Three biological replicates were carried out for each time point. Representative sets of 2DGE gels and Western blots are shown in Fig. 4. Immunogenic spots appearing on two of the three replicates were excised and analyzed by MALDI-TOF/TOF MS. Based on the MALDI-TOF/ TOF MS results, 28 proteins were identified (Table 2). Of these proteins, 15 were expressed under both planktonic and biofilm growth conditions, 7 were upregulated under planktonic conditions, and 6 were upregulated during biofilm growth. DISCUSSION
While several studies have shown the presence of a biofilm during a group A streptococcus infection in vivo, none have done so in a mammalian model of deep-tissue infection. Not only did this study demonstrate the presence of a GAS biofilm during a deeptissue infection model, it represents the first animal model of streptococcal osteomyelitis. This model recreated the conditions that might occur during an orthopedic-implant-associated infection in humans. This was evident by the bone remodeling, inflam-
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mation, and involucrum formation that occurred in the infected tibia. Also, similarly to many orthopedic-implant-associated infections, the infection was persistent but remained localized. After 28 days, the host was able to reduce the bacterial populations to only a single log below the number of bacteria initially injected into the tibia. This suggests that the bacteria had established a niche that allowed them to survive, protected from the rabbit’s immune system. By the use of several different visualization techniques, GAS biofilm could be seen in the infected tibia. As shown in Fig. 2, the dextran beads injected into the rabbit could be seen under a light microscope with extensive inflammatory infiltrate and chains of cocci. When examined using confocal microscopy with PNAFISH probes, the presence of a dense GAS biofilm could be seen on the surface of the beads (Fig. 3A). Additionally, GAS microcolonies growing in a dense extracellular matrix could be seen adhering to bone in a manner independent of the beads (Fig. 3B and C). Together, all of the visual evidence supports the idea that GAS biofilms are playing a role in this persistent, localized osteomyelitis model. As the formation of a biofilm has often been found to be associated with persistent infections (8), it was not surprising to find evidence of a biofilm in the histological samples. In addition to demonstrating the presence of a biofilm in a group A streptococcus deep-tissue model, this study aimed to examine protein expression in GAS biofilms during an infection. Since host proteins vastly outweigh bacterial proteins in vivo, the GAS proteins produced in vivo could not be determined through proteomics. Instead, an immunoproteomic approach was employed. Convalescent-phase serum from the infected rabbit was used to identify S. pyogenes antigens from in vitro-grown cultures. The benefits of this approach are 4-fold. First, since proteins must be expressed in vivo in order to trigger the production of antibodies that recognize the protein, any immunogenic proteins identified will have been expressed during the infection. Second, the proteins are readily identified following isolation from their respective 2DGE gels and then subsequent MALDI-TOF/TOF MS. Third, this method identifies only S. pyogenes proteins that are immunogenic and thus likely to be candidate antigens for a vaccine. Lastly, the proteins are produced in vitro and in vivo and, as such, are more amenable to recombinant protein expression. In this screen, only the S. pyogenes cell wall protein fraction was screened for immunogenic proteins. We specifically chose to ex-
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S. pyogenes microcolonies (arrows) within damaged bone.
In Vivo Expression of S. pyogenes Antigens
Downloaded from http://iai.asm.org/ on August 14, 2014 by guest FIG 3 Histologic sections from infected rabbit tibia stained with PNA-FISH FITC-labeled Universal bacterial probe. (A) Dextran bead (asterisk) surrounded by S. pyogenes biofilm (arrows). (B and C) S. pyogenes microcolonies and chains of cocci (arrows) adherent to lacunae within bone.
clude the secreted protein fraction in our analysis. Secreted proteins shown to be involved in GAS virulence include streptococcal pyrogenic exotoxins such as SpeA and SpeC, the cysteine protease SpeB, secreted inhibitor of complement (sic), streptokinase, streptolysins, hyaluronidase, and DNases (31, 32). However, in spite of the important role of secreted proteins in virulence, we felt that proteins attached to the cell surface were most likely to be good candidates for in vivo diagnostics or for a protective subunit vaccine that induces opsonophagocytosis. While our strategy limits
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the number of proteins that can be identified, it does increase the odds that identified proteins will be relevant surface-exposed antigens. Some potential antigens are also likely missed due to our method of 2DGE. The majority of S. pyogenes proteins have an isoelectric point (pI) value of approximately 5.5, but there are also a minority of proteins with a pI value greater than 9 (33). As is evident from the immunogenic spots appearing on the right side of the 2D gels that were not identifiable by mass spectrometry, some immunogenic spots with pI values greater than 7 were not
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found. However, it would appear that our approach was still able to identify a majority of the immunogenic cell wall proteins. To verify that the proteins identified were, in fact, cell wallassociated proteins, the amino acid sequences of the identified proteins were analyzed using pSORTb (34). This confirmed only a small subset of the proteins to be noncytoplasmic. However, this result was due to the fact that many of the proteins identified were anchorless surface proteins and lacked the LPXTG motif and other signal sequences that are traditionally used to identify cell wall-associated proteins. The cell wall-associated proteins that were identified in this study were validated by other such studies in S. pyogenes. Two other studies both identified a similar set of cell wall-associated proteins from GAS (4, 35). Although those studies focused on planktonic cultures, 20 of the 22 proteins we identified as being found in planktonic cultures were also identified as such by Cole et al. (35). Additionally, a number of the proteins we identified were homologous to cell wall proteins identified from Staphylococcus aureus using a similar immunoproteomic approach (24). Several proteins in particular stand out from the list of the 28 immunogenic proteins. Although most immunogenic proteins identified were expressed in both planktonic and biofilm samples, some of the more immunogenic proteins were the ones that were
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expressed only under one condition. This suggests that both the planktonic and biofilm growth conditions are involved in the host immune response. The presence of three ABC transporter components (lipoprotein, ABC transporter substrate-binding protein, and ABC transporter metal binding protein) among the proteins upregulated in a biofilm is not entirely surprising. Bacterial ABC transporters have a very important role in, among other things, nutrient uptake (36, 37). Within a biofilm, there are various niches where bacteria experience nutrient deprivation, because the bacteria have limited access to nutrient-rich fluids (38–43). By increasing the expression of ABC transporters, the biofilm bacteria can enhance their ability to scavenge the limited resources. The three biofilm-specific ABC transporter proteins identified in this study are all lipoprotein components of ABC transporters for three different substrate types. GAS lipoproteins have been previously identified as possible vaccine targets, as they have been shown to have the potential to trigger the production of bactericidal antibodies (44). One of these biofilm-specific ABC transporter proteins, mtsA (SPy453), is part of the mtsABC transporter system which has been shown to be important for the function of the Mn-dependent superoxide dismutase and for full virulence in GAS (45). Additionally, the homolog of mtsA in Streptococcus pneumoniae, psaA,
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FIG 4 Representative 2DGE and Western blots of group A streptococcus cell wall protein. (A) 2DGE— biofilm cell wall protein. (B) Western blot— biofilm cell wall protein. (C) 2DGE—planktonic cell wall protein. (D) Western blot—planktonic cell wall protein. Note that some spots listed in Table 2 are not present, as not all spots were visible in every sample at every time point.
In Vivo Expression of S. pyogenes Antigens
TABLE 2 Identities of cell wall antigensa Accession no.b
SPy no.b,c
Function(s)d
pIe
MWb
Planktonic or biofilm expression
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28
15675606 161378148 15675235 15675444 15675256 15675442 15674367 15675236 15675439 15674453 15675837 15675699 15674582 15675326 15675522 15675128 15675062 13622630 15675343 15675650 15674481 15675799 15675599 15675192 15674335 15674482 15675028 15674573
SPy_1760 SPy_1676 SPy_1282 SPy_1547 SPy_1306 SPy_1544 SPy_0160 SPy_1283 SPy_1541 SPy_0274 SPy_2079 SPy_1881 SPy_0463 SPy_1406 SPy_1651 SPy_1151 SPy_1070 SPy_1542 SPy_1429 SPy_1821 SPy_0316 SPy_2018 SPy_1491 SPy_1228 SPy_0115 SPy_0317 SPy_1031 SPy_0453
Chaperone Metal ion binding; transketolase activity Carbohydrate degradation; glycolysis Amino acid degradation Maltose ABC transporter activity Amino acid degradation De novo purine nucleotide biosynthesis Carbohydrate degradation; glycolysis Amino acid biosynthesis Glycolysis; binds human plasminogen Oxidative stress response Carbohydrate degradation; glycolysis Translational termination Destroys superoxide anion radicals Aminopeptidase activity Glycolysis; fermentation Proteolysis Proteolysis Glycolysis Protein biosynthesis Transcription regulation Virulence factor (54) Fatty acid biosynthesis Nucleoside-binding ABC transporter aminopeptidase activity amino acid ABC transporter Cell redox homeostasis Zn, Fe, and Cu ABC transporter
4.445 4.729 4.704 4.841 5.351 5.072 5.388 5.282 4.54 5.267 4.493 4.649 5.576 4.723 4.899 5.019 4.717 4.58 4.939 4.678 4.318 6.581 6.174 8.066 5.029 7.326 4.747 6.577
64.789 71.222 54.404 46.166 45.422 37.8 47.331 35.617 33.087 35.812 20.352 41.999 20.441 22.552 50.733 35.141 51.215 48.95 25.952 20.3336 25.757 54.09 34.083 36.265 38.448 30.491 62.336 34.227
Both Both Both Both Both Both Both Both Both Both Both Both Both Both Both Planktonic Planktonic Planktonic Planktonic Planktonic Planktonic Planktonic Biofilm Biofilm Biofilm Biofilm Biofilm Biofilm
DnaK chaperone protein Transketolase Pyruvate kinase Arginine deiminase Maltose/maltodextrin-binding protein Ornithine carbamoyltransferase Adenylosuccinate synthetase 6-Phosphofructokinase Carbamate kinase Glyceraldehyde-3-phosphate dehydrogenase Alkyl hydroperoxide reductase Phosphoglycerate kinase Ribosome-recycling factor Superoxide dismutase (FE/Mn) Cysteine aminopeptidase C L-Lactate dehydrogenase Dipeptidase PepV putative Xaa-His dipeptidase Phosphoglyceromutase Elongation factor P Hypothetical protein M protein ACP–S-malonyl transferase Lipoprotein Glutamyl-aminopeptidase ABC transporter substrate-binding protein Dihydrolipoyl dehydrogenase ABC transporter metal binding protein
a
ACP, acyl carrier protein; ID, identification; MW, molecular weight. The information was obtained from the NCBI Protein Database (http://www.ncbi.nlm.nih.gov/protein). SPy numbers refer to Streptococcus pyogenes M1 GAS strain SF370. d Function data were derived from the UniProt database (www.uniprot.org), unless noted otherwise. e Values were derived from the Isoelectric Point Calculator (http://isoelectric.ovh.org). b c
has been shown to provide protection from S. pneumoniae when injected as a vaccine in mice (46, 47), and the homologous protein in Staphylococcus aureus has been successful utilized in a multivalent vaccine (48). Notably absent from the list of proteins expressed in the biofilm samples is M protein. M protein is a major S. pyogenes virulence factor and a highly antigenic surface protein (49). Since SpeB is known to cleave M protein (50) and SpeB has been shown to be downregulated in biofilms (17, 51), M protein would have been expected to actually be overexpressed in a biofilm instead of being absent. However, a previous study that looked at the role of M protein in biofilms found that while an M protein mutant was unable to form a biofilm, the expression of the M protein gene was downregulated during in vitro biofilm growth (18). That study used an M14 strain, as opposed to the M1 strain used in this study, suggesting that lack of expression of the M protein is not due solely to serotype-specific regulation. It is also possible that the method of growing in vitro S. pyogenes biofilms employed in this study caused a downregulation of M protein that could not be identified via immunoproteomics. However, we do not believe this to be the case. Instead, we feel it is more likely that the M protein is required for initial biofilm attachment but is quickly downregulated, as early as 16 h after inoculation. As the M protein has already been shown to be involved in attachment and adherence (52), it is conceivable that it is expressed in a biofilm only initially. Once a
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biofilm is formed, it could be advantageous for the biofilm bacteria to hide such an immunogenic protein if the M protein is no longer required to protect the bacteria from the host immune system. Once the prime target for a vaccine against GAS, M protein has been largely abandoned due to its tendency to trigger antibodies that cross-react with human cardiac myosin, leading to rheumatic heart disease (53). There are some studies still pursuing an M protein-based vaccine using a subunit approach that appears to alleviate concerns about cross-reactivity (54, 55). However, if the M protein is downregulated early in the formation of a biofilm during an infection, it raises questions about its role as a vaccine target. A vaccine based solely on the M protein might have an inherent gap in protection if it cannot recognize an infection once it has reached the biofilm stage. Whatever the cause, the absence of the M protein in the biofilm cell wall fractions is worthy of further investigation as it has the potential to cause a reevaluation of the role of the M protein in group A streptococcus pathogenesis. At least one protein expressed under both growth conditions has already been tested as a protective antigen. Arginine deiminase was shown to provide protection when used to vaccinate mice prior to a challenge with S. pyogenes (4, 56). The fact that, of the proteins identified by this study, the one protein besides M protein that has been tested as a vaccine candidate has provided some degree of protection further validates this method for identifying
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ID no. Protein name
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ACKNOWLEDGMENTS These studies were funded by the National Institute of Allergy and Infectious Diseases, National Institutes of Health grant R01 AI69568, and The University of Maryland, College Park (UMD) and Baltimore (UMB) Research and Innovation Seed Grant Program.
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vaccine targets. It is possible that several of the proteins identified in this study could be utilized together to make a multivalent vaccine that is sufficient to protect against S. pyogenes. As has been shown with many other pathogens, the biofilm growth phenotype is an important part of understanding pathogenesis in a GAS infection. The significance of group A streptococcus biofilms in an infection can be seen both directly in infected tissue and through the immunoproteomic fingerprint that they leave. Any vaccine strategy designed to target GAS should take both its planktonic and biofilm growth phases into account. To that extent, this study has identified several immunogenic cell wall antigens expressed under both planktonic and biofilm conditions that have the potential to be useful vaccine antigens. This work will hopefully contribute to the successful identification of components of a future vaccine and diagnostic targets against group A streptococcus.
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