RESEARCH LETTER
Identification of high immunoreactive proteins from Streptococcus agalactiae isolates recognized by human serum antibodies Monika Brzychczy-Wloch1, Sabina Gorska2, Ewa Brzozowska2, Andrzej Gamian2, Piotr B. Heczko1 & Malgorzata Bulanda3 1
Department of Bacteriology, Microbial Ecology and Parasitology, Jagiellonian University Medical College, Krakow, Poland; 2Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland; and 3Department of Infection Epidemiology, Jagiellonian University Medical College, Krakow, Poland
Correspondence: Monika Brzychczy-Wloch, Department of Bacteriology, Microbial Ecology and Parasitology, Jagiellonian University Medical College, 18 Czysta Street, 31-121 Krakow, Poland. Tel.: +4812 633 25 67; fax: +4812 423 39 24; e-mail: [email protected] Received 28 May 2013; revised 30 July 2013; accepted 29 September 2013. Final version published online 24 October 2013.
MICROBIOLOGY LETTERS
DOI: 10.1111/1574-6968.12292 Editor: Akio Nakane Keywords Streptococcus agalactiae (GBS); immunogenic proteins; pregnant women; vaccination.
Abstract The aim of the studies was to identify immunogenic proteins of Streptococcus agalactiae (group B streptococcus; GBS) isolates. Investigation of the immunoreactivity with human sera allowed us to determine major immunogenic proteins which might be potential candidates for the development of vaccine. For the study, we have selected 60 genetically different, well-characterized GBS clinical isolates. The proteins immunoreactivity with 24 human sera from patients with GBS infections, carriers, and control group without GBS was detected by SDS-PAGE and Western blotting. As a result, some major immunogenic proteins were identified, of which four proteins with molecular masses of about 45 to 50 kDa, which exhibited the highest immunoreactivity features, were analyzed by LC-MS/MS. The proteins were identified by comparative analysis of peptides masses using MASCOT and statistical analysis. The results showed known molecules such as enolase (47.4 kDa), aldehyde dehydrogenase (50.6 kDa), and ones not previously described such as trigger factor (47 kDa) and elongation factor Tu (44 kDa). The preliminary results indicated that some GBS proteins that elicit protective immunity hold promise not only as components in a vaccine as antigens but also as carriers or adjuvants in polysaccharide conjugate vaccines, but more studies are needed.
Introduction Streptococcus agalactiae (group B streptococcus, GBS) is one of the most common causes of life-threatening bacterial infections in infants, which is associated with high mortality, and is also an emerging pathogen among adult humans, especially in the elderly, immunocompromised individuals and diabetic adults. In recent years, both in Europe and in the Polish clinical centers, there has been a significant increase in the disease rate caused by GBS (de la Rosa Fraile et al., 2001; Kowalska et al., 2003; Colbur & Gilbert, 2007; Strus et al., 2009; RodriguezGranger et al., 2012). Moreover, more and more frequently, S. agalactiae exhibits resistance to macrolides, lincosamides, and streptogramins B, which significantly reduces the effectiveness of antibiotics (Gherardi et al., FEMS Microbiol Lett 349 (2013) 61–70
2007; Brzychczy-Wloch et al., 2010). The presence of GBS in the maternal genital or gastrointestinal tract causes neonatal infection in most cases (Schuchat, 2000; Schrag et al., 2002). In Europe, the rate of colonization with group B streptococcus in pregnant women ranges from 6.6% in Greece to 36% in Denmark. In Poland, depending on the area and the methodology used, the rate of GBS-colonized pregnant women reaches 30% (BrzychczyWloch et al., 2012). It was shown that the risk of GBS transmission from mother to child reaches 70%, and the incidence of infections is 2–4 per 1000 live births. In newborns, streptococcus may cause the so-called earlyonset disease – EOD, which develops in the first week of life, or late-onset disease – LOD, developing between the 7th and the 90th day of life. GBS infections usually take the form of pneumonia and sepsis (80% of cases) and ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
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meningitis (10%), which is associated with high mortality among infants (Schuchat, 2000; Schrag et al., 2002; Johri et al., 2006). So far, ten GBS serotypes have been described (Ia, Ib, II-IX), distinguished on the basis of the differences in the structure of surface polysaccharides, also called capsular polysaccharides (CPS). In the United States and in Europe, serotypes Ia, III, and V are dominant, while in Asia the most common are serotypes VI and VIII (Poyart et al., 2007; Rodriguez-Granger et al., 2012). In Poland, serotypes Ia, III, and V represent over 70% of all the GBS strains from pregnant carriers and from newborns (Brzychczy-Wloch et al., 2012). Given the partial efficacy of antibiotic prophylaxis against S. agalactiae infections in neonates and increasing infection rates among the elderly or immunocompromised, as well as problems with growing resistance of GBS strains to antibiotics, the best long-term solution to prevent infections caused by group B streptococci would be constituted by vaccination (Baker & Edwards, 2003; Lin et al., 2004; Johri et al., 2006; He et al., 2010; Heath, 2011). Research on effective immunization against S. agalactiae infections has lasted from the time when in the 1970s Professor Rebecca Lancefield demonstrated in a mouse model that protective antibodies directed against the capsular polysaccharides of group B streptococci are able to prevent from the occurrence of invasive GBS infections; however, we still lack an effective vaccine (Baker & Edwards, 2003; Johri et al., 2006; Heath, 2011). It was shown that a natural and effective factor in protecting infants from infection is provided by IgG class antibodies, directed against GBS surface antigens, transferred with mother’s blood through the placenta (Lin et al., 2004; Palmeiro et al., 2011). In recent years, in the United States, several polyvalent vaccines based on surface polysaccharides of S. agalactiae were developed (Edwards et al., 2012). They are currently the subject of clinical trials, but it is already known that due to the strong variation of CPS, such a vaccine must contain at least a few different types of S. agalactiae, or various isolated polysaccharides representing different serotypes most prevalent in a given geographical area (Baker & Edwards, 2003; Johri et al., 2006; Heath, 2011). Because the capsule-based vaccines showed low immunogenicity, interest has shifted toward S. agalactiae proteins as vaccine antigens. The most interesting as regards designing highly efficacious synthetic vaccines are protective epitopes of surface proteins (Berzofsky et al., 2001; Johri et al., 2006; He et al., 2010). The first identified surface protein antigen of GBS was the C antigen composed of two protein components: a protein (alpha C protein, ACP) and b protein. The best potential candidates for vaccines are the members of the Alp family, such as a protein, Rib, Alp3, or Alp2, and ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
these have so far been extensively studied (StalhammarCarlemalm et al., 1993; Baker & Edwards, 2003; Johri et al., 2006; Heath, 2011). In a mouse model, Rib and a protein would protect against the majority of S. agalactiae infections, but any surface antigen that is a target for protective immunity in natural infections may exhibit antigenic variation, necessitating the development of multivalent vaccines (Larsson et al., 1996). Other important proteins of interest for vaccine development are Sip protein, which is highly conserved surface protein, and surface-localized enzyme C5a peptidase (Brodeur et al., 2000; Santillan et al., 2008). The list of GBS surface proteins is long and fully valuable, as a vaccine giving complete protection may be composed of two or, even, a few immunogenic proteins (Johri et al., 2006). The aim of the studies was to identify immunogenic proteins of well-characterized Streptococcus agalactiae (group B streptococcus, GBS) strains isolated from pregnant women and newborns and to investigate their immunoreactivity with human sera (cord blood serum, serum from patients with GBS infections, carriers and from control group without GBS) to determine major proteins which might be potential candidates for carriers in the development of vaccine against S. agalactiae infections.
Materials and methods Study population and specimen collection
Streptococcus agalactiae strains and serum samples came from the Jagiellonian University, Medical College’s Department of Bacteriology, Microbial Ecology and Parasitology’s own collection, which was being gathered from 2006 until 2010 within the framework of two projects financed by the Polish Ministry of Research and Higher Education and no. 3PO5E08425 and NN401042337. The 60 selected GBS strains represented different groups of patients (pregnant and nonpregnant women, newborns), different clinical status (infection, carrier state), various clinical materials (blood, vagina and anus swabs, newborn ear swab, urine), different serotypes (Ia, Ib, II-V), various genes encoding proteins of the Alp family (bca, alp 2, alp 3, epsilon, rib), different macrolide resistance phenotypes (cMLSB, iMLSB, and M), various sequence types (ST) and also different regions in Poland (Table 1). The reference GBS strains 2134 (Deutsche Sammlung von Mikroorganismen und Zellkulturen, DSMZ), 12403, and BAA-611 (American Type Culture Collection, ATCC) were used. Serum samples
Twenty-four serum samples were used. Group I – tested sera – TS (n = 16); serum samples collected in the 3rd FEMS Microbiol Lett 349 (2013) 61–70
Clinical number
7/P/2a 25/P/1a 306735 CM169 5279/08 5303/08 13445/07 11277/08 13695/08 2337/08 5338/08 9/0/2a 13608/08 D121 2107/08 CM184 305245 10/P/3a 23/P/3a CM47 13073/08 9353/08 13640/07 14041/07 14191/07 2341/08 D120 D126 20/0/3a 28/0/3a 5886/09 3634/08 13723/07 L1-181 4504/08 D136 W2/18 CM173 CM176 CM185
GBS no.
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 29 30 31 32 33 34 35 36 37 38 39 40
Ia Ia Ia Ia Ia Ia Ia Ia Ia Ia Ia Ib Ib Ib Ib Ib II II II II II II II II II II II II III III III III III III III III III III III III
Serotypes
Resistance phenotype; gene – – – – – – – – – – – – – – – – – – M; mefA/E cMLSB; ermB – – – – – – M; mefA/E – – – – – – – – – – cMLSB; ermB – iMLSB
Alp genes epsilon epsilon epsilon bca epsilon epsilon epsilon rib epsilon epsilon epsilon bca bca epsilon bca bca bca rib rib rib bca rib bca rib bca rib epsilon epsilon alp2 rib rib rib rib rib rib epsilon alp2 bca rib rib nt nt ST-23 ST-220 nt nt nt nt nt nt nt nt nt nt nt nt ST-106 nt nt ST-19 ST-12 nt ST-10 nt nt nt nt nt nt nt ST-17 ST-17 ST-358 ST-17 ST-19 nt ST-23 ST-22 ST-220 ST-410
Sequence type (ST) Vaginal swab from pregnant Vaginal swab from pregnant Blood of newborn Blood of newborn Newborn urine Newborn urine Swab from the newborn mouth Newborn ear swab Newborn urine Swab from the newborn mouth Swab from the newborn mouth Pregnant anal swab Newborn urine Cervical swab from nonpregnant Swab from the newborn mouth Vaginal swab from pregnant Blood of newborn Vaginal swab from pregnant Vaginal swab from pregnant Blood of newborn Blood of newborn Newborn urine Content bronchial newborn Swab from the newborn mouth Newborn ear swab Swab from the newborn mouth Vaginal swab from nonpregnant Vaginal swab from nonpregnant Vaginal swab from pregnant Vaginal swab from pregnant Blood of newborn Blood of newborn Newborn ear swab Blood of newborn Blood of newborn Vaginal swab from nonpregnant Blood of newborn Swab from the newborn mouth Swab from the newborn mouth Newborn ear swab
Clinical material
Table 1. Characterization of 60 genetically different, well-characterized GBS clinical isolates, chosen to identify their surface proteins
Carrier Carrier Newborn sepsis (EOD) Newborn sepsis (EOD) Bacteriuria (≥ 105 CFU mL Bacteriuria (≥ 105 CFU mL Colonization Colonization Bacteriuria (≥ 105 CFU mL Colonization Colonization Carrier Bacteriuria (≥ 105 CFU mL Inflammation Colonization Carrier Newborn sepsis (EOD) Carrier Carrier Newborn sepsis (EOD)* Newborn sepsis (EOD) Bacteriuria (≥ 105 CFU mL Pneumonia Colonization Colonization Colonization Inflammation Inflammation Carrier Carrier Newborn sepsis (EOD) Newborn sepsis (EOD) Colonization Newborn sepsis (EOD) Newborn sepsis (EOD) Inflammation Newborn sepsis (EOD) Newborn sepsis (EOD)* Newborn sepsis (EOD)* Newborn sepsis (EOD)
Clinical signs
) )
FEMS Microbiol Lett 349 (2013) 61–70
)
1
)
1
)
1
1
1
Southern Poland Southern Poland Northern Poland Southern Poland Western Poland Western Poland Western Poland Western Poland Western Poland Western Poland Western Poland Southern Poland Western Poland Southern Poland Western Poland Southern Poland Northern Poland Southern Poland Southern Poland Southern Poland Western Poland Western Poland Western Poland Western Poland Western Poland Western Poland Southern Poland Southern Poland Southern Poland Southern Poland Western Poland Western Poland Western Poland Central Poland Western Poland Southern Poland Central Poland Southern Poland Southern Poland Southern Poland
Geographical area
Immunogenic GBS Proteins
63
ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
CM28 3A-012 CM49 306723 CM87 CM3 3/P/2a 42/P/3a 14/P/3a S1/4 D156 3514/08 14030/08 1716/08 1736/08 2992/08 13793/08 12403 2134 BAA-611
41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
III III III III III IV V V V V V V V V V V V III II V
Serotypes
Resistance phenotype; gene – – – – – – cMLSB; ermB – cMLSB; ermB – – – M; mefA/E – cMLSB; ermB – cMLSB; ermB – – –
Alp genes rib rib rib alp2 rib epsilon alp3 alp2 alp3 alp3 epsilon rib rib alp2 alp2 rib alp3 alp2 rib rib ST-286 ST-447 ST-148 ST-23 ST-19 nt nt nt Nt ST-1 nt ST-220 nt nt nt nt nt nt nt nt
Sequence type (ST) Blood of newborn Vaginal swab from pregnant Vaginal swab from pregnant Newborn urine Newborn ear swab Vaginal swab from pregnant Vaginal swab from pregnant Vaginal swab from pregnant Vaginal swab from pregnant Blood of newborn Vaginal swab Blood of newborn Blood of newborn Newborn urine Newborn urine Newborn urine Newborn urine GBS standard (ATCC) GBS standard (DSMZ) GBS standard (ATCC)
Clinical material
Newborn sepsis (EOD) Carrier Carrier Bacteriuria (≥ 105 CFU mL Colonization Carrier Carrier Carrier Carrier Newborn sepsis (EOD)* Inflammation Newborn sepsis (EOD) Newborn sepsis (EOD) Bacteriuria (≥ 105 CFU mL Bacteriuria (≥ 105 CFU mL Bacteriuria (≥ 105 CFU mL Bacteriuria (≥ 105 CFU mL Fetal septicemia No data Clinical specimen
Clinical signs
1
) ) 1 ) 1 )
1
)
1
Southern Poland Southern Poland Southern Poland Northern Poland Southern Poland Southern Poland Southern Poland Southern Poland Southern Poland Northern Poland Southern Poland Western Poland Western Poland Western Poland Western Poland Western Poland Western Poland Germany USA Germany
Geographical area
*Death of newborn. cMLSB, constitutive resistant to macrolides, lincosamides, and streptogramin B encoded by ermB gene; iMLSB, inducible resistant to macrolides, lincosamides, and streptogramin B; M, resistant to macrolides, but not lincosamides or streptogramin B, conferred by mefA/E gene; EOD, early-onset diseases; nt, not tested.
Clinical number
GBS no.
Table 1. Continued
64 M. Brzychczy-Wloch et al.
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Immunogenic GBS Proteins
trimester of pregnancy from pregnant women colonized with group B streptococcus (n = 10); serum from umbilical cord blood from pregnant women colonized with group B streptococcus (n = 1); serum samples from the studied women during puerperium (n = 3) collected 8 weeks after labor; serum from neonates with GBS EOS sepsis (n = 2). Group II – control sera – CS (n = 8); serum samples collected in the 3rd trimester of pregnancy from pregnant women without GBS colonization, which was confirmed throughout three trimesters of their pregnancy. Molecular characterization of the GBS strains
Most of the tested strains were carefully characterized in the previous study (Brzychczy-Wloch et al., 2010, 2012). The detection of genes encoding capsular polysaccharides Ia, Ib, II-VIII was investigated using the multiplex PCR method with specific primers (Genomed) as previously described (Poyart et al., 2007). To detect the surface protein genes alp2, alp3, alp4, bca, epsilon, and rib, multiplex PCR was used with specific primers (Genomed), according to the procedure proposed by Creti et al. (2004) and Gherardi et al. (2007). Macrolide resistance phenotypes cMLSB (constitutive), iMLSB (inducible), and M were determined by the double-disk test with erythromycin (15 lg) and clindamycin (2 lg; Oxoid). The erm(A), erm (B), erm(C), and mef(A/E) resistance determinants were detected by multiplex PCR with adequate four pairs of primers (Genomed) according to Sutcliffe et al. (1996). MLST analysis was performed as described by Jones et al. (2003) with the use of oligonucleotide primer pairs (Genomed) specific for the seven housekeeping loci selected for the GBS MLST. The online database (http://pubmlst. org/sagalactiae) was used for assigning alleles for seven loci, and each isolate was defined by the sequence type (ST). Protein isolation
Bacterial strains were cultured on brain-heart infusion broth (BHI, Biocorp) for 24 h at 37 °C. Bacteria from the plates were suspended in phosphate-buffered saline (PBS, Institute of Immunology and Experimental Therapy, PAN) to reach the final density of bacteria in the solution A600 = 1.0. After centrifuging (3312 g per min), the bacterial mass was suspended in Tris-HCl (Merck) buffer containing different concentrations of SDS (1–2%; Sigma-Aldrich) or directly in the buffer for electrophoresis according to Heilmann et al. (1996). The next steps were sonication (three times for 5 min) and centrifugation (13 248 g per min). Proteins were precipitated from the resulting supernatant using 3 volumes of cold 95% FEMS Microbiol Lett 349 (2013) 61–70
ethanol (POCH). After overnight incubation at 4 °C, the precipitated proteins were centrifuged (12 000 r.p.m. per min) and dissolved in water. Protein concentration was analyzed using the BCA method (Smith et al., 1985). SDS-PAGE and immunoblotting
Samples (10 lg of protein) were applied to SDS-PAGE gels using 5% to 12.5% gels according to Laemmli (1970). After electrophoresis, proteins were stained with Coomassie Brilliant Blue (Serva) or transferred to a polyvinylidene difluoride membrane (Millipore) for immunoblotting. The membranes were blocked in PBS containing 1% of bovine serum albumin (BSA, KPL) for 1 h. After washing three times with PBS containing 0.25% Tween 20 (SigmaAldrich), PBS-T (Institute of Immunology and Experimental Therapy, PAN), the membranes were incubated in a selected human sera in 1% BSA for 2 h at 37 °C. We tested different dilutions of sera (from 1 : 150 to 1 : 15000 in doubly increasing amounts), but the most effective was 1 : 3000 (reproducible results, without the Hook effect). Next, the membranes were washed three times with PBS-T. After washing, the membranes were incubated for 1 h in alkaline phosphatase-conjugated goat-anti-human IgG antibodies (Sigma) diluted 1 : 5000. Finally, the membranes were washed as described above, and solution containing nitroblue tetrazolium (NBT, Roth), 5-bromo4-chloro-3-indolyl phosphate (BCIP, Roth), and MgCl2 (POCh) was added for 5 s to visualize reaction. Protein identification
Immunogenic proteins were separated and purified by preparative electrophoresis in denaturing condition (SDSPAGE) using Prep-Cell apparatus (Model 491 Bio-Rad). Bands of interest were cut out and digested using a proteolytic enzyme such as trypsin (Roche) to obtain a mixture of peptides. Then, the peptides were separated by liquid chromatography (LC), and mass fragments were measured using mass spectrometer LC-MS/MS Orbitrap (Thermo). Proteins were identified by comparative analysis of peptides masses (NCBI, UniProt databases) using MASCOT (http://www.matrixscience.com/) and statistical analysis.
Results Immunogenic proteins were shown to be present in all 60 tested GBS strains. The protein concentration in the studied extracts was significantly different and varied, ranging from 0.2 to 2.7 mg mL 1 with an average value equal to 0.749 mg mL 1. We observed some significant variations in an average amount of proteins concentration ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
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in relation to different GBS serotypes. We demonstrated that strains with V serotype produced the highest concentration of the proteins with an average amount equal 1.18 1.12 mg mL 1 of extract, while Ia serotype had the lowest concentration of the protein with an average amount equal 0.5 0.29 mg mL 1. For the other serotypes, the following values were obtained: Ib (0.51 0.31), II (0.86 0.95), and III (0.62 0.57). Samples were subjected to SDS-PAGE, and protein bands were visualized with Coomassie Brilliant Blue. The representative results are shown in Fig. 1. It could be demonstrated that protein samples of all serotypes share some predominant bands, but it was also shown that despite common protein bands, the protein pattern varies between different serotypes. For the analysis of immunoreactivity of serum samples, protein samples were transferred to a polyvinylidene difluoride membrane for immunoblotting. The immunoreactivity of tested sera (TS) from women with GBS carriers was comparable to that of cord blood serum and sera from women during puerperium and from infants with GBS infection with respect to the number and intensity of bands. The banding patterns were heterogeneous and support the previous statement that pattern of proteins varies between serotypes. However, it was observed that in most cases, among the 60 S. agalactiae isolates, the proteins with molecular mass of about 45–50 kDa are most immunoreactive with umbilical cord blood (Fig. 2). The antigenic properties were observed also for proteins with molecular mass of about 40, 60, and 90 kDa. Interestingly, we observed that antibodies of serum from a newborn can recognize the protein antigens of S. agalactiae species on the same level as mother’s serum (Fig. 3) and also that the level of specific anti-GBS proteins antibodies increases significantly from third trimester to puerperium (Fig. 4). The immunoreactivity of the control sera (CS) from healthy pregnant women without GBS
(a)
colonization was also investigated. There was no reaction (data not shown). To identify the most immunogenic proteins, electrophoretic preparation of isolated proteins from S. agalactiae strain no. 57 (V serotype) was made using Prep-Cell Apparatus (491 model Bio-Rad). Four proteins with molecular masses of about 45 to 50 kDa showing immunoreactivity features were cut from the gel and analyzed in LC-MS/MS (Fig. 5). Proteins were identified by comparative analysis of peptides masses (NCBI, UniProt databases) using MASCOT and statistical analysis. Trigger factor with molecular mass of 47 kDa (89% protein sequence coverage), enolase with molecular mass of 47.4 kDa (81% protein sequence coverage), elongation factor Tu with molecular mass of 44 kDa (76% protein sequence coverage), and aldehyde dehydrogenase with molecular mass of 50.6 kDa (71% protein sequence coverage) were identified.
Discussion Group B streptococci, despite the introduction of adequate perinatal prophylaxis in the United States and many European countries, still constitute the main cause of neonatal morbidity and mortality in developed countries (Schuchat, 2000; de la Rosa Fraile et al., 2001; Schrag et al., 2002; Rodriguez-Granger et al., 2012). Major efforts are therefore under way to find new methods to prevent and treat infections caused by these pathogens, to analyze pathogenic mechanisms, and to develop efficient vaccines. A substantial difficulty in developing group B streptococcal vaccines is the existence of a multiplicity of serotypes with different geographical distributions. A vaccine suitable for Asian or European populations may not be appropriate for African populations (Johri et al., 2006; Poyart et al., 2007; Rodriguez-Granger et al., 2012). The use of GBS preventive vaccination would
(b)
Fig. 1. SDS-PAGE profile of proteins isolated from selected Streptococcus agalactiae strains. Lane: STD – molecular mass marker (GE Healthcare); 1 – 2337/08; 2 – 5338/08; 3 – 9/0/2a; 4 – 13608/08; 5 – D121; 6 – 2107/08; 7 – CM184; 8 – 305245; 9 – 10/P/3a; 10 – 23/P/3a; 11 – CM47; 12 – 13073/08; 13 – 9353/08; 14 – 13640/07; 15 – 14041/07; 16 – 14191/07; 17 – 2341/08; 18 – D120.
ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
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Immunogenic GBS Proteins
(a)
(b)
Fig. 2. (a) SDS-PAGE profile of proteins extracted from chosen Streptococcus agalactiae strains (b) Immunoblotting of proteins extracted from chosen Streptococcus agalactiae strains with cord blood serum (TS/1b). Lane: STD – molecular mass marker (GE Healthcare); 1 – 13073/08; 2 – 3A-012; 3 – 3/P/2a; 4 – 1716/08; 5 – 1736/08; 6 – 2992/08; 7 – 13793/08.
(a)
(b)
Fig. 3. Immunoblotting of proteins extracted from chosen Streptococcus agalactiae strains with (a) serum (TS/4a) from a GBS-carrying mother and (b) serum (TS/4b) from her baby with GBS sepsis. Lane: 1 – CM185; 2 – CM28; 3 – 3A-012; 4 – CM49; 5 – 306723; 6 – CM87; 7 – CM3; 8 – 3/P/2a; 9 – 42/P/3a.
(a)
(b)
Fig. 4. Immunoblotting of proteins from chosen Streptococcus agalactiae strains with (a) serum from a GBS-carrying woman in third trimester (TS/2a) and (b) serum from the same woman during puerperium (TS/2b). Lane: 1 – CM3; 2 – 3/P/2a; 3 – 42/P/3a; 4 – 14/P/3a; 5 – S1/4; 6 – D156; 7 – 3514/08; 8 – 14030/08; 9 – 1716/08.
make it possible to eliminate the problem of carriage in the population of pregnant women, reduce the number of infections in both the early- and late-onset neonatal group, as well as among the elderly and the immunocompromised; it would additionally limit the use of antibiotics and the problems of increasing resistance of bacteria, and finally, preventive vaccination would eliminate the side effects of antibiotic treatment, such as allergy and FEMS Microbiol Lett 349 (2013) 61–70
anaphylaxis (Baker & Edwards, 2003; Johri et al., 2006; He et al., 2010; Heath, 2011). A number of candidates, including capsular polysaccharides and proteins, have the potential to be a component of new vaccines. The capsular polysaccharides (CPS) from all ten currently identified GBS serotypes were found to elicit serotype-specific protective immunity in animal models, but showed low immunogenicity if not conjugated to a ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
68
Fig. 5. SDS-PAGE profile of a separated immunoreactive protein isolated from Streptococcus agalactiae 13793/08 using Prep-Cell apparatus.
protein carrier. It is noteworthy that memory B cells are not produced in response to most polysaccharide vaccines, therefore lacking the ability of inducing a booster response. Furthermore, plain polysaccharide vaccines are not generally immunogenic in infants, and their use is not allowed as vaccines to prevent disease in children caused by polysaccharide-encapsulated bacteria that have their highest incidence in the first year of life (Johri et al., 2006; Palmeiro et al., 2011). Therefore, a more recent approach to developing GBS vaccines is to use proteins (Fluegge et al., 2004; Santillan et al., 2008; Papasergi et al., 2013). The search for GBS immunogenic proteins allowed the discovery of many proteins that induced protective antibodies. Purified GBS proteins could be used as effective carriers for conjugate CPS vaccines while simultaneously inducing protective immunity against GBS (Johri et al., 2006). In this study, we were able to identify some major proteins of S. agalactiae that are immunoreactive and may suggest that they could play a potential role in the development of vaccine against GBS infections. Although the protein patterns observed for the 60 different GBS isolates studied showed many similarities, some differences between serotypes and source of clinical materials were observed. We found four immunoreactive proteins present in all 60 strains tested: enolase, dehydrogenase, trigger ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
M. Brzychczy-Wloch et al.
factor, and elongation factor Tu. All of these proteins are cytoplasmic protein and belong to cell surface-associated or surface-exposed proteins (Pancholi & Fischetti, 1998; Severin et al., 2007; Kolberg et al., 2008; Feng et al., 2009; Sharma et al., 2013). Surface enolase and dehydrogenase have been described as multifunctional proteins of streptococcal bacteria (Pancholi & Fischetti, 1998). Enolase, with its plasminogen binding activity, is involved in invasion and adherence of S. pyogenes and S. pneumonia to human pharyngeal cells (Severin et al., 2007). Streptococcal surface dehydrogenase is also a virulence factor. Structurally and functionally, it is related to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). It can bind to mammalian fibronectin or cytoskeletal proteins such as myosin and possesses ADP-ribosylating activities (Pancholi & Fischetti, 1998). It was shown that streptococcal surface-associated dehydrogenase recognizes pharyngeal plasminogen activator receptor, which contributes to bacterial adherence and plays a crucial role in pathogenesis (Severin et al., 2007). The elongation factor Tu has been found on S. pyogenes surface, and its possible function is bacteria attachment to mammalian cells. Elongation factor Tu is also present in cytoplasmic fraction of bacterial cells. It is one of the most abundant proteins in prokaryotes, representing even 10% of the total amount of proteins in Escherichia coli (Kolberg et al., 2008). High cytoplasmic content of elongation factor raises the question whether detection of this protein in bacterial membrane preparations might represent a contamination by cytosolic one during their preparation. It could be true, but this does not change the fact that the epitope localized on this protein is highly immunoreactive and is recognized by naturally produced antibodies. The presence of antibodies indicates that this protein had to be exposed to the outside of bacteria cell during the infection process. Trigger factor is ribosome-associated chaperone with a peptidyl-prolyl cis-trans isomerase activity. The protein is important for SpeB cysteine proteins secreting. Trigger factor has been detected in cytoplasm and on the S. pyogenes surface (Severin et al., 2007). The most frequent concept of immunoreactive proteins identification is based on standard biochemical and microbiological methods such as animal immunization using cell surface components and then immune testing. In our study, we used natural sera containing originally produced antibodies after streptococcal infection, and it was not an artificially induced reaction. It must be emphasized, because during infection process certain changes in the surface proteins expression occur and, consequently, the profile of proteins exposed on bacterial surface is different than the profile of the surface proteins extracted from bacterial cell after cultivation. It was shown that glyceraldehyde-3-phosphate dehydrogenase FEMS Microbiol Lett 349 (2013) 61–70
Immunogenic GBS Proteins
(GAPDH), which is involved in host–cell interaction, is expressed only during infection. It is suggested that differential upregulation of all GBS virulence genes occurs (Sharma et al., 2013). Results of our study show that human immune response to streptococcal pathogens is not directed against the most abundant cell surface proteins such as M protein or C5a but also against other proteins which seem to be better immunoreactants (Severin et al., 2007; Santillan et al., 2008). In our study, we used natural human model of immunization during bacterial infection, and we showed which protein antigens of streptococci are involved in immune response. So far, vaccine candidates have been constituted by capsular polysaccharides and surface proteins such as C protein, whereas our study suggests that proteins involved in bacterial cell–host interaction during the infection process should be considered vaccine candidates against the GBS infection. Further investigation and identification of these proteins may be of value for determining the role played by the proteins in GBS infections. Because protective antibodies, as we have shown, are transferred transplacentally, a vaccine intended for protection against neonatal disease should be administered to pregnant women or teenage girls. An active immunization of mothers during the third trimester of pregnancy to elicit an antibody response and passively immunize the newborns represents an attractive strategy to protect the neonates from GBS infection.
Conclusions The preliminary results indicated that some S. agalactiae immunogenic proteins that elicit protective immunity hold promise not only as components in a vaccine as an antigen but also as carriers or adjuvants in polysaccharide conjugate vaccines; however, more studies are needed.
Acknowledgements The study was supported by a grant from the Polish Ministry of Research and Higher Education no NN401042 337. The study was approved by Jagiellonian University Bioethical Committee decisions no. KBET/143/B/2007. The authors declare that they have no competing interests.
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Kolberg J, Hammerschmidt S, Frank R, Jonak J, Sanderova H & Aase A (2008) The surface–associated elongation factor Tu is concealed for antibody binding on viable pneumococci and meningococci. FEMS Immunol Med Microbiol 53: 222–230. Kowalska B, Niemiec T & Drejewicz H (2003) The incidence of hemolytic streptococcal colonization of the B group streptococcus of pregnant women and newborns estimated from screening patients Polyclinic and Department of Obstetrics and Gynecology, Institute of Mother and Child, a pilot study. Ginekol Pol 74: 1223–1227. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685. Larsson C, Stalhammar-Carlemalm M & Lindahl G (1996) Experimental vaccination against group B streptococcus, an encapsulated bacterium, with highly purified preparations of cell surface proteins Rib and alpha. Infect Immun 64: 3518–3523. Lin FY, Weisman LE, Azimi PH et al. (2004) Level of maternal IgG anti-Group B Streptococcus type III antibody correlated with protection of neonates against early-onset disease caused by this pathogen. JID 190: 928–934. Palmeiro JK, De Carvalho NS, Botelho AC, Fracalanzza SE, Madeira HM & Dalla-Costa LM (2011) Maternal group B streptococcal immunization: capsular polysaccharide (CPS)-based vaccines and their implications on prevention. Vaccine 29: 3729–3730. Pancholi V & Fischetti VA (1998) a-Enolase, a novel strong plasmin(ogen) binding protein on the surface of pathogenic streptococci. J Biol Chem 273: 14503– 14515. Papasergi S, Galbo R, Lanza-Cariccio V et al. (2013) Analysis of the Streptococcus agalactiae exoproteome. J Proteomics 14: 154–164. Poyart C, Tazi A, Reglier-Poupet H, Billo€et A, Tavares N, Raymond J & Trieu-Cuot P (2007) Multiplex PCR assay for rapid and accurate capsular typing of group B streptococci. J Clin Microbiol 45: 1985–1988.
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Rodriguez-Granger J, Alvargonzalez JC, Berardi A et al. (2012) Prevention of group B streptococcal neonatal disease revisited. The DEVANI European project. Eur J Clin Microbiol Infect Dis 31: 2097–2104. Santillan DA, Andracki ME & Hunter SK (2008) Protective immunization in mice against group B streptococci using encapsulated C5a peptidase. Am J Obstet Gynecol 198: 114 e1–114 e6. Schrag S, Gorwitz R & Fultz-Butts K (2002) Prevention of perinatal group B streptococcal disease. Revised guidelines from CDC. MMWR Recomm Rep 15: 1–22. Schuchat A (2000) Neonatal group B streptococcal disease: screening and prevention. N Engl J Med 343: 209–210. Severin A, Nickbarg E, Wooters J, Quazi SA, Matsuka YV, Murphy E, Moutsatsos IK, Zagursky RJ & Olmsted SB (2007) Proteomic analysis and identification of Streptococcus pyogenes surface-associated proteins. J Bacteriol 189: 1514–1522. Sharma P, Lata H, Ayra DK, Kashyap A, Pathak HK, Dua M, Ali A & Johri AK (2013) Role of pili proteins in adherence and invasion of Streptococcus agalactiae to the lung and cervical epithelial cells. J Biol Chem 288: 4023–4034. Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ & Klenk DC (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150: 76–85. Stalhammar-Carlemalm M, Stenberg L & Lindahl G (1993) Protein rib: a novel group B streptococcal cell surface protein that confers protective immunity and is expressed by most strains causing invasive infections. J Exp Med 177: 1593–1603. Strus M, Pawlik D, Brzychczy-Wloch M, Gosiewski T, Rytlewski K, Lauterbach R & Heczko PB (2009) Group B streptococcus (GBS) colonization of pregnant women and their children observed on obstetrical and neonatal wards of the University Hospital in Cracow, Poland. J Med Microbiol 58: 228–233. Sutcliffe J, Grebe T, Tait-Kamradt A & Wondrack L (1996) Detection of erythromycin-resistant determinants by PCR. Antimicrob Agents Chemother 40: 2562–2566.
FEMS Microbiol Lett 349 (2013) 61–70
Identification of high immunoreactive proteins from Streptococcus agalactiae isolates recognized by human serum antibodies Monika Brzychczy-Wloch1, Sabina Gorska2, Ewa Brzozowska2, Andrzej Gamian2, Piotr B. Heczko1 & Malgorzata Bulanda3 1
Department of Bacteriology, Microbial Ecology and Parasitology, Jagiellonian University Medical College, Krakow, Poland; 2Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland; and 3Department of Infection Epidemiology, Jagiellonian University Medical College, Krakow, Poland
Correspondence: Monika Brzychczy-Wloch, Department of Bacteriology, Microbial Ecology and Parasitology, Jagiellonian University Medical College, 18 Czysta Street, 31-121 Krakow, Poland. Tel.: +4812 633 25 67; fax: +4812 423 39 24; e-mail: [email protected] Received 28 May 2013; revised 30 July 2013; accepted 29 September 2013. Final version published online 24 October 2013.
MICROBIOLOGY LETTERS
DOI: 10.1111/1574-6968.12292 Editor: Akio Nakane Keywords Streptococcus agalactiae (GBS); immunogenic proteins; pregnant women; vaccination.
Abstract The aim of the studies was to identify immunogenic proteins of Streptococcus agalactiae (group B streptococcus; GBS) isolates. Investigation of the immunoreactivity with human sera allowed us to determine major immunogenic proteins which might be potential candidates for the development of vaccine. For the study, we have selected 60 genetically different, well-characterized GBS clinical isolates. The proteins immunoreactivity with 24 human sera from patients with GBS infections, carriers, and control group without GBS was detected by SDS-PAGE and Western blotting. As a result, some major immunogenic proteins were identified, of which four proteins with molecular masses of about 45 to 50 kDa, which exhibited the highest immunoreactivity features, were analyzed by LC-MS/MS. The proteins were identified by comparative analysis of peptides masses using MASCOT and statistical analysis. The results showed known molecules such as enolase (47.4 kDa), aldehyde dehydrogenase (50.6 kDa), and ones not previously described such as trigger factor (47 kDa) and elongation factor Tu (44 kDa). The preliminary results indicated that some GBS proteins that elicit protective immunity hold promise not only as components in a vaccine as antigens but also as carriers or adjuvants in polysaccharide conjugate vaccines, but more studies are needed.
Introduction Streptococcus agalactiae (group B streptococcus, GBS) is one of the most common causes of life-threatening bacterial infections in infants, which is associated with high mortality, and is also an emerging pathogen among adult humans, especially in the elderly, immunocompromised individuals and diabetic adults. In recent years, both in Europe and in the Polish clinical centers, there has been a significant increase in the disease rate caused by GBS (de la Rosa Fraile et al., 2001; Kowalska et al., 2003; Colbur & Gilbert, 2007; Strus et al., 2009; RodriguezGranger et al., 2012). Moreover, more and more frequently, S. agalactiae exhibits resistance to macrolides, lincosamides, and streptogramins B, which significantly reduces the effectiveness of antibiotics (Gherardi et al., FEMS Microbiol Lett 349 (2013) 61–70
2007; Brzychczy-Wloch et al., 2010). The presence of GBS in the maternal genital or gastrointestinal tract causes neonatal infection in most cases (Schuchat, 2000; Schrag et al., 2002). In Europe, the rate of colonization with group B streptococcus in pregnant women ranges from 6.6% in Greece to 36% in Denmark. In Poland, depending on the area and the methodology used, the rate of GBS-colonized pregnant women reaches 30% (BrzychczyWloch et al., 2012). It was shown that the risk of GBS transmission from mother to child reaches 70%, and the incidence of infections is 2–4 per 1000 live births. In newborns, streptococcus may cause the so-called earlyonset disease – EOD, which develops in the first week of life, or late-onset disease – LOD, developing between the 7th and the 90th day of life. GBS infections usually take the form of pneumonia and sepsis (80% of cases) and ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
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meningitis (10%), which is associated with high mortality among infants (Schuchat, 2000; Schrag et al., 2002; Johri et al., 2006). So far, ten GBS serotypes have been described (Ia, Ib, II-IX), distinguished on the basis of the differences in the structure of surface polysaccharides, also called capsular polysaccharides (CPS). In the United States and in Europe, serotypes Ia, III, and V are dominant, while in Asia the most common are serotypes VI and VIII (Poyart et al., 2007; Rodriguez-Granger et al., 2012). In Poland, serotypes Ia, III, and V represent over 70% of all the GBS strains from pregnant carriers and from newborns (Brzychczy-Wloch et al., 2012). Given the partial efficacy of antibiotic prophylaxis against S. agalactiae infections in neonates and increasing infection rates among the elderly or immunocompromised, as well as problems with growing resistance of GBS strains to antibiotics, the best long-term solution to prevent infections caused by group B streptococci would be constituted by vaccination (Baker & Edwards, 2003; Lin et al., 2004; Johri et al., 2006; He et al., 2010; Heath, 2011). Research on effective immunization against S. agalactiae infections has lasted from the time when in the 1970s Professor Rebecca Lancefield demonstrated in a mouse model that protective antibodies directed against the capsular polysaccharides of group B streptococci are able to prevent from the occurrence of invasive GBS infections; however, we still lack an effective vaccine (Baker & Edwards, 2003; Johri et al., 2006; Heath, 2011). It was shown that a natural and effective factor in protecting infants from infection is provided by IgG class antibodies, directed against GBS surface antigens, transferred with mother’s blood through the placenta (Lin et al., 2004; Palmeiro et al., 2011). In recent years, in the United States, several polyvalent vaccines based on surface polysaccharides of S. agalactiae were developed (Edwards et al., 2012). They are currently the subject of clinical trials, but it is already known that due to the strong variation of CPS, such a vaccine must contain at least a few different types of S. agalactiae, or various isolated polysaccharides representing different serotypes most prevalent in a given geographical area (Baker & Edwards, 2003; Johri et al., 2006; Heath, 2011). Because the capsule-based vaccines showed low immunogenicity, interest has shifted toward S. agalactiae proteins as vaccine antigens. The most interesting as regards designing highly efficacious synthetic vaccines are protective epitopes of surface proteins (Berzofsky et al., 2001; Johri et al., 2006; He et al., 2010). The first identified surface protein antigen of GBS was the C antigen composed of two protein components: a protein (alpha C protein, ACP) and b protein. The best potential candidates for vaccines are the members of the Alp family, such as a protein, Rib, Alp3, or Alp2, and ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
these have so far been extensively studied (StalhammarCarlemalm et al., 1993; Baker & Edwards, 2003; Johri et al., 2006; Heath, 2011). In a mouse model, Rib and a protein would protect against the majority of S. agalactiae infections, but any surface antigen that is a target for protective immunity in natural infections may exhibit antigenic variation, necessitating the development of multivalent vaccines (Larsson et al., 1996). Other important proteins of interest for vaccine development are Sip protein, which is highly conserved surface protein, and surface-localized enzyme C5a peptidase (Brodeur et al., 2000; Santillan et al., 2008). The list of GBS surface proteins is long and fully valuable, as a vaccine giving complete protection may be composed of two or, even, a few immunogenic proteins (Johri et al., 2006). The aim of the studies was to identify immunogenic proteins of well-characterized Streptococcus agalactiae (group B streptococcus, GBS) strains isolated from pregnant women and newborns and to investigate their immunoreactivity with human sera (cord blood serum, serum from patients with GBS infections, carriers and from control group without GBS) to determine major proteins which might be potential candidates for carriers in the development of vaccine against S. agalactiae infections.
Materials and methods Study population and specimen collection
Streptococcus agalactiae strains and serum samples came from the Jagiellonian University, Medical College’s Department of Bacteriology, Microbial Ecology and Parasitology’s own collection, which was being gathered from 2006 until 2010 within the framework of two projects financed by the Polish Ministry of Research and Higher Education and no. 3PO5E08425 and NN401042337. The 60 selected GBS strains represented different groups of patients (pregnant and nonpregnant women, newborns), different clinical status (infection, carrier state), various clinical materials (blood, vagina and anus swabs, newborn ear swab, urine), different serotypes (Ia, Ib, II-V), various genes encoding proteins of the Alp family (bca, alp 2, alp 3, epsilon, rib), different macrolide resistance phenotypes (cMLSB, iMLSB, and M), various sequence types (ST) and also different regions in Poland (Table 1). The reference GBS strains 2134 (Deutsche Sammlung von Mikroorganismen und Zellkulturen, DSMZ), 12403, and BAA-611 (American Type Culture Collection, ATCC) were used. Serum samples
Twenty-four serum samples were used. Group I – tested sera – TS (n = 16); serum samples collected in the 3rd FEMS Microbiol Lett 349 (2013) 61–70
Clinical number
7/P/2a 25/P/1a 306735 CM169 5279/08 5303/08 13445/07 11277/08 13695/08 2337/08 5338/08 9/0/2a 13608/08 D121 2107/08 CM184 305245 10/P/3a 23/P/3a CM47 13073/08 9353/08 13640/07 14041/07 14191/07 2341/08 D120 D126 20/0/3a 28/0/3a 5886/09 3634/08 13723/07 L1-181 4504/08 D136 W2/18 CM173 CM176 CM185
GBS no.
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 29 30 31 32 33 34 35 36 37 38 39 40
Ia Ia Ia Ia Ia Ia Ia Ia Ia Ia Ia Ib Ib Ib Ib Ib II II II II II II II II II II II II III III III III III III III III III III III III
Serotypes
Resistance phenotype; gene – – – – – – – – – – – – – – – – – – M; mefA/E cMLSB; ermB – – – – – – M; mefA/E – – – – – – – – – – cMLSB; ermB – iMLSB
Alp genes epsilon epsilon epsilon bca epsilon epsilon epsilon rib epsilon epsilon epsilon bca bca epsilon bca bca bca rib rib rib bca rib bca rib bca rib epsilon epsilon alp2 rib rib rib rib rib rib epsilon alp2 bca rib rib nt nt ST-23 ST-220 nt nt nt nt nt nt nt nt nt nt nt nt ST-106 nt nt ST-19 ST-12 nt ST-10 nt nt nt nt nt nt nt ST-17 ST-17 ST-358 ST-17 ST-19 nt ST-23 ST-22 ST-220 ST-410
Sequence type (ST) Vaginal swab from pregnant Vaginal swab from pregnant Blood of newborn Blood of newborn Newborn urine Newborn urine Swab from the newborn mouth Newborn ear swab Newborn urine Swab from the newborn mouth Swab from the newborn mouth Pregnant anal swab Newborn urine Cervical swab from nonpregnant Swab from the newborn mouth Vaginal swab from pregnant Blood of newborn Vaginal swab from pregnant Vaginal swab from pregnant Blood of newborn Blood of newborn Newborn urine Content bronchial newborn Swab from the newborn mouth Newborn ear swab Swab from the newborn mouth Vaginal swab from nonpregnant Vaginal swab from nonpregnant Vaginal swab from pregnant Vaginal swab from pregnant Blood of newborn Blood of newborn Newborn ear swab Blood of newborn Blood of newborn Vaginal swab from nonpregnant Blood of newborn Swab from the newborn mouth Swab from the newborn mouth Newborn ear swab
Clinical material
Table 1. Characterization of 60 genetically different, well-characterized GBS clinical isolates, chosen to identify their surface proteins
Carrier Carrier Newborn sepsis (EOD) Newborn sepsis (EOD) Bacteriuria (≥ 105 CFU mL Bacteriuria (≥ 105 CFU mL Colonization Colonization Bacteriuria (≥ 105 CFU mL Colonization Colonization Carrier Bacteriuria (≥ 105 CFU mL Inflammation Colonization Carrier Newborn sepsis (EOD) Carrier Carrier Newborn sepsis (EOD)* Newborn sepsis (EOD) Bacteriuria (≥ 105 CFU mL Pneumonia Colonization Colonization Colonization Inflammation Inflammation Carrier Carrier Newborn sepsis (EOD) Newborn sepsis (EOD) Colonization Newborn sepsis (EOD) Newborn sepsis (EOD) Inflammation Newborn sepsis (EOD) Newborn sepsis (EOD)* Newborn sepsis (EOD)* Newborn sepsis (EOD)
Clinical signs
) )
FEMS Microbiol Lett 349 (2013) 61–70
)
1
)
1
)
1
1
1
Southern Poland Southern Poland Northern Poland Southern Poland Western Poland Western Poland Western Poland Western Poland Western Poland Western Poland Western Poland Southern Poland Western Poland Southern Poland Western Poland Southern Poland Northern Poland Southern Poland Southern Poland Southern Poland Western Poland Western Poland Western Poland Western Poland Western Poland Western Poland Southern Poland Southern Poland Southern Poland Southern Poland Western Poland Western Poland Western Poland Central Poland Western Poland Southern Poland Central Poland Southern Poland Southern Poland Southern Poland
Geographical area
Immunogenic GBS Proteins
63
ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
CM28 3A-012 CM49 306723 CM87 CM3 3/P/2a 42/P/3a 14/P/3a S1/4 D156 3514/08 14030/08 1716/08 1736/08 2992/08 13793/08 12403 2134 BAA-611
41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
III III III III III IV V V V V V V V V V V V III II V
Serotypes
Resistance phenotype; gene – – – – – – cMLSB; ermB – cMLSB; ermB – – – M; mefA/E – cMLSB; ermB – cMLSB; ermB – – –
Alp genes rib rib rib alp2 rib epsilon alp3 alp2 alp3 alp3 epsilon rib rib alp2 alp2 rib alp3 alp2 rib rib ST-286 ST-447 ST-148 ST-23 ST-19 nt nt nt Nt ST-1 nt ST-220 nt nt nt nt nt nt nt nt
Sequence type (ST) Blood of newborn Vaginal swab from pregnant Vaginal swab from pregnant Newborn urine Newborn ear swab Vaginal swab from pregnant Vaginal swab from pregnant Vaginal swab from pregnant Vaginal swab from pregnant Blood of newborn Vaginal swab Blood of newborn Blood of newborn Newborn urine Newborn urine Newborn urine Newborn urine GBS standard (ATCC) GBS standard (DSMZ) GBS standard (ATCC)
Clinical material
Newborn sepsis (EOD) Carrier Carrier Bacteriuria (≥ 105 CFU mL Colonization Carrier Carrier Carrier Carrier Newborn sepsis (EOD)* Inflammation Newborn sepsis (EOD) Newborn sepsis (EOD) Bacteriuria (≥ 105 CFU mL Bacteriuria (≥ 105 CFU mL Bacteriuria (≥ 105 CFU mL Bacteriuria (≥ 105 CFU mL Fetal septicemia No data Clinical specimen
Clinical signs
1
) ) 1 ) 1 )
1
)
1
Southern Poland Southern Poland Southern Poland Northern Poland Southern Poland Southern Poland Southern Poland Southern Poland Southern Poland Northern Poland Southern Poland Western Poland Western Poland Western Poland Western Poland Western Poland Western Poland Germany USA Germany
Geographical area
*Death of newborn. cMLSB, constitutive resistant to macrolides, lincosamides, and streptogramin B encoded by ermB gene; iMLSB, inducible resistant to macrolides, lincosamides, and streptogramin B; M, resistant to macrolides, but not lincosamides or streptogramin B, conferred by mefA/E gene; EOD, early-onset diseases; nt, not tested.
Clinical number
GBS no.
Table 1. Continued
64 M. Brzychczy-Wloch et al.
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Immunogenic GBS Proteins
trimester of pregnancy from pregnant women colonized with group B streptococcus (n = 10); serum from umbilical cord blood from pregnant women colonized with group B streptococcus (n = 1); serum samples from the studied women during puerperium (n = 3) collected 8 weeks after labor; serum from neonates with GBS EOS sepsis (n = 2). Group II – control sera – CS (n = 8); serum samples collected in the 3rd trimester of pregnancy from pregnant women without GBS colonization, which was confirmed throughout three trimesters of their pregnancy. Molecular characterization of the GBS strains
Most of the tested strains were carefully characterized in the previous study (Brzychczy-Wloch et al., 2010, 2012). The detection of genes encoding capsular polysaccharides Ia, Ib, II-VIII was investigated using the multiplex PCR method with specific primers (Genomed) as previously described (Poyart et al., 2007). To detect the surface protein genes alp2, alp3, alp4, bca, epsilon, and rib, multiplex PCR was used with specific primers (Genomed), according to the procedure proposed by Creti et al. (2004) and Gherardi et al. (2007). Macrolide resistance phenotypes cMLSB (constitutive), iMLSB (inducible), and M were determined by the double-disk test with erythromycin (15 lg) and clindamycin (2 lg; Oxoid). The erm(A), erm (B), erm(C), and mef(A/E) resistance determinants were detected by multiplex PCR with adequate four pairs of primers (Genomed) according to Sutcliffe et al. (1996). MLST analysis was performed as described by Jones et al. (2003) with the use of oligonucleotide primer pairs (Genomed) specific for the seven housekeeping loci selected for the GBS MLST. The online database (http://pubmlst. org/sagalactiae) was used for assigning alleles for seven loci, and each isolate was defined by the sequence type (ST). Protein isolation
Bacterial strains were cultured on brain-heart infusion broth (BHI, Biocorp) for 24 h at 37 °C. Bacteria from the plates were suspended in phosphate-buffered saline (PBS, Institute of Immunology and Experimental Therapy, PAN) to reach the final density of bacteria in the solution A600 = 1.0. After centrifuging (3312 g per min), the bacterial mass was suspended in Tris-HCl (Merck) buffer containing different concentrations of SDS (1–2%; Sigma-Aldrich) or directly in the buffer for electrophoresis according to Heilmann et al. (1996). The next steps were sonication (three times for 5 min) and centrifugation (13 248 g per min). Proteins were precipitated from the resulting supernatant using 3 volumes of cold 95% FEMS Microbiol Lett 349 (2013) 61–70
ethanol (POCH). After overnight incubation at 4 °C, the precipitated proteins were centrifuged (12 000 r.p.m. per min) and dissolved in water. Protein concentration was analyzed using the BCA method (Smith et al., 1985). SDS-PAGE and immunoblotting
Samples (10 lg of protein) were applied to SDS-PAGE gels using 5% to 12.5% gels according to Laemmli (1970). After electrophoresis, proteins were stained with Coomassie Brilliant Blue (Serva) or transferred to a polyvinylidene difluoride membrane (Millipore) for immunoblotting. The membranes were blocked in PBS containing 1% of bovine serum albumin (BSA, KPL) for 1 h. After washing three times with PBS containing 0.25% Tween 20 (SigmaAldrich), PBS-T (Institute of Immunology and Experimental Therapy, PAN), the membranes were incubated in a selected human sera in 1% BSA for 2 h at 37 °C. We tested different dilutions of sera (from 1 : 150 to 1 : 15000 in doubly increasing amounts), but the most effective was 1 : 3000 (reproducible results, without the Hook effect). Next, the membranes were washed three times with PBS-T. After washing, the membranes were incubated for 1 h in alkaline phosphatase-conjugated goat-anti-human IgG antibodies (Sigma) diluted 1 : 5000. Finally, the membranes were washed as described above, and solution containing nitroblue tetrazolium (NBT, Roth), 5-bromo4-chloro-3-indolyl phosphate (BCIP, Roth), and MgCl2 (POCh) was added for 5 s to visualize reaction. Protein identification
Immunogenic proteins were separated and purified by preparative electrophoresis in denaturing condition (SDSPAGE) using Prep-Cell apparatus (Model 491 Bio-Rad). Bands of interest were cut out and digested using a proteolytic enzyme such as trypsin (Roche) to obtain a mixture of peptides. Then, the peptides were separated by liquid chromatography (LC), and mass fragments were measured using mass spectrometer LC-MS/MS Orbitrap (Thermo). Proteins were identified by comparative analysis of peptides masses (NCBI, UniProt databases) using MASCOT (http://www.matrixscience.com/) and statistical analysis.
Results Immunogenic proteins were shown to be present in all 60 tested GBS strains. The protein concentration in the studied extracts was significantly different and varied, ranging from 0.2 to 2.7 mg mL 1 with an average value equal to 0.749 mg mL 1. We observed some significant variations in an average amount of proteins concentration ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
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in relation to different GBS serotypes. We demonstrated that strains with V serotype produced the highest concentration of the proteins with an average amount equal 1.18 1.12 mg mL 1 of extract, while Ia serotype had the lowest concentration of the protein with an average amount equal 0.5 0.29 mg mL 1. For the other serotypes, the following values were obtained: Ib (0.51 0.31), II (0.86 0.95), and III (0.62 0.57). Samples were subjected to SDS-PAGE, and protein bands were visualized with Coomassie Brilliant Blue. The representative results are shown in Fig. 1. It could be demonstrated that protein samples of all serotypes share some predominant bands, but it was also shown that despite common protein bands, the protein pattern varies between different serotypes. For the analysis of immunoreactivity of serum samples, protein samples were transferred to a polyvinylidene difluoride membrane for immunoblotting. The immunoreactivity of tested sera (TS) from women with GBS carriers was comparable to that of cord blood serum and sera from women during puerperium and from infants with GBS infection with respect to the number and intensity of bands. The banding patterns were heterogeneous and support the previous statement that pattern of proteins varies between serotypes. However, it was observed that in most cases, among the 60 S. agalactiae isolates, the proteins with molecular mass of about 45–50 kDa are most immunoreactive with umbilical cord blood (Fig. 2). The antigenic properties were observed also for proteins with molecular mass of about 40, 60, and 90 kDa. Interestingly, we observed that antibodies of serum from a newborn can recognize the protein antigens of S. agalactiae species on the same level as mother’s serum (Fig. 3) and also that the level of specific anti-GBS proteins antibodies increases significantly from third trimester to puerperium (Fig. 4). The immunoreactivity of the control sera (CS) from healthy pregnant women without GBS
(a)
colonization was also investigated. There was no reaction (data not shown). To identify the most immunogenic proteins, electrophoretic preparation of isolated proteins from S. agalactiae strain no. 57 (V serotype) was made using Prep-Cell Apparatus (491 model Bio-Rad). Four proteins with molecular masses of about 45 to 50 kDa showing immunoreactivity features were cut from the gel and analyzed in LC-MS/MS (Fig. 5). Proteins were identified by comparative analysis of peptides masses (NCBI, UniProt databases) using MASCOT and statistical analysis. Trigger factor with molecular mass of 47 kDa (89% protein sequence coverage), enolase with molecular mass of 47.4 kDa (81% protein sequence coverage), elongation factor Tu with molecular mass of 44 kDa (76% protein sequence coverage), and aldehyde dehydrogenase with molecular mass of 50.6 kDa (71% protein sequence coverage) were identified.
Discussion Group B streptococci, despite the introduction of adequate perinatal prophylaxis in the United States and many European countries, still constitute the main cause of neonatal morbidity and mortality in developed countries (Schuchat, 2000; de la Rosa Fraile et al., 2001; Schrag et al., 2002; Rodriguez-Granger et al., 2012). Major efforts are therefore under way to find new methods to prevent and treat infections caused by these pathogens, to analyze pathogenic mechanisms, and to develop efficient vaccines. A substantial difficulty in developing group B streptococcal vaccines is the existence of a multiplicity of serotypes with different geographical distributions. A vaccine suitable for Asian or European populations may not be appropriate for African populations (Johri et al., 2006; Poyart et al., 2007; Rodriguez-Granger et al., 2012). The use of GBS preventive vaccination would
(b)
Fig. 1. SDS-PAGE profile of proteins isolated from selected Streptococcus agalactiae strains. Lane: STD – molecular mass marker (GE Healthcare); 1 – 2337/08; 2 – 5338/08; 3 – 9/0/2a; 4 – 13608/08; 5 – D121; 6 – 2107/08; 7 – CM184; 8 – 305245; 9 – 10/P/3a; 10 – 23/P/3a; 11 – CM47; 12 – 13073/08; 13 – 9353/08; 14 – 13640/07; 15 – 14041/07; 16 – 14191/07; 17 – 2341/08; 18 – D120.
ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
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Immunogenic GBS Proteins
(a)
(b)
Fig. 2. (a) SDS-PAGE profile of proteins extracted from chosen Streptococcus agalactiae strains (b) Immunoblotting of proteins extracted from chosen Streptococcus agalactiae strains with cord blood serum (TS/1b). Lane: STD – molecular mass marker (GE Healthcare); 1 – 13073/08; 2 – 3A-012; 3 – 3/P/2a; 4 – 1716/08; 5 – 1736/08; 6 – 2992/08; 7 – 13793/08.
(a)
(b)
Fig. 3. Immunoblotting of proteins extracted from chosen Streptococcus agalactiae strains with (a) serum (TS/4a) from a GBS-carrying mother and (b) serum (TS/4b) from her baby with GBS sepsis. Lane: 1 – CM185; 2 – CM28; 3 – 3A-012; 4 – CM49; 5 – 306723; 6 – CM87; 7 – CM3; 8 – 3/P/2a; 9 – 42/P/3a.
(a)
(b)
Fig. 4. Immunoblotting of proteins from chosen Streptococcus agalactiae strains with (a) serum from a GBS-carrying woman in third trimester (TS/2a) and (b) serum from the same woman during puerperium (TS/2b). Lane: 1 – CM3; 2 – 3/P/2a; 3 – 42/P/3a; 4 – 14/P/3a; 5 – S1/4; 6 – D156; 7 – 3514/08; 8 – 14030/08; 9 – 1716/08.
make it possible to eliminate the problem of carriage in the population of pregnant women, reduce the number of infections in both the early- and late-onset neonatal group, as well as among the elderly and the immunocompromised; it would additionally limit the use of antibiotics and the problems of increasing resistance of bacteria, and finally, preventive vaccination would eliminate the side effects of antibiotic treatment, such as allergy and FEMS Microbiol Lett 349 (2013) 61–70
anaphylaxis (Baker & Edwards, 2003; Johri et al., 2006; He et al., 2010; Heath, 2011). A number of candidates, including capsular polysaccharides and proteins, have the potential to be a component of new vaccines. The capsular polysaccharides (CPS) from all ten currently identified GBS serotypes were found to elicit serotype-specific protective immunity in animal models, but showed low immunogenicity if not conjugated to a ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
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Fig. 5. SDS-PAGE profile of a separated immunoreactive protein isolated from Streptococcus agalactiae 13793/08 using Prep-Cell apparatus.
protein carrier. It is noteworthy that memory B cells are not produced in response to most polysaccharide vaccines, therefore lacking the ability of inducing a booster response. Furthermore, plain polysaccharide vaccines are not generally immunogenic in infants, and their use is not allowed as vaccines to prevent disease in children caused by polysaccharide-encapsulated bacteria that have their highest incidence in the first year of life (Johri et al., 2006; Palmeiro et al., 2011). Therefore, a more recent approach to developing GBS vaccines is to use proteins (Fluegge et al., 2004; Santillan et al., 2008; Papasergi et al., 2013). The search for GBS immunogenic proteins allowed the discovery of many proteins that induced protective antibodies. Purified GBS proteins could be used as effective carriers for conjugate CPS vaccines while simultaneously inducing protective immunity against GBS (Johri et al., 2006). In this study, we were able to identify some major proteins of S. agalactiae that are immunoreactive and may suggest that they could play a potential role in the development of vaccine against GBS infections. Although the protein patterns observed for the 60 different GBS isolates studied showed many similarities, some differences between serotypes and source of clinical materials were observed. We found four immunoreactive proteins present in all 60 strains tested: enolase, dehydrogenase, trigger ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
M. Brzychczy-Wloch et al.
factor, and elongation factor Tu. All of these proteins are cytoplasmic protein and belong to cell surface-associated or surface-exposed proteins (Pancholi & Fischetti, 1998; Severin et al., 2007; Kolberg et al., 2008; Feng et al., 2009; Sharma et al., 2013). Surface enolase and dehydrogenase have been described as multifunctional proteins of streptococcal bacteria (Pancholi & Fischetti, 1998). Enolase, with its plasminogen binding activity, is involved in invasion and adherence of S. pyogenes and S. pneumonia to human pharyngeal cells (Severin et al., 2007). Streptococcal surface dehydrogenase is also a virulence factor. Structurally and functionally, it is related to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). It can bind to mammalian fibronectin or cytoskeletal proteins such as myosin and possesses ADP-ribosylating activities (Pancholi & Fischetti, 1998). It was shown that streptococcal surface-associated dehydrogenase recognizes pharyngeal plasminogen activator receptor, which contributes to bacterial adherence and plays a crucial role in pathogenesis (Severin et al., 2007). The elongation factor Tu has been found on S. pyogenes surface, and its possible function is bacteria attachment to mammalian cells. Elongation factor Tu is also present in cytoplasmic fraction of bacterial cells. It is one of the most abundant proteins in prokaryotes, representing even 10% of the total amount of proteins in Escherichia coli (Kolberg et al., 2008). High cytoplasmic content of elongation factor raises the question whether detection of this protein in bacterial membrane preparations might represent a contamination by cytosolic one during their preparation. It could be true, but this does not change the fact that the epitope localized on this protein is highly immunoreactive and is recognized by naturally produced antibodies. The presence of antibodies indicates that this protein had to be exposed to the outside of bacteria cell during the infection process. Trigger factor is ribosome-associated chaperone with a peptidyl-prolyl cis-trans isomerase activity. The protein is important for SpeB cysteine proteins secreting. Trigger factor has been detected in cytoplasm and on the S. pyogenes surface (Severin et al., 2007). The most frequent concept of immunoreactive proteins identification is based on standard biochemical and microbiological methods such as animal immunization using cell surface components and then immune testing. In our study, we used natural sera containing originally produced antibodies after streptococcal infection, and it was not an artificially induced reaction. It must be emphasized, because during infection process certain changes in the surface proteins expression occur and, consequently, the profile of proteins exposed on bacterial surface is different than the profile of the surface proteins extracted from bacterial cell after cultivation. It was shown that glyceraldehyde-3-phosphate dehydrogenase FEMS Microbiol Lett 349 (2013) 61–70
Immunogenic GBS Proteins
(GAPDH), which is involved in host–cell interaction, is expressed only during infection. It is suggested that differential upregulation of all GBS virulence genes occurs (Sharma et al., 2013). Results of our study show that human immune response to streptococcal pathogens is not directed against the most abundant cell surface proteins such as M protein or C5a but also against other proteins which seem to be better immunoreactants (Severin et al., 2007; Santillan et al., 2008). In our study, we used natural human model of immunization during bacterial infection, and we showed which protein antigens of streptococci are involved in immune response. So far, vaccine candidates have been constituted by capsular polysaccharides and surface proteins such as C protein, whereas our study suggests that proteins involved in bacterial cell–host interaction during the infection process should be considered vaccine candidates against the GBS infection. Further investigation and identification of these proteins may be of value for determining the role played by the proteins in GBS infections. Because protective antibodies, as we have shown, are transferred transplacentally, a vaccine intended for protection against neonatal disease should be administered to pregnant women or teenage girls. An active immunization of mothers during the third trimester of pregnancy to elicit an antibody response and passively immunize the newborns represents an attractive strategy to protect the neonates from GBS infection.
Conclusions The preliminary results indicated that some S. agalactiae immunogenic proteins that elicit protective immunity hold promise not only as components in a vaccine as an antigen but also as carriers or adjuvants in polysaccharide conjugate vaccines; however, more studies are needed.
Acknowledgements The study was supported by a grant from the Polish Ministry of Research and Higher Education no NN401042 337. The study was approved by Jagiellonian University Bioethical Committee decisions no. KBET/143/B/2007. The authors declare that they have no competing interests.
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