Streptococcus pneumoniae capsular polysaccharide is linked to peptidoglycan via a direct glycosidic bond to β-D-N-acetylglucosamine.

Proc Natl Acad Sci U S A. 2017 May 30;114(22):5695-5700. doi: 10.1073/pnas.1620431114. Epub 2017 May 11.

Streptococcus pneumoniae capsular polysaccharide is linked to peptidoglycan via a direct glycosidic bond to β-D-N-acetylglucosamine.

Larson TR1, Yother J2.

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Abstract

For many bacteria, including those important in pathogenesis, expression of a surface-localized capsular polysaccharide (CPS) can be critical for survival in host environments. In Gram-positive bacteria, CPS linkage is to either the cytoplasmic membrane or the cell wall. Despite the frequent occurrence and essentiality of these polymers, the exact nature of the cell wall linkage has not been described in any bacterial species. Using the Streptococcus pneumoniae serotype 2 CPS, which is synthesized by the widespread Wzy mechanism, we found that linkage occurs via the reducing end glucose of CPS and the β-D-N-acetylglucosamine (GlcNAc) residues of peptidoglycan (PG). Hydrofluoric acid resistance, 31P-NMR, and 32P labeling demonstrated the lack of phosphodiester bonds, which typically occur in PG-polysaccharide linkages. Component sugar analysis of purified CPS-PG identified only CPS and PG sugars in the appropriate ratios, suggesting the absence of an oligosaccharide linker. Time of flight mass spectrometry confirmed a direct glycosidic linkage between CPS and PG and showed that a single CPS repeat unit can be transferred to PG. The linkage was acetolysis susceptible, indicative of a 1,6 glycosidic bond between CPS and the GlcNAc C-6. The acetylation state of GlcNAc did not affect linkage. A direct glycosidic linkage to PG was also demonstrated for serotypes 8 and 31, whose reducing end sugars are glucose and galactose, respectively. These results provide the most detailed descriptions of CPS-PG linkages for any microorganism. Identification of the linkage is a first step toward identifying the linking enzyme and potential inhibitors of its activity.

KEYWORDS:

Gram positive; Wzy; capsule; glycobiology; pneumococcus

PMID: 28495967 PMCID: PMC5465879 [Available on 2017-11-30] DOI: 10.1073/pnas.1620431114

Genome Evolution to Penicillin Resistance in Serotype 3 Streptococcus pneumoniae by Capsular Switching.

Antimicrob Agents Chemother. 2017 Jun 19. pii: AAC.00478-17. doi: 10.1128/AAC.00478-17. [Epub ahead of print]

Genome Evolution to Penicillin Resistance in Serotype 3 Streptococcus pneumoniae by Capsular Switching.

Chiba N1,2, Murayama SY3, Morozumi M1, Iwata S1, Ubukata K4.

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Abstract

Streptococcus pneumoniae of serotype 3 was collected from cases of invasive pneumococcal disease (n = 124) throughout Japan from April 2010 to March 2013. A penicillin-resistant S. pneumoniae (PRSP) isolate from an adult patient, KK0981 of serotype 3, was identified among these strains. Whole-genome analysis characterized this PRSP as a recombinant strain derived from PRSP of serotype 23F with the cps-locus (20.3 kb) replaced by that of a penicillin-susceptible strain of serotype 3.

Copyright © 2017 American Society for Microbiology.

PMID: 28630198 DOI: 10.1128/AAC.00478-17

High-throughput CRISPRi phenotyping identifies new essential genes in Streptococcus pneumoniae.

Mol Syst Biol. 2017 May 10;13(5):931. doi: 10.15252/msb.20167449.

High-throughput CRISPRi phenotyping identifies new essential genes in Streptococcus pneumoniae.

Liu X1,2, Gallay C1, Kjos M1,3, Domenech A1, Slager J1, van Kessel SP1, Knoops K4, Sorg RA1, Zhang JR2, Veening JW5,6.

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Abstract

Genome-wide screens have discovered a large set of essential genes in the opportunistic human pathogen Streptococcus pneumoniae However, the functions of many essential genes are still unknown, hampering vaccine development and drug discovery. Based on results from transposon sequencing (Tn-seq), we refined the list of essential genes in S. pneumoniae serotype 2 strain D39. Next, we created a knockdown library targeting 348 potentially essential genes by CRISPR interference (CRISPRi) and show a growth phenotype for 254 of them (73%). Using high-content microscopy screening, we searched for essential genes of unknown function with clear phenotypes in cell morphology upon CRISPRi-based depletion. We show that SPD_1416 and SPD_1417 (renamed to MurT and GatD, respectively) are essential for peptidoglycan synthesis, and that SPD_1198 and SPD_1197 (renamed to TarP and TarQ, respectively) are responsible for the polymerization of teichoic acid (TA) precursors. This knowledge enabled us to reconstruct the unique pneumococcal TA biosynthetic pathway. CRISPRi was also employed to unravel the role of the essential Clp-proteolytic system in regulation of competence development, and we show that ClpX is the essential ATPase responsible for ClpP-dependent repression of competence. The CRISPRi library provides a valuable tool for characterization of pneumococcal genes and pathways and revealed several promising antibiotic targets.

© 2017 The Authors. Published under the terms of the CC BY 4.0 license.

KEYWORDS:

DNA replication; bacterial cell wall; competence; gene essentiality; teichoic acid biosynthesis

PMID: 28490437 PMCID: PMC5448163

Tokai J Exp Clin Med. 2017 Apr 20;42(1):37-40.

Performance Evaluation of a Newly Developed and Fully Automated Bacteriological Analyzer "RAISUS ANY" for Antimicrobial Susceptibility Testing of Fastidious Bacteria Haemophilus influenzae and Streptococcus pneumoniae.

Ohshima T1, Nomiya S, Yamamoto Y, Miyazawa M, Ohsuga J, Hisada A, Iwawaki K, Asai S, Miyachi H.

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Abstract

OBJECTIVE:

Antimicrobial susceptibility testing for fastidious bacteria, such as Haemophilus influenzae (H. influenzae) and Streptococcus pneumoniae (S. pneumoniae) has been performed manually. We evaluated the performance of a newly developed fully automated system for rapid bacterial identification and antimicrobial susceptibility testing "RAISUS ANY" (Nissui Pharmaceutical Co., Ltd.).

METHODS:

We evaluated the performance of "RAISUS ANY" for measurement of minimal inhibitory concentrations (MICs) of H. influenzae and S. pneumoniae, in comparison with the manual method (DP34, Eiken Chem. Co., Ltd.). The repeatability of MICs was studied using the reference strain of these bacteria, obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

RESULTS:

The comparison with the manual method for 35 and 36 clinical strains of H. influenzae and S. pneumonia showed 62.9-100% and 86.1-100% agreement, respectively. Five of 35 H. influenzae strains that showed a trailing effect were stably and accurately measured for MICs without a variation among the examiners.

CONCLUSION:

In conclusion, the automated system "RAISUS ANY" provided a reliable MICs data for H. influenzae and S. pneumonia, suggesting its improvement in performance and reliability for routine antimicrobial susceptibility testing in clinical bacteriological laboratories.

PMID: 28413870

[Indexed for MEDLINE]

Antimicrobial Resistant Streptococcus pneumoniae: Prevalence, Mechanisms, and Clinical Implications.

Am J Ther. 2017 May;24(3):e361-e369. doi: 10.1097/MJT.0000000000000551.

Antimicrobial Resistant Streptococcus pneumoniae: Prevalence, Mechanisms, and Clinical Implications.

Cherazard R1, Epstein M, Doan TL, Salim T, Bharti S, Smith MA.

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Abstract

BACKGROUND:

Streptococcus pneumoniae is a major cause of pneumonia, meningitis, sepsis, bacteremia, and otitis media. S. pneumoniae has developed increased resistance to multiple classes of antibiotics.

STUDY DESIGN:

Systematic literature review of prevalence, mechanisms, and clinical implications in S. pneumoniae resistance.

AREAS OF UNCERTAINTY:

Since S. pneumoniae resistance to penicillin was first reported with subsequent development of resistance to other classes of drugs, selection of appropriate antibiotic treatment is challenging.

DATA SOURCES:

We searched PubMed (English language) for citations to antibiotic resistance in S. pneumoniae published before March 1, 2016.

RESULTS:

We present a review of S. pneumoniae resistance to beta-lactams, macrolides, lincosamides, fluoroquinolones, tetracyclines, and trimethoprim-sulfamethoxazole (TMP-SMX). There has been a steady decline in susceptibility of S. pneumoniae to commonly used beta-lactams. Phenotypic expression of penicillin resistance occurs as a result of a genetic structural modification in penicillin-binding proteins. Between 20% and 40% of S. pneumoniae isolates are resistant to macrolides. Macrolide resistance mechanisms include ribosomal target site alteration, alteration in antibiotic transport, and modification of the antibiotic. Approximately 22% of S. pneumoniae isolates are resistant to clindamycin. Similar to macrolide resistance, clindamycin involves a target site alteration. The prevalence of fluoroquinolone resistance is low, although increasing. S. pneumoniae resistance to fluoroquinolones occurs by accumulated mutations within the bacterial genome, increased efflux, or acquisition of plasmid-encoded genes. S. pneumoniae resistance has also increased for the tetracyclines. The primary mechanism is mediated by 2 genes that confer ribosomal protection. The prevalence of TMP-SMX resistance is around 35%. As with fluoroquinolones, resistance to TMP-SMX is secondary to mutations in the bacterial genome.

CONCLUSIONS:

Effective treatment of resistant S. pneumoniae is a growing concern. New classes of drugs, newer formulations of older drugs, combination antibiotic therapy, nonantibiotic modalities, better oversight of antibiotic usage, and enhanced preventive measures hold promise.

PMID: 28430673 DOI: 10.1097/MJT.0000000000000551

TIGR4 strain causes more severe disease than WU2 strain in a mouse model of Streptococcus pneumoniae meningitis: a common pathogenic role for interferon-γ.

Microbes Infect. 2017 Apr 22. pii: S1286-4579(17)30062-X. doi: 10.1016/j.micinf.2017.04.002. [Epub ahead of print]

TIGR4 strain causes more severe disease than WU2 strain in a mouse model of Streptococcus pneumoniae meningitis: a common pathogenic role for interferon-γ.

Yau B1, Too LK1, Ball HJ1, Hunt NH2.

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Abstract

Streptococcus pneumoniae (S. pneumoniae) meningitis causes debilitating neurological symptoms and acute fatalities in patients, and long-term neurological sequelae in some survivors. Current vaccines do not protect against all 94 known S. pneumoniae capsular serotypes, many of which are capable of causing pneumococcal meningitis (PM). We here compare the pathogenic outcomes of two clinically virulent isolates of S. pneumoniae, serotype 3 strain WU2 and serotype 4 strain TIGR4, in a murine model of PM. At an identical infectious dosage of 103 CFU administered via the intracerebroventricular route, significantly greater mortality, interleukin (IL)1β and IL6 production, and blood-brain barrier dysfunction occurred in TIGR4-induced PM compared to PM caused by WU2. Higher bacterial counts in the cerebrospinal fluid and nitrite/nitrate in serum were observed 40 h post inoculation with TIGR4 compared to mice infected with WU2. Similar to our previous findings in WU2 PM, interferon-γ was an essential driver of the pathogenesis of TIGR4 PM, suggesting that this cytokine may be a common pathogenic agent across a range of pneumococcal meningitides and, thus, a potential therapeutic target for intervention.

Copyright © 2017. Published by Elsevier Masson SAS.

KEYWORDS:

Cytokines; Inflammation; Interferon-gamma; Pneumococcal meningitis; Serotype; Streptococcus pneumoniae; TIGR4; WU2

PMID: 28438705 DOI: 10.1016/j.micinf.2017.04.002

Role of neural barriers in the pathogenesis and outcome of Streptococcus pneumoniae meningitis.

Exp Ther Med. 2017 Mar;13(3):799-809. doi: 10.3892/etm.2017.4082. Epub 2017 Jan 24.

Role of neural barriers in the pathogenesis and outcome of Streptococcus pneumoniae meningitis.

Prager O1,2, Friedman A1,2,3, Nebenzahl YM4.

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Abstract

Bacterial meningitis is an inflammatory disease of the meninges of the central nervous system (CNS). Streptococcus pneumoniae (S. pneumoniae), Neisseria meningitidis, and Haemophilus influenzae are the major bacterial pathogens causing meningitis with S. pneumoniae being responsible for two thirds of meningitis cases in the developed world. To reach the CNS following nasopharyngeal colonization and bacteraemia, the bacteria traverse from the circulation across the blood brain barrier (BBB) and choroid plexus. While the BBB has a protective role in healthy individuals by shielding the CNS from neurotoxic substances circulating in the blood and maintaining the homeostasis within the brain environment, dysfunction of the BBB is associated with the pathophysiology of numerous neurologic disorders, including bacterial meningitis. Inflammatory processes, including release of a broad range of cytokines and free radicals, further increase vascular permeability and contribute to the excessive neural damage observed. Injury to the cerebral microvasculature and loss of blood flow auto-regulation promote increased intracranial pressure and may lead to vascular occlusion. Other common complications commonly associated with meningitis include abnormal neuronal hyper-excitability (e.g., seizures) and loss of hearing. Despite the existence of antibiotic treatment and adjuvant therapy, the relatively high mortality rate and the severe outcomes among survivors of pneumococcal meningitis in developing and developed countries increase the urgency in the requirement of discovering novel biomarkers for the early diagnosis as well as novel treatment approaches. The present review aimed to explore the changes in the brain vascular barriers, which allow S. pneumoniae to invade the CNS, and describe the resultant brain injuries following bacterial meningitis.

KEYWORDS:

S. pneumoniae; blood brain barrier; central nervous system; inflammation; innate immunity; meningitis; pathogenesis; virulence factors

PMID: 28450902 PMCID: PMC5403536 DOI: 10.3892/etm.2017.4082

The accessory Sec system (SecY2A2) in Streptococcus pneumoniae is involved in export of pneumolysin toxin, adhesion and biofilm formation.

Microbes Infect. 2017 Apr 26. pii: S1286-4579(17)30064-3. doi: 10.1016/j.micinf.2017.04.003. [Epub ahead of print]

The accessory Sec system (SecY2A2) in Streptococcus pneumoniae is involved in export of pneumolysin toxin, adhesion and biofilm formation.

Bandara M1, Skehel JM2, Kadioglu A3, Collinson I4, Nobbs AH5, Blocker AJ6, Jenkinson HF7.

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Abstract

In Streptococcus pneumoniae TIGR4, genes encoding a SecY2A2 accessory Sec system are present within a locus encoding a serine-rich repeat surface protein PsrP. Mutant strains deleted in secA2 or psrP were deficient in biofilm formation, while the ΔsecA2 mutant was reduced in binding to airway epithelial cells. Cell wall protein (CWP) fractions from the ΔsecA2 mutant, but not from the ΔpsrP mutant, were reduced in haemolytic (pneumolysin) activity. Contact-dependent pneumolysin (Ply) activity of wild type TIGR4 cells was ten-fold greater than that of ΔsecA2 mutant cells suggesting that Ply was not active at the ΔsecA2 cell surface. Ply protein was found to be present in the CWP fraction from the ΔsecA2 mutant, but showed aberrant electrophoretic migration indicative of protein modification. Proteomic analyses led to the discovery that the ΔsecA2 mutant CWP fraction was deficient in two glycosidases as well as other enzymes involved in carbohydrate metabolism. Taken collectively the results suggest that positioning of Ply into the cell wall compartment in active form, together with glycosyl hydrolases and adhesins, requires a functional accessory Sec system.

Copyright © 2017 The Author(s). Published by Elsevier Masson SAS.. All rights reserved.

KEYWORDS:

O-glycosidase; Streptococcus pneumoniae; biofilms; export; pneumolysin; secA2

PMID: 28456649 DOI: 10.1016/j.micinf.2017.04.003

Streptococcus pneumoniae-induced ototoxicity in organ of Corti explant cultures.

Hear Res. 2017 Apr 25;350:100-109. doi: 10.1016/j.heares.2017.04.012. [Epub ahead of print]

Streptococcus pneumoniae-induced ototoxicity in organ of Corti explant cultures.

Perny M1, Solyga M2, Grandgirard D3, Roccio M2, Leib SL4, Senn P5.

Author information

Abstract

Hearing loss remains the most common long-term complication of pneumococcal meningitis (PM) reported in up to 30% of survivors. Streptococcus pneumoniae have been shown to possess different ototoxic properties. Here we present a novel ex vivo experimental setup to examine in detail the pattern of hair cell loss upon exposure to different S. pneumoniae strains, therefore recapitulating pathogen derived aspects of PM-induced hearing loss. Our results show a higher susceptibility towards S. pneumoniae-induced cochlear damage for outer hair cells (OHC) compared to inner hair cells (IHC), which is consistent with in vivo data. S. pneumoniae-induced hair cell loss was both time and dose-dependent. Moreover, we have found significant differences in the level of cell damage between tissue from the basal and the apical turns. This shows that the higher vulnerability of hair cells located at high frequency regions observed in vivo cannot be explained solely by the spatial organisation and bacterial infiltration from the basal portion of the cochlea. Using a wild type D39 strain and a mutant defective for the pneumolysin (PLY) gene, we also have shown that the toxin PLY is an important factor involved in ototoxic damages. The obtained results indicate that PLY can cause both IHC and OHC loss. Finally, we are reporting here for the first time a higher vulnerability of HC located at the basal and middle cochlear region to pneumolysin-induced damage. The detailed description of the susceptibility of hair cells to Streptococcus pneumoniae provided in this report can in the future determine the choice and the development of novel otoprotective therapies during pneumococcal meningitis.

Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

KEYWORDS:

Hair cells; Organ of Corti; Ototoxicity; Pneumolysin; Streptococcus pneumoniae

PMID: 28460251 DOI: 10.1016/j.heares.2017.04.012

Dendritic cell-targeting DNA-based nasal adjuvants for protective mucosal immunity to Streptococcus pneumoniae.

Microbiol Immunol. 2017 May 2. doi: 10.1111/1348-0421.12487. [Epub ahead of print]

Dendritic cell-targeting DNA-based nasal adjuvants for protective mucosal immunity to Streptococcus pneumoniae.

Kataoka K1, Fukuyama Y2, Briles DE2, Miyake T1, Fujihashi K2.

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Abstract

In order to develop safe vaccines for effective mucosal immunity to major pulmonary bacterial infections, one must consider appropriate vaccine antigens (Ags), delivery systems and nontoxic molecular adjuvants. Such vaccine constructs can induce Ag-specific immune responses which provide effective protection from mucosal infections. In particular, it has been shown that adjuvant-based mucosal vaccine preparations are relatively easy to construct by simply mixing the adjuvant with the bacterial Ag, and the resulting vaccine can elicit protective immunity. We have studied DNA-based nasal adjuvants targeting mucosal dendritic cells (DCs) in order to induce Ag-specific mucosal and systemic immune responses that provide essential protection against microbial pathogens which invade our mucosal surfaces. In this review, we initially introduce a plasmid encoding the cDNA of Flt3 ligand (pFL), a molecule which is a growth factor for DCs as an effective adjuvant for mucosal immunity to pneumococcal infections. Next, we discuss the potential of adding unmethylated CpG oligodeoxynucleotide together with pFL together with a pneumococcal Ag for protection from pneumococcal infections. To do this, we have used pneumococcal surface protein A as vaccine for the restoration of mucosal immunity in aging. Further, we have also used our nasal pFL adjuvant system with phosphorylcholine-keyhole limpet hemocyanin (PC-KLH) in pneumococcal vaccine development, to successfully induce complete protection from nasal carriage by Streptococcus pneumoniae. Finally, we discuss the possibility that anti-PC antibodies induced by nasal delivery of pFL plus PC-KLH may play a protective role for prevention of atherogenesis and thus block the subsequent development of cardiovascular disease.

© 2017 The Societies and Wiley Publishing Asia Pty Ltd.

KEYWORDS:

DNA-based adjuvants; Dendritic cells; Nasal vaccination; Streptococcus pneumoniae

PMID: 28463465 DOI: 10.1111/1348-0421.12487

Braz J Infect Dis. 2017 May 1. pii: S1413-8670(16)30619-5. doi: 10.1016/j.bjid.2017.03.011. [Epub ahead of print]

Serotype changes and antimicrobial nonsusceptibility rates of invasive and non-invasive Streptococcus pneumoniae isolates after implementation of 10-valent pneumococcal nontypeable Haemophilus influenzae protein D conjugate vaccine (PHiD-CV) in Bulgaria.

Setchanova L1, Murdjeva M2, Stancheva I3, Alexandrova A4, Sredkova M5, Stoicheva T6, Yoneva M7, Kurchatova A8, Mitov I4.

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Abstract

The 10-valent pneumococcal conjugate vaccine (PCV10) has been included in Bulgarian Childhood Immunization Program since 2010. This study aimed to assess serotype distribution and antimicrobial resistance of 198 invasive and non-invasive Streptococcus pneumoniae strains that had been isolated in Bulgaria during 2011-2016 from patients with invasive (IPD) and non-invasive (NIPD) pneumococcal diseases. The most common invasive serotypes were 3 (10.1%), 19F (4.0%), and 7F (3.0%). A significant decrease in the proportion of invasive vaccine types (VTs) from 64.2% to 35.2% was found in comparison with pre-vaccine era. The most common serotypes among middle ear fluids were 3, 19A and 19F (5.6% each), and VTs fell down from 66.4% to 40.0% in post-PCV10 period. Among respiratory isolates, the most prevalent serotypes were some emergent serotypes such as 15A/B/C (5.0%), 19A, and 6C (4.0% each). VTs decreased significantly (16.3%) among vaccinated children compared to unvaccinated children and adults (44.0%). Two non-VTs (19A and 6C) have increased significantly more (p<0.05) in vaccinated children than in unvaccinated patients. The rates of antibiotic nonsusceptible S. pneumoniae in Bulgaria remained high in post-PCV10 era. Among all source of isolates, antimicrobial nonsusceptibility rates were: oral penicillin - 46.5%, trimethoprim-sulfamethoxazole - 45.4%, erythromycin - 43.9%, tetracycline - 37.4%, and multidrug-resistance (MDR) was 44%. The most common MDR serotypes were 19F, 19A, 6A/C, 15A/B/C and 23A. Our results proved that PCV10 vaccination substantially reduced VTs pneumococcal IPD and NIPD. There has been a shift in the distribution of S. pneumoniae serotypes mostly in vaccinated children but also in the whole population and strong serotype-specific antibiotic resistance was observed after vaccine implementation. Therefore, it is important to continue monitoring serotype changes and pneumococcal resistance among all patient ages in addition to aid in determining the long-term effectiveness of PCV10 interventions.

Copyright © 2017 Sociedade Brasileira de Infectologia. Published by Elsevier Editora Ltda. All rights reserved.

KEYWORDS:

Antimicrobial nonsusceptibility; Invasive and non-invasive isolates; Serotypes; Streptococcus pneumoniae

PMID: 28472614 DOI: 10.1016/j.bjid.2017.03.011

Role of Streptococcus pneumoniae Proteins in Evasion of Complement-Mediated Immunity.

Front Microbiol. 2017 Feb 20;8:224. doi: 10.3389/fmicb.2017.00224. eCollection 2017.

Role of Streptococcus pneumoniae Proteins in Evasion of Complement-Mediated Immunity.

Andre GO1, Converso TR2, Politano WR1, Ferraz LF1, Ribeiro ML3, Leite LC4, Darrieux M1.

Author information

Abstract

The complement system plays a central role in immune defense against Streptococcus pneumoniae. In order to evade complement attack, pneumococci have evolved a number of mechanisms that limit complement mediated opsonization and subsequent phagocytosis. This review focuses on the strategies employed by pneumococci to circumvent complement mediated immunity, both in vitro and in vivo. At last, since many of the proteins involved in interactions with complement components are vaccine candidates in different stages of validation, we explore the use of these antigens alone or in combination, as potential vaccine approaches that aim at elimination or drastic reduction in the ability of this bacterium to evade complement.

KEYWORDS:

Streptococcus pneumoniae; complement system; pneumococcal moonlighting proteins; protein-based vaccines; virulence factors pneumococcal surface proteins

PMID: 28265264 PMCID: PMC5316553 DOI: 10.3389/fmicb.2017.00224