A genetic interaction screen in Streptococcus pneumoniae identifies functionally redundant vaccine candidate proteins CbpC and CbpJ

Streptococcus pneumoniae is a Gram-positive bacterium that asymptomatically colonizes the nasopharynx and can disseminate to sterile sites resulting in pneumococcal diseases such as pneumonia, otitis media, bacteremia, and meningitis. Due to increased incidence of invasive disease caused by serotypes that are not included in available polysaccharide vaccines, there is a need for a broadly protective protein vaccine to complement the polysaccharide based vaccines. To limit immune escape such a vaccine would ideally target proteins that are essential for virulence. However, the genetic robustness of S. pneumoniae results in few surface exposed proteins being essential for virulence. Here we carried out a genetic interaction screen to identify functionally redundant surface protein pairs that could be used as bivalent protein vaccines, based on the observation that together, these protein pairs are essential for virulence. We identified four pairs of functionally redundant surface proteins that displayed a significant competitive disadvantage during murine pneumococcal pneumonia. Immunization with the most attenuated pair, CbpC and CbpJ, resulted in production of high titers of specific antibodies and a modest increased median survival times of mice challenged with pneumococcal pneumonia. This study demonstrates a method to identify essential pairs of surface-associated virulence proteins that could be widely applied to many bacterial pathogens.

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The Contribution of PspC to Pneumococcal Virulence Varies between Strains and Is Accomplished by Both Complement Evasion and Complement-Independent Mechanisms

Infection and Immunity ◽

10.1128/iai.00543-06 ◽

2006 ◽

Vol 74 (9) ◽

pp. 5319-5324 ◽

Cited By ~ 36

Author(s):

Alison R. Kerr ◽

Gavin K. Paterson ◽

Jackie McCluskey ◽

Francesco Iannelli ◽

Marco R. Oggioni ◽

...

Keyword(s):

Streptococcus Pneumoniae ◽

Protein C ◽

Pneumococcal Pneumonia ◽

Surface Protein ◽

Murine Models ◽

Wild Type ◽

Survival Times ◽

Pneumococcal Strain ◽

Wild Type Parent

ABSTRACTPneumococcal surface protein C (PspC) is a virulence factor ofStreptococcus pneumoniaepreviously shown to play a role in bacterial adherence, invasion, and evasion of complement. We investigated the role of this protein in our murine models of pneumococcal pneumonia with different pneumococcal strains. The deletion ofpspCin strains of serotypes 2, 3, and 19F did not significantly alter host survival times in the pneumonia model. In contrast,pspCdeletion significantly reduced the virulence of the serotype 4 strain, TIGR4, in both the pneumonia and bacteremia models. Therefore,pspCis a systemic and pulmonary virulence determinant forS. pneumoniae, but its effects are influenced by the pneumococcal strain. Finally, pneumonia infection of complement-deficient (C3−/−) mice enhancedpspCvirulence, illustrating that PspC-mediated complement evasion contributes to virulence. However, other functions of PspC also contribute to virulence, as demonstrated by the finding that thepspC-deficient TIGR4 mutant was still attenuated relative to the wild-type parent, even in the absence of C3.

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Immunization of Mice with Combinations of Pneumococcal Virulence Proteins Elicits Enhanced Protection against Challenge with Streptococcus pneumoniae

Infection and Immunity ◽

10.1128/iai.68.5.3028-3033.2000 ◽

2000 ◽

Vol 68 (5) ◽

pp. 3028-3033 ◽

Cited By ~ 151

Author(s):

A. David Ogunniyi ◽

Rebekah L. Folland ◽

David E. Briles ◽

Susan K. Hollingshead ◽

James C. Paton

Keyword(s):

Streptococcus Pneumoniae ◽

Metal Binding ◽

Protein A ◽

Surface Protein ◽

Active Immunization ◽

Survival Times ◽

Virulence Proteins ◽

Virulent Type ◽

Vaccine Potential ◽

High Doses

ABSTRACT The vaccine potential of a combination of three pneumococcal virulence proteins was evaluated in an active-immunization–intraperitoneal-challenge model in BALB/c mice, using very high challenge doses of Streptococcus pneumoniae. The proteins evaluated were a genetic toxoid derivative of pneumolysin (PdB), pneumococcal surface protein A (PspA), and a 37-kDa metal-binding lipoprotein referred to as PsaA. Mice immunized with individual proteins or combinations thereof were challenged with high doses of virulent type 2 or type 4 pneumococci. The median survival times for mice immunized with combinations of proteins, particularly PdB and PspA, were significantly longer than those for mice immunized with any of the antigens alone. A similar effect was seen in a passive protection model. Thus, combinations of pneumococcal proteins may provide the best non-serotype-dependent protection against S. pneumoniae.

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Antigenic Variation inStreptococcus pneumoniaePspC Promotes Immune Escape in the Presence of Variant-Specific Immunity

mBio ◽

10.1128/mbio.00264-18 ◽

2018 ◽

Vol 9 (2) ◽

Cited By ~ 9

Author(s):

M. Georgieva ◽

L. Kagedan ◽

Ying-Jie Lu ◽

C. M. Thompson ◽

M. Lipsitch

Keyword(s):

Streptococcus Pneumoniae ◽

Sequence Variation ◽

Antigenic Variation ◽

Biological Function ◽

Immune Escape ◽

Surface Protein ◽

Factor H ◽

Sequence Diversity ◽

Protein Antigens ◽

Specific Immunity

ABSTRACTGenomic analysis reveals extensive sequence variation and hot spots of recombination in surface proteins ofStreptococcus pneumoniae. While this phenomenon is commonly attributed to diversifying selection by host immune responses, there is little mechanistic evidence for the hypothesis that diversification of surface protein antigens produces an immune escape benefit during infection withS. pneumoniae. Here, we investigate the biological significance of sequence variation within theS. pneumoniaecell wall-associated pneumococcal surface protein C (PspC) protein antigen. UsingpspCallelic diversity observed in a large pneumococcal collection, we produced variant-specific protein constructs that span the sequence variability within thepspClocus. We show that antibodies raised against these PspC constructs are variant specific and prevent association between PspC and the complement pathway mediator, human factor H. We found that PspC variants differ in their capacity to bind factor H, suggesting that sequence variation withinpspCreflects differences in biological function. Finally, in an antibody-dependent opsonophagocytic assay,S. pneumoniaeexpressing a PspC variant matching the antibody specificity was killed efficiently. In contrast, killing efficacy was not evident againstS. pneumoniaeexpressing mismatched PspC variants. Our data suggest that antigenic variation within the PspC antigen promotes immune evasion and could confer a fitness benefit during infection.IMPORTANCELoci encoding surface protein antigens inStreptococcus pneumoniaeare highly polymorphic. It has become a truism that these polymorphisms are the outcome of selective pressure onS. pneumoniaeto escape host immunity. However, there is little mechanistic evidence to support the hypothesis that diversifying protein antigens produces a benefit for the bacteria. Using the highly diversepspClocus, we have now characterized the functional and immune implications of sequence diversity within the PspC protein. We have characterized the spectrum of biological function among diverse PspC variants and show thatpspCsequence diversity reflects functional differences. Further, we show that sequence variation in PspC confers an immune escape benefit in the presence of anti-PspC variant-specific immunity. Overall, the results of our studies provide insights into the functional implications of protein sequence diversity and the role of variant-specific immunity in its maintenance.

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Contributions of Pneumolysin, Pneumococcal Surface Protein A (PspA), and PspC to Pathogenicity of Streptococcus pneumoniae D39 in a Mouse Model

Infection and Immunity ◽

10.1128/iai.01384-06 ◽

2007 ◽

Vol 75 (4) ◽

pp. 1843-1851 ◽

Cited By ~ 66

Author(s):

Abiodun D. Ogunniyi ◽

Kim S. LeMessurier ◽

Rikki M. A. Graham ◽

James M. Watt ◽

David E. Briles ◽

...

Keyword(s):

Streptococcus Pneumoniae ◽

Systemic Disease ◽

Protein A ◽

Virulence Gene ◽

Surface Protein ◽

Invasive Disease ◽

Upper Respiratory Tract ◽

Virulence Proteins ◽

Pneumococcal Surface Protein A ◽

Genes Encoding

ABSTRACTSuccessful colonization of the upper respiratory tract byStreptococcus pneumoniaeis an essential first step in the pathogenesis of pneumococcal disease. However, the bacterial and host factors that provoke the progression from asymptomatic colonization to invasive disease are yet to be fully defined. In this study, we investigated the effects of single and combined mutations in genes encoding pneumolysin (Ply), pneumococcal surface protein A (PspA), and pneumococcal surface protein C (PspC, also known as choline-binding protein A) on the pathogenicity ofStreptococcus pneumoniaeserotype 2 (D39) in mice. Following intranasal challenge with D39, stable colonization of the nasopharynx was maintained over a 7-day period at a level of approximately 105bacteria per mouse. The abilities of the mutant deficient in PspA to colonize the nasopharynx and to cause lung infection and bacteremia were significantly reduced. Likewise, the PspC mutant and, to a lesser extent, the Ply mutant also had reduced abilities to colonize the nasopharynx. As expected, the double mutants colonized less well than the parent to various degrees and had difficulty translocating to the lungs and blood. A significant additive attenuation was observed for the double and triple mutants in pneumonia and systemic disease models. Surprisingly, the colonization profile of the derivative lacking all three proteins was similar to that of the wild type, indicating virulence gene compensation. These findings further demonstrate that the mechanism of pneumococcal pathogenesis is highly complex and multifactorial but ascribes a role for each of these virulence proteins, alone or in combination, in the process.

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Streptococcus pneumoniae Surface Protein PcpA Elicits Protection against Lung Infection and Fatal Sepsis

Infection and Immunity ◽

10.1128/iai.01126-07 ◽

2008 ◽

Vol 76 (6) ◽

pp. 2767-2776 ◽

Cited By ~ 68

Author(s):

David T. Glover ◽

Susan K. Hollingshead ◽

David E. Briles

Keyword(s):

Streptococcus Pneumoniae ◽

Protein A ◽

Surface Protein ◽

Murine Models ◽

Wild Type ◽

Survival Times ◽

High Manganese ◽

Subcutaneous Immunization ◽

Fatal Sepsis ◽

Manganese Concentrations

ABSTRACTPrevious studies have suggested that pneumococcal choline binding protein A (PcpA) is important for the full virulence ofStreptococcus pneumoniae, and its amino acid sequence suggests that it may play a role in cellular adherence. PcpA is under the control of a manganese-dependent regulator and is only expressed at low manganese concentrations, similar to those found in the blood and lungs. PcpA expression is repressed under high manganese concentrations, similar to those found in secretions. In this study, we have demonstrated that PcpA elicits statistically significant protection in murine models of pneumonia and sepsis. In the model of pneumonia with each of four challenge strains, statistically fewerS. pneumoniaecells were recovered from the lungs of mice immunized with PcpA and alum versus mice immunized with alum only. The immunizations reduced the median CFU by 4- to 400-fold (average of 28-fold). In the model of sepsis using strain TIGR4, PcpA expression resulted in shorter times to become moribund and subcutaneous immunization with PcpA increased survival times of mice infected with wild-type PcpA-expressing pneumococci.

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PspK of Streptococcus pneumoniae Increases Adherence to Epithelial Cells and Enhances Nasopharyngeal Colonization

Infection and Immunity ◽

10.1128/iai.00755-12 ◽

2012 ◽

Vol 81 (1) ◽

pp. 173-181 ◽

Cited By ~ 50

Author(s):

L. E. Keller ◽

C. V. Jones ◽

J. A. Thornton ◽

M. E. Sanders ◽

E. Swiatlo ◽

...

Keyword(s):

Streptococcus Pneumoniae ◽

Epithelial Cells ◽

Immunoglobulin A ◽

Surface Protein ◽

Invasive Disease ◽

Factor H ◽

Host Cells ◽

Content Type ◽

Capsule Locus ◽

Complement Regulator

Streptococcus pneumoniae(the pneumococcus) colonizes the human nasopharynx and can cause invasive disease aided by the pneumococcal capsule. Group II nontypeableS. pneumoniae(NTSp) lacks a polysaccharide capsule, and a subgroup of NTSp carriage isolates has been found to have a novel gene, pneumococcal surface protein K (pspK), which replaces the capsule locus. A recent rise in the number of NTSp isolates colonizing the human nasopharynx has been observed, but the colonization factors of NTSp have not been well studied. PspK has been shown to play a role in mouse colonization. We therefore examined PspK-mediated immune evasion along with adherence to host cells and colonization. PspK bound human secretory immunoglobulin A (sIgA) but not the complement regulator factor H and did not decrease C3b deposition on the pneumococcal surface. PspK increased binding of pneumococci to epithelial cells and enhanced pneumococcal colonization independently of the genetic background. Understanding how NTSp colonizes and survives within the nasopharynx is important due to the increase in NTSp carriage. Our data suggest that PspK may aid in the persistence of NTSp within the nasopharynx but is not involved in invasion.

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Intranasal Vaccination with Pneumococcal Surface Protein A and Interleukin-12 Augments Antibody-Mediated Opsonization and Protective Immunity against Streptococcus pneumoniae Infection

Infection and Immunity ◽

10.1128/iai.69.11.6718-6724.2001 ◽

2001 ◽

Vol 69 (11) ◽

pp. 6718-6724 ◽

Cited By ~ 113

Author(s):

Bernard P. Arulanandam ◽

Joyce M. Lynch ◽

David E. Briles ◽

Susan Hollingshead ◽

Dennis W. Metzger

Keyword(s):

Streptococcus Pneumoniae ◽

Bacterial Colonization ◽

Protein A ◽

Flow Cytometric Analysis ◽

Surface Protein ◽

Invasive Disease ◽

Interleukin 12 ◽

Opsonic Activity ◽

Pneumococcal Surface Protein A ◽

Optimal Approach

ABSTRACT Streptococcus pneumoniae is a major pathogen in humans that enters the host primarily through the respiratory tract. Targeting mucosal surfaces directly may therefore be an optimal approach for vaccination to prevent bacterial colonization and invasive disease. We have previously demonstrated the effectiveness of interleukin-12 (IL-12) delivered intransally (i.n.) as an antiviral respiratory adjuvant. In this study, we examined the effects of i.n. IL-12 treatment on induction of protective humoral immunity against S. pneumoniae. Immunization i.n. with pneumococcal surface protein A (PspA) and IL-12 resulted in enhanced lung IL-10 mRNA expression and marked augmentation of respiratory and systemic immunoglobulin G1 (IgG1), IgG2a, and IgA antibody levels compared to those in animals receiving PspA alone. In addition, i.n. vaccination with PspA and IL-12 provided increased protection against nasopharyngeal carriage. Flow cytometric analysis revealed a threefold increase in antibody-mediated, complement-independent opsonic activity in the sera of PspA- and IL-12-treated animals, which was mainly contributed by IgG2a and, to a lesser extent, IgA. Passive transfer of these immune sera conferred complete protection from death upon systemic pneumococcal challenge. These findings demonstrate the effectiveness of combining PspA and IL-12 at mucosal sites to achieve optimal antibody-mediated opsonization and killing of S. pneumoniae.

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The Ami-AliA/AliB Permease of Streptococcus pneumoniae Is Involved in Nasopharyngeal Colonization but Not in Invasive Disease

Infection and Immunity ◽

10.1128/iai.72.7.3902-3906.2004 ◽

2004 ◽

Vol 72 (7) ◽

pp. 3902-3906 ◽

Cited By ~ 54

Author(s):

A. R. Kerr ◽

P. V. Adrian ◽

S. Estevão ◽

R. de Groot ◽

G. Alloing ◽

...

Keyword(s):

Streptococcus Pneumoniae ◽

Invasive Disease ◽

Wild Type ◽

Survival Times ◽

Triple Mutant ◽

Nasopharyngeal Colonization ◽

Atp Binding Cassette Transporter ◽

Pneumococcal Colonization ◽

Oligopeptide Permease

ABSTRACT The Ami-AliA/AliB oligopeptide permease is an ATP-binding cassette transporter which is found in Streptococcus pneumoniae and which is involved in nutrient uptake. We investigated the role of the three paralogous oligopeptide-binding lipoproteins AmiA, AliA, and AliB by using murine models of pneumococcal colonization and invasive disease. A series of mutants lacking aliA, aliB, and amiA either alone or in combination as double or triple mutations were used. Inoculation of the nasopharynx with a mixture of the obl (oligopeptide-binding lipoprotein-negative) triple-mutant and wild-type (D39) bacteria resulted in significantly smaller numbers of obl bacteria colonizing the nasopharynx. The use of a mixture of individual mutants and wild-type pneumococci revealed that AmiA, AliA, and AliB were all required for successful colonization of the nasopharynx. The obl mutant was more attenuated than the aliB mutant but not the aliA or amiA mutant. Therefore, there is some redundancy in the Ami-AliA/AliB complex in terms of nasopharyngeal colonization, with AliA and AmiA being able to compensate for the removal of AliB. Animals with invasive disease caused by these mutants had survival times, bacterial loads, and inflammatory cytokine production levels similar to those of animals infected with wild-type pneumococci. Our results show that although the Ami-AliA/AliB complex is not required for virulence during pneumococcal pneumonia, it does play a role in colonization of the nasopharynx.

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Same, Same, but Different: Molecular Analyses of Streptococcus pneumoniae Immune Evasion Proteins Identifies new Domains and Reveals Structural Differences between PspC and Hic Variants

10.1101/2020.08.11.246066 ◽

2020 ◽

Author(s):

Shanshan Du ◽

Claudia Vilhena ◽

Samantha King ◽

Alfredo Sahagun ◽

Sven Hammerschmidt ◽

...

Keyword(s):

Streptococcus Pneumoniae ◽

Immune Evasion ◽

Antigenic Variation ◽

Immune Escape ◽

Functional Characterization ◽

Surface Proteins ◽

Surface Distribution ◽

Domain Composition ◽

Structural Differences ◽

Domain Level

AbstractPspC and Hic proteins of Streptococcus pneumoniae are some of the most variable microbial immune evasion proteins identified to date. Due to structural similarities and conserved binding profiles it was assumed over a long time that these pneumococcal surface proteins represent a protein family, comprising eleven subgroups. Recently, however, by evaluating more proteins larger diversity of individual proteins became apparent. In contrast to previous assumptions a pattern evaluation of six PspC and five Hic variants, each representing one of the previously defined subgroups, revealed distinct structural and likely functionally regions of the proteins, and identified nine new domains and new domain alternates. Several domains are unique to PspC and Hic variants, while other domains are shared with other S. pneumoniae and bacterial virulent determinants. This understanding improved pattern evaluation on the level of full-length proteins, allowed a sequence comparison on the domain level and furthermore identified domains with a modular composition. This novel concept allows a better characterization of variability, and modular domain composition of individual proteins, enables a structural and functional characterization at the domain level and furthermore shows substantial structural differences between PspC and Hic proteins. Such knowledge will also be useful for molecular strain typing, characterizing PspC and Hic proteins from new clinical S. pneumoniae strains, including those derived from patients who present with pneumococcal hemolytic uremic syndrome. Furthermore this analysis explains the role of multifaceted intact PspC and Hic proteins in pathogen host interactions. and can provide a basis for rational vaccine design.Author SummaryThe human pathobiont Streptococcus pneumoniae expresses highly polymorphic PspC or Hic proteins, which bind a repertoire of host immune regulators and combine antigenic variation with conserved immune evasion features. Understanding domain composition of each protein encoded by more than 60 000 pspC or hic genes deposited in the data banks defines their diversity, a role in immune escape and can furthermore delineate structure function approach for single protein domains. PspC and Hic proteins show variable domain composition and sequence diversity, which explain differences in binding of human regulators and likely in immune escape. The results of our analyses provide insights in the domain composition of these diverse immune evasion proteins, identifies new domains, defines domains which are unique to PspC or Hic variants, and identifies domains which are shared with other bacterial immune evasion proteins. These data have implication on cell wall attachment, surface distribution and in immune escape.

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Survival of Streptococcus pneumoniae in sputum from patients with pneumonia

Journal of Clinical Microbiology ◽

10.1128/jcm.7.1.3-5.1978 ◽

1978 ◽

Vol 7 (1) ◽

pp. 3-5

Author(s):

S G Williams ◽

C A Kauffman

Keyword(s):

Streptococcus Pneumoniae ◽

Pneumococcal Pneumonia ◽

Room Temperature ◽

Survival Times ◽

Isolation Rate

The isolation rate of Streptococcus pneumoniae in sputum cultures from patients with pneumococcal pneumonia is low. An investigation was made to determine whether this low yield might be due to loss of pneumocci and/or overgrowth by pharyngeal flora before the specimen is plated. Pneumococcal survival times and pharyngeal overgrowth at 4 degrees C and at room temperature were determined in sputum obtained from 42 patients with pneumococcal pneumonia. It was found that pneumococci survived for long periods in sputum--2.2 +/- 1.4 days at room temperature and 9.5 +/- 3.6 days at 4 degrees C. Overgrowth by pharyngeal flora occurred in only 6 of 42 specimens kept at 4 degrees C and 31 of 42 specimens kept at room temperature. The low yield of S. pneumoniae in sputum from patients with pneumococcal pneumonia is not explained by decreased viability of the organism.

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