The Pneumococcus Blog: September 2014

Tuesday, September 23, 2014

Streptococcus pneumoniae Translocates into the Myocardium and Forms Unique Microlesions That Disrupt Cardiac Function.

PLoS Pathog. 2014 Sep 18;10(9):e1004383. doi: 10.1371/journal.ppat.1004383. eCollection 2014.

Streptococcus pneumoniae Translocates into the Myocardium and Forms Unique Microlesions That Disrupt Cardiac Function.

Brown AO1, Mann B2, Gao G2, Hankins JS3, Humann J2, Giardina J2, Faverio P4, Restrepo MI5, Halade GV6, Mortensen EM7, Lindsey ML8, Hanes M9, Happel KI10, Nelson S10, Bagby GJ10, Lorent JA11, Cardinal P11, Granados R11, Esteban A11, LeSaux CJ12, Tuomanen EI2, Orihuela CJ1.

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Abstract

Hospitalization of the elderly for invasive pneumococcal disease is frequently accompanied by the occurrence of an adverse cardiac event; these are primarily new or worsened heart failure and cardiac arrhythmia. Herein, we describe previously unrecognized microscopic lesions (microlesions) formed within the myocardium of mice, rhesus macaques, and humans during bacteremic Streptococcus pneumoniae infection. In mice, invasive pneumococcal disease (IPD) severity correlated with levels of serum troponin, a marker for cardiac damage, the development of aberrant cardiac electrophysiology, and the number and size of cardiac microlesions. Microlesions were prominent in the ventricles, vacuolar in appearance with extracellular pneumococci, and remarkable due to the absence of infiltrating immune cells. The pore-forming toxin pneumolysin was required for microlesion formation but Interleukin-1β was not detected at the microlesion site ruling out pneumolysin-mediated pyroptosis as a cause of cell death. Antibiotic treatment resulted in maturing of the lesions over one week with robust immune cell infiltration and collagen deposition suggestive of long-term cardiac scarring. Bacterial translocation into the heart tissue required the pneumococcal adhesin CbpA and the host ligands Laminin receptor (LR) and Platelet-activating factor receptor. Immunization of mice with a fusion construct of CbpA or the LR binding domain of CbpA with the pneumolysin toxoid L460D protected against microlesion formation. We conclude that microlesion formation may contribute to the acute and long-term adverse cardiac events seen in humans with IPD.

PMID: 25232870 [PubMed - in process]

Wednesday, September 10, 2014

The Impact of Pneumolysin on the Macrophage Response to Streptococcus pneumoniae is Strain-Dependent.

PLoS One. 2014 Aug 8;9(8):e103625. doi: 10.1371/journal.pone.0103625. eCollection 2014.

The Impact of Pneumolysin on the Macrophage Response to Streptococcus pneumoniae is Strain-Dependent.

Harvey RM1, Hughes CE1, Paton AW1, Trappetti C1, Tweten RK2, Paton JC1.

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Abstract

Streptococcus pneumoniae is the world's leading cause of pneumonia, bacteremia, meningitis and otitis media. A major pneumococcal virulence factor is the cholesterol-dependent cytolysin, which has the defining property of forming pores in cholesterol-containing membranes. In recent times a clinically significant and internationally successful serotype 1 ST306 clone has been found to express a non-cytolytic variant of Ply (Ply306). However, while the pneumococcus is a naturally transformable organism, strains of the ST306 clonal group have to date been virtually impossible to transform, severely restricting efforts to understand the role of non-cytolytic Ply in the success of this clone. In this study isogenic Ply mutants were constructed in the D39 background and for the first time in the ST306 background (A0229467) to enable direct comparisons between Ply variants for their impact on the immune response in a macrophage-like cell line. Strains that expressed cytolytic Ply were found to induce a significant increase in IL-1β release from macrophage-like cells compared to the non-cytolytic and Ply-deficient strains in a background-independent manner, confirming the requirement for pore formation in the Ply-dependent activation of the NLRP3 inflammasome. However, cytolytic activity in the D39 background was found to induce increased expression of the genes encoding GM-CSF (CSF2), p19 subunit of IL-23 (IL23A) and IFNβ (IFNB1) compared to non-cytolytic and Ply-deficient D39 mutants, but had no effect in the A0229467 background. The impact of Ply on the immune response to the pneumococcus is highly dependent on the strain background, thus emphasising the importance of the interaction between specific virulence factors and other components of the genetic background of this organism.

PMID: 25105894 [PubMed - in process] PMCID: PMC4126675

Competence for Genetic Transformation in Streptococcus pneumoniae: Mutations in σA Bypass the comW Requirement.

J Bacteriol. 2014 Aug 11. pii: JB.01933-14. [Epub ahead of print]

Competence for Genetic Transformation in Streptococcus pneumoniae: Mutations in σA Bypass the comW Requirement.

Tovpeko Y1, Morrison DA2.

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Abstract

Competence for genetic transformation in the genus streptococcus depends on an alternative sigma factor, σX, for coordinated synthesis of 23 proteins, which together establish the X state by permitting lysis of incompetent streptococci, uptake of DNA fragments, and integration of strands of that DNA into the resident genome. Initiation of transient accumulation of high levels of σX is coordinated between cells by transcription factors linked to peptide pheromone signals. In S. pneumoniae, elevated σX is insufficient for development of full competence without co-expression of a second competence-specific protein, ComW. ComW, shared by six species in the mitis and anginosus groups, is regulated by the same pheromone circuit that controls σX, but its role in expression of the σX regulon is unknown. Using the strong, but not absolute, dependence of transformation on comW as a selective tool, we collected 27 independent comW bypass mutations and mapped them to ten single-base transitions, all within rpoD, encoding the primary sigma factor subunit of RNA polymerase, σA. Eight mapped to sites in rpoD region 4 that are implicated in interaction with the core β subunit, indicating that ComW may act to facilitate competition of the alternative sigma factor σX for access to core polymerase.

PMID: 25112479 [PubMed - as supplied by publisher]

The Oral Commensal Streptococcus mitis Shows a Mixed Memory Th Cell Signature That Is Similar to and Cross-Reactive with Streptococcus pneumoniae.

PLoS One. 2014 Aug 13;9(8):e104306. doi: 10.1371/journal.pone.0104306. eCollection 2014.

The Oral Commensal Streptococcus mitis Shows a Mixed Memory Th Cell Signature That Is Similar to and Cross-Reactive with Streptococcus pneumoniae.

Engen SA1, Valen Rukke H1, Becattini S2, Jarrossay D2, Blix IJ3, Petersen FC1, Sallusto F2, Schenck K1.

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Abstract

BACKGROUND:

Carriage of and infection with Streptococcus pneumoniae is known to predominantly induce T helper 17 (Th17) responses in humans, but the types of Th cells showing reactivity towards commensal streptococci with low pathogenic potential, such as the oral commensals S. mitis and S. salivarius, remain uncharacterized.

METHODS:

Memory CD4+ T helper (Th) cell subsets were isolated from healthy human blood donors according to differential expression of chemokine receptors, expanded in vitro using polyclonal stimuli and characterized for reactivity against different streptococcal strains.

RESULTS:

Th cells responding to S. mitis, S. salivarius and S. pneumoniae were predominantly in a CCR6+CXCR3+ subset and produced IFN-γ, and in a CCR6+CCR4+ subset and produced IL-17 and IL-22. Frequencies of S. pneumoniae-reactive Th cells were higher than frequencies of S. mitis- and S. salivarius-specific Th cells. S. mitis and S. pneumoniae isogenic capsule knock-out mutants and a S. mitis mutant expressing the serotype 4 capsule of S. pneumoniae showed no different Th cell responses as compared to wild type strains. S. mitis-specific Th17 cells showed cross-reactivity with S. pneumoniae.

CONCLUSIONS:

As Th17 cells partly control clearance of S. pneumoniae, cross-reactive Th17 cells that may be induced by commensal bacterial species may influence the immune response, independent of capsule expression.

PMID: 25119879 [PubMed - in process] PMCID: PMC4131883

Characterisation of a collection of Streptococcus pneumoniae isolates from patients suffering from acute exacerbations of chronic bronchitis: In vitro susceptibility to antibiotics and biofilm formation in relation to antibiotic efflux and serotypes/serogroups.

Int J Antimicrob Agents. 2014 Sep;44(3):209-17. doi: 10.1016/j.ijantimicag.2014.05.016. Epub 2014 Jul 7.

Characterisation of a collection of Streptococcus pneumoniae isolates from patients suffering from acute exacerbations of chronic bronchitis: In vitro susceptibility to antibiotics and biofilm formation in relation to antibiotic efflux and serotypes/serogroups.

Vandevelde NM1, Tulkens PM2, Diaz Iglesias Y1, Verhaegen J3, Rodriguez-Villalobos H4, Philippart I5, Cadrobbi J6, Coppens N7, Boel A8, Van Vaerenbergh K8, Francart H9, Vanhoof R10, Liistro G11, Jordens P12, d'Odemont JP13, Valcke Y14, Verschuren F15, Van Bambeke F1.

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Abstract

The correlation between Streptococcus pneumoniae serotypes, biofilm production, antibiotic susceptibility and drug efflux in isolates from patients suffering from acute exacerbations of chronic bronchitis (AECB) remains largely unexplored. Using 101 isolates collected from AECB patients for whom partial (n=51) or full (n=50) medical details were available, we determined serotypes (ST)/serogroups (SG) (Quellung reaction), antibiotic susceptibility patterns [MIC (microdilution) using EUCAST and CLSI criteria] and ability to produce biofilm in vitro (10-day model; crystal violet staining). The majority of patients were 55-75 years old and <5% were vaccinated against S. pneumoniae. Moreover, 54% showed high severity scores (GOLD 3-4), and comorbidities were frequent including hypertension (60%), cancer (24%) and diabetes (20%). Alcohol and/or tobacco dependence was >30%. Isolates of SG6-11-15-23, known for large biofilm production and causing chronic infections, were the most prevalent (>15% each), but other isolates also produced biofilm (SG9-18-22-27 and ST8-20 being most productive), except SG7, SG29 and ST5 (<2% of isolates each). Resistance (EUCAST breakpoints) was 8-13% for amoxicillin and cefuroxime, 35-39% for macrolides, 2-8% for fluoroquinolones and 2% for telithromycin. ST19A isolates showed resistance to all antibiotics, ST14 to all except moxifloxacin, and SG9 and SG19 to all except telithromycin, moxifloxacin and ceftriaxone (SG19 only). Solithromycin and telithromycin MICs were similar. No correlation was observed between biofilm production and MIC or efflux (macrolides, fluoroquinolones). S. pneumoniae serotyping may improve AECB treatment by avoiding antibiotics with predictable low activity, but it is not predictive of biofilm production.

KEYWORDS:

Acute exacerbations of chronic bronchitis (AECB); Biofilm; Efflux; Serotype; Streptococcus pneumoniae; Susceptibility

PMID: 25123808 [PubMed - in process]

Streptococcus pneumoniae and influenza: dynamic changes in the pneumococcal transcriptome during transition from biofilm formation to invasive disease.

Infect Immun. 2014 Aug 18. pii: IAI.02225-14. [Epub ahead of print]

Streptococcus pneumoniae and influenza: dynamic changes in the pneumococcal transcriptome during transition from biofilm formation to invasive disease.

Pettigrew MM1, Marks LR2, Kong Y3, Gent JF4, Roche-Hakansson H2, Hakansson AP5.

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Abstract

Streptococcus pneumoniae is a leading cause of infectious disease globally. Nasopharyngeal colonization occurs in biofilms and precedes infection. Prior studies indicate that biofilm derived pneumococci are avirulent. However, influenza A virus (IAV) infection releases virulent pneumococci from biofilms in vitro and in vivo. Triggers of dispersal include IAV-induced changes in the nasopharynx such as increased temperature (fever) and extracellular ATP (tissue damage). We used RNA-seq to compare the S. pneumoniae transcriptome in biofilms, bacteria dispersed from biofilms after exposure to IAV, febrile-range temperature, or ATP, and planktonic cells grown at 37°C. Compared with biofilm bacteria, actively dispersed S. pneumoniae, which were more virulent in invasive disease, up-regulated genes involved in carbohydrate metabolism. Enzymatic assays for ATP and lactate production confirmed that dispersed pneumococci exhibited increased metabolism compared to those in biofilms. Dispersed pneumococci also up-regulated genes associated with production of bacteriocins and down-regulated colonization-associated genes related to competence, fratricide, and the transparent colony phenotype. IAV had the largest impact on the pneumococcal transcriptome. Similar transcriptional differences were also observed when actively dispersed bacteria were compared with avirulent planktonic bacteria. Our data demonstrate complex changes in the pneumococcal transcriptome in response to IAV-induced changes in the environment. Our data suggest that disease is caused by pneumococci that are primed to move to tissue sites with altered nutrient availability and to protect themselves from the nasopharyngeal microflora and host immune response. These data help explain pneumococcal virulence after IAV infection and have important implications for studies of S. pneumoniae pathogenesis.

PMID: 25135685 [PubMed - as supplied by publisher]

MicroRNA-155 is required for the clearance of Streptococcus pneumoniae from the nasopharynx.

Infect Immun. 2014 Aug 25. pii: IAI.02251-14. [Epub ahead of print]

MicroRNA-155 is required for the clearance of Streptococcus pneumoniae from the nasopharynx.

Verschoor CP1, Dorrington MG1, Novakowski KE1, Kaiser J2, Radford K3, Nair P3, Anipindi V1, Kaushic C1, Surette MG2, Bowdish DM4.

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Abstract

Pneumonia caused by Streptococcus pneumoniae is a major cause of death and economic burden worldwide. S. pneumoniae is an intermittent colonizer of the human upper respiratory tract and the ability to control asymptomatic colonization determines the likelihood of developing invasive disease. Recognition of S. pneumoniae by resident macrophages via TLR-2 and MARCO and the presence of IL-17 secreting CD4+ T-cells are required for macrophage recruitment and bacterial clearance. Despite the fact that the primary cellular effectors needed for bacterial clearance have been identified, much of the underlying regulatory mechanisms are unknown. Herein we demonstrate that the small, non-coding RNA, microRNA-155 (mir-155) is critical for the effective clearance of S. pneumoniae. Our studies show that mir-155 deficient mice maintain the ability to prevent acute, invasive pneumococcal infection, but have significantly higher bacterial burdens following colonization, independent of macrophage recognition by TLR-2, MARCO expression, or bactericidal capacity. The observed defects in bacterial clearance parallel reduced IL-17A and IFN-gamma CD4+ T-cell responses in vivo, lower IL-17A mRNA in the nasopharynx and a reduced capacity to induce Th17 cell polarization. Given that knockout mice are also limited in their capacity to generate high titre S. pneumoniae specific antibodies, we conclude that mir-155 is a critical mediator of the cellular effectors needed to clear primary and secondary S. pneumoniae colonization.

PMID: 25156727 [PubMed - as supplied by publisher]

A point mutation in cpsE renders Streptococcus pneumoniae nonencapsulated and enhances its growth, adherence and competence.

BMC Microbiol. 2014 Aug 28;14(1):210. doi: 10.1186/s12866-014-0210-x.

A point mutation in cpsE renders Streptococcus pneumoniae nonencapsulated and enhances its growth, adherence and competence.

Schaffner TO, Hinds J, Gould KA, Wüthrich D, Bruggmann R, Küffer M, Mühlemann K, Hilty M, Hathaway LJ1.

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Abstract

BACKGROUND:

The polysaccharide capsule is a major virulence factor of the important human pathogen Streptococcus pneumoniae. However, S. pneumoniae strains lacking capsule do occur.

RESULTS:

Here, we report a nasopharyngeal isolate of Streptococcus pneumoniae composed of a mixture of two phenotypes; one encapsulated (serotype 18C) and the other nonencapsulated, determined by serotyping, electron microscopy and fluorescence isothiocyanate dextran exclusion assay.By whole genome sequencing, we demonstrated that the phenotypes differ by a single nucleotide base pair in capsular gene cpsE (C to G change at gene position 1135) predicted to result in amino acid change from arginine to glycine at position 379, located in the cytoplasmic, enzymatically active, region of this transmembrane protein. This SNP is responsible for loss of capsule production as the phenotype is transferred with the capsule operon. The nonencapsulated variant is superior in growth in vitro and is also 117-fold more adherent to and more invasive into Detroit 562 human epithelial cells than the encapsulated variant.Expression of six competence pathway genes and one competence-associated gene was 11 to 34-fold higher in the nonencapsulated variant than the encapsulated and transformation frequency was 3.7-fold greater.

CONCLUSIONS:

We identified a new single point mutation in capsule gene cpsE of a clinical S. pneumoniae serotype 18C isolate sufficient to cause loss of capsule expression resulting in the co-existence of the encapsulated and nonencapsulated phenotype. The mutation caused phenotypic changes in growth, adherence to epithelial cells and transformability. Mutation in capsule gene cpsE may be a way for S. pneumoniae to lose its capsule and increase its colonization potential.

PMID: 25163487 [PubMed - in process]

TLR2 Signaling Decreases Transmission of Streptococcus pneumoniae by Limiting Bacterial Shedding in an Infant Mouse Influenza A Co-infection Model.

PLoS Pathog. 2014 Aug 28;10(8):e1004339. doi: 10.1371/journal.ppat.1004339. eCollection 2014.

TLR2 Signaling Decreases Transmission of Streptococcus pneumoniae by Limiting Bacterial Shedding in an Infant Mouse Influenza A Co-infection Model.

Richard AL1, Siegel SJ1, Erikson J2, Weiser JN1.

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Abstract

While the importance of transmission of pathogens is widely accepted, there is currently little mechanistic understanding of this process. Nasal carriage of Streptococcus pneumoniae (the pneumococcus) is common in humans, especially in early childhood, and is a prerequisite for the development of disease and transmission among hosts. In this study, we adapted an infant mouse model to elucidate host determinants of transmission of S. pneumoniae from inoculated index mice to uninfected contact mice. In the context of co-infection with influenza A virus, the pneumococcus was transmitted among wildtype littermates, with approximately half of the contact mice acquiring colonization. Mice deficient for TLR2 were colonized to a similar density but transmitted S. pneumoniae more efficiently (100% transmission) than wildtype animals and showed decreased expression of interferon α and higher viral titers. The greater viral burden in tlr2-/- mice correlated with heightened inflammation, and was responsible for an increase in bacterial shedding from the mouse nose. The role of TLR2 signaling was confirmed by intranasal treatment of wildtype mice with the agonist Pam3Cys, which decreased inflammation and reduced bacterial shedding and transmission. Taken together, these results suggest that the innate immune response to influenza virus promotes bacterial shedding, allowing the bacteria to transit from host to host. These findings provide insight into the role of host factors in the increased pneumococcal carriage rates seen during flu season and contribute to our overall understanding of pathogen transmission.

PMID: 25166617 [PubMed - in process] PMCID: PMC4148449

Chemical Interference with Iron Transport Systems to Suppress Bacterial Growth of Streptococcus pneumoniae.

PLoS One. 2014 Aug 29;9(8):e105953. doi: 10.1371/journal.pone.0105953. eCollection 2014.

Chemical Interference with Iron Transport Systems to Suppress Bacterial Growth of Streptococcus pneumoniae.

Yang XY1, Sun B2, Zhang L1, Li N1, Han J1, Zhang J1, Sun X1, He QY1.

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Abstract

Iron is an essential nutrient for the growth of most bacteria. To obtain iron, bacteria have developed specific iron-transport systems located on the membrane surface to uptake iron and iron complexes such as ferrichrome. Interference with the iron-acquisition systems should be therefore an efficient strategy to suppress bacterial growth and infection. Based on the chemical similarity of iron and ruthenium, we used a Ru(II) complex R-825 to compete with ferrichrome for the ferrichrome-transport pathway in Streptococcus pneumoniae. R-825 inhibited the bacterial growth of S. pneumoniae and stimulated the expression of PiuA, the iron-binding protein in the ferrichrome-uptake system on the cell surface. R-825 treatment decreased the cellular content of iron, accompanying with the increase of Ru(II) level in the bacterium. When the piuA gene (SPD_0915) was deleted in the bacterium, the mutant strain became resistant to R-825 treatment, with decreased content of Ru(II). Addition of ferrichrome can rescue the bacterial growth that was suppressed by R-825. Fluorescence spectral quenching showed that R-825 can bind with PiuA in a similar pattern to the ferrichrome-PiuA interaction in vitro. These observations demonstrated that Ru(II) complex R-825 can compete with ferrichrome for the ferrichrome-transport system to enter S. pneumoniae, reduce the cellular iron supply, and thus suppress the bacterial growth. This finding suggests a novel antimicrobial approach by interfering with iron-uptake pathways, which is different from the mechanisms used by current antibiotics.

PMID: 25170896 [PubMed - in process] PMCID: PMC4149436

Preparation and testing of a Vi conjugate vaccine using pneumococcal surface protein A (PspA) from Streptococcus pneumoniae as the carrier protein.

Vaccine. 2014 Aug 27. pii: S0264-410X(14)01164-5. doi: 10.1016/j.vaccine.2014.08.041. [Epub ahead of print]

Preparation and testing of a Vi conjugate vaccine using pneumococcal surface protein A (PspA) from Streptococcus pneumoniae as the carrier protein.

Kothari N1, Genschmer KR2, Kothari S3, Kim JA3, Briles DE2, Rhee DK4, Carbis R5.

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Abstract

In the current study pneumococcal surface protein A (PspA) was conjugated to Vi capsular polysaccharide from Salmonella Typhi to make available a vaccine against typhoid fever that has the potential to also provide broad protection from Streptococcus pneumoniae. High yielding production processes were developed for the purification of PspAs from families 1 and 2. The purified PspAs were conjugated to Vi with high recovery of both Vi and PspA. The processes developed especially for PspA family 2 could readily be adapted for large scale production under cGMP conditions. Previously we have shown that conjugation of diphtheria toxoid (DT) to Vi polysaccharide improves the immune response to Vi but can also enhance the response to DT. In this study it was shown that conjugation of PspA to Vi enhanced the anti-PspA response and that PspA was a suitable carrier protein as demonstrated by the characteristics of a T-cell dependent response to the Vi. We propose that a bivalent vaccine consisting of PspA from families 1 and 2 bound to Vi polysaccharide would protect against typhoid fever and has the potential to also protect against pneumococcal disease and should be considered for use in developing countries.

KEYWORDS:

Conjugate vaccine; Pneumococcal surface protein A (PspA); Typhoid fever vaccine; Vaccine development; Vi polysaccharide

PMID: 25171842 [PubMed - as supplied by publisher]

TLR-Mediated Inflammatory Responses to Streptococcus pneumoniae Are Highly Dependent on Surface Expression of Bacterial Lipoproteins.

J Immunol. 2014 Aug 29. pii: 1401413. [Epub ahead of print]

TLR-Mediated Inflammatory Responses to Streptococcus pneumoniae Are Highly Dependent on Surface Expression of Bacterial Lipoproteins.

Tomlinson G1, Chimalapati S2, Pollard T2, Lapp T1, Cohen J3, Camberlein E2, Stafford S2, Periselneris J2, Aldridge C4, Vollmer W4, Picard C5, Casanova JL6, Noursadeghi M1, Brown J7.

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Abstract

Streptococcus pneumoniae infections induce inflammatory responses that contribute toward both disease pathogenesis and immunity, but the host-pathogen interactions that mediate these effects are poorly defined. We used the surface lipoprotein-deficient ∆lgt pneumococcal mutant strain to test the hypothesis that lipoproteins are key determinants of TLR-mediated immune responses to S. pneumoniae. We show using reporter assays that TLR2 signaling is dependent on pneumococcal lipoproteins, and that macrophage NF-κB activation and TNF-α release were reduced in response to the ∆lgt strain. Differences in TNF-α responses between Δlgt and wild-type bacteria were abrogated for macrophages from TLR2- but not TLR4-deficient mice. Transcriptional profiling of human macrophages revealed attenuated TLR2-associated responses to ∆lgt S. pneumoniae, comprising many NF-κB-regulated proinflammatory cytokine and chemokine genes. Importantly, non-TLR2-associated responses were preserved. Experiments using leukocytes from IL-1R-associated kinase-4-deficient patients and a mouse pneumonia model confirmed that proinflammatory responses were lipoprotein dependent. Our data suggest that leukocyte responses to bacterial lipoproteins are required for TLR2- and IL-1R-associated kinase-4-mediated inflammatory responses to S. pneumoniae.

PMID: 25172490 [PubMed - as supplied by publisher]

Adenylate kinase from Streptococcus pneumoniae is essential for growth through its catalytic activity.

FEBS Open Bio. 2014 Jul 8;4:672-82. doi: 10.1016/j.fob.2014.07.002. eCollection 2014.

Adenylate kinase from Streptococcus pneumoniae is essential for growth through its catalytic activity.

Thach TT1, Luong TT2, Lee S1, Rhee DK2.

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Abstract

Streptococcus pneumoniae (pneumococcus) infection causes more than 1.6 million deaths worldwide. Pneumococcal growth is a prerequisite for its virulence and requires an appropriate supply of cellular energy. Adenylate kinases constitute a major family of enzymes that regulate cellular ATP levels. Some bacterial adenylate kinases (AdKs) are known to be critical for growth, but the physiological effects of AdKs in pneumococci have been poorly understood at the molecular level. Here, by crystallographic and functional studies, we report that the catalytic activity of adenylate kinase from S . pneumoniae (SpAdK) serotype 2 D39 is essential for growth. We determined the crystal structure of SpAdK in two conformations: ligand-free open form and closed in complex with a two-substrate mimic inhibitor adenosine pentaphosphate (Ap5A). Crystallographic analysis of SpAdK reveals Arg-89 as a key active site residue. We generated a conditional expression mutant of pneumococcus in which the expression of the adk gene is tightly regulated by fucose. The expression level of adk correlates with growth rate. Expression of the wild-type adk gene in fucose-inducible strains rescued a growth defect, but expression of the Arg-89 mutation did not. SpAdK increased total cellular ATP levels. Furthermore, lack of functional SpAdK caused a growth defect in vivo. Taken together, our results demonstrate that SpAdK is essential for pneumococcal growth in vitro and in vivo.

KEYWORDS:

Adenylate kinase; Ap5A, adenosine pentaphosphate; Bacteria growth; Crystal structure; SpAdK, Streptococcus pneumoniae adenylate kinase; Streptococcus pneumoniae; Virulence factor

PMID: 25180151 [PubMed] PMCID: PMC4141199

Binding of vitronectin and Factor H to Hic contributes to immune evasion of Streptococcus pneumoniae serotype 3.

Thromb Haemost. 2014 Aug 28;112(6). [Epub ahead of print]

Binding of vitronectin and Factor H to Hic contributes to immune evasion of Streptococcus pneumoniae serotype 3.

Kohler S, Hallström T, Singh B, Riesbeck K, Sparta G, Zipfel PF, Hammerschmidt S1.

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Abstract

Streptococcus pneumoniae serotype 3 strains are highly resistant to opsonophagocytosis due to recruitment of the complement inhibitor Factor H via Hic, a member of the pneumococcal surface protein C (PspC) family. In this study, we demonstrated that Hic also interacts with vitronectin, a fluid-phase regulator involved in haemostasis, angiogenesis, and the terminal complement cascade as well as a component of the extracellular matrix. Blocking of Hic by specific antiserum or genetic deletion significantly reduced pneumococcal binding to soluble and immobilised vitronectin and to Factor H, respectively. In parallel, ectopic expression of Hic on the surface of Lactococcus lactis conferred binding to soluble and immobilised vitronectin as well as Factor H. Molecular analyses with truncated Hic fragments narrowed down the vitronectin-binding site to the central core of Hic (aa 151-201). This vitronectin-binding region is separate from that of Factor H, which binds to the N-terminus of Hic (aa 38-92). Binding of pneumococcal Hic was localised to the C-terminal heparin-binding domain (HBD3) of vitronectin. However, an N-terminal region to HBD3 was further involved in Hic-binding to immobilised vitronectin. Finally, vitronectin bound to Hic was functionally active and inhibited formation of the terminal complement complex. In conclusion, Hic interacts with vitronectin and simultaneously with Factor H, and both human proteins may contribute to colonisation and invasive disease caused by serotype 3 pneumococci.

KEYWORDS:

Complement; Hic; PspC11; Streptococcus pneumoniae; pathogenesis; serotype 3; vitronectin

PMID: 25181963 [PubMed - as supplied by publisher]

Recognition of Streptococcus pneumoniae and MDP by NOD2 results in a potent induction of MMP-9, which can be controlled by LPS stimulation.

Infect Immun. 2014 Sep 2. pii: IAI.02150-14. [Epub ahead of print]

Recognition of Streptococcus pneumoniae and MDP by NOD2 results in a potent induction of MMP-9, which can be controlled by LPS stimulation.

Vissers M1, Hartman Y1, Groh L1, de Jong DJ2, de Jonge MI1, Ferwerda G3.

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Abstract

Matrix metallopeptidase-9 (MMP-9) is a protease involved in the degradation of extracellular matrix collagen. Evidence suggests that MMP-9 is involved in the pathogenesis during a Streptococcus pneumoniae infection. However, not much is known about the induction of MMP-9 and the regulatory processes involved. We show that the Gram-positive bacteria used in this study induce high amounts of MMP-9 in contrast to the Gram-negative bacteria that were used. An important PAMP for Gram-positive bacteria is muramyl dipeptide (MDP). MDP is a very potent inducer of MMP-9 and showed a dose-dependent MMP-9 induction. Experiments using PBMCs from Crohn's disease patients with a non-functional NOD2 show that MMP-9 induction by Streptococcus pneumoniae and MDP is NOD2 dependent. Increasing amounts of LPS, an important PAMP for Gram-negative bacteria, resulted in decreasing amounts of MMP-9. Moreover, the induction of MMP-9 by MDP could be counteracted by simultaneously adding LPS. The inhibition of MMP-9 expression by LPS was found to be post-transcriptional regulated, independent of metalloproteinase-1 (TIMP-1), an endogenous inhibitor of MMP-9. Collectively, these data show that Streptococcus pneumoniae is able to induce high amounts of MMP-9. These high MMP-9 levels could potentially be involved in Streptococcus pneumoniae pathogenesis.

PMID: 25183734 [PubMed - as supplied by publisher]

Mouse, but Not Human, ApoB-100 Lipoprotein Cholesterol Is a Potent Innate Inhibitor of Streptococcus pneumoniae Pneumolysin.

PLoS Pathog. 2014 Sep 4;10(9):e1004353. doi: 10.1371/journal.ppat.1004353. eCollection 2014.

Mouse, but Not Human, ApoB-100 Lipoprotein Cholesterol Is a Potent Innate Inhibitor of Streptococcus pneumoniae Pneumolysin.

Wade KR1, Hotze EM1, Briles DE2, Tweten RK1.

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Abstract

Streptococcus pneumoniae produces the pore-forming toxin pneumolysin (PLY), which is a member of the cholesterol-dependent cytolysin (CDC) family of toxins. The CDCs recognize and bind the 3β-hydroxyl group of cholesterol at the cell surface, which initiates membrane pore formation. The cholesterol transport lipoproteins, which carry cholesterol in their outer monolayer, are potential off-pathway binding targets for the CDCs and are present at significant levels in the serum and the interstitial spaces of cells. Herein we show that cholesterol carried specifically by the ApoB-100-containing lipoprotein particles (CH-ApoB-100) in the mouse, but not that carried by human or guinea pig particles, is a potent inhibitor of the PLY pore-forming mechanism. Cholesterol present in the outer monolayer of mouse ApoB-100 particles is recognized and bound by PLY, which stimulates premature assembly of the PLY oligomeric complex thereby inactivating PLY. These studies further suggest that the vast difference in the inhibitory capacity of mouse CH-ApoB-100 and that of the human and the guinea pig is due to differences in the presentation of cholesterol in the outer monolayer of their ApoB-100 particles. Therefore mouse CH-ApoB-100 represents a significant innate CDC inhibitor that is absent in humans, which may underestimate the contribution of CDCs to human disease when utilizing mouse models of disease.

PMID: 25188225 [PubMed - in process] PMCID: PMC4154877

PLoS One. 2014 Sep 4;9(9):e107015. doi: 10.1371/journal.pone.0107015. eCollection 2014. Pronounced Metabolic Changes in Adaptation to Biofilm Growth by Streptococcus pneumoniae

PLoS One. 2014 Sep 4;9(9):e107015. doi: 10.1371/journal.pone.0107015. eCollection 2014.

Pronounced Metabolic Changes in Adaptation to Biofilm Growth by Streptococcus pneumoniae.

Allan RN1, Skipp P2, Jefferies J3, Clarke SC4, Faust SN5, Hall-Stoodley L6, Webb J7.

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Abstract

Streptococcus pneumoniae accounts for a significant global burden of morbidity and mortality and biofilm development is increasingly recognised as important for colonization and infection. Analysis of protein expression patterns during biofilm development may therefore provide valuable insights to the understanding of pneumococcal persistence strategies and to improve vaccines. iTRAQ (isobaric tagging for relative and absolute quantification), a high-throughput gel-free proteomic approach which allows high resolution quantitative comparisons of protein profiles between multiple phenotypes, was used to interrogate planktonic and biofilm growth in a clinical serotype 14 strain. Comparative analyses of protein expression between log-phase planktonic and 1-day and 7-day biofilm cultures representing nascent and late phase biofilm growth were carried out. Overall, 244 proteins were identified, of which >80% were differentially expressed during biofilm development. Quantitatively and qualitatively, metabolic regulation appeared to play a central role in the adaptation from the planktonic to biofilm phenotype. Pneumococci adapted to biofilm growth by decreasing enzymes involved in the glycolytic pathway, as well as proteins involved in translation, transcription, and virulence. In contrast, proteins with a role in pyruvate, carbohydrate, and arginine metabolism were significantly increased during biofilm development. Downregulation of glycolytic and translational proteins suggests that pneumococcus adopts a covert phenotype whilst adapting to an adherent lifestyle, while utilization of alternative metabolic pathways highlights the resourcefulness of pneumococcus to facilitate survival in diverse environmental conditions. These metabolic proteins, conserved across both the planktonic and biofilm phenotypes, may also represent target candidates for future vaccine development and treatment strategies. Data are available via ProteomeXchange with identifier PXD001182.

PMID: 25188255 [PubMed - in process] PMCID: PMC4154835

Quorum Sensing and Biofilms in the Pathogen, Streptococcus Pneumoniae.

Curr Pharm Des. 2014 Sep 5. [Epub ahead of print]

Quorum Sensing and Biofilms in the Pathogen, Streptococcus Pneumoniae.

Galante J, Ho AC, Tingey S, Charalambous BM1.

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Abstract

Bacteria are able to colonize and thrive in a variety of different environments as a biofilm, but only within the last half century new insights have been gained in this complex biosystem. Bacterial biofilms play a major role in human health by forming a defensive barrier against antibacterial chemical therapeutics and other potential pathogens, and in infectious disease when the bacteria invade normally sterile compartments. Quorum sensing is the signaling network for cell to cell communication and utilized by bacteria to regulate biofilms and other cellular processes. This review will describe recent advances in quorum sensing and biofilms. Initially, it will focus on Streptococcus pneumoniae biofilm regulation and the involvement of the ComABCDE pathway. As part of this review an original analysis of the genotypic and phenotypic variation of the signaling molecule, ComC and its cognate receptor ComD, firstly within the pneumococcal species and then within the genus Streptococcus will be presented. Additionally, a pathway similar to ComABCDE, the BlpABCSRH that regulates bacteriocin and immunity protein production that inhibit the growth of competing bacteria will be described. This review will then examine a third quorum sensing mechanism in the pneumococcus, the LuxS/AI-2, and present a novel gene and protein sequence comparative analysis that indicates its occurrence is more universal across bacterial genera compared with the Com pathway, with more sequence similarities between bacterial genera that are known to colonize the mucosal epithelium.

PMID: 25189864 [PubMed - as supplied by publisher]

Control of transcription elongation by GreA determines rate of gene expression in Streptococcus pneumoniae.

Nucleic Acids Res. 2014 Sep 4. pii: gku790. [Epub ahead of print]

Control of transcription elongation by GreA determines rate of gene expression in Streptococcus pneumoniae.

Yuzenkova Y1, Gamba P2, Herber M2, Attaiech L2, Shafeeq S2, Kuipers OP2, Klumpp S3, Zenkin N4, Veening JW5.

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Abstract

Transcription by RNA polymerase may be interrupted by pauses caused by backtracking or misincorporation that can be resolved by the conserved bacterial Gre-factors. However, the consequences of such pausing in the living cell remain obscure. Here, we developed molecular biology and transcriptome sequencing tools in the human pathogen Streptococcus pneumoniae and provide evidence that transcription elongation is rate-limiting on highly expressed genes. Our results suggest that transcription elongation may be a highly regulated step of gene expression in S. pneumoniae. Regulation is accomplished via long-living elongation pauses and their resolution by elongation factor GreA. Interestingly, mathematical modeling indicates that long-living pauses cause queuing of RNA polymerases, which results in 'transcription traffic jams' on the gene and thus blocks its expression. Together, our results suggest that long-living pauses and RNA polymerase queues caused by them are a major problem on highly expressed genes and are detrimental for cell viability. The major and possibly sole function of GreA in S. pneumoniae is to prevent formation of backtracked elongation complexes.

PMID: 25190458 [PubMed - as supplied by publisher]

Effects of recombinant IL-17F intranasal inoculation against Streptococcus pneumoniae infection in a murine model.

Biotechnol Appl Biochem. 2014 Sep 4. doi: 10.1002/bab.1286. [Epub ahead of print]

Effects of recombinant IL-17F intranasal inoculation against Streptococcus pneumoniae infection in a murine model.

Chen L1, Guo S, Wu LX, Hao CL, Xu WT, Zhang JH.

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Abstract

Interleukin (IL)-17F is an important member of IL-17 cytokine family, which plays important roles in host defense against microbial infections. Streptococcus pneumoniae (S. pneumoniae) is a common pathogen associated with several invasive and non-invasive pneumococcal diseases, and mucosal immune response plays crucial roles in defenses against pneumococcal infection. Thus, intranasal inoculation may be an alternative approach against pneumococci. In this study, BALB/c mice were intranasally inoculated with recombinant IL-17F (rIL-17F) prior to S. pneumoniae (ATCC 6303, serotype 3) infection. Compared to control group, the numbers of total leukocyte, neutrophil and macrophage in lung were significantly increased in mice inoculated with rIL-17F. The levels of macrophage inflammatory protein (MIP)-1α, MIP-2β and interferon (IFN)-γ were significantly increased in bronchoalveolar lavage fluid (BALF) and culture supernatant of splenocytes from mice inoculated with rIL-17F. rIL-17F inoculation also significantly elevated β-defensin-2 (Defb2) expression in lung tissue. Furthermore, compared to S. pneumoniae infection group, rIL-17F inoculation prior to infection significantly reduced S. pneumoniae colonization in lung. These findings demonstrated that rIL-17F intranasal inoculation strengthened host defense against pneumococci, which may be developed to prevent pneumococcal infection. This article is protected by copyright. All rights reserved.

KEYWORDS:

Streptococcus pneumoniae; inflammation; interleukin-17F; β-defensin-2

PMID: 25196250 [PubMed - as supplied by publisher]

Recent Approaches in Vaccine Development against Streptococcus pneumoniae.

J Mol Microbiol Biotechnol. 2014 Aug 30;24(4):215-227. [Epub ahead of print]

Recent Approaches in Vaccine Development against Streptococcus pneumoniae.

Tarahomjoo S.

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Abstract

Streptococcus pneumoniae is a major cause of morbidity and mortality among children under 5 years of age worldwide. Vaccines have long been used for protection against pneumococcal infections. Capsular polysaccharides of pneumococci are main antigenic components of these vaccines. However, pneumococcal polysaccharide-based vaccines are not able to elicit appropriate immunological responses in young children and cannot induce the immune memory. Thus, pneumococcal conjugate vaccines were developed through chemical coupling of an immunogenic carrier protein to the capsule. The currently available pneumococcal conjugate vaccines elicited protection against the bacterium efficiently. However, these vaccines are expensive to manufacture and have limited serotype coverage. In this mini-review, therefore, we describe approaches attempted by researchers to circumvent the shortcomings of the conjugate vaccines including specifying appropriate cultivation conditions for the production of S. pneumoniae capsular antigens, development of suitable expression systems for the frequently used carrier protein in the conjugate vaccines (cross-reacting material 197), construction of protein-based vaccines, whole-cell vaccines, DNA vaccines, and using antigen delivery vehicles. Future trends in this field are also discussed. © 2014 S. Karger AG, Basel.

PMID: 25196525 [PubMed - as supplied by publisher]