The impact of pneumolysin on the macrophage response to Streptococcus pneumoniae is strain-dependent

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

Genome analysis of a highly virulent serotype 1 strain of streptococcus pneumoniae from West Africa

Streptococcus pneumoniae is a leading cause of pneumonia, meningitis, and bacteremia, estimated to cause 2 million deaths annually. The majority of pneumococcal mortality occurs in developing countries, with serotype 1 a leading cause in these areas. To begin to better understand the larger impact that serotype 1 strains have in developing countries, we characterized virulence and genetic content of PNI0373, a serotype 1 strain from a diseased patient in The Gambia. PNI0373 and another African serotype 1 strain showed high virulence in a mouse intraperitoneal challenge model, with 20% survival at a dose of 1 cfu. The PNI0373 genome sequence was similar in structure to other pneumococci, with the exception of a 100 kb inversion. PNI0373 showed only15 lineage specific CDS when compared to the pan-genome of pneumococcus. However analysis of non-core orthologs of pneumococcal genomes, showed serotype 1 strains to be closely related. Three regions were found to be serotype 1 associated and likely products of horizontal gene transfer. A detailed inventory of known virulence factors showed that some functions associated with colonization were absent, consistent with the observation that carriage of this highly virulent serotype is unusual. The African serotype 1 strains thus appear to be closely related to each other and different from other pneumococci despite similar genetic content

Presence of Nonhemolytic Pneumolysin in Serotypes of Streptococcus pneumoniae Associated with Disease Outbreaks

Pneumolysin is an important virulence factor of the human pathogen Streptococcus pneumoniae. Sequence analysis of the ply gene from 121 clinical isolates of S. pneumoniae uncovered a number of alleles. Twenty-two strains were chosen for further analysis, and 14 protein alleles were discovered. Five of these had been reported previously, and the remaining 9 were novel. Cell lysates were used to determine the specific hemolytic activities of the pneumolysin proteins. Six strains showed no hemolytic activity, and the remaining 16 were hemolytic, to varying degrees. We report that the nonhemolytic allele reported previously in serotype 1, sequence type (ST) 306 isolates is also present in a number of pneumococcal isolates of serotype 8 that belong to the ST53 lineage. Serotype 1 and 8 pneumococci are known to be associated with outbreaks of invasive disease. The nonhemolytic pneumolysin allele is therefore associated with the dominant clones of outbreak-associated serotypes of S. pneumonia

Characterising virulence factors from pathogenic bacteria using fluorescent reporters

Protein translocation systems are invaluable to pathogenic bacteria, facilitating the display of virulence factors on their surface or their release into the extracellular environment. Some protein export systems are ubiquitous and essential to cell survival whereas others are horizontally acquired on prophages or pathogenicity islands (PAI), in many cases providing the bacterium with pathogenic advantages. For the majority of the known protein export systems, their structure, function and secreted substrates have been characterised, yet some proteins have been identified that are secreted via unknown mechanisms. Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 is an important cause of human foodborne disease worldwide. The pathogenesis of this bacterium is mainly attributed to the secretion of toxins and the presence of a Type III Secretion System (T3SS). The T3SS can translocate bacterial proteins, known as effectors, into the host cell which mediate an effect culminating in the formation of a characteristic attaching and effacing (A/E) lesion. This system is encoded on a horizontally acquired PAI termed the locus of enterocyte effacement (LEE). The LEE not only encodes the T3SS apparatus but also several effectors secreted by the system and transcription factors which regulate its expression. However, it was recently found that T3SS not only secretes LEE encoded effectors but can also secrete proteins encoded on other prophages present in the EHEC genome. Characterisation of these non-LEE encoded effectors is ongoing and this study investigates the expression, regulation and function of non-LEE encoded effector H1 (NleH1) and H2 (NleH2). NleH1 and NleH2 are secreted by the T3SS but are encoded on different prophages. This study demonstrates that expression of NleH1 and NleH2 is induced in the same in vitro conditions which stimulate the expression of the LEE but is diminished upon initial host cell contact in vitro. Transcription of nleH1 and nleH2 is dependant upon factors specific to E. coli O157:H7 and these factors are regulated by LEE encoded regulators Ler and GrlA, as they have a positive effect on nleH transcription. NleH1 and H2 are predicted serine/threonine protein kinases and are able to autophosphorylate. Yeast two hybrid screening and 2D differential gel electrophoresis did not elucidate a eukaryotic protein binding partner of NleH1 or NleH2. Transfection assays show that they do not have a significant effect upon NF-κB activation in vitro. Determining the expression, regulation and function of non-LEE encoded effectors contributes towards further understanding of how this pathogen causes disease. Streptococcus pneumoniae, also known as the pneumococcus, is another globally important human pathogen. It is a very diverse pathogen, with over 90 capsular serotypes and is naturally competent for DNA uptake. Pneumococcal pathogenesis is facilitated by the production of a pore-forming toxin, pneumolysin. Pneumolysin’s activities in pneumococcal pathogenesis extend beyond its cytolytic function as it can also activate the complement pathway and modulate the host cytoskeleton. Pneumolysin is a member of a conserved family of toxins known as the cholesterol dependant cytolysins but differs due to the lack of a secretion signal peptide within its sequence. This indicates that it is not secreted from the bacterium however it has been reported that some strains can release pneumolysin in a cell lysis-independent manner. Additional to this, pneumolysin can also localise to the cell wall, and this localisation is not strain dependent. This study characterised codon-optimised N-terminally labelled pneumolysin constructs and applied them to assess the localisation of pneumolysin. In addition, the importance of autolysin and genes which are co-transcribed with Ply upon the localisation/secretion of pneumolysin was investigated by construction of a pneumococcal strain carrying an autolysin-pneumolysin fusion which naturally occurs in equine strains. These genes were not required for the translocation of pneumolysin or its association with the cell wall. Growth of this strain, and its isogenic parent, in vitro at a low density and low temperature resulted in the pneumolysin being detected in the broth culture. This indicates that pneumolysin can be released from the cell wall and that this action is not dependant upon the genes which were deleted in the mutant. The distribution of pneumolysin on the pneumococcal surface was assessed with immunofluorescence, and LumioTM substrate fluorescence, microscopy and found to have a general distribution. As a contribution to future pneumococcal research, codon-optimised fluorescent protein reagents were developed and can be used as reporters for gene expression and protein localisation

Ecology of virulence genes in the human pathogen Streptococcus pneumoniae.

Streptococcus pneumoniae, also known as the pneumococcus, is an important human pathogen, with high burdens of disease and mortality worldwide. There are over 90 serotypes of this pathogen, demonstrating the vast amounts of diversity present. Currently, there are two pneumococcal vaccines, both targeting the polysaccharide capsule. However, one vaccine is ineffective in the paediatric population, whilst the other only targets a minority of disease-causing serotypes, and has increased disease caused by serotypes not present in the vaccine. One solution is a new pneumococcal vaccine targeting a protein virulence factor possessed by all pneumococci, which would afford cross-serotype protection. As a result, it is important to assess the diversity of pneumococcal virulence factors in order to determine their potential as vaccine candidates, as excess diversity present may prevent full serotype-independent protection of a vaccine. Furthermore, diversity studies offer important insight on pneumococcal biology, epidemiology and pathogenesis. The diversity in the toxin pneumolysin (Ply) was greater than previously thought, with 14 protein alleles discovered. However, diversity remained significantly lower than surface-exposed virulence factors, indicating this toxin may be more suitable as a vaccine candidate. Furthermore, all 14 alleles were recognised by polyclonal antibodies, showing the potential cross-serotype protection of a vaccine targeting this toxin. A novel non-haemolytic Ply allele was associated with clones recently expanding in the pneumococcal population, as well as serotypes associated with outbreaks of pneumococcal disease. The non-haemolytic toxin may therefore play a role in driving clonal expansion in certain genetic backgrounds, or be involved in establishing outbreaks of pneumococcal disease. The diversity in the neuraminidase A (NanA) enzyme was significantly higher than in Ply, with many point mutations, mosaic blocks and insertions regions present in 18 divergent alleles. This level of diversity should not be prohibitive to the use of this protein as a vaccine candidate, as polyclonal antibodies recognised 4 NanA alleles of significant diversity, indicating the possibility of cross-serotype protection. The role of NanA in pathogenesis of pneumococcal haemolytic uraemic syndrome (p-HUS) was investigated, although there was no correlation between p-HUS and NanA allele or activity. The novel discovery that pneumococcal NanA was inhibited by viral neuraminidase inhibitors of influenza allowed insight into the synergistic relationship between these two deadly pathogens, and showed for the first time that treatment with these drugs acts on both the primary and secondary pathogen. One of these inhibitors, Oseltamivir, was found to have potential in treating secondary pneumococcal pneumonia, which may help decrease the significant burden of this disease, as well as reducing the over-reliance on antibiotics for treating pneumococcal diseases. Homologues of Ply and NanA were identified and characterised in the related species Streptococcus mitis and Streptococcus pseudopneumoniae, giving insight into the evolutionary relationships between these species. Furthermore, the presence of these homologues in related species gives rise to the possibility of pneumococci acquiring altered genes through horizontal gene transfer. The selective pressure of a vaccine targeting these proteins may give evolutionary advantage to these pneumococci, resulting in evasion of a vaccine. Microarray studies have been used to assess pneumococcal diversity at a genome-wide level. Gene expression studies identified candidate genes which may play a role in p-HUS pathogenesis. Further studies into this area will improve the diagnosis and treatment of this disease. Furthermore, a large number of established pneumococcal virulence factors, many of which are vaccine candidates, were found to have homologues in closely related commensal species. These results must be taken into consideration for future protein-based pneumococcal vaccine design

Rapid Differentiation of Pneumococci And Viridans Group Streptococci by MALDI-TOF Mass Spectrometry And a Rapid Nucleic Acid Amplification Test in A Clinical Microbiology Laboratory

Streptococcus pneumoniae is one of the most significant human bacterial pathogens. It is a major causative agent of community-acquired pneumonia. It is also the most common cause of bacterial otitis media and among most common bacteria causing meningitis in children. The close relatives of S. pneumoniae, the Viridans group streptococci form a central part of the human oral microbiome, but they are also a leading cause of endocarditis. The fast and reliable identification of these bacteria from clinical specimens would therefore be of prime importance for a clinical microbiology laboratory, allowing for the laboratory to provide helpful information to clinicians for identifying the infection focus and targeting antibiotic treatment. The close relationship between S. pneumoniae and Viridans group streptococci, especially their subgroup Mitis group streptococci, complicates the reliable species level identification of these bacteria. The identification of streptococci by the traditional biochemical methods is both unreliable and time-consuming: the identification takes 1-2 days after bacterial growth has been detected on a plate or in a blood culture bottle. Even sequencing of the 16S ribosomal RNA gene commonly used as a reference method for bacterial identification cannot reliably differentiate between pneumococci and other Mitis group streptococci or identify other Mitis group streptococci to the species level. For the identification of pneumococci, detection of pneumococcal-specific genes is considered a “golden standard”. For the Viridans group streptococci, multi-locus sequence typing (MLST), based on the analysis of the sequences of several genes, is considered to be the most reliable method of identification. However, the MLST is too laborious and time-consuming for the identification of streptococci in a routine clinical microbiology laboratory. Within the last decade, MALDI-TOF mass spectrometry has enabled the fast and reliable identification of most bacterial species. However, exceptionally closely related bacterial groups pose a challenge even to the MALDI-TOF technology. This holds true even for pneumococci and Mitis group streptococci. In this study, the earlier database and algorithm versions of the commonly used MALDI-TOF systems were shown to be unable to reliably differentiate between pneumococci and Mitis group streptococci. Therefore, rapid molecular detection tests, such as GenomEra S. pneumoniae™ evaluated in this study, are still needed for rapid detection of pneumococci. However, it seems that the addition of more pneumococcal and Mitis group strains in the databases and new interpretation algorithms allow for reliable differentiation between pneumococci and the Mitis group streptococci. Altogether, the combination of rapid gene detection tests and MALDI-TOF technology with enhanced databases and identification algorithms enables fast, reliable and cost-effective differentiation between pneumococci and Mitis group streptococci and the reliable group level identification of other Mitis group streptococci. This is a sufficient level of identification in a clinical microbiology laboratory, enabling the clinician to evaluate the clinical significance of the finding and target further investigations and antibiotic treatment more specifically.Pneumokokki (Streptococcus pneumoniae) on merkittävimpiä ihmisen bakteeripatogeenejä: se on merkittävä avohoitosyntyisen keuhkokuumeen ja lasten välikorvatulehdusten aiheuttaja ja aiheuttaa myös suuren osan lasten aivokalvontulehduksista. Sen lähisukulaiset, viridans-ryhmän streptokokit, taas muodostavat merkittävän osan ihmisen suun normaalifloorasta, mutta ovat myös keskeisiä sydänkalvontulehduksen aiheuttajia. Pneumokokkien ja viridans-ryhmän streptokokkien nopea ja luotettava tunnistus kliinisen mikrobiologian laboratoriossa olisi kuitenkin ensiarvoisen tärkeää, koska tunnistustulos voi auttaa lääkäriä tunnistamaan infektiofokuksen ja suuntaamaan antibioottihoidon oikein. Pneumokokin ja viridans-ryhmän, erityisesti sen alaryhmän, mitis-ryhmän streptokokkien lähisukulaisuus vaikeuttaa näiden bakteerien luotettavaa erottamista toisistaan ja tunnistamista lajitasolle. Perinteisillä biokemiallisilla tunnistusmenetelmillä pneumokokkien ja viridans-ryhmän streptokokkien tunnistus vie yhdestä kahteen vuorokautta siitä, kun maljalla tai veriviljelypullossa on havaittu bakteerikasvua. Edes bakteerien tunnistuksessa yleisesti käytetty 16S rRNA-geenin sekvensointi ei kykene luotettavasti erottelemaan pneumokokkeja mitis-ryhmän streptokokeista eikä viridans-streptokokkeja lajitasolle. Pneumokokkien tunnistuksessa referenssimenetelmänä käytetäänkin pneumokokeille tyypillisten geenien monistusta. Viridans-ryhmän streptokokkien kohdalla tällaisena “kultaisena standardina” käytetään usean geenin nukleiinihappojärjestyksen analysointiin perustuvaa tyypitystä (MLST). Useiden geenien sekvenssianalyysiin perustuva tunnistus on kuitenkin liian kallista ja aikaa vievää kliinisen mikrobiologian laboratorion tarpeisiin. Viimeisen vuosikymmenen aikana MALDI-TOF-massaspektrometria on mahdollistanut useimpien bakteerien nopean ja luotettavan tunnistamisen. Toisilleen poikkeuksellisen läheistä sukua olevat bakteerit ovat kuitenkin haasteellisia tunnistettavia myös MALDI-TOF:lla. Tämä pätee myös pneumokokkeihin ja Mitis-ryhmän streptokokkeihin. Tässä työssä osoitettiin, että varhaisempia tietokantaversioita ja algoritmejä käytettäessä tavallisimmin käytetyt MALDI-TOF-laitteistot eivät kyenneet luotettavasti erottamaan pneumokokkeja Mitis-ryhmän streptokokeista. Tämän vuoksi geenimonistustestit kuten tässä työssä arvioitu GenomEra S. pneumoniae™ ovat tarpeen pneumokokkien nopeassa osoituksessa. Tämän tutkimuksen kolmas osatyö osoitti kuitenkin, että uusien pneumokokki- ja Mitis-ryhmän streptokokkikantojen lisääminen ja uusien tunnistusalgoritmien kehittäminen mahdollistavat pneumokokkien ja Mitis-ryhmän streptokokkien luotettavan erottamisen toisistaan myös MALDI-TOF:lla. MALDI-TOF:n ja nopean nukleiinihappotestin käyttö mahdollistavat pneumokokkien ja Viridans-streptokokkien nopean, luotettavan ja kustannustehokkaan erottamisen sekä Viridans-streptokokkien luotettavan tunnistuksen ryhmätasolle. Tämä tunnistustaso on riittävä kliinisen mikrobiologian laboratorion tarpeisiin, sillä hoitava lääkäri pystyy sen perusteella arvioimaan löydöksen kliinistä merkitystä ja kohdistamaan mahdollisia jatkotutkimuksia ja antibioottihoitoa tarkemmin

A cardinal role for cathepsin D in co-ordinating the host-mediated apoptosis of macrophages and killing of pneumococci

The bactericidal function of macrophages against pneumococci is enhanced by their apoptotic demise, which is controlled by the anti-apoptotic protein Mcl-1. Here, we show that lysosomal membrane permeabilization (LMP) and cytosolic translocation of activated cathepsin D occur prior to activation of a mitochondrial pathway of macrophage apoptosis. Pharmacological inhibition or knockout of cathepsin D during pneumococcal infection blocked macrophage apoptosis. As a result of cathepsin D activation, Mcl-1 interacted with its ubiquitin ligase Mule and expression declined. Inhibition of cathepsin D had no effect on early bacterial killing but inhibited the late phase of apoptosis-associated killing of pneumococci in vitro. Mice bearing a cathepsin D-/- hematopoietic system demonstrated reduced macrophage apoptosis in vivo, with decreased clearance of pneumococci and enhanced recruitment of neutrophils to control pulmonary infection. These findings establish an unexpected role for a cathepsin D-mediated lysosomal pathway of apoptosis in pulmonary host defense and underscore the importance of apoptosis-associated microbial killing to macrophage function

The importance of pneumococcal capsule in inate immunity and biofilm formation

Chapter 1 gives an overview about Streptococcus pneumoniae, its role as a human pathogen and its virulence factors. Additionally, biofilm development and its relevance in clinics are introduced, and the innate immune response to pneumococcus as well as bacterial-viral interactions in the upper respiratory tract are also discussed. Chapter 2 emphasizes the three main topics of this thesis: the role of capsule and pneumolysin in the immune response in the respiratory tract, biofilm formation of S. pneumoniae serotypes and commensal streptococci in vitro, and host innate immune responses to RSV and S. pneumoniae during in vitro co-infections. Aims and hypotheses are provided here. Chapter 3 is divided into two parts: First, the release of the pro-inflammatory cytokines CXCL8 and IL-6 from the human pharyngeal epithelial cell line Detroit 562 and from human bronchial epithelial cells (iHBEC) is described in response to S. pneumoniae. Capsule was shown to suppress the release of both cytokines in both cell lines tested, but release was much less from iHBEC cells. During intranasal colonization of mice, suppression of CXCL8 release by the capsule was also observed in vivo, but the effect was only measured in the absence of pneumolysin. Long term, stable nasopharyngeal carriage in a mouse model resulted in the dissemination of nonencapsulated pneumococci into the lungs, whereas encapsulated strains remained in the nasopharynx. The S. pneumoniae capsule thus plays a role in modulation of the pro-inflammatory immune response in the respiratory tract. Second, results on immunological cells and immune regulation in a long term, stable nasopharyngeal carriage mouse model are presented. Mice were infected with encapsulated or nonencapsulated pneumococcal strains, and after 1, 3, 8 and 15 days, were sacrificed to evaluate the numbers of CD45+ cells, neutrophils, macrophages, FoxP3+ regulatory T-cells and CD3+ T-cells in the nasal mucosa as well as the amount of secreted IL-10 in the nasopharynx. Nasopharyngeal colonization which is effectively silent resulted in the stimulation of FoxP3+ regulatory T-cells and IL-10 release associated with immune homeostasis, whereas lung infiltration was required to increase the number of neutrophils and macrophages resulting in a stronger innate immune response in the nasal mucosa. Chapter 4 contains results of mono- and co-stimulation using RSV and pneumococci or pneumococcal virulence factors on the human bronchial epithelial cell line BEAS-2B. An increase in CXCL8 and IL-6 levels was measured for mixed stimulations of RSV and pneumococcus when encapsulated bacteria were used. Increasing pneumolysin concentrations resulted in enhanced CXCL8 levels. Priming of bronchial epithelial cells with RSV opens the door for more severe pneumococcal infections. Chapter 5 is composed of two parts: The first part describes initial biofilm formation of serotypes 6B and 7F in a static model in vitro. Biofilms of both serotypes contained SCVs, but only serotype 6B increased in SCV formation between 16 and 65h of incubation. SCV stability was tested by passaging clones in complex medium, where SCV production is not associated with advantages in growth. Serotype 6B lost the SCV phenotype indicating a fast adaptation to a changing nutritional environment. Limitations of our in vitro model are discussed. The second part is about initial biofilm formation of mixed culture growth of S. pneumoniae with commensal streptococci. Competition dominates this process. S. oralis and pneumococcus compete for nutrients, whereas mixed species growth of S. mitis or S. pseudopneumoniae with S. pneumoniae is mainly influenced by other factors. In Chapter 6 the findings of chapters 3, 4 and 5 are discussed and an outlook for further studies is provided. Chapters 7, 8, 9, 10 and 11 contain the references, the acknowledgements, the curriculum vitae, the appendix and the declaration of originality