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CXC chemokine responses of respiratory epithelial cells to Streptococcus pneumoniae.Graham, Rikki Marie Ann January 2005 (has links)
Title page, table of contents and abstract only. The complete thesis in print form is available from the University of Adelaide Library. / Streptococcus pneumoniae (the pneumococcus) remains a major cause of morbidity and mortality worldwide, particularly in young children and the elderly. It is responsible for a spectrum of diseases ranging from otitis media, to potentially fatal conditions such as pneumonia and meningitis, and is estimated to cost health services billions of dollars each year. The interaction of S. pneumoniae with the host generally begins in the nasopharynx, and invasive disease is almost invariably preceded by nasopharyngeal colonisation. In some circumstances, S. pneumoniae may translocate from the nasopharynx to the lungs where pneumonia can develop, and inflammation is believed to play a role in this process. The presence of pneumococci in the lungs also triggers an inflammatory response, which is important for clearance of the bacteria. However, a prolonged inflammatory response leads to tissue damage, and is linked with a poor prognosis of disease. It has been shown that respiratory epithelial cells are able to play an active part in the response to respiratory pathogens by releasing chemokines that are responsible for neutrophil recruitment, and it has recently been shown that infection of type II pneumocytes with S. pneumoniae leads to the release of interleukin (IL)-8. In order to determine the role of specific pneumococcal factors in eliciting a CXC chemokine response from type II pneumocytes (A549) and nasopharyngeal cells (Detroit-562), monolayers of these cells were infected with wild type (WT) S. pneumoniae 039, or mutants deficient in choline binding protein A (CbpA), pneumococcal surface protein A (PspA), or pneumolysin (Ply), and the CXC chemokine mRNA response was measured by real-time RT-PCR. Release of IL-8 was also measured by ELISA. In response to WT D39, both A549 and Detroit-562 cells showed a significant increase in CXC chemokine mRNA, and IL-8 protein. This response was increased 2-fold when a CbpA-negative (ACbpA) mutant was used to infect cells, suggesting that CbpA may have an inhibitory effect on the CXC chemokine response of these cells. Further investigatiDn demonstrated that this activity is dependent on the N-terminal region of CbpA and that all three N-terminal domains are required for this effect, as deletion of any one of these domains had the same effect on the CXC chemokine response as removing CbpA altogether. Infection with a PspA-negative mutant (APspA) led to a 2-fold decrease in the CXC chemokine response of A549 cells, compared to infection with WT D39 at 2 h, but no difference was seen in the response of Detroit-562 cells to this mutant compared to WT D39. Thus, PspA appears to have the ability to stimulate an early CXC chemokine release from A549 cells. Deletion of the first of 2 regions of the N-terminal a-helical domain of PspA reduced the ability of S pneumoniae to elicit a chemokine response to the same degree as removing PspA altogether, indicating that it is this region that is responsible for the chemokine inducing ability of PspA. Ply appeared to have no effect on the CXC chemokine response of A549 cells with no obvious difference seen in the response of these cells to APly compared to WT D39. However, infection of Detroit-562 cells with APly led to a 2-fold decrease in IL-8 mRNA and protein release compared to WT D39. Using D39 strains producing mutant forms of Ply with reduced cytotoxicity and/or complement activating abilities, the role of the cytotoxic activity of Ply was demonstrated to be important in generation of a chemokine response from both cell lines. Infection of A549 or Detroit-562 cells with mutants producing Ply with only 0.02% or 0.1% haemolytic activity led to a 2-fold decrease in IL-8 release compared to that elicited by WT D39. The complement activating ability of Ply also appeared to be important in the generation of a CXC chemokine response from A549 cells. Cells infected with a mutant that produced Ply with no complement activating ability released significantly less IL-8 than cells infected with WT D39. This activity of Ply did not appear to have an effect on the CXC chemokine release of Detroit-562 cells. Thus all three virulence factors investigated had some role in the ability of S. pneumoniae to generate a CXC chemokine response from respiratory epithehal cells, although their roles and the cell lines that were affected differed. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1225410 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Sciences, 2005
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Evaluation of the random amplified polymorphic DNA technique for the epidemiological investigation of streptococcus pneumoniae outbreaks.Friedland, Hillel David January 1994 (has links)
A dissertation submitted to the Faculty of Medicine, University of the
Witwatersrand, Johannesburg, in fulfilment of the requirements for the
degree of Master of Medicine. / The emergence of strains of S. pneumoniae resistant to penicillin and to other
antibiotics, and the spread of that resistance over the world, have become
major concerns and increase the need for epidemiological surveillance. The
following typing methods have been used to detect strain variability in
pneumococci: Serotyping, antibiotic susceptibility profiles, multilocus
enzyme electrophoresis (MLEE), penicillin-binding protein (PBP) profiles,
pulse-field gel electrophoresis (pFGE), and ribotyping. Serotyping,
antibiograms, and MLEE only detect phenotypic variability. PBP gene profiles,
PFGE, and ribotyping detect genotypic differences but these techniques are
labour intensive and time consuming.
Random amplified polymorphic DNA (RAPD) is a new technique that bas
proved useful for typing bacteria, fungi, and parasites, but has not been.
studied using pneumococci. Unlike conventional polymerase chain reaction
(peR), RAPD utilizes single, short primers, usually 10 oligonucleotides in
length. As the primer is short and low astringency annealing temperatures are
used, there will be many complimentary sites scattered randomly throughout
a bacterium's genome, When such sites occur a few hundred base pairs away
from each other and on opposite DNA strands, the enclosed region can be
amplified by peR This results in numerous discrete target fragments which
can be separated by agarose-gel electrophoresis and ethidium bromide
staining. RAPD requires no sequence information and it scans the whole
genome rather than relying on hypervariability within one specific gene.
The aims of this study were: to determine strain variability using RAPD, to
determine the reproducibility ofRAPD, and to demonstrate intercontinental
spread of a multiresistant pneumococcal strain.
The following strains were evaluated: a) 10 strains from a day-care centre
(DCC), the index case being a 3 year old girl 'with otitis media. An aunt from
Spain had recently been staying with the family. The other strains were
isolated from class mates and siblings of the index case.; b) 18 clinical
isolates from Seoul, Korea; and c) 11 epidemiologically unrelated isolates
from South Africa, including the reference strain, R6.
Two DNA extraction methods were used. The first involving lysis with
sodmm-dodecyl-sulphaze and proteinase K. Proteins were removed with
phenol-chloroform, and the DNA precipitated with ethanol. The second
method involved incubating the cells at 95 0C for 15 microlitres, followed by
centrifugation. 2 microlitres of the supernatant was then used for each PCR
reaction, Three primers were evaluated.
After 01uimisation of the RAPDreaction for pneumococci, the final peR
mixtures per 50 ul was: primer (4 plY1), template (0.5 ng), nuc1eotides (300
pMeach), magnesium (4 mM~, and Taq polymerase (2 U). 35 cycles were
used with an annealing temperature of 35'C.
Both DNA extraction methods: gave reproducible results but were not
comparable to each other. All 10 strains from the DCC gave the same
banding pattern as the Spanish done for all 3 primers. 7 of the Korean strains
gave the same banding pattern as the Spanish clone using the first two
primers, however one strain showed an additional band using the third
primer. Of the remaining 22 strains, 21 different banding patterns were
obtained.
This study has shown that RAPD is a simple and rapid technique that can
distinguish strain variation among pneumococci. The reproducibility is
excellent within the same laboratory. Finally using RAPD. this study
identified a Spanish multiresistant 23F clone in South Africa and Korea. / Andrew Chakane 2018
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Clonagem, expressão e purificação das proteínas de superfície, PsaA e fragmentos de PspA de Streptococcus pneumoniae / Cloning, expression and purification of proteins of surface, PsaA and fragments of PspA from Streptococcus pneumoniaeSilva, Marcelo da 25 April 2005 (has links)
Streptococcus pneumoniae é o principal causador da pneumonia bacteriana. As vacinas atualmente disponíveis contêm polissacarídeo capsular conjugado ou não com proteínas carreadoras. No entanto, elas apresentam elevado custo ou proteção reduzida nos grupos de risco (crianças abaixo de 5 anos de idade e idosos). Proteínas de superfície de S. pneumoniae, como a PsaA e PspA, são consideradas fortes candidatas vacinais. Com o objetivo de se desenvolver uma vacina de ampla cobertura e baixo custo contra pneumococos, os genes psaA e pspA foram clonados em vetores de expressão em E. coli, pAE e pET e as proteínas expressas foram purificadas por cromatografias de afinidade e de troca aniônica. O rendimento de proteína recombinante obtido com a construção baseada em pET foi 3 vezes maior que o obtido com pAE. Condições de cultivo foram estabelecidas utilizando meio definido com indução por IPTG e/ou por lactose. As cepas recombinantes estão adequadas para serem usadas em estudos para escalonamento da produção em biorreatores. / Streptococcus pneumoniae is the main causative agent of bacterial pneumonia. The current vaccines available contain capsular polysaccharide conjugated or not with carrier proteins. However these are either too expensive or do not protect the high-risk groups. Surface proteins of S. pneumoniae, such as PsaA and PspA, are considered strong vaccine candidates. With the aim of developing a broad-coverage and low-cost vaccine against pneumococcus, the psaA and pspA genes were cloned in E. coli expression vectors, pAE and pET and the expressed proteins were purified through affinity and anion exchange chromatography. The yield of the recombinant protein obtained with the construction based in pET was 3-fold higher than that obtained with pAE. Culture conditions were established using defined media with IPTG and/or lactose induction. The recombinant strains are now ready to undergo studies for scale-up of production in bioreactors.
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Clonagem, expressão e purificação das proteínas de superfície, PsaA e fragmentos de PspA de Streptococcus pneumoniae / Cloning, expression and purification of proteins of surface, PsaA and fragments of PspA from Streptococcus pneumoniaeMarcelo da Silva 25 April 2005 (has links)
Streptococcus pneumoniae é o principal causador da pneumonia bacteriana. As vacinas atualmente disponíveis contêm polissacarídeo capsular conjugado ou não com proteínas carreadoras. No entanto, elas apresentam elevado custo ou proteção reduzida nos grupos de risco (crianças abaixo de 5 anos de idade e idosos). Proteínas de superfície de S. pneumoniae, como a PsaA e PspA, são consideradas fortes candidatas vacinais. Com o objetivo de se desenvolver uma vacina de ampla cobertura e baixo custo contra pneumococos, os genes psaA e pspA foram clonados em vetores de expressão em E. coli, pAE e pET e as proteínas expressas foram purificadas por cromatografias de afinidade e de troca aniônica. O rendimento de proteína recombinante obtido com a construção baseada em pET foi 3 vezes maior que o obtido com pAE. Condições de cultivo foram estabelecidas utilizando meio definido com indução por IPTG e/ou por lactose. As cepas recombinantes estão adequadas para serem usadas em estudos para escalonamento da produção em biorreatores. / Streptococcus pneumoniae is the main causative agent of bacterial pneumonia. The current vaccines available contain capsular polysaccharide conjugated or not with carrier proteins. However these are either too expensive or do not protect the high-risk groups. Surface proteins of S. pneumoniae, such as PsaA and PspA, are considered strong vaccine candidates. With the aim of developing a broad-coverage and low-cost vaccine against pneumococcus, the psaA and pspA genes were cloned in E. coli expression vectors, pAE and pET and the expressed proteins were purified through affinity and anion exchange chromatography. The yield of the recombinant protein obtained with the construction based in pET was 3-fold higher than that obtained with pAE. Culture conditions were established using defined media with IPTG and/or lactose induction. The recombinant strains are now ready to undergo studies for scale-up of production in bioreactors.
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Effect of nicotine on streptococcus mutansHuang, Ruijie 11 1900 (has links)
Indiana University-Purdue University Indianapolis (IUPUI) / Streptococcus mutans is a key contributor to dental caries. Smokers have increased caries, but the association between tobacco, nicotine, caries and S. mutans growth is little investigated. In the first section, seven S. mutans strains were used for screening. The minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and minimum biofilm inhibitory concentration (MBIC) were 16 mg/ml (0.1 M), 32 mg/ml (0.2 M), and 16 mg/ml (0.1 M), respectively, for most of the S. mutans strains. Growth of planktonic S. mutans cells was significantly repressed by 2.0-8.0 mg/ml nicotine concentrations. Biofilm formation and metabolic activity of S. mutans was increased in a nicotine-dependent manner up to 16.0 mg/ml. Scanning electron microscopy (SEM) revealed higher nicotine-treated S. mutans had thicker biofilm and more spherical bacterial cells than lower concentrations of nicotine. In the second section, confocal laser scanning microscopy (CLSM) results demonstrated that both biofilm bacterial cell numbers and extracellular polysaccharide (EPS) synthesis were increased by nicotine. Glucosyltransferase (Gtf) and glucan binding protein A (GbpA) protein expression of S. mutans planktonic cells were upregulated, while GbpB protein expression of biofilm cells were downregulated by nicotine. The mRNA expression of those genes were mostly consistent with their protein results. Nicotine was not directly involved in S. mutans LDH activity. However, since it increased the total number of bacterial cells in biofilm; total LDH activity of S. mutans biofilm was increased. In the third section, a PCR-based multiple species cell counting (PCR-MSCC) method was designed to investigate the effect of nicotine on S. mutans in a ten mixed species culture. The absolute S. mutans number in mixed biofilm culture was increased but the percentage of S. mutans in the total number of bacterial cells was not changed.
In conclusion, nicotine enhanced biofilm formation and biofilm metabolism of S. mutans, through stimulating S. mutans planktonic cell Gtfs and Gbps expression. This leads to more planktonic cells attaching to dental biofilm. Increased S. mutans cell numbers, in biofilms of single species or ten mixed species, resulted in higher overall LDH activity. More lactic acid may be generated and contribute to caries development in smokers.
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