New insights into the sociomicrobiology of Streptococcus pneumoniae : exploring biofilm formation

Tese de doutoramento, Ciências e Tecnologias da Saúde (Microbiologia), Universidade de Lisboa, Faculdade de Medicina, 2011During the last decade it has become more evident that Streptococcus pneumoniae (pneumococcus) holds a high genetic diversity throughout its population. Two important biological resources for achieving pneumococcalgenetic plasticity are competence/natural transformation and phage transduction. Both factors strongly contribute to genome modification and have a real impact in the capacity of survival and adaptation of this bacterium. Another important survival skill of the pneumococcus is its ability to form matrix‐enclosed biofilms. These microbial communities guarantee protection from environmental threats such as host immune defenses and antibiotics. Moreover, due to the fact that bacteria are believed to spend most of their lifetime in biofilms and that there is a higher physical proximity of bacteria within these structures, biofilms are probably the prefered stage for the occurrence of genetic exchange between pneumococci. This thesis contributes to a better understanding of the mechanisms that generate pneumococcal genetic diversity and the important bacterial ability to form biofilms. In the pursuit of this goal, two biological conditions were selected: a) prophage carriage and b) pherotype characterization, resulting in three independent studies: i) the study of the impact of prophage spontaneous activation on pneumococcal biofilm formation, ii) a molecular epidemiology study of the distribution of pherotypes and its contribution to pneumococcal genetic differentiation and iii) the study of the influence of pherotypes on pneumococcal biofilm growth and recombination efficiency. The study of the effect of prophage carriage and its spontaneously induced host lysis on pneumococcal biofilms reveal that, although limited phage induction results in the death of their bacterial hosts, the bacterial population as a whole benefits from prophage carriage and an enhancement in biofilm formation is observed. Moreover, this study shows a link between the external DNA (eDNA) that is released to the medium due to bacterial lysis and the growth of pneumococcal biofilms. The molecular epidemiology study performed showed that of the two dominants pherotypes (CSP1 and CSP2) the majority of the invasive isolates screened presented the CSP1 pherotype. Several associations with the pherotypes and other biological markers were observed indicating that pherotypes are not randomly distributed within the pneumococcal population. Associations with serotype, antimicrobial resistance and genetic lineage were unveiled; it was also detected that phage transduction may be indirectly arbitrated by pherotypes, implicating an uneven distribution of large genetic elements such as some genetic determinants of antibiotic resistance. The study also showed that strains that are phylogenetically closer have a higher likelihood of sharing the same pherotype. Severe limitations to inter‐pherotype communication may be leading towards an ongoing genetic drift, explaining the two genetically distinct subpopulations that were detected. Moving forward, we decided to explore the impact of the two major pherotypes on both the capacity to form biofilms and on recombination efficiency. Biofilms of strains presenting CSP1 had increased biofilm mass and were more densely packed. Also, the addition of synthetic cognate CSP amplifies the observed differences in biofilm growth between the pherotypes. The study also revealed that CSP1 strains transform more efficient both in the liquid medium and within the biofilm structure. Taken together this thesis work has shown that genetic exchanges between pneumococcal strains are occurring preferentially between strains sharing the same pherotypes and that the enhancement of biofilm formation detected both by prophage carriage and by CSP signaling have in common the positive impact of DNA release to the extracellular medium resulting from the lysis of a fraction of the bacteria inside the biofilm.A última década assistiu a um aumento das evidências reunidas sobre a elevada diversidade genética existente na população de Streptococcus pneumoniae.A competência associada à transformação natural e a transdução fágica são dois recursos biológicos importantes para a plasticidade genómica do pneumococo. Ambos contribuem de forma signficativa para a modificação genómica e têm um impacto visível na capacidade de adaptação e sobrevivência desta bactéria. Outra estratégia de sobrevivência do pneumococo é a sua capacidade de formação de biofilmes. Estas comunidades bacterianas constituem mecanismos de protecção face a ameaças ambientais tais como os mecanismos de defesa do sistema imunitário do hospedeiro e os antibióticos. Além disso, devido ao facto das bactérias existirem maioritamente sob a forma de biofilmes e de existir uma maior proximidade física entre as bactérias nestas estruturas, os biofilmes são provavelmente o palco principal para a ocorrência das trocas genéticas. Esta tese contribui para um maior conhecimento da relação entre mecanismos geradores de diversidade genética e a capacidade de formação de biofilmes em Streptococcus pneumoniae. Com esse intuito foram seleccionadas duas características biológicas: a) a presença de fagos lisogénicos no genoma bacteriano e b) a diversidade ferotípica, resultando em três estudos independentes: i) o impacto da activação espontânea de fagos lisogénicos na formação de biofilmes pneumocócicos, ii) o estudo de genética de populações sobre a distribuição dos ferótipos e a sua contribuição para a diferenção genética no pneumococo, iii) o estudo da influência dos ferótipos na formação de biofilmes pneumocócicos e na respectiva eficiência de recombinação. O estudo do efeito da presença de fagos lisogénicos no genoma bacteriano e da sua lise espontânea nos biofilmes pneumocócicos mostrou que, apesar da indução fágica resultar na morte das respectivas bactérias hospedeiras, a população bacteriana como um todo beneficia da presença dos fagos lisogénicos observando‐se um aumento na capacidade de formação de biofilmes. Este estudo permitiu ainda mostrar a existência de uma ligação entre a libertação de DNA e o aumento dos biofilmes pneumocócicos. O estudo de genética de populações permitiu mostrar que, de entre os dois ferótipos dominantes no pneumococo (CSP1 e CSP2), a maioria das estirpes invasivas caracterizadas apresenta o ferótipo CSP1. Várias associações entre os ferótipos e outros marcadores biológicos foram identificadas indicando que os ferótipos não estão aleatoriamente distribuídos na população pneumocócica. Foram detectadas associações com o serótipo, a resistência antimicrobiana e a linhagem genética; os dados obtidos sugerem igualmente que a transdução fágica pode ser indirectamente arbitrada pelo ferótipo, implicando uma distribuição heterogénea de elementos genéticos de maior dimensão, como é o caso de alguns determinantes genéticos de resistência antimicrobiana. O estudo permitiu ainda mostrar que estirpes filogeneticamente mais próximas têm uma maior probabilidade de partilhar o mesmo ferótipo. Limitações severas à comunicação entre ferótipos parecem estar a conduzir a população pneumocócica para uma situação de afastamento genético continuado, explicando deste modo as duas populações geneticamente distintas que foram detectadas ao longo deste estudo. No âmbito desta tese foi ainda explorado o impacto dos dois ferótipos dominantes na capacidade de formação de biofilmes e na respectiva eficiência de transformação. As estirpes com CSP1 apresentam biofilmes mais densos e com mais biomassa. Além disso, a adição de CSP sintético respectivo amplifica as diferenças observadas no crescimento dos biofilmes entre ferótipos. Este estudo mostra também que estirpes CSP1 são mais eficientes no processo de transformação em meio líquido e em biofilme. Em conjunto, os resultados obtidos nesta tese mostram que as trocas genéticas entre estirpes pneumocócicas estão a ocorrer preferencialmente entre estirpes que partilham o mesmo ferótipo e que o aumento na formação de biofilme detectado quer como consequência da presença do fago lisógenico quer pela via de sinalização do CSP, têm em comum o impacto positivo da libertação de DNA para o meio extracelular resultante da lise de uma fracção de bactérias dentro do biofilme

Pherotypes are driving genetic differentiation within Streptococcus pneumoniae

<p>Abstract</p> <p>Background</p> <p>The boundaries of bacterial species and the mechanisms underlying bacterial speciation are matters of intense debate. Theoretical studies have shown that recombination acts as a strong cohesive force preventing divergence in bacterial populations. <it>Streptococcus pneumoniae </it>populations have the telltale signs of high recombination with competence implicated as the major driving force behind gene exchange. Competence in <it>S. pneumoniae </it>is triggered by a quorum-sensing mechanism controlled by the competence-stimulating peptide pheromone.</p> <p>Results</p> <p>We studied the distribution of the two major pherotypes in the pneumococcal population and their association with serotype, antimicrobial resistance and genetic lineage. Using multilocus sequence data we evaluated pherotype influence on the dynamics of horizontal gene transfer. We show that pherotype is a clonal property of pneumococci. Standard population genetic analysis and multilocus infinite allele model simulations support the hypothesis that two genetically differentiated populations are defined by the major pherotypes.</p> <p>Conclusion</p> <p>Severe limitations to gene flow can therefore occur in bacterial species in the absence of geographical barriers and within highly recombinogenic populations. This departure from panmixia can have important consequences for our understanding of the response of pneumococci to human imposed selective pressures such as vaccination and antibiotic use.</p

The impact of exercise training on liver transplanted familial amyloidotic polyneuropathy (FAP) patients

Liver transplantation is nowadays the only effective answer to adjourn the outcome of functional limitations associated with familial amyloidotic polyneuropathy (FAP), a neurodegenerative disease characterized by sensory and motor polyneuropathies. Nevertheless, there is a detrimental impact associated with the after-surgery period on the fragile physical condition of these patients. Exercise training has been proven to be effective on reconditioning patients after transplantation. However, the effects of exercise training in liver transplanted FAP patients have not been scrutinized yet

Distribution of pherotype-characterized strains according to the ability to form <i>in vitro</i> biofilms.

<p>A total of 90 strains with pherotype CSP1 (n = 67) or CSP2 (n = 23) were screened for their ability to form biofilms in 96-well plates without the addition of synthetic CSP. After 24h of incubation, the resulting biofilm was measured by crystal violet staining. Each plotted value is an average of 9 replicates normalized by the average biofilm mass measured for TIGR4 strain (OD_595nm = 0.147). Horizontal lines represent the relative biofilm formation of R36A, TIGR4 and their isogenic switched-pherotype mutant strains.</p

Hill function parameters estimated by non-linear regression for the transformation efficiency assay of Δ<i>comC</i> strains.

<p>* <i>T</i> were normalized by the maximal transformation efficiency obtained for TIGR4Δ<i>comC</i> (10^7.62 CFU/ml).</p

Comparison of intra-pherotype and inter-pherotype recombination in biofilms and liquid cultures.

<p>Each bar represents the fraction of double resistant CFUs in the total number of CFUs, normalized by the same fraction obtained for TIGR4 intrapherotype transformation (0.048 in biofilm and 4.1×10⁻⁶ in liquid culture). Experiments were performed both in biofilm (panel A) and in liquid culture (panel B) by mixing equal amounts of two strains, each with one antibiotic resistance marker (novobiocin and streptomycin). In each experiment, the two leftmost bars represent mixtures of strains with the same pherotype and on the right pherotypes were mixed to evaluate inter-pherotype recombination. White bars represent mixtures of CSP1 producing strains, grey bars CSP2 producing strains and hatched bars mixtures of CSP1 and CSP2 producing strains. In the left slanted hatched bars the CSP1 producing strain carried the novobiocin resistance marker, while the CSP2 producing strain carried the streptomycin resistance marker. In the right slanted hatched bars the resistance markers on the strains were reversed. Results show that in biofilms intra-pherotype recombination is more frequent than inter-pherotype recombination. The opposite relationship is observed in liquid culture. Error bars represent the 95% confidence intervals of the mean of 3 or 9 (biofilms and liquid culture, respectively) independent experiments. Each independent biofilm experiment consisted of 3 technical replicates. ANOVA was applied to identify significant differences in the fraction of transformants between the four types of mixed cultures. Significant differences were found (p<10⁻⁴) for both R36A and TIGR4 backgrounds and in both biofilm and liquid culture. Groups with significant differences according to post-hoc analysis are identified in the figure by horizontal brackets. In all cases p<10⁻³, except for the difference in transformants obtained in liquid culture with the R36A background between CSP1+CSP1 and CSP2+CSP2 mixtures, where p<10⁻².</p

eDNA quantification and DNA impact on biofilm mass.

<p><b>A</b>) Extracellular DNA was isolated from the biofilm matrices of R36A, R36AΔ<i>lytA</i>, R36AP, R36APΔ<i>svl</i>, R36APΔ<i>lytA</i> and R36APΔ<i>lytA</i>Δ<i>svl</i> and quantitative real-time PCR of two chromosomal genes, <i>spi</i> and <i>gdh</i>, was done. The relative biomass was quantified at OD_{595 nm} and the eDNA measurements were normalized to total biofilm mass. <b>B</b>) The effect of salmon sperm DNA (1000 ng/ml) added from seeding on biofilm biomass at 24 h was tested. R36APΔ<i>svl</i>, R36APΔ<i>lytA</i>, R36AΔ<i>lytA</i> and R36APΔ<i>lytA</i>Δ<i>svl</i> are mutants in which the phage lysin (Svl), the bacterial autolysin (LytA) or both were deleted. <b>C</b>) The same experiments described in panel B were done with the encapsulated wild type host of phage SV1, strain SVMC28, and its mutants. SVMC28Δ<i>svl</i>, SVMC28Δ<i>lytA</i> and SVMC28Δ<i>lytA</i>Δ<i>svl</i> are mutants in which the phage lysin (Svl), the bacterial autolysin (LytA) or both were deleted. In all panels, the results are an average of 9 independent replicates and error bars represent 95% confidence intervals for the sample mean.</p

Confocal laser scanning microscopy images of R3A6P and R36A biofilms.

<p>Staining was done with Syto 9/PI (Live/Dead BacLight Bacterial Viability kit) and images were acquired at 630×amplification. Only live cells internalize Syto 9 (fluorescing green) whereas dead cells allow the uptake of PI (fluorescing red). The large images are optical sections of top views and the small images to the right and above are optical sections of side views. The depth of the biofilm is indicated by the height of the <i>z</i>-stack. The inset scale bar represents 5 µm. <b>A</b>) Biofilm formed by the lysogenic strain R36AP. <b>B</b>) Biofilm of the non-lysogenic strain R36A. <b>C</b>) The biofilm was grown in the presence of salmon sperm DNA at 1000 ng/ml. <b>D</b>) The biofilm was grown in medium supplemented with DNase I at 50 µg/ml. In all panels the results are representative images of 3 independent experiments and biofilm growth was evaluated at 24 h.</p

Genistein-supplemented diet decreases malaria liver infection in mice and constitutes a potential prophylactic strategy

Contains fulltext : 70187.pdf (publisher's version ) (Open Access)In tropical regions millions of people still live at risk of malaria infection. Indeed the emergence of resistance to chloroquine and other drugs in use in these areas reinforces the need to implement alternative prophylactic strategies. Genistein is a naturally occurring compound that is widely used as a food supplement and is thought to be effective in countering several pathologies. Results presented here show that genistein inhibits liver infection by the Plasmodium parasite, the causative agent of malaria. In vitro, genistein decreased the infection rates of both mouse and human hepatoma cells by inhibiting the early stages of the parasite's intracellular development. Oral or intraperitoneal administration of genistein decreased the liver parasite load of P. berghei-infected mice. Moreover, mice fed on a genistein-supplemented diet showed a significant reduction in Plasmodium liver infection as well as a reduced blood parasitemia and partial protection from severe disease. Since genistein is a safe, low-cost, natural compound that can be used permanently in a diet, we propose its use as a prophylactic agent against malaria for endemic populations and long-time travelers