The photosynthetic bacterial reaction center in native and artificial envirnoments: effects on light-induced electron transfer

In this thesis we focussed on the characterization of the reaction center (RC) protein purified from the photosynthetic bacterium Rhodobacter sphaeroides. In particular, we discussed the effects of native and artificial environment on the light-induced electron transfer processes. The native environment consist of the inner antenna LH1 complex that copurifies with the RC forming the so called core complex, and the lipid phase tightly associated with it. In parallel, we analyzed the role of saccharidic glassy matrices on the interplay between electron transfer processes and internal protein dynamics. As a different artificial matrix, we incorporated the RC protein in a layer-by-layer structure with a twofold aim: to check the behaviour of the protein in such an unusual environment and to test the response of the system to herbicides. By examining the RC in its native environment, we found that the light-induced charge separated state P+QB - is markedly stabilized (by about 40 meV) in the core complex as compared to the RC-only system over a physiological pH range. We also verified that, as compared to the average composition of the membrane, the core complex copurifies with a tightly bound lipid complement of about 90 phospholipid molecules per RC, which is strongly enriched in cardiolipin. In parallel, a large ubiquinone pool was found in association with the core complex, giving rise to a quinone concentration about ten times larger than the average one in the membrane. Moreover, this quinone pool is fully functional, i.e. it is promptly available at the QB site during multiple turnover excitation of the RC. The latter two observations suggest important heterogeneities and anisotropies in the native membranes which can in principle account for the stabilization of the charge separated state in the core complex. The thermodynamic and kinetic parameters obtained in the RC-LH1 complex are very close to those measured in intact membranes, indicating that the electron transfer properties of the RC in vivo are essentially determined by its local environment. The studies performed by incorporating the RC into saccharidic matrices evidenced the relevance of solvent-protein interactions and dynamical coupling in determining the kinetics of electron transfer processes. The usual approach when studying the interplay between internal motions and protein function consists in freezing the degrees of freedom of the protein at cryogenic temperature. We proved that the “trehalose approach” offers distinct advantages with respect to this traditional methodology. We showed, in fact, that the RC conformational dynamics, coupled to specific electron transfer processes, can be modulated by varying the hydration level of the trehalose matrix at room temperature, thus allowing to disentangle solvent from temperature effects. The comparison between different saccharidic matrices has revealed that the structural and dynamical protein-matrix coupling depends strongly upon the sugar. The analyses performed in RCs embedded in polyelectrolyte multilayers (PEM) structures have shown that the electron transfer from QA - to QB, a conformationally gated process extremely sensitive to the RC environment, can be strongly modulated by the hydration level of the matrix, confirming analogous results obtained for this electron transfer reaction in sugar matrices. We found that PEM-RCs are a very stable system, particularly suitable to study the thermodynamics and kinetics of herbicide binding to the QB site. These features make PEM-RC structures quite promising in the development of herbicide biosensors. The studies discussed in the present thesis have shown that, although the effects on electron transfer induced by the native and artificial environments tested are markedly different, they can be described on the basis of a common kinetic model which takes into account the static conformational heterogeneity of the RC and the interconversion between conformational substates. Interestingly, the same distribution of rate constants (i.e. a Gamma distribution function) can describe charge recombination processes in solutions of purified RC, in RC-LH1 complexes, in wet and dry RC-PEM structures and in glassy saccharidic matrices over a wide range of hydration levels. In conclusion, the results obtained for RCs in different physico-chemical environments emphasize the relevance of the structure/dynamics solvent/protein coupling in determining the energetics and the kinetics of electron transfer processes in a membrane protein complex.

Effects of solar radiation on marine bacterial and phytoplankton heterotrophic activities

Programa de doctorado: Oceanografía (Bienio 2006-2008). Universidad de Las Palmas de Gran Canaria, Departamento de Biología y Institut de Ciéncies del Mar.

New insight on a Group A Streptococcus protective antigen contributing to bacterial cell division

Bioinformatic analysis of Group A Streptococcus (GAS) genomes aiming at the identification of new vaccine antigens, revealed the presence of a gene coding for a putative surface-associated protein, named GAS40, inducing protective antibodies in an animal model of sepsis. The aim of our study was to unravel the involvement of GAS40 in cell division processes and to identify the putative interactor. Firstly, bioinformatic analysis showed that gas40 shares homology with ezrA, a gene coding for a negative regulator of Z-ring formation during cell division process. Both scanning and transmission electron microscopy indicated morphological differences between wild-type and the GAS40 knock-out mutant strain, with the latter showing an impaired capacity to divide resulting in the formation of very long chains. Moreover, when the localization of the antigen on the bacterial surface was analyzed, we found that in bacteria grown at exponential phase GAS40 specifically localized at septum, indicating a possible role in cell division. Furthermore, by ELISA and co-sedimentation assays, we found that GAS40 is able to interact with FtsZ, a protein involved in Z-ring formation during cell division process. These data together with the co-localization of GAS40/FtsZ at bacterial septum demonstrated by by confocal microscopy, strongly support the hypothesis for a key role of GAS40 in bacterial cell division.

Clostridium difficile toxins facilitate bacterial colonization by modulating the fence and gate function of colonic epithelium

The contribution of Clostridium difficile toxin A and B (TcdA and TcdB) to cellular intoxication has been extensively studied, but their impact on bacterial colonization remains unclear. By setting-up two- and three-dimensional in vitro models of polarized gut epithelium, we investigated how C. difficile infection is affected by host cell polarity and whether TcdA and TcdB contribute to such events. Indeed, we observed that C. difficile adhesion and penetration of the epithelial barrier is substantially enhanced in poorly polarized or EGTA-treated cells, indicating that bacteria bind preferentially to the basolateral cell surface. In this context, we demonstrated that sub-lethal concentrations of C. difficile TcdA are able to alter cell polarity by causing redistribution of plasma membrane components between distinct surface domains. Taken together, the data suggest that toxin-mediated modulation of host cell organization may account for the capacity of this opportunistic pathogen to gain access to basolateral receptors leading to a successful colonization of the colonic mucosa.

Analysis of Two Component Systems in Group B Streptococcus Shows that RgfAC and the Novel FspSR Modulate Virulence and Bacterial Fitness

Group B Streptococcus (GBS), in its transition from commensal to pathogen, will encounter diverse host environments and thus require coordinately controlling its transcriptional responses to these changes. This work was aimed at better understanding the role of two component signal transduction systems (TCS) in GBS pathophysiology through a systematic screening procedure. We first performed a complete inventory and sensory mechanism classification of all putative GBS TCS by genomic analysis. Five TCS were further investigated by the generation of knock-out strains, and in vitro transcriptome analysis identified genes regulated by these systems, ranging from 0.1-3% of the genome. Interestingly, two sugar phosphotransferase systems appeared differently regulated in the knock-out mutant of TCS-16, suggesting an involvement in monitoring carbon source availability. High throughput analysis of bacterial growth on different carbon sources showed that TCS-16 was necessary for growth of GBS on fructose-6-phosphate. Additional transcriptional analysis provided further evidence for a stimulus-response circuit where extracellular fructose-6-phosphate leads to autoinduction of TCS-16 with concomitant dramatic up-regulation of the adjacent operon encoding a phosphotransferase system. The TCS-16-deficient strain exhibited decreased persistence in a model of vaginal colonization and impaired growth/survival in the presence of vaginal mucoid components. All mutant strains were also characterized in a murine model of systemic infection, and inactivation of TCS-17 (also known as RgfAC) resulted in hypervirulence. Our data suggest a role for the previously unknown TCS-16, here named FspSR, in bacterial fitness and carbon metabolism during host colonization, and also provide experimental evidence for TCS-17/RgfAC involvement in virulence.

Influence of plant structure, cultural pratices and environmental conditions on the development of the bacterial canker of kiwifruits

Italy has a preeminent rank in kiwifruit industry, being the first exporter and the second largest producer after China. However, in the last years kiwifruit yields and the total cultivated area considerably decreased, due to the pandemic spread of the bacterial canker caused by Pseudomonas syringae pv. actinidiae (Psa). Several climatic conditions and cultural practices affect the development of the bacterial canker. This research work focused on the impact of agricultural practices and microclimate conditions on the incidence and epidemiology of Psa in the orchard. Therefore, the effect of fertilization, irrigation, use of bio-regulators, rootstock, training system and pruning were examined. The effect of different tunnel systems was analyzed as well, to study the plant-pathogen interaction. Considering the importance of insects as vectors in other pathosystems, the role of Metcalfa pruinosa in the spread of the bacterial canker was investigated in controlled conditions. In addition, quality and storage properties of fruits from infected plants were assessed. The study of all these aspects of the agronomic practices is useful to define a strategy to limit the bacterial diffusion in the orchard. Overall, excess nitrogen fertilization, water stress, stagnant water supplies, pruning before summer and the high number of Metcalfa pruinosa increased the Psa incidence. In contrast, tunnel covers may be useful for the control of the disease, with special attention to the kind of material.

Stochastic modeling of bacterial protein domains distribution to predict horizontal gene transfer

In questo elaborato, abbiamo tentato di modellizzare i processi che regolano la presenza dei domini proteici. I domini proteici studiati in questa tesi sono stati ottenuti dai genomi batterici disponibili nei data base pubblici (principalmente dal National Centre for Biotechnology Information: NCBI) tramite una procedura di simulazione computazionale. Ci siamo concentrati su organismi batterici in quanto in essi la presenza di geni trasmessi orizzontalmente, ossia che parte del materiale genetico non provenga dai genitori, e assodato che sia presente in una maggiore percentuale rispetto agli organismi più evoluti. Il modello usato si basa sui processi stocastici di nascita e morte, con l'aggiunta di un parametro di migrazione, usato anche nella descrizione dell'abbondanza relativa delle specie in ambito delle biodiversità ecologiche. Le relazioni tra i parametri, calcolati come migliori stime di una distribuzione binomiale negativa rinormalizzata e adattata agli istogrammi sperimentali, ci induce ad ipotizzare che le famiglie batteriche caratterizzate da un basso valore numerico del parametro di immigrazione abbiano contrastato questo deficit con un elevato valore del tasso di nascita. Al contrario, ipotizziamo che le famiglie con un tasso di nascita relativamente basso si siano adattate, e in conseguenza, mostrano un elevato valore del parametro di migrazione. Inoltre riteniamo che il parametro di migrazione sia direttamente proporzionale alla quantità di trasferimento genico orizzontale effettuato dalla famiglia batterica.

The role of the interplay between polymer architecture and bacterial surface properties on the microbial adhesion to polyoxazoline-based ultrathin films

Surface platforms were engineered from poly(L-lysine)-graft-poly(2-methyl-2-oxazoline) (PLL-g-PMOXA) copolymers to study the mechanisms involved in the non-specific adhesion of Escherichia coli (E. coli) bacteria. Copolymers with three different grafting densities (PMOXA chains/Lysine residue of 0.09, 0.33 and 0.56) were synthesized and assembled on niobia (Nb O ) surfaces. PLL-modified and bare niobia surfaces served as controls. To evaluate the impact of fimbriae expression on the bacterial adhesion, the surfaces were exposed to genetically engineered E. coli strains either lacking, or constitutively expressing type 1 fimbriae. The bacterial adhesion was strongly influenced by the presence of bacterial fimbriae. Non-fimbriated bacteria behaved like hard, charged particles whose adhesion was dependent on surface charge and ionic strength of the media. In contrast, bacteria expressing type 1 fimbriae adhered to the substrates independent of surface charge and ionic strength, and adhesion was mediated by non-specific van der Waals and hydrophobic interactions of the proteins at the fimbrial tip. Adsorbed polymer mass, average surface density of the PMOXA chains, and thickness of the copolymer films were quantified by optical waveguide lightmode spectroscopy (OWLS) and variable-angle spectroscopic ellipsometry (VASE), whereas the lateral homogeneity was probed by time-of-flight secondary ion mass spectrometry (ToF-SIMS). Streaming current measurements provided information on the charge formation of the polymer-coated and the bare niobia surfaces. The adhesion of both bacterial strains could be efficiently inhibited by the copolymer film only with a grafting density of 0.33 characterized by the highest PMOXA chain surface density and a surface potential close to zero.

Is detection of bacterial DNA in ascitic fluid of clinical relevance?

In patients with cirrhosis, bacterial DNA has been found in ascites reflecting bacterial translocation. However, the clinical relevance of this finding is ill-defined especially compared with the standard diagnostics for detection of spontaneous bacterial peritonitis (SBP). Furthermore, other DNA tests have not been sufficiently evaluated.

Air suctioning during colon biopsy forceps removal reduces bacterial air contamination in the endoscopy suite

Bacterial contamination of endoscopy suites is of concern; however studies evaluating bacterial aerosols are lacking. We aimed to determine the effectiveness of air suctioning during removal of biopsy forceps in reducing bacterial air contamination.

Bacterial colonization and infection of electrophysiological cardiac devices detected with sonication and swab culture

Electrophysiological cardiac devices are increasingly used. The frequency of subclinical infection is unknown. We investigated all explanted devices using sonication, a method for detection of microbial biofilms on foreign bodies.

Factors predicting protracted improvement after pallidal DBS for primary dystonia: the role of age and disease duration

In many patients, optimal results after pallidal deep brain stimulation (DBS) for primary dystonia may appear over several months, possibly beyond 1 year after implant. In order to elucidate the factors predicting such protracted clinical effect, we retrospectively reviewed the clinical records of 44 patients with primary dystonia and bilateral pallidal DBS implants. Patients with fixed skeletal deformities, as well as those with a history of prior ablative procedures, were excluded. The Burke-Fahn-Marsden Dystonia Rating Scale (BFMDRS) scores at baseline, 1 and 3 years after DBS were used to evaluate clinical outcome. All subjects showed a significant improvement after DBS implants (mean BFMDRS improvement of 74.9% at 1 year and 82.6% at 3 years). Disease duration (DD, median 15 years, range 2-42) and age at surgery (AS, median 31 years, range 10-59) showed a significant negative correlation with DBS outcome at 1 and 3 years. A partition analysis, using DD and AS, clustered subjects into three groups: (1) younger subjects with shorter DD (n = 19, AS < 27, DD ? 17); (2) older subjects with shorter DD (n = 8, DD ? 17, AS ? 27); (3) older subjects with longer DD (n = 17, DD > 17, AS ? 27). Younger patients with short DD benefitted more and faster than older patients, who however continued to improve 10% on average 1 year after DBS implants. Our data suggest that subjects with short DD may expect to achieve a better general outcome than those with longer DD and that AS may influence the time necessary to achieve maximal clinical response.

Intravaginal practices, bacterial vaginosis, and HIV infection in women: individual participant data meta-analysis

Background Identifying modifiable factors that increase women's vulnerability to HIV is a critical step in developing effective female-initiated prevention interventions. The primary objective of this study was to pool individual participant data from prospective longitudinal studies to investigate the association between intravaginal practices and acquisition of HIV infection among women in sub-Saharan Africa. Secondary objectives were to investigate associations between intravaginal practices and disrupted vaginal flora; and between disrupted vaginal flora and HIV acquisition. Methods and Findings We conducted a meta-analysis of individual participant data from 13 prospective cohort studies involving 14,874 women, of whom 791 acquired HIV infection during 21,218 woman years of follow-up. Data were pooled using random-effects meta-analysis. The level of between-study heterogeneity was low in all analyses (I2 values 0.0%–16.1%). Intravaginal use of cloth or paper (pooled adjusted hazard ratio [aHR] 1.47, 95% confidence interval [CI] 1.18–1.83), insertion of products to dry or tighten the vagina (aHR 1.31, 95% CI 1.00–1.71), and intravaginal cleaning with soap (aHR 1.24, 95% CI 1.01–1.53) remained associated with HIV acquisition after controlling for age, marital status, and number of sex partners in the past 3 months. Intravaginal cleaning with soap was also associated with the development of intermediate vaginal flora and bacterial vaginosis in women with normal vaginal flora at baseline (pooled adjusted odds ratio [OR] 1.24, 95% CI 1.04–1.47). Use of cloth or paper was not associated with the development of disrupted vaginal flora. Intermediate vaginal flora and bacterial vaginosis were each associated with HIV acquisition in multivariable models when measured at baseline (aHR 1.54 and 1.69, p<0.001) or at the visit before the estimated date of HIV infection (aHR 1.41 and 1.53, p<0.001), respectively. Conclusions This study provides evidence to suggest that some intravaginal practices increase the risk of HIV acquisition but a direct causal pathway linking intravaginal cleaning with soap, disruption of vaginal flora, and HIV acquisition has not yet been demonstrated. More consistency in the definition and measurement of specific intravaginal practices is warranted so that the effects of specific intravaginal practices and products can be further elucidated.