S. pneumoniae pode causar infecções do trato respiratório. Uma alternativa às vacinas conjugadas, que conferem proteção sorotipo-específica, seria uma vacina baseada em epitopos de diferentes variantes de antígenos como PspA e PspC. O objetivo deste trabalho foi a identificação de epitopos de variantes de PspA e PspC, utilizando a técnica peptide array, visando a síntese de um antígeno composto por múltiplos epitopos. Os resultados obtidos indicaram que anticorpos contra epitopos lineares de PspA reconhecem as regiões inicial N-terminal e rica em prolinas, mas estes epitopos parecem ser apenas marcadores de exposição ao pneumococo e epitopos conformacionais seriam os de fato protetores, como observado nos experimentos realizados com os Frags de 100 aminoácidos construídos a partir do PspARx1, especialmente aqueles presentes nas regiões dos Frags 2 e 4. Epitopos lineares de PspC parecem ser importantes na região de ligação à sIgA e FH. Assim, uma vacina proteica de ampla cobertura, poderia conter as regiões do Frag 2 de PspA e de ligação de sIgA e FH de PspC. / S. pneumoniae can cause respiratory tract infections. An alternative to the conjugate vaccines, which confer serotype-specific protection, would be a vaccine based on epitopes of variants of antigens such as PspA and PspC. The objective of this study was to identify epitopes of variants of PspA and PspC, using the peptide array technique, aiming at the synthesis of a multi-epitope antigen. The results obtained with peptide arrays indicated that antibodies against linear epitopes of PspA recognize the initial N-terminal and proline-rich regions, but these epitopes seem to be only markers of exposure to pneumococcus and conformational epitopes would be in fact protective, as observed in the experiments with fragments of 100 amino acids constructed from PspARx1, especially those present in Frags 2 and 4. Linear epitopes of PspC seem to be important in the regions of sIgA and FH binding. Thus, a protein-based vaccine with broad coverage could contain the regions of Frag 2 of PspA and the regions of sIgA and FH binding of PspC.

S. pneumoniae pode causar infecções do trato respiratório. Uma alternativa às vacinas conjugadas, que conferem proteção sorotipo-específica, seria uma vacina baseada em epitopos de diferentes variantes de antígenos como PspA e PspC. O objetivo deste trabalho foi a identificação de epitopos de variantes de PspA e PspC, utilizando a técnica peptide array, visando a síntese de um antígeno composto por múltiplos epitopos. Os resultados obtidos indicaram que anticorpos contra epitopos lineares de PspA reconhecem as regiões inicial N-terminal e rica em prolinas, mas estes epitopos parecem ser apenas marcadores de exposição ao pneumococo e epitopos conformacionais seriam os de fato protetores, como observado nos experimentos realizados com os Frags de 100 aminoácidos construídos a partir do PspARx1, especialmente aqueles presentes nas regiões dos Frags 2 e 4. Epitopos lineares de PspC parecem ser importantes na região de ligação à sIgA e FH. Assim, uma vacina proteica de ampla cobertura, poderia conter as regiões do Frag 2 de PspA e de ligação de sIgA e FH de PspC. / S. pneumoniae can cause respiratory tract infections. An alternative to the conjugate vaccines, which confer serotype-specific protection, would be a vaccine based on epitopes of variants of antigens such as PspA and PspC. The objective of this study was to identify epitopes of variants of PspA and PspC, using the peptide array technique, aiming at the synthesis of a multi-epitope antigen. The results obtained with peptide arrays indicated that antibodies against linear epitopes of PspA recognize the initial N-terminal and proline-rich regions, but these epitopes seem to be only markers of exposure to pneumococcus and conformational epitopes would be in fact protective, as observed in the experiments with fragments of 100 amino acids constructed from PspARx1, especially those present in Frags 2 and 4. Linear epitopes of PspC seem to be important in the regions of sIgA and FH binding. Thus, a protein-based vaccine with broad coverage could contain the regions of Frag 2 of PspA and the regions of sIgA and FH binding of PspC.

The complement system is a major component of innate immunity and an effector of antibody-mediated immune responses. Unlike the other two activation pathways of the complement system, the alternative pathway is permanently switched on. Discrimination by complement between self and foreign is therefore achieved by selective protection of healthy host tissue and cells. This study investigated the alternative pathway regulator factor H (FH), which is crucial for protection of self surfaces from complement. FH engages via its N- and C- terminal ends with activation-specific fragments of C3, C3b and C3d. The middle region of FH has no binding sites for complement components. It presumably ensures that the binding sites at either end of the extended and flexible FH molecule cooperate in recognizing C3b in fluid phase or on self surfaces, but not on foreign targets. This study was aimed at achieving an atomic level understanding of the structure of the middle portion of FH, thereby testing hypotheses as to how it promotes the overall biological efficacy of the intact protein. High-resolution NMR-derived structures of two module pairs FH-10-11 and FH-11-12 were solved and combined with SAXS data to produce a model of FH-10-12. This was combined, in silico, with the previously solved FH-12-13 structure, then the model of FH-10-13 was used to revisit SAXS data for FH-10-15 and FH-8-15. A unique structure emerged, unlike any other encountered previously in the family of complement regulators, in which CCPs 13, 14 and 15 have a highly compacted organization that has repercussions for function. While devoid of binding affinity for host ligands, this central region is a binding site for PspC, a virulence factor of S. pneumoniae. It has been speculated that the bacteria use this interaction to sequester FH in a conformation that resembles the one adopted by FH on self cells and makes it particularly good at regulating complement. Structural and functional investigations of this interaction were performed to establish the molecular basis of the use of FH by this pathogen in order to avoid complement-mediated elimination. It was found that PspC and FH form a near-irreversible complex, while FH-8-15 binds PspC almost as tightly as intact protein. When bound to PspC, FH has a higher affinity for some of its targets, supporting the theory that this bacterial protein stabiles a particularly active conformation of the regulator.

One of the oldest and most exciting questions in science is: are we alone in the universe? During the last four billion years of Earth’s history, countless organisms have inhabited almost every environmental niche on the planet, from the deepest sea to driest deserts. However, so far no extraterrestrial life has been found. Studying the propensity for life on our neighboring planet, Mars,helps us understanding its potential for past and present life, and guides future missions. Liquid water is a prerequisite for life as we know it and recently, evidence of transient night time liquidbrines on the surface of present day Mars have been theorized. These brines may be hyper-salinewith high ionic strengths and varying pH-values. Halobacterium salinarum is an extremophilic (saltloving) halophilic archaeon whose natural habitat includes hyper-saline brines, desiccating conditions and exposure to high fluences of solar UV radiation. Herein, we report the response of Hbt.salinarum following exposure to simulated Martian conditions, with regard to survival and DNAintegrity. The simulated conditions include the synthetic Martian Brine Analogues (MBAs), diurnalnocturnaltemperature cycling, prolonged desiccation and Mars-like solar UV (200-400 nm) radiation.We also addressed the prolific space hardware contaminant, Bacillus subtilis whose endospores show substantial resistance against space conditions. The ambition was to investigate potentia linterplanetary forward contamination by Hbt. salinarum, should it have bacterial spores available as nutrients in the Martian brines. Halophiles are some of our best candidates for studying unicellular life on Mars and other bodies where liquid water is also stabilized by high salt concentrations.Moreover, Hbt. salinarum was able to survive over one month in the Martian brines, albeit with growth limited to one particularly hospitable brine. It displayed survival in the brines at relevant temperatures and with diurnal-nocturnal cycling but only when first desiccated to remove preventwater crystal formation. The radiation resistance was highly dependent on the choice of brine inwhich Hbt. salinarum was confined and desiccated. Even in the hospitable brines, the halophile lost over 90% of its viable population following irradiation equal to one Martian day, in our experimentalsetup. The inter-brine difference in DNA fragmentation following irradiation confirmed the differencein survival. Hbt. salinarum was subsequently unable to digest B. subtilis endospores for nutrient exploit and responded no differently than when nutrient-deprived. Surprisingly, the addition of otherwise available nutrients in the brines caused a hurried decrease in survival, with the exception of the hospitable brine. Despite its extremophilic and polyextremotolerant character, Hbt. salinarumis unlikely to survive, not to mention thrive, in a combination of all tested stressors.

Maintaining cell envelope integrity is of vital importance for all microorganisms. Not surprisingly, evolution has shaped conserved protein protection networks that connect stress perception, transmembrane signal transduction, and mediation of cellular responses upon cell envelope stress. The phage shock protein (Psp) stress response is one such conserved protection network. Most knowledge about the Psp response derives from studies in the Gram-negative model bacterium Escherichia coli, where the Psp system consists of several well-defined protein components. Homologous systems were identified in representatives of the Proteobacteria, Actinobacteria, and Firmicutes. However, the Psp system distribution in the microbial world remains largely unknown. By carrying out a large-scale, unbiased comparative genomics analysis, we found components of the Psp system in many bacterial and archaeal phyla and describe that the predicted Psp systems deviate dramatically from the known prototypes. The core proteins PspA and PspC have been integrated into various (often phylum-specifically) conserved protein networks during evolution. Based on protein domain-based and gene neighborhood analyses of pspA and pspC homologs, we built a natural classification system for Psp networks in bacteria and archaea. We validate our approach by performing a comprehensive in vivo protein interaction study of Psp domains identified in the Gram-positive model organism Bacillus subtilis and found a strong interconnected protein network. Our study highlights the diversity of Psp domain organizations and potentially diverse functions across the plethora of the microbial landscape, thus laying the ground for studies beyond known Psp functions in underrepresented organisms.