Daniela Fadda

Control of cell division in Streptococcus pneumoniae by the conserved Ser/Thr protein kinase StkP

How the human pathogen Streptococcus pneumoniae coordinates cell-wall synthesis during growth and division to achieve its characteristic oval shape is poorly understood. The conserved eukaryotic-type Ser/Thr kinase of S. pneumoniae, StkP, previously was reported to phosphorylate the cell-division protein DivIVA. Consistent with a role in cell division, GFP-StkP and its cognate phosphatase, GFP-PhpP, both localize to the division site. StkP localization depends on its penicillin-binding protein and Ser/Thr-associated domains that likely sense uncross-linked peptidoglycan, because StkP and PhpP delocalize in the presence of antibiotics that target the latest stages of cell-wall biosynthesis and in cells that have stopped dividing. Time-lapse microscopy shows that StkP displays an intermediate timing of recruitment to midcell: StkP arrives shortly after FtsA but before DivIVA. Furthermore, StkP remains at midcell longer than FtsA, until division is complete. Cells mutated for stkP are perturbed in cell-wall synthesis and display elongated morphologies with multiple, often unconstricted, FtsA and DivIVA rings. The data show that StkP plays an important role in regulating cell-wall synthesis and controls correct septum progression and closure. Overall, our results indicate that StkP signals information about the cell-wall status to key cell-division proteins and in this way acts as a regulator of cell division.

Characterization of divIVA and Other Genes Located in the Chromosomal Region Downstream of the dcw Cluster in Streptococcus pneumoniae

We analyzed the chromosome region of Streptococcus pneumoniae located downstream of the division and cell wall (dcw) cluster that contains the homolog of the Bacillus subtilis cell division gene divIVA and some genes of unknown function. Inactivation of divIVA in S. pneumoniae resulted in severe growth inhibition and defects in cell shape, nucleoid segregation, and cell division. Inactivation of the ylm genes resulted in some morphological and/or division abnormalities, depending on the inactivated gene. Transcriptional analysis revealed a relationship between these genes and the ftsA and ftsZ cell division genes, also indicating that the connection between the dcw cluster and the divIVA region is more extensive than just chromosomal position and gene organization.

Streptococcus pneumoniae DivIVA: Localization and Interactions in a MinCD-Free Context

To clarify the function of DivIVA in Streptococcus pneumoniae, we localized this protein in exponentially growing cells by both immunofluorescence microscopy and immunoelectron microscopy and found that S. pneumoniae DivIVA (DivIVA(SPN)) had a unique localization profile: it was present simultaneously both as a ring at the division septum and as dots at the cell poles. Double-immunofluorescence analysis suggested that DivIVA is recruited to the septum at a later stage than FtsZ and is retained at the poles after cell separation. All the other cell division proteins that we tested were localized in the divIVA null mutant, although the percentage of cells having constricted Z rings was significantly reduced. In agreement with its localization profile and consistent with its coiled-coil nature, DivIVA interacted with itself and with a number of known or putative S. pneumoniae cell division proteins. Finally, a missense divIVA mutant, obtained by allelic replacement, allowed us to correlate, at the molecular level, the specific interactions and some of the facets of the divIVA mutant phenotype. Taken together, the results suggest that although the possibility of a direct role in chromosome segregation cannot be ruled out, DivIVA in S. pneumoniae seems to be primarily involved in the formation and maturation of the cell poles. The localization and the interaction properties of DivIVA(SPN) raise the intriguing possibility that a common, MinCD-independent function evolved differently in the various host backgrounds.

Division protein interaction web: identification of a phylogenetically conserved common interactome between Streptococcus pneumoniae and Escherichia coli

The ability of each of the 11 Streptococcus pneumoniae division proteins to interact with itself and with each of the remaining proteins was studied in 66 combinations of protein pairs, using a bacterial two-hybrid system. Interactions (homo- or hetero-dimerizations) were detected between 37 protein pairs, whereas 29 protein pairs did not interact. In some cases, positive interactions of the S. pneumoniae proteins were confirmed by co-immunoprecipitation experiments in Escherichia coli. Comparison between the S. pneumoniae division protein interaction web and that of E. coli, the only micro-organisms for which the whole division interactome has been described systematically, was also performed. At least nine division proteins, ZapA, FtsZ, FtsA, FtsK, FtsQ/DivIB, FtsB/DivIC, FtsL, FtsI and FtsW, are believed to have a conserved function between these bacteria and thus we may say that a significant part of the interactions are conserved. Out of 45 protein pairs tested in both bacteria, 30 showed the same behaviour: 23 interacted while seven did not. In agreement with these results, cross-interactions between S. pneumoniae proteins and the corresponding E. coli orthologues were observed. Taken together, these results suggest a phylogenetically conserved minimal common interactome of the division proteins.

Roles of the essential protein FtsA in cell growth and division in Streptococcus pneumoniae

Streptococcus pneumoniae is an ovoid-shaped Gram-positive bacterium that grows by carrying out peripheral and septal peptidoglycan (PG) synthesis, analogous to model bacilli, such as Escherichia coli and Bacillus subtilis In the model bacilli, FtsZ and FtsA proteins assemble into a ring at midcell and are dedicated to septal PG synthesis but not peripheral PG synthesis; hence, inactivation of FtsZ or FtsA results in long filamentous cells unable to divide. Here, we demonstrate that FtsA and FtsZ colocalize at midcell in S. pneumoniae and that partial depletion of FtsA perturbs septum synthesis, resulting in elongated cells with multiple FtsZ rings that fail to complete septation. Unexpectedly, complete depletion of FtsA resulted in the delocalization of FtsZ rings and ultimately cell ballooning and lysis. In contrast, depletion or deletion of gpsB and sepF, which in B. subtilis are synthetically lethal with ftsA, resulted in enlarged and elongated cells with multiple FtsZ rings, with deletion of sepF mimicking partial depletion of FtsA. Notably, cell ballooning was not observed, consistent with later recruitment of these proteins to midcell after Z-ring assembly. The overproduction of FtsA stimulates septation and suppresses the cell division defects caused by the deletion of sepF and gpsB under some conditions, supporting the notion that FtsA shares overlapping functions with GpsB and SepF at later steps in the division process. Our results indicate that, in S. pneumoniae, both GpsB and SepF are involved in septal PG synthesis, whereas FtsA and FtsZ coordinate both peripheral and septal PG synthesis and are codependent for localization at midcell.IMPORTANCEStreptococcus pneumoniae (pneumococcus) is a clinically important human pathogen for which more therapies against unexploited essential targets, like cell growth and division proteins, are needed. Pneumococcus is an ovoid-shaped Gram-positive bacterium with cell growth and division properties that have important distinctions from those of rod-shaped bacteria. Gaining insights into these processes can thus provide valuable information to develop novel antimicrobials. Whereas rods use distinctly localized protein machines at different cellular locations to synthesize peripheral and septal peptidoglycans, we present evidence that S. pneumoniae organizes these two machines at a single location in the middle of dividing cells. Here, we focus on the properties of the actin-like protein FtsA as an essential orchestrator of peripheral and septal growth in this bacterium.

Validation of an Italian Version of the Oxford Happiness Inventory in Adolescence

An Italian adaptation of the Oxford Happiness Inventory was administered to 782 adolescents. Exploratory structural equation modeling (ESEM) was used to examine the first- and second-order factorial structure of the scale and its invariance across gender; internal consistency and construct validity were also investigated. ESEM underlined a 5-factor structure (mastery and self-fulfillment, satisfaction with life, vigor, social interest, and social cheerfulness) that measures positive psychological functioning. These dimensions form a single latent construct of general psychological well-being. The scale showed adequate internal consistency values and strong measurement invariance across gender. Finally, regarding convergent validity, both total scale and subscales were positively correlated with extraversion and self-esteem, were negatively correlated with neuroticism, and displayed no correlation with psychoticism.

Roles of Pneumococcal DivIB in Cell Division

DivIB, also known as FtsQ in gram-negative organisms, is a division protein that is conserved in most eubacteria. DivIB is localized at the division site and forms a complex with two other division proteins, FtsL and DivIC/FtsB. The precise function of these three bitopic membrane proteins, which are central to the division process, remains unknown. We report here the characterization of a divIB deletion mutant of Streptococcus pneumoniae, which is a coccus that divides with parallel planes. Unlike its homologue FtsQ in Escherichia coli, pneumococcal DivIB is not required for growth in rich medium, but the Delta divIB mutant forms chains of diplococci and a small fraction of enlarged cells with defective septa. However, the deletion mutant does not grow in a chemically defined medium. In the absence of DivIB and protein synthesis, the partner FtsL is rapidly degraded, whereas other division proteins are not affected, pointing to a role of DivIB in stabilizing FtsL. This is further supported by the finding that an additional copy of ftsL restores growth of the Delta divIB mutant in defined medium. Functional mapping of the three distinct alpha, beta, and gamma domains of the extracellular region of DivIB revealed that a complete beta domain is required to fully rescue the deletion mutant. DivIB with a truncated beta domain reverts only the chaining phenotype, indicating that DivIB has distinct roles early and late in the division process. Most importantly, the deletion of divIB increases the susceptibility to beta-lactams, more evidently in a resistant strain, suggesting a function in cell wall synthesis.

Suppression and Synthetic-Lethal Genetic Relationships of ΔgpsB Mutations Indicate That GpsB Mediates Protein Phosphorylation and Penicillin-Binding Protein Interactions in Streptococcus pneumoniae D39.

GpsB regulatory protein and StkP protein kinase have been proposed as molecular switches that balance septal and peripheral (side‐wall like) peptidoglycan (PG) synthesis in Streptococcus pneumoniae (pneumococcus); yet, mechanisms of this switching remain unknown. We report that ΔdivIVA mutations are not epistatic to ΔgpsB division‐protein mutations in progenitor D39 and related genetic backgrounds; nor is GpsB required for StkP localization or FDAA labeling at septal division rings. However, we confirm that reduction of GpsB amount leads to decreased protein phosphorylation by StkP and report that the essentiality of ΔgpsB mutations is suppressed by inactivation of PhpP protein phosphatase, which concomitantly restores protein phosphorylation levels. ΔgpsB mutations are also suppressed by other classes of mutations, including one that eliminates protein phosphorylation and may alter division. Moreover, ΔgpsB mutations are synthetically lethal with Δpbp1a, but not Δpbp2a or Δpbp1b mutations, suggesting GpsB activation of PBP2a activity. Consistent with this result, co‐IP experiments showed that GpsB complexes with EzrA, StkP, PBP2a, PBP2b and MreC in pneumococcal cells. Furthermore, depletion of GpsB prevents PBP2x migration to septal centers. These results support a model in which GpsB negatively regulates peripheral PG synthesis by PBP2b and positively regulates septal ring closure through its interactions with StkP‐PBP2x.

Contribution of personal and environmental factors on positive psychological functioning in adolescents

This study examined self-esteem as mediator in the relations of personal (extraversion, neuroticism) and environmental (maternal, paternal, peer-relationships) variables with domains of positive psychological functioning (PPF) in adolescence (Satisfaction with life, Mastery, Vigor, Social Interest, Social Cheerfulness). We compared one-sided and multidimensional models using a sample of 1193 high school students (592 males and 601 females). We examined variations in adolescent PPF as a function of parenting styles via independent examination of maternal and paternal bonding. Results supported the multidimensional models, which indicated direct effects of personality traits, maternal care and peer relationships, as well as indirect effects, mediated by self-esteem, of all predictors on most PPF dimensions. Overall, our study provided a broader picture of personal and environmental predictors on different dimensions of PPF, which supported the mediating role of self-esteem and emphasized the importance of considering multidimensional models to characterize PPF in adolescents.

Neuroticism as a Moderator of Direct and Mediated Relationships Between Introversion-Extraversion and Well-Being

Among personality traits, extraversion has received major theoretical and empirical attention as predictor of subjective well-being (SWB), whereas the role of emotional stability-neuroticism has been partially neglected. The present study aims to study the role of neuroticism in the relationship between introversion-extraversion and SWB. In particular, we explored if the trait of neuroticism moderates the relationships between introversion-extraversion and SWB dimensions (Satisfaction with life, Mastery, Vigour, Social Cheerfulness), directly and by mediation of self-esteem. Indeed, previous studies have suggested that self-esteem is positively associated with high extraversion and low neuroticism and that it positively mediates the relationship between SWB and personality traits in adolescents. For this purpose, a sample of high school students (N = 1173) completed the Oxford Happiness Inventory, the Rosenberg Self-Esteem Scale and the Big Five Questionnaire. In a latent variable model, we examined the interaction effects (direct and indirect) of extraversion and neuroticism on SWB dimensions. Our results showed that the nature of differences between introverts and extraverts on SWB could be related to the level of neuroticism in relation to Satisfaction with life. Moreover, self-esteem mediated the relationship between personality traits and SWB. In particular, mediated moderation effect analysis showed that self-esteem mediates completely the relationship between the interaction term (extraversion x neuroticism) and Mastery, and partially the relationship with Satisfaction with life. Moreover, moderated mediation effect analysis showed that high levels of neuroticism moderate the effect of extraversion on Satisfaction with life and Mastery through the mediation of self-esteem. In conclusion, our results suggest that although extraversion has a cardinal role on SWB dimensions related to Vigour and Social Cheerfulness, neuroticism and the mediating role of self-esteem should more properly considered in relation to Satisfaction with life and Mastery.