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The widespread emergence of antimicrobial resistance (AMR) is a serious threat to global public health and among Gram-positive cocci, Streptococcus pneumoniae constitutes a priority in the list of AMR-threatening pathogens. To counteract this fundamental problem, the bacterial cell division cycle and the crucial proteins involved in this process emerged as novel attractive targets. FtsZ is an essential cell division protein, and FtsZ inhibitors, especially the benzamide derivatives, have been exploited in the last decade. In this work, we identified, for the first time, some benzodioxane–benzamide inhibitors capable of targeting FtsZ in Streptococcus pneumoniae, in addition to their previously demonstrated activity against other bacteria. These promising benzamides, with minimal inhibitory concentrations (MICs) ranging from 25 to 80 µg/mL, demonstrated bactericidal activity against S. pneumoniae. This was evidenced by their ability to dramatically affect growth and viability, further supported by the morphological changes observed through microscopy. Moreover, the compounds were characterized in vitro, combining turbidity measurements and confocal imaging, and significant alteration of a GTP-induced FtsZ assembly was found, in line with our previous data from other microorganisms. [ABSTRACT FROM AUTHOR]

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.

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.

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 (DivIV[A.sub.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 DivIV[A.sub.SPN] raise the intriguing possibility that a common, MinCD-independent function evolved differently in the various host backgrounds.

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.

An 87-bp deletion in the penicillin-binding protein five (PBP five) synthesis repressor (psr) of Enterococcus hirae caused significant changes in cell wall characteristics. The sensitivity to lysozyme-catalyzed protoplasts fromation of exponential-phase cells was increased due to the overproduction of PBP five and deletion of psr. The variations on the amount of methylpentose rhamnose in the cell walls of the microbes with 85-bp deletion indicated that the psr gene is part of a regulatory system for the properties of cell walls.

Purpose: The aim of this triple-blinded randomised controlled trial was to test if argon plasma cleaning/sterilisation of customised abutments can affect peri-implant marginal bone levels when compared to 5 seconds of steam cleaning. Materials and methods: A total of 20 consecutive periodontally healthy patients requiring single implant-supported restorations in the maxillary premolar or anterior area were selected. All patients received a single implant. At abutment connection, customised abutments were randomly allocated to control (subjected only to usually adopted steam cleaning, CG) and test groups (subjected to plasma cleaning/sterilisation, TG). Abutments were screwed in at 32 Ncm, provisional restorations adapted and periapical radiographs were taken using customised film holders. Two weeks later, definitive restorations were placed. Patients were followed-up for 2 years post-loading. Outcome measures were implant/crown success, complications, periapical marginal bone level changes on periapical standardised radiographs, and microbiological analyses of the abutments after customisation and cleaning procedures but before connection. Comparisons between groups were performed by independent sample t tests (significance threshold of P ≤ 0.05). Results: No patient dropped out 2 years after loading. The presence of bacterial growth (staphylococci, including Staphylococcus aureus) was observed only on the CG abutments. No implant failed and no complications occurred. After 2 years of prosthetic loading, radiographic analysis revealed a statistically significantly higher mean bone loss for the CG group (mean difference 0.4 mm; 95% CI 0.08-0.73; P = 0.018). Conclusions: This study suggests that removal of contaminants from titanium abutments using plasma of argon can allow for better bone level maintenance when compared to 5-second steam cleaning of titanium abutments. It is therefore important to use cleaned and sterilised customised abutments in patients. [ABSTRACT FROM AUTHOR]

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 Δ divIVA mutations are not epistatic to Δ gpsB 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 Δ gpsB mutations is suppressed by inactivation of PhpP protein phosphatase, which concomitantly restores protein phosphorylation levels. Δ gpsB mutations are also suppressed by other classes of mutations, including one that eliminates protein phosphorylation and may alter division. Moreover, Δ gpsB mutations are synthetically lethal with Δ pbp1a, but not Δ pbp2a or Δ pbp1b 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. [ABSTRACT FROM AUTHOR]