Regulering av ekspresjon, rensing og strukturbestemmelse av PcsB, enzymet som kløyver septal cellevegg hos Streptococcus pneumoniae

The human pathogen Streptococcus pneumoniae causes approximately 1.6 million deaths per year. Hence, it is of great concern that the bacterium`s resistance against antibiotics has increased dramatically in recent decades. If this development continues, currently prescribed antibiotics might become useless. For this reason, it is important to identify new drug targets and antibiotics that can be used to fight pneumococcal infections. The cell wall synthesis machinery and the cell division apparatus are attractive targets for development of new antimicrobial agents. However, much remains to be learned about these processes in S. pneumoniae and other bacteria. Further research on bacterial cell division and cell wall synthesis is therefore needed. The integrity of the cell wall protects bacterial cells from turgor pressure-induced lysis during cell division. The synthesis and splitting of the new cross wall are two operations that must be carefully coordinated. The two-component regulatory system WalRK is believed to play a central coordinating role in these processes. The gene pcsB, which is under WalRK control, encodes an enzyme that has been predicted to split the septal cross wall during cell division.Den humanpatogene Streptococcus pneumoniae forårsaker omtrent 1,6 millioner dødsfall hvert år. Derfor er det knyttet stor bekymring til at bakteriens resistens mot antibiotika har økt dramatisk de siste tiårene. Dersom denne utviklingen fortsetter vil trolig dagens antibiotika ikke lenger fungere. For å kunne behandle infeksjoner forårsaket av bakterien i fremtiden, er det derfor viktig å identifisere nye mål for antibiotika. Cellevegg-syntesemaskineriet og celledelingsapparatet er attraktive mål i utviklingen av nye antibiotika. Dessverre er disse prosessene fremdeles dårlig karakterisert i S. pneumoniae og i andre bakterier. Derfor trengs det mer forskning på celledeling og celleveggsyntese i bakterier. Celleveggen beskytter bakteriene fra lysis forårsaket av turgor trykket under celledeling. Derfor må syntese og kløyving av septal cellevegg være to finkoordinerte prosesser. To-komponent systemet WalRK spiller trolig en sentral rolle i denne koordineringen. Genet pcsB, som er regulert av WalRK, koder for et enzym som er antatt å kløyve septal cellevegg under celledeling

Overexpression of the fratricide immunity protein ComM leads to growth inhibition and morphological abnormalities in Streptococcus pneumoniae

The important human pathogen Streptococcus pneumoniae is a naturally transformable species. When developing the competent state, it expresses proteins involved in DNA-uptake, DNA-processing and homologous recombination. In addition to the proteins required for the transformation process, competent pneumococci express proteins involved in a predatory DNA-acquisition-mechanism termed fratricide. This is a mechanism by which the competent pneumococci secrete a muralytic fratricin termed CbpD, which lyse susceptible sister cells or closely related streptococcal species. The released DNA can then be taken up by the competent pneumococci and be integrated into their genomes. To avoid committing suicide, competent pneumococci produce an integral membrane protein, ComM, which protects them against CbpD by an unknown mechanism. In the present study we show that overexpression of ComM results in growth inhibition and development of severe morphological abnormalities, such as cell elongation, misplacement of the septum and inhibition of septal cross-wall synthesis. The toxic effect of ComM is tolerated during competence because it is not allowed to accumulate in the competent cells. We provide evidence that an intramembrane protease called RseP is involved in the process of controlling the ComM levels, since Delta-rseP mutants produce higher amounts of ComM compared to wild type cells. The data presented here indicate that ComM mediates immunity against CbpD by a mechanism that is detrimental to the pneumococcus if exaggerated.acceptedVersionpublishedVersio

Prevention of EloR/KhpA heterodimerization by introduction of site-specific amino acid substitutions renders the essential elongasome protein PBP2b redundant in Streptococcus pneumoniae

acceptedVersio

Penicillin-binding protein PBP2a provides variable levels of protection towards different β-lactams in Staphylococcus aureus RN4220

Methicillin-resistant Staphylococcus aureus (MRSA) is resistant to most β-lactams due to the expression of an extra penicillin-binding protein, PBP2a, with low β-lactam affinity. It has long been known that heterologous expression of the PBP2a-encoding mecA gene in methicillin-sensitive S. aureus (MSSA) provides protection towards β-lactams, however, some reports suggest that the degree of protection can vary between different β-lactams. To test this more systematically, we introduced an IPTGinducible mecA into the MSSA laboratory strain RN4220. We confirm, by growth assays as well as single-cell microfluidics time-lapse microscopy experiments, that PBP2a expression protects against β-lactams in S. aureus RN4220. By testing a panel of ten different β-lactams, we conclude that there is also a great variation in the level of protection conferred by PBP2a. Expression of PBP2a resulted in an only fourfold increase in minimum inhibitory concentration (MIC) for imipenem, while a 32-fold increase in MIC was observed for cefaclor and cephalexin. Interestingly, in our experimental setup, PBP2a confers the highest protection against cefaclor and cephalexin—two β-lactams that are known to have a high specific affinity toward the transpeptidase PBP3 of S. aureus. Notably, using a single-cell microfluidics setup we demonstrate a considerable phenotypic variation between cells upon β-lactam exposure and show that mecA-expressing S. aureus can survive β-lactam concentrations much higher than the minimal inhibitory concentrations. We discuss possible explanations and implications of these results including important aspects regarding treatment of infection

Penicillin‐binding protein PBP2a provides variable levels of protection toward different β‐lactams in Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) is resistant to most β-lactams due to the expression of an extra penicillin-binding protein, PBP2a, with low β-lactam affinity. It has long been known that heterologous expression of the PBP2a-encoding mecA gene in methicillin-sensitive S. aureus (MSSA) provides protection towards β-lactams, however, some reports suggest that the degree of protection can vary between different β-lactams. To test this more systematically, we introduced an IPTGinducible mecA into the MSSA laboratory strain RN4220. We confirm, by growth assays as well as single-cell microfluidics time-lapse microscopy experiments, that PBP2a expression protects against β-lactams in S. aureus RN4220. By testing a panel of ten different β-lactams, we conclude that there is also a great variation in the level of protection conferred by PBP2a. Expression of PBP2a resulted in an only fourfold increase in minimum inhibitory concentration (MIC) for imipenem, while a 32-fold increase in MIC was observed for cefaclor and cephalexin. Interestingly, in our experimental setup, PBP2a confers the highest protection against cefaclor and cephalexin—two β-lactams that are known to have a high specific affinity toward the transpeptidase PBP3 of S. aureus. Notably, using a single-cell microfluidics setup we demonstrate a considerable phenotypic variation between cells upon β-lactam exposure and show that mecA-expressing S. aureus can survive β-lactam concentrations much higher than the minimal inhibitory concentrations. We discuss possible explanations and implications of these results including important aspects regarding treatment of infection

Structural basis of PcsB-mediated cell separation in Streptococcus pneumoniae

43 p.-5 fig.Separation of daughter cells during bacterial cell division requires splitting of the septal cross wall by peptidoglycan hydrolases. In Streptococcus pneumoniae, PcsB is predicted to perform this operation. Recent evidence shows that PcsB is recruited to the septum by the transmembrane FtsEX complex, and that this complex is required for cell division. However, PcsB lacks detectable catalytic activity in vitro, and while it has been proposed that FtsEX activates PcsB, evidence for this is lacking. Here we demonstrate that PcsB has muralytic activity, and report the crystal structure of full-length PcsB. The protein adopts a dimeric structure in which the V-shaped coiled–coil (CC) domain of each monomer acts as a pair of molecular tweezers locking the catalytic domain of each dimeric partner in an inactive configuration. This suggests that the release of the catalytic domains likely requires an ATP-driven conformational change in the FtsEX complex, conveyed towards the catalytic domains through coordinated movements of the CC domain.This work was supported by grants BFU2011-25326 and S2010/BMD-2457 and grants from the Research Council of Norway.Peer reviewe

Class A PBPs have a distinct and unique role in the construction of the pneumococcal cell wall.

acceptedVersio

A CozE Homolog Contributes to Cell Size Homeostasis of Streptococcus pneumoniae

publishedVersio