Biology subjects

Stamsas, G. A.

Publications and source records attributed to Stamsas, G. A..

4 recordsLinked to original sources

bioRxiv2021

SmdA is a novel cell morphology determinant in Staphylococcus aureus

Cell division and cell wall synthesis in staphylococci need to be precisely coordinated and controlled to allow the cell to multiply while maintaining their nearly spherical shape. The mechanisms ensuring correct placement of the division plane and synthesis of new cell wall have been studied intensively, however, hitherto unknown factors and proteins are likely to play key roles in this complex interplay. We here identified and investigated a protein with major influence on cell morphology in Staphylococcus aureus. The protein, named SmdA (for staphylococcal morphology determinant A), is a membrane-protein with septum-enriched localization. By CRISPRi knockdown and overexpression combined with different microscopy techniques, we demonstrate that proper levels of SmdA is necessary for cell division, including septum formation and cell splitting. We also identified conserved residues in SmdA that are critical for its functionality. Pulldown- and bacterial two-hybrid interaction experiments showed that SmdA interacts with several known cell division- and cell wall synthesis proteins, including penicillin binding proteins (PBPs) and EzrA. Notably, SmdA also affects susceptibility to cell wall targeting antibiotics, particularly in methicillin-resistant S. aureus (MRSA). Together, our results show that S. aureus is dependent on balanced amounts of membrane-attached SmdA in order to carry out proper cell division. ImportanceStaphylococcus aureus is an important human and animal pathogen. Antibiotic resistance is a major problem in treatment of staphylococcal infections, and cell division and cell wall synthesis factors have previously been shown to modulate susceptibility to antibiotics in this species. In the current work we investigated the function of an essential protein named SmdA, which was identified based on its septal localization and knockdown phenotype resulting in defective cellular morphologies. We demonstrate that this protein is critical for normal cell division in S. aureus. Depletion of SmdA sensitize resistant staphylococci to {beta}-lactam antibiotics. This work thus reveals a new staphylococcal cell division factor and a potential future target for narrow spectrum antimicrobials or compounds to resensitize antibiotic resistant staphylococcal strains.

microbiology↗

bioRxiv2019

Spatio-temporal control of DNA replication by the pneumococcal cell cycle regulator CcrZ

Most bacteria replicate and segregate their DNA concomitantly while growing, before cell division takes place. How bacteria synchronize these different cell cycle events to ensure faithful chromosome inheritance is poorly understood. Here, we identified a conserved and essential protein in pneumococci and related Firmicutes named CcrZ (for Cell Cycle Regulator protein interacting with FtsZ) that couples cell division with DNA replication by controlling the activity of the master initiator of DNA replication, DnaA. The absence of CcrZ causes mis-timed and reduced initiation of DNA replication, which subsequently results in aberrant cell division. We show that CcrZ from Streptococcus pneumoniae directly interacts with the cytoskeleton protein FtsZ to place it in the middle of the newborn cell where the DnaA-bound origin is positioned. Together, this work uncovers a new mechanism for the control of the bacterial cell cycle in which CcrZ controls DnaA activity to ensure that the chromosome is replicated at the right time during the cell cycle.

microbiology↗

bioRxiv2019

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

In oval shaped Streptococcus pneumoniae, septal and longitudinal peptidoglycan synthesis is performed by independent functional complexes; the divisome and the elongasome. Penicillin binding proteins (PBPs) were long considered as the key peptidoglycan synthesizing enzymes in these complexes. Among these were the bifunctional class A PBPs, which are both glycosyltransferases and transpeptidases, and monofunctional class B PBPs with only transpeptidase activity. Recently, however, it was established that the monofunctional class B PBPs work together with non-PBP glycosyltransferases (FtsW and RodA) to make up the core peptidoglycan synthesizing machineries within the pneumococcal divisome (FtsW/PBP2x) and elongasome (RodA/PBP2b). The function of class A PBPs is therefore now an open question. Here we utilize the peptidoglycan hydrolase CbpD to show that class A PBPs have an autonomous role during cell wall synthesis in S. pneumoniae. Purified CbpD was shown to target the septum of S. pneumoniae cells. Using assays to specifically inhibit PBP2x, we demonstrate that CbpD specifically target nascent peptidoglycan synthesized by the divisome. Notably, class A PBPs could process this nascent peptidoglycan from a CbpD-sensitive to a CbpD-resistant form. The class A PBP mediated processing was independent of divisome and elongasome activities. Class A PBPs thus constitute an autonomous functional entity which processes or repairs nascent peptidoglycan synthesized by FtsW/PBP2x. Our results support a model in which pneumococcal peptidoglycan is made by three functional entities, the divisome, the elongasome and a peptidoglycan-repairing or -remodelling complex consisting of bifunctional PBPs. To our knowledge this is the first time a specific function has been identified for class A PBPs in bacterial cell wall synthesis.

microbiology↗

bioRxiv2018

CozEa and CozEb play overlapping and essential roles in controlling cell division in Staphylococcus aureus

Staphylococcus aureus needs to control the position and timing of cell division and cell wall synthesis to maintain its spherical shape. We identified two membrane proteins, named CozEa and CozEb, which together are important for proper cell division in S. aureus. CozEa and CozEb are homologs of the cell elongation regulator CozESpn of Streptococcus pneumoniae. While cozEa and cozEb were not essential individually, the {Delta}cozEa{Delta}cozEb double mutant was lethal. To study the functions of cozEa and cozEb, we constructed a CRISPR interference (CRISPRi) system for S. aureus, allowing transcriptional knockdown of essential genes. CRISPRi knockdown of cozEa in the {Delta}cozEb strain (and vice versa) causes cell morphological defects and aberrant nucleoid staining, showing that cozEa and cozEb have overlapping functions and are important for normal cell division. We found that CozEa and CozEb interact with the cell division protein EzrA, and that EzrA-GFP mislocalizes in the absence of CozEa and CozEb. Furthermore, the CozE-EzrA interaction is conserved in S. pneumoniae, and cell division is mislocalized in cozESpn-depleted S. pneumoniae cells. Together, our results show that CozE proteins mediate control of cell division in S. aureus and S. pneumoniae, likely via interactions with key cell division proteins such as EzrA.

microbiology↗