EzrA Contributes to the Regulation of Cell Size in Staphylococcus aureus

EzrA is a negative regulator of FtsZ in Bacillus subtilis, involved in the coordination between cell growth and cell division and in the control of the cell elongation–division cycle. We have now studied the role of the Staphylococcus aureus homologue of the B. subtilis EzrA protein and shown that it is not essential for cell viability. EzrA conditional and null mutants have an overall increase of the average cell size, compared to wild type strains. In the larger ezrA mutant S. aureus cells, cell division protein FtsZ and the cell wall synthesizing Penicillin Binding Proteins (PBPs) are not properly localized. This suggests that there may be a maximum cell diameter that allows formation of a Z-ring capable of recruiting the other components of the divisome and of driving cytokinesis. We propose that the major role of EzrA in S. aureus is in cell size homeostasis

Study on international cooperation in the Marie Skłodowska-Curie actions

The objective of this study is to provide a clear view and comprehensive analysis of the structure and results of third countries’ involvement in the MSCA under FP7 and Horizon 2020, covering both inward and outward mobility of researcher. The study also addresses the impacts of international cooperation in the MSCA under Horizon 2020 (2014-2020) and the predecessor programme (Marie Curie Actions) under the FP7 People Specific Programme (2007-2013). The findings of the study are expected to feed into the final evaluation of H2020, but in particular into the preparation for the future international dimension of the MSCA under Horizon Europe. Before this study, only anecdotal evidence was available for understanding the reasons and factors influencing the involvement of third countries in the MSCA. In addition, there was no comprehensive qualitative or quantitative assessment, which would allow an informed decision to be taken as to which cooperation should be reinforced to ensure more effective brain circulation. This study provides the first in-depth analysis by taking stock of the structure and impacts of international cooperation within the MSCA. The outcomes of the study and its recommendations will allow the European Commission to better decide on the focus, funding streams and rules regarding third country involvement in the MSCA. Furthermore, the research conducted will help to look beyond the MSCA and embed the MSCA in the wider context of European R&I international cooperation policies. The study concludes that the programme is and will remain bottom-up, but at the same time efforts should be made to better align it with the EU external policy objectives, given its strong international dimension and the growing role of science diplomacy in foreign policies and strategies. As an overarching line of action, the study therefore recommends that MSCA cooperation issues be systematically addressed during formal S&T policy dialogues held with national authorities from third countries or regional fora, and that the results of these discussions be reflected in corresponding bilateral and regional cooperation roadmaps. The study provides 13 detailed recommendations that should be implemented as part of this overarching framework

The ClpXP protease is dispensable for degradation of unfolded proteins in <i>Staphylococcus aureus</i>

Abstract In living cells intracellular proteolysis is crucial for protein homeostasis, and ClpP proteases are conserved between eubacteria and the organelles of eukaryotic cells. In Staphylococcus aureus, ClpP associates to the substrate specificity factors, ClpX and ClpC forming two ClpP proteases, ClpXP and ClpCP. To address how individual ClpP proteases impact cell physiology, we constructed a S. aureus mutant expressing ClpX with an I265E substitution in the ClpP recognition tripeptide of ClpX. This mutant cannot degrade established ClpXP substrates confirming that the introduced amino acid substitution abolishes ClpXP activity. Phenotypic characterization of this mutant showed that ClpXP activity controls cell size and is required for growth at low temperature. Cells expressing the ClpXI265E variant, in contrast to cells lacking ClpP, are not sensitive to heat-stress and do not accumulate protein aggregates showing that ClpXP is dispensable for degradation of unfolded proteins in S. aureus. Consistent with this finding, transcriptomic profiling revealed strong induction of genes responding to protein folding stress in cells devoid of ClpP, but not in cells lacking only ClpXP. In the latter cells, highly upregulated loci include the urease operon, the pyrimidine biosynthesis operon, the betA-betB operon, and the pathogenicity island, SaPI5, while virulence genes were dramatically down-regulated

A comparative genomics approach for identifying host-range determinants in <i>Streptococcus thermophilus</i> bacteriophages

The authors would like to thank: Mimi Birkelund, Ditte E. Christiansen, Goncalo Covas, and Denise Coutinho for their help in collecting data; Ana Rute Neves for valuable discussions; Witold Kot and Lars H. Hansen for sequencing of phage genomes at Aarhus University (Roskilde, Denmark). This work is part of an Industrial PhD Project funded by Innovation Fund Denmark and Chr. Hansen A/S, grant no. 4135-00104B.Comparative genomics has proven useful in exploring the biodiversity of phages and understanding phage-host interactions. This knowledge is particularly useful for phages infecting Streptococcus thermophilus, as they constitute a constant threat during dairy fermentations. Here, we explore the genetic diversity of S. thermophilus phages to identify genetic determinants with a signature for host specificity, which could be linked to the bacterial receptor genotype. A comparative genomic analysis was performed on 142 S. thermophilus phage genomes, 55 of which were sequenced in this study. Effectively, 94 phages were assigned to the group cos (DT1), 36 to the group pac (O1205), six to the group 5093, and six to the group 987. The core genome-based phylogeny of phages from the two dominating groups and their receptor binding protein (RBP) phylogeny corresponded to the phage host-range. A role of RBP in host recognition was confirmed by constructing a fluorescent derivative of the RBP of phage CHPC951, followed by studying the binding of the protein to the host strain. Furthermore, the RBP phylogeny of the cos group was found to correlate with the host genotype of the exocellular polysaccharide-encoding operon. These findings provide novel insights towards developing strategies to combat phage infections in dairies.publishersversionpublishe

Cell division protein FtsK coordinates bacterial chromosome segregation and daughter cell separation in Staphylococcus aureus

Funding Information: We thank Nathalie Reichmann and Leendert Hamoen (University of Amsterdam) for critical reading of the manuscript, Ana Velic (Proteome Center Tübingen) for help with proteome analysis and Mike VanNieuwenhze (Indiana University) for the generous gift of HADA. This study was funded by the European Research Council through grant ERC‐2017‐CoG‐771709 (to MGP), by national funds through FCT– Fundação para a Ciência e a Tecnologia, PTDC/BIA‐MIC/6982/2020 (to HV); PTDC/BIA‐PLA/3432/2012 (to SRF); FCT through MOSTMICRO‐ITQB R&D Unit (UIDB/04612/2020, UIDP/04612/2020) and LS4FUTURE Associated Laboratory (LA/P/0087/2020) and FCT fellowship SFRH/BD/147052/2019 (to BMS); by the Swiss National National Foundation through P300P3_155346 (to AJ); by the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska‐Curie grant agreement No 839596 (to SS) and by the European Molecular Biology Organization through award ALTF 673‐2018 (to SS). Figure 6D and Appendix Fig S7 were created with Biorender.com . Funding Information: We thank Nathalie Reichmann and Leendert Hamoen (University of Amsterdam) for critical reading of the manuscript, Ana Velic (Proteome Center Tübingen) for help with proteome analysis and Mike VanNieuwenhze (Indiana University) for the generous gift of HADA. This study was funded by the European Research Council through grant ERC-2017-CoG-771709 (to MGP), by national funds through FCT– Fundação para a Ciência e a Tecnologia, PTDC/BIA-MIC/6982/2020 (to HV); PTDC/BIA-PLA/3432/2012 (to SRF); FCT through MOSTMICRO-ITQB R&D Unit (UIDB/04612/2020, UIDP/04612/2020) and LS4FUTURE Associated Laboratory (LA/P/0087/2020) and FCT fellowship SFRH/BD/147052/2019 (to BMS); by the Swiss National National Foundation through P300P3_155346 (to AJ); by the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 839596 (to SS) and by the European Molecular Biology Organization through award ALTF 673-2018 (to SS). Figure 6D and Appendix Fig S7 were created with Biorender.com. Publisher Copyright: © 2023 The Authors. Published under the terms of the CC BY NC ND 4.0 license.Unregulated cell cycle progression may have lethal consequences and therefore, bacteria have various mechanisms in place for the precise spatiotemporal control of cell cycle events. We have uncovered a new link between chromosome replication/segregation and splitting of the division septum. We show that the DNA translocase domain-containing divisome protein FtsK regulates cellular levels of a peptidoglycan hydrolase Sle1, which is involved in cell separation in the bacterial pathogen Staphylococcus aureus. FtsK interacts with a chaperone (trigger factor, TF) and establishes a FtsK-dependent TF concentration gradient that is higher in the septal region. Trigger factor binds Sle1 and promotes its preferential export at the septal region, while also preventing Sle1 degradation by the ClpXP proteolytic machinery. Upon conditions that lead to paused septum synthesis, such as DNA damage or impaired DNA replication/segregation, TF gradient is dissipated and Sle1 levels are reduced, thus halting premature septum splitting.publishersversionpublishe

Quinone oxidoreductase from Staphylococcus aureus

Funding Information: Helena Gaspar is acknowledged for the HPLC analyses and Bruno Victor for advice on modelling. F.M.S. and M.S.S. are recipients of fellowships by Fundação para a Ciência e a Tecnologia (PD/BD/128213/2016 and PD/BD/128202/2016, respectively, both within the scope of the PhD program Molecular Biosciences PD/00133/2012). A.B. is recipient of a fellowship by Fundação para a Ciência e a Tecnologia UI/BD/153052/2022. The work was funded by Fundação para a Ciência e a Tecnologia ( PTDC/BIA-BQM/2599/2021 to M.M.P). The project was further supported by UIDB/04046/2020 and UIDP/04046/2020 Centre grants from FCT , Portugal (to BioISI), by LISBOA-01-0145-FEDER-007660 cofunded by FEDER through COMPETE2020-POCI and by Fundação para a Ciência e a Tecnologia and by UIDB/04612/2020 and UIDP/04612/2020 research unit grants from FCT (to Mostmicro). The NMR spectrometers are part of the National NMR Network (PTNMR) and are supported by Infrastructure Project N° 022161 (co-financed by FEDER through COMPETE 2020, POCI, and PORL and FCT through PIDDAC). Funding Information: Helena Gaspar is acknowledged for the HPLC analyses and Bruno Victor for advice on modelling. F.M.S. and M.S.S. are recipients of fellowships by Fundação para a Ciência e a Tecnologia (PD/BD/128213/2016 and PD/BD/128202/2016, respectively, both within the scope of the PhD program Molecular Biosciences PD/00133/2012). A.B. is recipient of a fellowship by Fundação para a Ciência e a Tecnologia UI/BD/153052/2022. The work was funded by Fundação para a Ciência e a Tecnologia (PTDC/BIA-BQM/2599/2021 to M.M.P). The project was further supported by UIDB/04046/2020 and UIDP/04046/2020 Centre grants from FCT, Portugal (to BioISI), by LISBOA-01-0145-FEDER-007660 cofunded by FEDER through COMPETE2020-POCI and by Fundação para a Ciência e a Tecnologia and by UIDB/04612/2020 and UIDP/04612/2020 research unit grants from FCT (to Mostmicro). The NMR spectrometers are part of the National NMR Network (PTNMR) and are supported by Infrastructure Project N° 022161 (co-financed by FEDER through COMPETE 2020, POCI, and PORL and FCT through PIDDAC). Publisher Copyright: © 2022 The Author(s)Staphylococcus aureus is an opportunistic pathogen and one of the most frequent causes for community acquired and nosocomial bacterial infections. Even so, its energy metabolism is still under explored and its respiratory enzymes have been vastly overlooked. In this work, we unveil the dihydroorotate:quinone oxidoreductase (DHOQO) from S. aureus, the first example of a DHOQO from a Gram-positive organism. This protein was shown to be a FMN containing menaquinone reducing enzyme, presenting a Michaelis-Menten behaviour towards the two substrates, which was inhibited by Brequinar, Leflunomide, Lapachol, HQNO, Atovaquone and TFFA with different degrees of effectiveness. Deletion of the DHOQO coding gene (Δdhoqo) led to lower bacterial growth rates, and effected in cell morphology and metabolism, most importantly in the pyrimidine biosynthesis, here systematized for S. aureus MW2 for the first time. This work unveils the existence of a functional DHOQO in the respiratory chain of the pathogenic bacterium S. aureus, enlarging the understanding of its energy metabolism.publishersversionpublishe

Staphylococcus aureus Survives with a Minimal Peptidoglycan Synthesis Machine but Sacrifices Virulence and Antibiotic Resistance

Many important cellular processes are performed by molecular machines, composed of multiple proteins that physically interact to execute biological functions. An example is the bacterial peptidoglycan (PG) synthesis machine, responsible for the synthesis of the main component of the cell wall and the target of many contemporary antibiotics. One approach for the identification of essential components of a cellular machine involves the determination of its minimal protein composition. Staphylococcus aureus is a Gram-positive pathogen, renowned for its resistance to many commonly used antibiotics and prevalence in hospitals. Its genome encodes a low number of proteins with PG synthesis activity (9 proteins), when compared to other model organisms, and is therefore a good model for the study of a minimal PG synthesis machine. We deleted seven of the nine genes encoding PG synthesis enzymes from the S. aureus genome without affecting normal growth or cell morphology, generating a strain capable of PG biosynthesis catalyzed only by two penicillin-binding proteins, PBP1 and the bi-functional PBP2. However, multiple PBPs are important in clinically relevant environments, as bacteria with a minimal PG synthesis machinery became highly susceptible to cell wall-targeting antibiotics, host lytic enzymes and displayed impaired virulence in a Drosophila infection model which is dependent on the presence of specific peptidoglycan receptor proteins, namely PGRP-SA. The fact that S. aureus can grow and divide with only two active PG synthesizing enzymes shows that most of these enzymes are redundant in vitro and identifies the minimal PG synthesis machinery of S. aureus. However a complex molecular machine is important in environments other than in vitro growth as the expendable PG synthesis enzymes play an important role in the pathogenicity and antibiotic resistance of S. aureus

Chemical Genetic Analysis and Functional Characterization of Staphylococcal Wall Teichoic Acid 2-Epimerases Reveals Unconventional Antibiotic Drug Targets

Here we describe a chemical biology strategy performed in Staphylococcus aureus and Staphylococcus epidermidis to identify MnaA, a 2-epimerase that we demonstrate interconverts UDP-GlcNAc and UDP-ManNAc to modulate substrate levels of TarO and TarA wall teichoic acid (WTA) biosynthesis enzymes. Genetic inactivation of mnaA results in complete loss of WTA and dramatic in vitro β-lactam hypersensitivity in methicillin-resistant S. aureus (MRSA) and S. epidermidis (MRSE). Likewise, the β-lactam antibiotic imipenem exhibits restored bactericidal activity against mnaA mutants in vitro and concomitant efficacy against 2-epimerase defective strains in a mouse thigh model of MRSA and MRSE infection. Interestingly, whereas MnaA serves as the sole 2-epimerase required for WTA biosynthesis in S. epidermidis, MnaA and Cap5P provide compensatory WTA functional roles in S. aureus. We also demonstrate that MnaA and other enzymes of WTA biosynthesis are required for biofilm formation in MRSA and MRSE. We further determine the 1.9Å crystal structure of S. aureus MnaA and identify critical residues for enzymatic dimerization, stability, and substrate binding. Finally, the natural product antibiotic tunicamycin is shown to physically bind MnaA and Cap5P and inhibit 2-epimerase activity, demonstrating that it inhibits a previously unanticipated step in WTA biosynthesis. In summary, MnaA serves as a new Staphylococcal antibiotic target with cognate inhibitors predicted to possess dual therapeutic benefit: as combination agents to restore β-lactam efficacy against MRSA and MRSE and as non-bioactive prophylactic agents to prevent Staphylococcal biofilm formation.publishe

DeepBacs for multi-task bacterial image analysis using open-source deep learning approaches

This work demonstrates and guides how to use a range of state-of-the-art artificial neural-networks to analyse bacterial microscopy images using the recently developed ZeroCostDL4Mic platform. We generated a database of image datasets used to train networks for various image analysis tasks and present strategies for data acquisition and curation, as well as model training. We showcase different deep learning (DL) approaches for segmenting bright field and fluorescence images of different bacterial species, use object detection to classify different growth stages in time-lapse imaging data, and carry out DL-assisted phenotypic profiling of antibiotic-treated cells. To also demonstrate the ability of DL to enhance low-phototoxicity live-cell microscopy, we showcase how image denoising can allow researchers to attain high-fidelity data in faster and longer imaging. Finally, artificial labelling of cell membranes and predictions of super-resolution images allow for accurate mapping of cell shape and intracellular targets. Our purposefully-built database of training and testing data aids in novice users' training, enabling them to quickly explore how to analyse their data through DL. We hope this lays a fertile ground for the efficient application of DL in microbiology and fosters the creation of tools for bacterial cell biology and antibiotic research.DeepBacs guides users without expertise in machine learning methods to leverage state-of-the-art artificial neural networks to analyse bacterial microscopy images

DeepBacs for multi-task bacterial image analysis using open-source deep learning approaches

This work demonstrates and guides how to use a range of state-of-the-art artificial neural-networks to analyse bacterial microscopy images using the recently developed ZeroCostDL4Mic platform. We generated a database of image datasets used to train networks for various image analysis tasks and present strategies for data acquisition and curation, as well as model training. We showcase different deep learning (DL) approaches for segmenting bright field and fluorescence images of different bacterial species, use object detection to classify different growth stages in time-lapse imaging data, and carry out DL-assisted phenotypic profiling of antibiotic-treated cells. To also demonstrate the ability of DL to enhance low-phototoxicity live-cell microscopy, we showcase how image denoising can allow researchers to attain high-fidelity data in faster and longer imaging. Finally, artificial labelling of cell membranes and predictions of super-resolution images allow for accurate mapping of cell shape and intracellular targets. Our purposefully-built database of training and testing data aids in novice users’ training, enabling them to quickly explore how to analyse their data through DL. We hope this lays a fertile ground for the efficient application of DL in microbiology and fosters the creation of tools for bacterial cell biology and antibiotic research