Identification of novel cell cycle proteins in Staphylococcus aureus

Antimicrobial resistance (AMR) is an increasing problem globally. Previously harmless infections can in the future become life threatening due to lack of effective antibiotics for treatment. Due to the increase in AMR, we need in depth understanding of the lives of pathogenic bacteria, in order to suggest novel methods for treatment. In this project we have studied cell division in bacteria, with special focus on staphylococci (Staphylococcus aureus). Staphylococci can cause a range of different infections, from skin abscesses to bacteraemia, in both humans and animals. Particularly problematic are the so-called MRSA (methicillin-resistant Staphylococcus aureus) bacteria which are resistant to many of the most-used antibiotics. Cell wall synthesis, cell division and DNA replication (the cell cycle) are known targets for antimicrobials. These are essential processes for the bacterial cells. The staphylococcal cell cycle has been studied for many years, but much is still not understood. For example, very little is known about how DNA replication and cell division are coordinated in these bacterial cells. Such knowledge is important both to understand the molecular mechanisms underlying antimicrobial resistance, and to be able to identify new antimicrobial targets. In this project we have identified and functionally characterized so-far unknown cell division proteins in staphylococci. We have done this by first developing novel genetic and cell biology tools for S. aureus. These tools include a CRISPR interference (CRISPRi) system for efficient gene knock-down and optimized methods for subcellular protein localization in S. aureus. These new methods have been used to search for new cell cycle proteins. During the course of the project we have performed in-depth studies of four novel staphylococcal cell cycle proteins. The functional studies of these proteins were performed using a range of different biochemical, genetic and bioinformatic methods. CozEa and CozEa are two homologous membrane proteins which were found to have overlapping functions during cell division in S. aureus. CcrZ is a cytoplasmic protein important for coordination of cell division and DNA replication. These proteins are also present in other related pathogenic bacteria, such as pneumococci. As part of the project, we have therefore also performed functional studies of the proteins in related bacterial species. We have also identified a cell division protein which is unique to staphylococci. This protein, which is also critical for proper cell division, is particularly interesting, since MRSA bacteria become more sensitive to beta-lactam antibiotics when this protein is inhibited. This protein may thus be a novel antibiotic target which also can help extend the lifetime of existing antibiotics. The results from the project have been or are in the process of being published in scientific papers, in addition to presentations at different meetings and in popularized science articles.

Prosjektet har ført til utvikling av nye molekylære verktøy og ny innsikt i cellebiologien hos Staphylococcus aureus og relaterte bakteriearter. Dette har gitt grunnlag før økt samarbeid med forskergrupper internasjonalt og nasjonalt. Det nye molekylære verktøyene har blitt tatt i bruk av andre forskergrupper og gitt grunnlag for nye prosjekter og prosjektsøknader. Prosjektleder, som hadde midlertidige stilling ved prosjektets start, har nå gått over i en innstegstilling. De øvrige prosjektdeltakerne har fått mulighet til fortsatte arbeidet innen samme forskningsfelt etter at prosjektet ble avsluttet. Et av proteinene studert i denne studien er et potensielt nytt antimikrobielt target for å bekjempe MRSA. Videre forskning basert på resultatene fra dette prosjektet er nødvendig.

In Staphylococcus aureus, a human pathogen which poses serious health threats due to increasing antimicrobial resistance issues, the underlying mechanisms and coordination of essential cell cycle processes remain poorly understood. Knowledge about the cell cycle, including cell division and chromosome segregation, is very limited in spherical cocci, such as S. aureus, since the majority of studies dealing with these issues have been performed in bacteria with elongated cell shapes. It is therefore likely that S. aureus use proteins and mechanisms which not yet have been discovered in other bacteria. Our major goal in this research is to identify novel proteins involved in the S. aureus cell cycle. From screens for essential proteins in S. aureus, it has been shown that there over hundred essential genes with unknown function. These genes not only represent a reservoir for antibiotic targets, but we also hypothesize that some of them play essential roles in the cell cycle. In the proposed research we will identify essential genes involved in the S. aureus cell cycle using a combination of next generation sequencing approaches, single cell analysis and classical genetics/biochemistry techniques. Furthermore, as part of the project, we aim to develop novel single cell tools to study cell division and chromosome segregation in S. aureus, and thereby characterize the chromosome organization in this pathogen. The tools we develop will be useful for the S. aureus research community. Results from the proposed research will provide valuable insights into fundamental aspects of the bacterial cell cycle, and this may potentially lead to the identification of novel target sites for antimicrobials. Subsequently, this may assist in the design of novel antimicrobials or help prolong the lifetime of antimicrobials already in use.