Unlocking the biology of bovine mastitis pathogens with novel molecular tools
Bovine mastitis, an inflammation of the udder, is among the most costly and persistent diseases in the dairy industry. It compromises cow welfare, reduces milk yield and quality, and drives high antibiotic use. Two important bacterial culprits are Staphylococcus aureus and Streptococcus dysgalactiae. Despite decades of research, these pathogens remain difficult to control because we still lack detailed knowledge of how they adapt and persist within the mammary gland. My work focused on developing new genetic tools to study these bacteria, providing insight into their biology and pointing to possible avenues for better treatment and prevention of mastitis.
Studying S. aureus fitness in milk with CRISPRi-seq
For pathogens to cause mastitis, they must survive and proliferate within the mammary gland. For example, milk is a central growth environment for pathogens during mastitis, yet little is known about the genetic requirements of S. aureus for growth in such infection relevant environments. To address this, we developed and applied a so-called CRISPR interference sequencing (CRISPRi-seq) – a high-throughput genetic screening technology that silences genes and identifies those required for growth and survival.
In the thesis, we used CRISPRi-seq to determine the genetic requirements for S. aureus to proliferate in bovine milk. The study revealed that S. aureus relies heavily on purine biosynthesis, the folate cycle, and metal acquisition pathways for growth in milk. Cell wall components such as teichoic acids, typically critical for S. aureus in other conditions, were less important in milk. Additional CRISPRi-seq screens under exposure to antibiotics (penicillin G and trimethoprim-sulfamethoxazole) highlighted genetic factors that modulate antibiotic susceptibility in milk. Our findings illustrate how milk reshapes bacterial physiology and antibiotic response. Furthermore, we demonstrated the usefulness of CRISPRi-seq for studying pathogens in infection-relevant niches in future works.
Expanding the toolkit for Streptococcus dysgalactiae to identify host-adaptations and a novel antimicrobial candidate
Genetic studies of Str. dysgalactiae have long been limited by the lack of reliable tools. I established a method for gene modification in this species based on the process of natural transformation. As proof-of-concept, we deleted genes involved in lactose metabolism. This showed that bovine-associated strains depend on the beta-galactosidase LacG for growth on lactose, a gene lacking in human-adapted Str. dysgalactiae strains, which are thus unable to use lactose as a carbon source. These differences reflect host-specific adaptations.
As part of this work, we also identified a novel antimicrobial protein produced by bovine-derived Str. dysgalactiae. The protein, which we named ScrM displayed antimicrobial activity against other streptococci associated with bovine mastitis as well as other infections. In the thesis, we also study the antimicrobial mechanism and mechanisms of immunity towards ScrM. This system improves our understanding of bacterial competition and survival in the bovine udder, while also highlighting ScrM as a potential novel antimicrobial candidate for mastitis control.
More broadly, natural transformation provides a platform for investigating genetic factors underlying adaptation and virulence and may help identify new treatment strategies.
Looking forward
This work demonstrates how molecular tools, such as CRISPRi-seq and natural transformation, provide a platform for investigating genetic factors underlying adaptation and virulence in bovine mastitis pathogens to help identify new treatment strategies. Specifically, the studies provide new insight into the physiology and adaptation of S. aureus and Str. dysgalactiae highlight genetic differences between bovine- and human-associated strains, as well as novel anti-streptococcal agent. Together, this thesis thus contributes to a better understanding of the biology of these pathogens and may inform the development of more targeted mastitis control strategies.
Marita Torrissen Mårli was born in 1994 in Bodø, northern Norway. She holds a bachelor’s degree in molecular biology from the University of Bergen, including an exchange semester at the University of California, Berkeley. She earned her master’s and PhD in molecular microbiology at NMBU under the supervision of Professor Morten Kjos and is currently employed as a senior engineer in molecular microbiology at Nordlandssykehuset, Bodø.
