Investigating functions of the staphylococcal type VII secretion systems

Secondary Supervisor(s): Professor David Roper

University of Registration: University of Warwick

BBSRC Research Themes:

• Understanding the Rules of Life (Microbiology, Structural Biology)
• Sustainable Agriculture and Food (Animal Health and Welfare)

Project Outline

Staphylococcus aureus, a highly versatile bacterial pathogen, is a leading cause of infections in animals and humans. It causes a range of serious conditions in humans including pneumonia and endocarditis, and is major cause of hospital-associated infections. The high rates of recurrent infection and a rise in antibiotic resistance has made staphylococcal infection harder to treat and has resulted in increased healthcare associated costs. In animals, it causes mastitis, a severe infection of the mammary glands in dairy cows. S. aureus is responsible for 10%-20% of the clinical and subclinical mastitis cases studied. Decreased milk production and increased expenditure due to mastitis have resulted in huge financial losses in the dairy industry worldwide. Subclinical mastitis is a chronic condition common in dairy herds with no visible clinical symptoms. Persistent sub clinical infections are very hard to eradicate.

S. aureus expresses an arsenal of virulence factors, most of which are secreted through conventional secretion systems. S. aureus also encodes a specialised type VII Esat-6 secretion system (Ess or T7SS), a system that was originally described in the intracellular pathogen, Mycobacterium tuberculosis and demonstrated to be crucial for intracellular survival within macrophages (1). The staphylococcal T7SS encodes virulence factors required for staphylococcal virulence and persistence in murine staphylococcal infection models. Our recent work demonstrated that Ess substrates interfere with host cell death pathways during intracellular staphylococcal infection, as well as modulating cytokine responses. The molecular mechanisms underlying Ess-mediated modulation of cellular processes and the precise role of Ess during persistent staphylococcal infection is currently unclear.

Recent studies from our group have also shown a role for the T7SS proteins protection from antimicrobial agents including fatty acids, and implicate a role for this system in bacterial membrane homeostasis (2). While some T7SS proteins have already been considered as vaccine targets, these proteins may also have good potential as drug targets.

The goal of this project is to understand mechanisms of action of the T7SS during S. aureus infection. Roles of individual T7SS components will be investigated employing isogenic mutants and in vitro cell infection assays. T7SS-associated local immune and cellular responses will be studied in murine models of acute and chronic infection. The project will span across cellular microbiology and biochemistry and will involve a range of techniques including infection assays, confocal microscopy, live imaging and immunological assays.

References

Studying the role of clocks in infection

Secondary Supervisor(s): Dr Robert Dallmann

University of Registration: University of Warwick

BBSRC Research Themes:

• Understanding the Rules of Life (Microbiology)
• Sustainable Agriculture and Food (Animal Health and Welfare)

Project Outline

Circadian rhythms impact many key biological functions in virtually all life. They play a critical role in coordinating a range of cellular processes in multicellular as well as unicellular organisms (Brown, et al 2020). In prokaryotes, although circadian clocks have long been reported in photosynthetic cyanobacteria, clocks were only recently discovered in non-photosynthetic bacteria (Zheng et al, 2021). Unlike in eukaryotes, however, the molecular cog wheels of this clock have not been elucidated, nor has this observation been confirmed in any clinically relevant bacterial pathogens. Importantly, the possible impact of circadian clocks on infection outcomes have not been explored. While there are studies have reported that the host circadian clocks can regulate immune cell responses to a limited number of pathogens (Tognini et al 2017, Keller et al 2009), molecular interactions between host and bacterial clocks during infection are poorly understood.

Gene expression and formation of bacterial biofilms by Bacillus were shown to respond to 24 hour light/ dark and temperature cycles indicating the existence of a “true” circadian clocks in free living bacteria (Zheng et al, 2021). Furthermore, recent studies have suggested that circadian clocks may exist in the human gut microbiota members like Enterobacter aerogenes. Rhythmic swarming behaviours induced by gastrointestinal hormone melatonin, was reported in Enterobacter, and its circadian clock could be entrained to temperature cycles (Paulose et al 2019). In the context of bacterial infection, given the close interplay of bacteria with host cells, it is likely of direct medical relevance to understand how bacterial pathogens interact with the host clock, and if/how bacterial clocks are altered by the host. In light of the increasing antimicrobial resistance (AMR) globally and the urgent need for novel solutions to address it, it is timely and pertinent that the functional importance of circadian clocks is investigated in antimicrobial resistant pathogens.

One such highly antibiotic resistant pathogen is Staphylococcus aureus which causes infections ranging from minor skin infections to serious pneumonias. S. aureus is a leading cause of healthcare and community-associated infections, which place a huge economic burden on healthcare systems worldwide. Methicillin resistant staphylococcus aureus (MRSA) were responsible for 12,878 cases of bacteraemia in the UK between 2018-2019, with ~26% 30-day mortality rates. A rise in highly drug-resistant strains with reduced susceptibility to vancomycin and high recurrence rates have made it challenging to effectively treat infections, resulting in increased healthcare costs. During infection, S. aureus closely associates with the host cells through adhering and invading cells, or forming biofilm communities on tissues employing a variety of bacterial factors. Intracellular survival and biofilms contribute to persistent or recurrent infections, which further complicates treatment. The circadian properties of S. aureus have not been investigated and the role of the host clock or any time-of-day differences after infection with S. aureus have not been examined to date.

The broad goal of this project is to explore the functional role of the circadian clock in S. aureus physiology and host cell infection, and to understand mechanisms that impact molecular clock function during staphylococcal infection. An important consequence of circadian bacterial physiology might be the circadian differences of the capacity of antibiotics to resolve staphylococcal infection. We will study the clocks in S. aureus physiology and study the impact of infection on host clocks using cellular in vitro and murine in vivo models. In addition, we will investigate effectiveness of antibiotic treatments in vitro and in vivo S. aureus infection models.