Proteomic analysis of mycobacterial biofilms enables global overview of key mechanisms regulating antibiotic resistance.

A new collaborative study by researchers in Finland and Norway – including NAPI Project Manager Tuula Nyman – provides novel insights into how mycobacteria can escape elimination by antibiotics. These findings could have important implications for treating mycobacteria-driven diseases such as Tuberculosis.

Tuberculosis (TB) is an infectious disease most commonly caused by the bacterium Mycobacterium tuberculosis (Mtb). This disease represents a major global health concern, with approximately 10 million new cases and 1.4 million deaths in 2019. Around one quarter of the human population is thought to carry Mtb as an asymptomatic infection, but 5-10% of infected people will develop TB, which primarily affects the lungs.

Antibiotic resistant mycobacteria

Treatment of TB involves the use of several antimicrobial drugs over a period of at least 6 months. Whilst this can be curative for many patients, the emergence of drug-resistant Mtb strains is a major health concern.

The publication follows collaborative work between researchers from Norway and Finland. Left: first author Kirsi Savijoki from the University of Helsinki, Finland. Middle: corresponding author Mataleena Parikka from Tampere University, Finland. Right: NAPI Project Manager and co-author Tuula Nyman from the University of Oslo/Oslo University Hospital.

Indeed, studies have shown that even those patients who have been effectively ‘cured’ using antibiotics (i.e. disease symptoms were alleviated) can still possess viable and infectious Mtb in their lungs and sputum.

A clear understanding of all mechanisms that allow mycobacteria such as Mtb to escape cell death by antibiotics is lacking. However, it is known that one of the key factors involves a strong physical barrier provided by the bacterium’s so-called ‘biofilm’. This is a thick, complex matrix that sits outside the bacterial cell and is made up of a range of molecules including proteins, polysaccharides and DNA/RNA. The biofilm provides mycobacteria with protection against both host defence systems (e.g. human immune cells) and therapeutic antibiotics, as these factors struggle to cut through the biofilm and access the bacterial cell.

Proteomic analysis of biofilms

In a recent publication in the journal mSystems, researchers from Finland and Norway attempted to gain more insight into how the various stages of biofilm production in mycobacteria help to avoid elimination by antibiotics. To achieve this, the researchers used mass spectrometry-based proteomics to perform a systems-wide analysis of the biofilm components of Mycobacterium marinum (Mmr). This is a commonly used model for Mtb infections, and allows researchers to investigate TB-like latent and chronic stages of infection without the health and safety risks associated with working with Mtb itself.

Dr Tuula Nyman is the Project Manager of NAPI, and Head of the NAPI Proteomics Core Facility at University of Oslo/Oslo University Hospital where the mycobacterial biofilms were analysed in this study. Explaining the approach in more detail, Dr Nyman said:

“Our colleagues in Finland cultured an Mmr strain named ATCC 927 to create in vitro biofilms at different times over a 3-month culture period. As a control, they also isolated cell surface proteins from mycobacteria under non-infectious planktonic-type growth, which enabled the identification of proteins that are exclusive to biofilms from infectious bacteria. They then imaged these biofilms using microscopy to visualise structural properties, but also sent samples to our facility in Oslo so that we could investigate the protein components by mass spectrometry. Together, these analyses revealed some interesting patterns of protein composition across different stages of infection.”

Microscopy images of two different subtypes of Mmr biofilms (see text below for more details) taken at two and three weeks of culture. The researchers found important morphological differences between the two subtypes, both of which changed over time. Images generated by Dr. Teemu Ihalainen, Tampere University, Finland.

New insights into antibiotic resistance mechanisms

This approach represents the first study to directly monitor mycobacterial biofilm components (via proteomics) and architecture (via microscopy) over a prolonged period. The researchers were able to gain valuable new insights related to key differences between two distinct biofilm subtypes known to function at different stages of Mmr growth,named floating/pellicle-type biofilms (PBFs) and submerged-type biofilms (SBFs; see image above). The two subtypes arose at different stages in the growth of the ATCC 927 strain, with SBFs detected at day 2 and PBFs arising after 2 weeks, and various proteins were identified that could dictate the main structural properties of each subtype. Importantly, the researchers also identified a subpopulation of mycobacterial cells within both biofilm subtypes that were highly resistant to the therapeutic antibiotic rifampicin, including in only 1 week-old SBFs. Follow-up analyses indicated that the key factors driving development of such resistant cells may include subtype-dependent changes in cell division, cell wall synthesis, and certain metabolic activities, but further studies will be necessary to provide conclusive evidence.

Collectively, the findings of this study show that the two biofilm subtypes differ in the mechanisms used to regulate virulence, antibiotic tolerance and infection persistence. This highlights the fact that both subtypes need to be targeted individually in curative therapies – something that is not the case today.

Important clinical implications

As corresponding author of the publication, Mataleena Parikka, concludes, the impact of these findings could be far-reaching:

“An important next step will be to determine whether the same molecular events dictate the progression and treatment of TB in human patients. If so, these findings could have important clinical implications that benefit a great number of patients worldwide.”

For further details, you can read the article in full on the mSystems website.

You can read more about the Infection Biology Research Group led by corresponding author Mataleena Parikka on the Tampere University webpages.

The first author of the publication, Kirsi Savijoki, is based at the University of Helsinki. More information about Kirsi, including contact details and other publications, is available of the University of Helsinki website.


Published July 2, 2021 9:57 AM - Last modified July 2, 2021 10:00 AM