MECHANISMS OF STREPTOCOCCUS PNEUMONIAE BIOFILM FORMATION; THE ROLE OF PSRP AND DIFFERENT CARBON SOURCES

Abstract

Respiratory tract infections (RTIs) are a major global health concern. Several studies indicate that most respiratory bacterial pathogens are capable of asymptomatic colonization and find their primary ecological niche within the nasopharynx of healthy individuals as harmless commensals that form biofilms. However, in response to external factors, such as concomitant virus infections, colonizing bacteria may sometimes disseminate from the nasopharynx, leading to a transition from asymptomatic carriage to infection.

Streptococcus pneumoniae is a leading cause of respiratory and systemic infections, even though they most often reside in healthy individuals asymptomatically. Pneumococcal colonization requires bacterial proteins that bind to host cells and proteins involved in bacterial aggregation, as well as the ability to retrieve and utilize the energy sources available in the colonizing niche. Of the many adherence factors expressed by the bacterium, the pneumococcal serine-rich repeat protein (PsrP) is a nasopharynx-specific virulence factor whose functional binding region (BR) binds to keratin-10 (KRT10) and DNA and promotes pneumococcal colonization and biofilm formation. Interfering with this type of proteins is expected to either block biofilm formation, to induce dispersal of intact biofilms, or both, leading to decreased colonization rates.

In this project, we evaluated the ability of both immunoglobulins as well as non-immunoglobulin affinity proteins, known as affibodies, to bind to PsrP and potentially block the formation of pneumococcal biofilms. We show that immunoglobulins bound to PsrP on selected strains but did not interfere with biofilm formation. We also investigated the role of niche-specific carbon sources on biofilm formation and function. We show that pneumococcal strain D39, when grown in the presence of galactose (that is present in the nasopharynx) rather than glucose (present in the bloodstream) sustain growth of biofilms and modulates the bacteria to be more resistant to antibiotics. This indicates a role for physiologically relevant carbon sources for optimal biofilm formation in the nasopharynx, the colonizing niche where S. pneumoniae forms biofilms.

Popular Abstract

Respiratory tract infections (RTIs) remain a major global health concern. Several studies now indicate that most bacterial pathogens are capable of asymptomatic colonization and find their primary ecological niche within the respiratory tract of healthy individuals, being a harmless commensal by forming biofilms. However, in response to a concomitant virus infection, colonizing bacterial biofilms may sometimes disperse, leading to a transition from asymptomatic carriage of the bacteria to infection of otherwise uninfected sites.

Streptococcus pneumoniae is a leading cause of respiratory and systemic disease even though they reside in healthy individuals without causing symptoms. Pneumococcal colonization requires bacterial proteins that bind to host cells and proteins involved in bacterial aggregation. The pneumococcal serine-rich repeat protein (PsrP) is a nasopharynx-specific virulence factor whose functional binding region (BR) binds to keratin-10 (KRT10) and DNA and promotes pneumococcal biofilm formation. Interfering with this type of proteins is expected to either block biofilm formation or induce dispersal of intact biofilms, or both.

Affibody are small non-immunoglobulin affinity proteins generated by combinatorial protein engineering, which are capable of selectively binding to desired target molecules. They act by blocking protein interactions, inhibiting peptide aggregation and targeted delivery. Therefore, these affibodies can play an important role in preventing the binding of the surface proteins to the extracellular DNA and inhibiting the formation of the biofilm, thereby potentially preventing the disease.

In this project, the pneumococcal biofilm bacteria were subjected to the affibodies and anti-PsrP IgG antibodies which unfortunately did not seem to inhibit the biofilm formation, also the affibodies and the antibodies did not seem to bind with high affinity to PsrP, on the surface of the bacteria.
It is known that in our nasopharynx, the concentration of glucose is negligible while the carbohydrates like galactose, sialic acid and hyaluronic acid are present in abundance. Therefore, we wanted to investigate if there is any difference in the growth rate, antibiotic resistance, and biofilm formation of the pneumococcal strains if they are subjected to different sugar sources. Experiments were performed using glucose and galactose initially. Galactose uptake was slow in comparison to glucose in some pneumococcal strains. Difference was also observed in the antibiotic resistance between galactose grown and glucose grown planktonic bacteria. Some pneumococcal strains grown in galactose were less sensitive to gentamycin compared to glucose. The biofilm bacteria, on the other hand did not show any significant difference, when grown in presence of either of the carbohydrates. We speculate this might be due to the slow metabolism rate of galactose that the amount of gentamycin taken up by the bacteria per unit time is less thereby making the bacteria less sensitive to antibiotic compared to glucose.
We also saw differences in the biofilms formed with the glucose or galactose supplemented media. We observed that despite similarity in the structural organization of the biofilms in both the sugars, galactose grown biofilms made less matrix and more bare bacteria were visible compared to the glucose grown biofilms. We hypothesize that glucose metabolism is faster than galactose which lead to a greater number of dead cells adding to the matrix formation. To have a better understanding of different sugar metabolism and its relation to the biofilm formation and virulence, experiments including gene regulation will be performed further.

Master’s Degree Project in Molecular Biology in Microbiology, 60 credits, 2020
Department of Biology, Lund University

Advisor: Anders P Håkansson
Department of Translational Medicine, Experimental Infection Medicine,
Lund University, Malmö

• author: De, Supradipta
• supervisor: Anders P Håkansson
• organization: Degree Projects in Molecular Biology
• course: MOBN03 20192
• year: 2020
• type: H2 - Master's Degree (Two Years)
• subject: Biology and Life Sciences
• language: English
• id: 9022077
• date added to LUP: 2020-06-24 13:34:37
• date last changed: 2020-06-24 13:34:37