Activation pathways involved in HAMLET induced death of Streptococcus pneumoniae

Abstract

Antibiotic resistant bacteria are emerging worldwide and have endangered the effectiveness of antibiotics. This problematic development has accelerated infections with antibiotic resistant bacteria and to counter this, the World Health Organization (WHO) recently published a report listing twelve antibiotic resistant pathogens that required the most attention. One of those is Streptococcus pneumoniae (the pneumococcus), a gram-positive, respiratory tract pathogen. Pneumococci cause many types of infections, some of which are serious and life-threatening, affecting the lungs, blood, brain, sinuses or middle ears. Increasing emergence of antibiotic resistant pneumococci poses a threat to treatment of these infections. Human milk contains multiple bioactive proteins that confer antimicrobial activity apart from its nutritional and growth stimulatory activity in infants. HAMLET is a protein-lipid complex of human α-lactalbumin and oleic acid that has a bactericidal activity against certain bacteria. To understand HAMLET’s bactericidal activity, we used S. pneumoniae in our studies. HAMLET caused dissipation of the pneumococcal membrane polarity and membrane integrity and killed the bacteria. Depolarization was accompanied by calcium and sodium transport and HAMLET’s activity was effectively inhibited with transport inhibitors and potentiated with ionophores. Inhibition of glycolysis with 2-deoxyglucose, sensitized the bacteria to HAMLET and suggested a role for ATP in maintaining membrane polarity. We also observed that kinase activity was required for HAMLET-induced depolarization and death, and that HAMLET modulated kinase activity in pneumococci. The relative importance of each of these events during HAMLET-induced bacterial death could lead to identifying novel targets for future antimicrobial targets.

Popular Abstract

New paths into HAMLET pneumococci treatment

Antibiotic resistant bacteria are a major threat to human health. In the US alone, antibiotic resistant organisms have caused approximately 2.9 million infections resulting in 35,000 deaths. In Europe, deaths from antibiotic resistant organisms increased from 2007 to 2015 by almost 250%. Additionally, misuse and overuse of antibiotics are accelerating the spread of resistant organisms compared to past decades and has increased the burden on health care systems around the world.

In response to the present situation, the World Health Organization (WHO) in collaboration with pharmaceutical companies, recently compiled and presented a list of twelve highly problematic pathogens. These pathogens were prioritized according to their antibiotic resistance level, and their prevalence in society. Since then, governments around the world, including Sweden, have invested into solving the problem of antibiotic resistance. One of the twelve pathogens listed by the WHO is Streptococcus pneumoniae, also known as pneumococcus. S. pneumoniae is one of the many pathogens located in the respiratory tract, usually as bacterial communities called biofilms, where a majority of the spread of resistance genes occur. Pneumococcal infection is often triggered through interactions with respiratory viruses that lead to secondary bacterial infections. The Spanish flu is one example of this interaction, where millions died from a secondary pneumococcal infection due to a novel influenza A virus. Currently, pneumococci cause nearly 500 million illnesses resulting in 1.6 million deaths each year, and an increasing portion of these infection are caused by antibiotic resistant organisms. As antibiotics are becoming less effective, novel strategies to treat and prevent infections with antibiotic resistant strains are needed.

Infants, through breast feeding, are protected from bacterial and viral infections, in both the gastrointestinal and respiratory tracts. In human milk, a protein-fatty acid molecule was identified, and named ‘Human Alpha-lactalbumin Made LEthal to Tumor cells’, or in short - HAMLET. HAMLET is antibacterial, but also has the ability to weaken bacterial resistance to antibiotics through re-sensitization of bacteria. This makes it an interesting candidate for medical treatment. However, the mechanisms of HAMLET-induced bacterial death and antibiotic-sensitization are not completely clear.

In our studies, we investigated HAMLETs effect on the pneumococcal membrane. As previously shown, HAMLET caused membrane depolarization and rupturing, we also verified that calcium and sodium were involved in the process. We also tested HAMLET’s activity on glucose starved pneumococci, as glucose is its main energy source, efficiency of HAMLET on starved pneumococci was higher. We also verified that a ‘kinase’ protein, known to be involved in bacterial death, was involved in HAMLET’s activity using a kinase inhibitor. Surprisingly, it was found that calcium and sodium were not required in kinase activation with HAMLET. This has implications on future research, as it presents new paths of inquiry into improved treatments of pneumococcal infections.

Master’s Degree Project in Molecular Biology - 30 credits, 2021
Department of Biology, Lund University

Advisor: Anders P Håkansson, Department of Translational Medicine, Malmö

• author: Ganganna, Karthik
• supervisor: Anders P Håkansson
• organization: Degree Projects in Molecular Biology
• course: MOBM02 20211
• year: 2021
• type: H2 - Master's Degree (Two Years)
• subject: Biology and Life Sciences
• language: English
• id: 9059284
• date added to LUP: 2021-06-28 11:26:13
• date last changed: 2021-06-28 11:26:13