HAMLET-induced death and sensitization of antibiotic-resistant bacteria

Streptococcus pneumoniae (pneumococcus) is one of the leading causes of morbidity and mortality from respiratory and invasive infections in children and the elderly worldwide. Pneumococci effectively colonize the human nasopharynx and cause infections such as pneumonia, acute otitis media (AOM), meningitis and sepsis by disseminating to otherwise sterile sites. The pneumococcal capsular conjugate vaccine that was introduced in the late 1990s effectively prevents invasive disease, but has had little impact on total episodes of AOM due to increased infection rates with non-vaccine serotypes. This data together with emergence of antibiotic-resistant pneumococcal strains, emphasizes the need for continued efforts to develop effective preventive and therapeutic agents against pneumococcal disease.

More information on pneumococcal disease
More information about human milk and infections

During my Ph.D studies in Lund, Sweden, we discovered a protein complex from human milk (HAMLET) that induced apoptosis in tumor cells without affecting healthy cells (click here for more information about the anti-tumor project). HAMLET was also found to kill certain species of bacteria with the highest activity against the respiratory tract pathogen Streptococcus pneumoniae (Hakansson et al 2000). Recently we have shown that HAMLET can also sensitize both HAMLET-sensitive and HAMLET-resistant bacteira to antibiotics (Marks PLoS One 2012 and Marks et al PLoS One 2013). This is an exciting new avenue to increase the treatment efficacy of antibiotic-resistant organisms.

ALA

Figure 1. Structure of human alpha-lactalbumin.

HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) is a partially unfolded form of human alpha-lactlabumin stabilized with human milk-specific fatty acids.

The long-term goal of this project is to characterize the mechanism of HAMLET-induced killing of pneumococci, and HAMLET's ability to sensitize antibiotic resistant bacteira against the antibiotics they are resistant against. We are currently pursuing several subprojects in the lab:

A. MECHANISTIC STUDIES

1. Receptor characterization.
S. pneumoniae requires choline for growth. Choline is incorporated in the lipoteichoic and teichoic acids of the cell wall and it has recently been shown that the flipase (TacF) exporting the teichoic acid repeats requires choline to be incorporated for transport to occur. Pneumococci grown in the presence of the choline analog ethanolamine will grow normally but will lack certain choline-dependent functions. Preliminary information suggest that ethanolamine-grown pneumococci are less sensitive to HAMLET and that lipoteichoic acid form pneumococci can inhibit the bacteriacidal actiivty of HAMLET. We are pursuing these interactions in collaborations with Moon Nahm at UAB.

2. Signaling.
Preliminary data suggest that HAMLET induces signals in the bacteria that are similar to what happens during HAMLET-induced apoptosis of tumor cells (see Hakansson PLoS One 2011). Like in tumor cell mitochondria, HAMLET induces dissipation of the membrane potential also in bacteria that is dependent both on the inactivation of the Hydrogen ATPase and a sodium-dependent calcium influx (Clementi JBC 2012). We have also shown that this results in the activation of a novel Ser/Thr kinase in the bacteria and that HAMLET targets glycolysis proteins to turn off ATP production in parallel. We are currently trying to identify the channels and other structures involved in this signaling in bacteria. We know that this signalling is required also for sensitization of bacteria to antibiotics (Marks et al PLoS One 2013).

3. Execution.
In tumor cells exectution of apoptosis involved caspases and caspase-independent pathways that lead to cell death. Caspases induces degradation of structural proteins as well as activation of enzymes and proteins. All in all this results in the programmed and well orchestrated cell death that normally happens in teh body from physiologic stimuli. Caspases are not required for this program to be exectuted but other molecules (proteases and DNAses) have been shown to execute a similar phenotype.
In bacteira we have shown that execution required serine protease activity and as we see similar DNA cleavage patterns we assume that DNAse activity is also involved in cell death of bacteria (Hakansson PLoS One 2011). We are currently identifying molecules that are involved in HAMLET induced cell death.


Figure 2. (1) HAMLET acts by blocking the hydrogen ATPase and inhibiting the proton motive force. This leads to an influx of calcium(2) through a sodium dependent mechanisms and calcium activates a ser/thr kinase (3) that is potentially invovlved in the activation of cell death or sensitizing bacteria to antibiotics. In parallel, HAMLET blocks glycolysis and ATP production (4), which further inhibits the ATPase activity.

B. TREATMENT EXPERIMENTS

We have shown that HAMLET effectively kills Streptococcus pneumoniae as well as other respiratory pathogens. This killing is more effective in vivo in the presence of antibiotics as HAMLET acts synergistically with antibiotics to kill these bacteria. Interestingly, HAMLET can also sensitize bacteira that are not killed by HAMLET alone. One example is Methicillin-resistant Staphylococcus aureus (MRSA) that are effectievly kiled by methicillin in the presence of HAMLET (Marks et al PLoS One 2013). HAMLET and methicillin effectively eradicates MRSA biofilms and also decreaseds the bacteiral burden in animals.

Future experiments are aimed at testing the synergistic effects of HAMLET with antibiotics against other multi-drug resistant bacteria in various model systems in vitro and in vivo.

We believe that HAMLET kills S. pneumoniae using a mechanism similar to that activated during HAMLET-induced tumor cell death and that this mechanism is also used during sensitization of bacteira to antibiotics. Characterization of the processes and molecules activated by HAMLET has the potential to lead to the development of better therapeutic agents against pneumococcal disease, with less risk for resistance development.