Infectious Diseases Conf 2019: ATP synthase as a molecular drug target to combat antibiotic resistant microbial infections

Introduction & Aim: Antibiotic resistance is posing an existential threat, as it will result in 10 million additional deaths worldwide per year by 2050. Currently, about 700,000 people die every year from microbial infections. Thus, microbial superbugs will become the top global killer, surpassing cancer. The impact of this public health crisis on the global economy is projected to cost $ 100 trillion. The World Health Organization???s global report on surveillance of antimicrobial resistance estimated the yearly cost to the US health system to reach $ 34 billion. Fast-encroaching antibiotic resistance by microbes in general and E. coli in particular is the main reason for this situation. Thus, finding alternative ways to kill microbes is of paramount importance. Selective inhibition of microbial ATP synthase provides an effective and efficient way to combat antibiotic resistant microbial infections. ATP synthase is the fundamental source of cellular energy production for almost all organisms. Inhibition of ATP synthase can deprive cells of required energy leading to cell death. A wide variety of inhibitors including phytochemicals and peptides are known to bind and inhibit ATP synthase. These phytochemicals and peptides bind to the specific binding pockets on ATP synthase. These binding pockets are flanked by many variable amino acids in different organisms. Our lab is identifying and characterizing phytochemicals and peptides as potent and selective inhibitors of ATP synthase to combat the antibiotic resistant microbial infections using E. coli as a model organism.
Antimicrobial resistance (AMR or AR) is a microbe's ability to resist the effects of medication that could once successfully treat the microbe. The term antibiotic resistance (AR or ABR) is a subset of AMR, as it only applies to bacteria that become antibiotic resistant. Resistant microbes are harder to treat which require alternative medicines or higher doses of antimicrobials. These approaches can be costlier, more toxic or both. Multi-antimicrobial resistant microbes are called multi-drug resistant (MDR). Those considered to be highly drug resistant (XDR) or completely drug resistant (TDR) are sometimes referred to as "superbugs." Resistance occurs by one of three mechanisms: natural resistance in some forms of bacteria, genetic mutation, or resistance of one species from another. Resistance may develop in all classes of microbes. Fungi build resistance to antimicrobials. Viruses develop resistance to the antivirals. Protozoa develop resistance to protozoa, and bacteria develop resistance to antibiotics. Due to random mutations resistance will appear spontaneously. Extended use of antimicrobials, however, tends to promote mutation selection which may make antimicrobials ineffective. Preventive steps involve the use of antibiotics only when appropriate, thus preventing the abuse of antibiotics or antimicrobials. Where appropriate, narrow-spectrum antibiotics are favored over broad-spectrum antibiotics, since successful and precise targeting of specific species is less likely to cause resistance and side effects. Education regarding proper use is important for people who are taking these drugs at home. Health care providers can reduce the spread of resistant infections by using good sanitation and hygiene, including hand washing and disinfecting between patients, and should promote patients , visitors, and family members alike. Increasing drug resistance is caused mainly by human and other animal use of antimicrobials, and the spread of resistant strains between the two. Increasing resistance has also been associated with the dumping from the pharmaceutical industry of inadequately treated effluents, particularly in countries where bulk drugs are made. Antibiotics increase selective pressure in populations of bacteria, causing the death of susceptible bacteria; this increases the amount of resistant bacteria that continues to develop. Resistant bacteria can have a growth advantage even at very low levels of antibiotics, and grow faster than vulnerable bacteria. Resistance to antimicrobials is growing globally due to greater access to antibiotic drugs in developing countries. According to figures, 700,000 to several million deaths annually occur. In the U.S., at least 2.8 million people get infected with antibiotic-resistant bacteria each year, resulting in at least 35,000 deaths. There are public calls for collective global action to address the threat which includes proposals for antimicrobial resistance international treaties. Worldwide antibiotic resistance is not completely known but more is being caused by developing countries with weaker healthcare systems. The WHO describes antimicrobial resistance as the resistance of a microorganism to an antimicrobial medication that could once cure an infection from that microorganism. One person can't become antibiotic resistant. Resistance is a feature of the microbe, not a microbially infected human or other organism. Antibiotic resistance is a subset of resistance to the antimicrobials.

Method: Wild type, null and mutant E. coli growth properties are being tested on fermentable glucose and non-fermentable succinate carbon sources. Wild type and mutant enzymes were isolated by harvesting cells in the minimal media. Inhibitory studies are performed on membrane bound F1Fo ATP synthase. Structural modifications of inhibitors are made through replacement or re-positioning of the functional groups (???OH, ???COOH, ???NH2, ???NO2, ???PO4) on phytochemicals or addition of positive charges on the peptides. Wild type and mutant cell growth assays are tested in presence and absence of inhibitors along with null control.

Results: We found that phytochemicals and peptides cause variable degree of inhibition of ATP synthase. Modification of inhibitors augments extent of inhibition. In phytochemicals, re-positioning and addition of new functional groups and for peptides, addition of a c-terminal NH2 group enhances the inhibitory potency. We also observed that incremental addition of positively charged residues in peptides augments the inhibitory effects of peptides by about 100-fold. Growth of E. coli strains in presence and absence inhibitors suggest that ATP synthase is a potent molecular drug target to combat microbial infections. It is also explored the synergistic inhibitory effects of phytochemicals and peptides on microbial ATP synthase.

Conclusion: It is concluded that ATP synthase is a potent molecular drug target and selective inhibition of microbial ATP synthase by phytochemicals and peptides can be used to combat drug resistant microbial infections.