|weeks #||TOPICS||LECTURE SUMMARIES|
Watch a video about antibiotics
|2||Penicillin Discovery and Mode of Action|| |
Organisms competing for limited resources can lead to the evolution of defensive mechanisms, such as the production of toxic chemicals with simultaneous tolerance for these chemicals. It was in 1928 when humans noticed the result of this competition. The discovery of the first antibiotic, penicillin, was one of the medical sciences' biggest breakthroughs. Sir Alexander Fleming, a microbiologist who made the discovery, stated: "When I woke up just after dawn on September 28, 1928, I certainly didn't plan to revolutionize all medicine by discovering the world's first antibiotic, or bacteria killer. But I guess that was exactly what I did." For our first paper we will go back in time and review this discovery. Next we will discuss Penicillin-binding Proteins (PBPs)—the targets of penicillin inhibition. In the second paper by Chen et al., we will compare the crystal structures of PBP6 from Escherichia coli when it is bound with a fragment of its natural substrate, peptidoglycan, or with an antibiotic, penicillin.
Watch a video that explains penicillin mode of action
A short biography of Alexander Fleming and his experience with "Germ Painting"
Time-lapse microscopy video of penicillin-treated bacteria
|3||Molecular Basis for Penicillin Resistance|| |
Shortly after penicillin was introduced in the clinic for the treatment of bacterial infections, Penicillin-resistant bacteria began to emerge. Many of these bacteria acquired resistance through mutations in Penicillin-binding Proteins (PBPs), which are essential bacterial enzymes that build peptidoglycan—the cell wall of bacteria. PBPs are the targets of penicillin and other β-lactam antibiotics. Intriguingly, PBP-dependent resistance involves mutations in PBPs that cause reduced penicillin binding affinity, but preserved peptidoglycan biosynthesis activity. This week, we will discuss two papers that highlight the mechanism of penicillin resistance using Structure-function analysis and by determining the molecular basis for biosynthetic changes that occur in the cell, specifically those that cause changes in peptidoglycan composition. In the first paper, by Powell et al., X-ray crystallographic structures of PBP2 from Penicillin-susceptible and-resistant strains of Neisseria gonorrhoeae were determined and used to analyze small, but significant, structural changes that impart antibiotic resistance. The second paper, by Filipe et al., focuses on the downstream effects caused by resistance mutations in PBPs using peptidoglycan analysis techniques and genetic mutations.
|4||Vancomycin Discovery and Mode of Action|| |
As a result of the emergence of Penicillin-resistant bacteria in the 1940s, the demand for alternative antibiotics increased. One alternative drug, vancomycin, was first isolated from the soil bacterium Streptomyces orientalis in the 1950s. Vancomycin is a glycopeptide that inhibits the biosynthesis of the cell wall of Gram-positive bacteria by binding to the D-Ala-D-Ala termini of peptidoglycan precursors. It took an army of scientists and a number of experiments to identify a mode of action for this antibiotic. This week we will discuss a paper by Perkins and Nieto in which one possible mode of action by vancomycin is investigated. The authors examine the distribution of radiolabeled vancomycin in living cells using cell fractionation analysis. Next, we will discuss a paper published in 1990 by Knox and Pratt that provides structural information about binding of vancomycin to the cell wall.
Watch a very short movie about vancomycin mode of action.
|5||Molecular Basis for Vancomycin Resistance|| |
Because of its high toxicity and the availability of alternative antibiotics in the early 1960s, vancomycin was considered a Last-resort drug and used primarily for the treatment of serious bacterial infections. However, vancomycin became more extensively used after the emergence of Methicillin-resistant Stapholococcus aureus (MRSA), a multidrug resistant bacterium that causes community and hospital acquired infections. The increased use of vancomycn is thought to be the reason for the rise of vancomycin resistant stapholococci and enterococci. To date, five types of acquired vancomycin resistance have been characterized. The papers this week are about two of the five mechanisms, VanA-type and VanB-type, which involve the acquisition of a D-Ala-D-Ala ligase with alternative substrate specificity. The study conducted by Bugg et al. uses biochemical techniques, such as protein purification and enzyme kinetic assays, to understand the molecular basis for VanA-type resistance. The paper by Foucault et al. uses isogeneic strains of the bacterium Enterococcus faecium to assess the fitness cost of VanB-type resistance and how enterococci have evolved to maintain vancomycin resistance.
|6||Macrolides: Ribosome-binding Antibiotics and Development of Bacterial Resistance|| |
Macrolide antibiotics were first discovered in the 1950s when scientists isolated erythromycin from the soil bacterium Streptomyces erythraeus. These compounds contain a macrocylic lactone ring decorated with deoxy sugar moieties and are used to treat infections caused by Gram-positive and a limited number of Gram-negative bacteria. In contrast to Gram-positive bacteria, which contain one lipid bilayer membrane and a thick extracellular peptidoglycan layer, Gram-negative bacteria have two lipid bilayers, with a thin peptidoglycan layer sandwiched between the two membranes. The mode of inhibition by macrolides remained unclear until recently solved X-ray crystallographic structures of macrolide antibiotics bound to the large ribosomal subunit were determined. Lincosamide, which contain a mycarose sugar, inhibit protein synthesis by mimicking the natural substrates, the Aminoacyl-and Peptidyl-tRNAs, and preventing nascent peptide bond formation. In contrast, macrolides such as erythromycin and streptogramin B inhibit the elongation of the polypeptide chain by causing premature dissociation of Peptidyl-tRNA from the ribosome through blocking the exit of the nascent peptide chain from the ribosome. In the first paper, Tenson et al. combine structural information with biochemical data to distinguish these two macrolide mechanisms of inhibition. This experimental system can be used to analyze the mechanism of action of novel macrolides as well as steps involved in protein synthesis at the molecular level. In the paper by Zaman et al., an erythromycin resistance mechanism is analyzed by examining mutations in proteins that form the peptide exit tunnel in the large ribosomal subunit.
Watch a video about mechanism of action of macrolides.
|7||Superbugs: Innate and Acquired Resistance|| |
Multidrug resistant bacteria or "superbugs" are currently a worldwide public health concern. The molecular basis of multidrug resistant bacteria is complex and multi-faceted. We will discuss two ways bacteria have evolved to resist a wide range of antibiotics. The first is through biofilm formation, a complex multicellular growth state with natural antimicrobial resistant properties. In the first paper by Mah et al., the authors used a transposon mutant library to identify a polymer produced by biofilms that is able to bind antibiotics and inhibit antibiotic function. Efflux pumps, protein complexes that extrude small molecules including antibiotics out of the cell, are the focus of the second paper by Lin et al. This study reports the identification of an efflux pump of the bacterium Campylobacter jejuni and characterizes its substrate specificity using susceptibility tests and small molecule accumulation assays.
Watch a video about mechanisms of bacterial resistance.
Watch a video that explains bacteria efflux pumps.
Watch a video about biofilm analysis using a Flow-cell apparatus.
|8||Field Trip|| |
We will tour the quality control chemistry lab and learn about the water treatment process and the techniques used to monitor water purity and the presence of antibiotics at the Cambridge Water Treatment Facility at Fresh Pond.
|9||Prevalence of Antibiotic Resistance in the Environment|| |
The widespread use of antibiotics for therapeutic and Non-therapeutic applications is thought to be a major factor in the proliferation of Antibiotic—resistant bacteria. Evidence for the direct impact of Non-therapeutic uses, such as in water treatment facilities and agriculture, are just beginning to surface. This week we will try to understand the mechanism by which antibiotic resistance is spread in the environment. First, a paper by Czekalski et al. looks at the prevalence of Antibiotic-resistant bacteria in the environment after water treatment at different steps of the process and at different locations after the treatment. We will also discuss the presence of Antibiotic-resistant bacteria in the soil following manure fertilization from cows treated with or without antibiotics, using a paper by Udikovic-Kolic et al.
Watch a video about development of bacterial resistance in a water treatment plant.
|10||Mode of Action of Antimicrobial Peptides|| |
Our diminishing ability to fight off infections caused by superbugs with traditional antibiotics stresses the serious need to develop other control agents. Advances in novel antimicrobial agents that act with less specificity to avoid potential resistance development are being made. One promising class of antimicrobial agents is antimicrobial peptides (AMPs), which are produced by eukaryotes and prokaryotes and have broad-spectrum activity against bacteria, viruses, and fungi. To use AMPs as potent antimicrobial agent, with low host toxicity, their mechanisms of action must be understood. First, we will discuss a paper by Sychev et al. wherein the mechanism of pore formation by two antimicrobial peptides in diverse lipid environments is assessed. The second paper by Deris et al., investigates polymixin lipopeptides, which are antibiotics targeting the bacterial outer membrane and are used as a last line of defense against infections caused by Multidrug-resistant Gram-negative bacteria. The authors developed a fluorescent polymyxin lipopeptide, an imaging probe, to analyze the mode of action and pharmacokinetics of polymyxin lipopeptides. This tool can be used to identify and characterize polymyxin lipopetides with activity against superbugs.
|11||New Methods for Antibiotic Discovery and Delivery|| |
With the continuous rise of Antibiotic-resistant bacteria, new strategies to combat bacterial infections are required. Research has focused on new ways to discover antibiotics and to more efficiently deliver these agents to target bacteria. This week we will discuss a recently discovered antibiotic, termed teixobactin, that was identified and isolated by Ling et al. using a new technique that allows the cultivation of previously uncultivable organisms. We will also examine a paper by Radovic-Moreno et al. wherein nanoparticles were developed for targeted drug delivery and to reduce therapeutic side effects. These nanoparticles are composed of polymers that are modified to encapsulate antibiotics and contain pH sensitive polymers that bind efficiently to bacteria.
Watch a video about a new technology, iChip, used to discover novel antibiotics.
|12||Non-traditional Methods to Treat Bacterial Infection: Fecal Transplants and Viruses|| |
The discovery of new natural and synthetic antibiotics can lead to treatments for patients with drug-resistant infections; however, history has shown that these solutions are only temporary, as bacteria will undoubtedly develop Antibiotic-resistance. For this reason, scientists continue to seek alternative approaches to treating infections. This week, we will discuss two promising methods to treat Antibiotic-resistant bacterial infections. The first approach is micobiome replacement therapy, which takes advantage of the resilient properties of bacteria present in the gut of healthy people to outcompete pathogenic species in diseased individuals. A clinical study by van Nood et al. demonstrates the utility of fecal transplants in fighting Clostridium difficile infections, which causes severe and recurrent gastroenteritis and are multidrug resistant. The second paper by Yosef et al. describes a strategy using bacteriophage to target and mutate the genomes of antibiotic-resistant bacteria. Both papers show promise for Non-traditional approaches for treating multidrug-resistant infections.
Watch a video about "Miracle Poop".
Watch a video that explains the CRISPR-Cas9 system described in Yosef et al.
|13||Presentations||This week will be devoted to student presentations. Each student will make a 15-minute presentation based on a preselected paper. The students should be prepared to answer questions during the presentation, and the entire group will engage in a discussion based on the presentations. During this class we also will reflect on key topics we learned about over the semester. Finally, we will ask for your impressions of the course and distribute course evaluation forms.|