..:: Cavanagh lab ::..

* Transition State Regulators - DNA binding proteins

When bacteria encounter any sort of stress they initially assess whether they should protect themselves. If the stress is persistent, a large morphological change is undertaken as the cell strives to survive. At first the bacteria enter a so-called transition state where processes required for continued growth are regulated simultaneously with those required for protection. This period is a 'cellular holding pattern'. It's where the bacteria decide what to do. It's likey bacteria spend most of their time in this state . Many processes are controlled at this time by, surprisingly, only a few regulatory general proteins. These are a relatively new class of proteins known as transition-state regulators. Transition-state regulators are DNA-binding proteins that recognize and interact with an enormous array of gene promoter regions. They are able to target seemingly disparate nucleotide sequences with little effort. On the other hand, they do not target all nucleotide sequences and different transition-state regulators recognize different targets. So, there is a balance between binding promiscuity and specificity. This is a very complex and subtle mechanism.

We have been comparing structurally identical transition-state regulators that recognize different targets to see if we can understand this DNA recognition process. It appears that structurally identical proteins possess different dynamic properties that may influence their binding targets. This constitutes an as yet unknown, novel DNA recognition mechanism. It's fundamentally different from anything else we're aware of. Our goal is to better understand this important binding model.

* The two-component system and response regulators

Continued exposure to stress causes the bacteria to go into a more self-protective mode. In our systems, this means the onset of biofilm formation, sporulation or virulence. The response is induced by a pair of so-called two-component systems. Two-component systems are ubiquitous signal transduction modules involved in a myriad of processes but have found particular notoriety by their critical role in the generation of bacterial pathogenesis and antibiotic resistance. They are found in all bacteria so far studied and are responsible for the ability of the cells to adapt to changing environments and ultimately to protect the bacteria in times of life threatening hostility. At some level they are responsible for the development of all bacterial virulence and protection. They consist of a histidine protein kinase and a response regulator. In times of stress the kinase becomes phosphorylated and subsequently passes the phosphoryl group onto the response regulator, which undergoes a conformational change, thereby allowing it to interact with its next target(s), be it DNA or another protein. Response regulators share similar structures and general function, however their specific mechanism of action is not well understood. Our recent work has defined much about the recognition processes of response regulators, showing that protein structure is only part of the answer. Protein dynamics and intra-protein communication networks greatly contribute to response regulator function. We are continuing these efforts in an array of model systems and systems that have direct human health impacts.

* Calbindin D28K and Alzhemier's Disease

Alzheimer's disease (AD) is characterized by synaptic loss and substantial neuronal degeneration in regions involved in memory and learning processes. It is generally accepted that the principal pathological features of AD are: (i) gradual accumulation of extracellular b-amyloid (Ab) in the form of senile plaques and (ii) intracellular neurofibrillary tangles (NFT's) formed by the microtubule-binding protein tau assembled mainly into paired helical filaments (PHF's).

It is also known that the protein caspase-3 performs numerous roles in the onset of Alzheimer's Disease. Importantly it known that amyloid production is increased by caspase-3 cleavage of mutant presenilins and that caspase-3 cleavage of the tau protein results in the increased production of pathogenic neurofibrillary tangles. For these reasons, selective inhibition of caspase-3 has been suggested as a potential therapeutic approach in AD. To date, the majority of caspase-3 inhibitors have been small peptidyl or chemical mimics of natural substrates. These compounds occupy the active site of caspase-3. Consequently these compounds are in competition with natural substrates for the active site of caspase-3.

From a therapeutic design standpoint, avoidance of such direct competition offers great benefits. With this in mind we are studying the structure and interactions of calbindin D28K in order to provide better leads for therapeutic development.

Calbindin D28K is a neuroprotective protein and an excellent natural inhibitor of caspase-3. Recent evidence suggests that it performs its role by both acting as a calcium buffer and also as by controlling caspase-3 function. It is known to interact directly to caspase-3 in a fashion that does not mimic natural substrate binding. It has also been suggested that calbindin D28K may interact with procaspase-3, the inactive form of caspase-3. Our general goals are to accurately determine the interaction site for calbindin D28K with both active caspase-3 and with the pro-domain from procaspase-3. Our goal is to derive peptides based on these interaction sites to be used as lead compounds in caspase-3 inhibition studies.

We are in a very strong position to perform these studies since we determined the high resolution NMR solution structure of fully calcium loaded calbindin D28K. In addition, we have accurately defined the region on calbindin D28K that binds to procaspase-3. We have also determined that calbindin D28K undergoes a large conformational change upon calcium loading and that as the calcium loads, the protein adopts discrete hydrophobic states. These states appear to dictate the efficiency with which calbindin D28K binds its targets. We have also made substantial progress in the NMR structure determination of apo-calbindin D28K.

* Development and Application of Novel, Non-Toxic, Anti-Biofilm Agents

We have been working closely with Christian Melander at NCSU in both developing and applying synthetically accessible, non-toxic small molecules that act as anti-biofilm agents with unprecedented effectiveness. We have recently discovered that these compounds also completely re-sensitize bacteria to conventional antibiotics.

Biofilms are formed when free floating planktonic bacteria accrete onto a surface in large colonies, engulfing themselves in a self-made extracellular polymeric substance (EPS). This EPS matrix serves as an adhesive and a protector of the colony against bactericidal agents. In fact, the infections induced by bacterial biofilms display more than a 1,000 fold increase in antimicrobial resistance than their planktonic counterparts and cause at least 80% of all bacterial infections. They are particularly problematic in: dental caries, periodontitis, otitis media, musculoskeletal infections, bacterial prostatitis, ophthalmic infections, cystic fibrosis, pneumonia and chronic urinary tract infections. Biofilms show preferential development on necrotic tissue, inert surfaces and appear very frequently on medical equipment, explaining the high frequency of nosocomial infections.

We have discovered that bromoageliferin - a marine natural product from the sponge Agelas conifera can be used as a base for developing compounds that eradicate biofilms. We have developed libraries of synthetically accessible analogs of bromoageliferin that have unprecendented activity against all bacterial biofilms so far tested. These compounds both inhibit and disperse pre-formed biofilms. Our compounds are the first to be active against gram-negative and gram-positive bacteria and across both bacterial order and class. Our lead compound library shows little toxicity against multi-cellular organisms. We have worked on the following bacterial biofilms with human health implications: Pseudomonas aeruginosa - infects the pulmonary tract and urinary tract. It is the most common cause of burn and external ear infections, and is the most frequent colonizer of medical devices. Cystic fibrosis patients are also predisposed to P. aeruginosa infection of the lungs. In addition, P. aeruginosa infections are responsible for countless deaths of cancer patients whose immune systems are compromised due to chemotherapy and radiation treatment. Acinetobacter baumannii, an opportunistic pathogen prevalent in hospitals. A. baumannii enters the body through wounds, catheters and breathing tubes. There have been many reports of multi-drug-resistant A. baumannii infections among American soldiers in Iraq. Bordetella pertussis - whooping cough. Vibrio vulnificus - infection after eating seafood; the bacteria also enter the body through wounds when swimming or wading in infected waters. V. vulnificus claimed lives in Hurricane Katrina. Staphylococcus epidermidis infections from indwelling catheters and medical devices. Strains produce a biofilm that allows them to adhere to the surfaces of prostheses. Staphylococcus aureus, illnesses from minor skin infections to pneumonia, meningitis, endocraditis, toxic shock syndrome and septicemia.

In addition, we have recently found that these compounds also re-sensitize bacteria to conventional antibiotics - effectively overcoming resistance traits. We will develop further analogues of our compounds and will detail their effects, as adjuvant therapies, on the performance of many current antibiotics against multiple bacterial strains. We will also develop new toxicology screens and initiate differential array work to begin determining the mode of action of our compounds.