Survey for antimicrobials effective against carbapenem-resistant Gram-negative bacteria

Principal Investigator: Dr Branko Jovcic 

            Among all of the bacterial drug resistance problems, Gram-negative pathogens are particularly worrisome, because they are becoming resistant to nearly all drugs that are being considered for the treatment. Causative agents of the most serious gram-negative infections (due to limited treatment options and a high mortality rates) are carbapenem-resistant Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter. Globally, urgent actions are recognized as necessary in order to find novel antimicrobials that will diminish the threat of multidrug-resistant Gram-negative bacteria to global public health. So far, novel antibiotic producers were screened among soil Actinomycetales. However, we are now aware that novel ecological niches must be exploited and screening should be expended towards other bacterial species as potential antibiotic producers. Sediments of glacial lakes from Western Balkans are unexplored natural treasure and represent a challenge regarding their microbial genomic and metabolic potential. Remote high mountain lakes, being far from habitation and located in extreme environments, receive less impact from human activities but magnify the effects of global climate changes, and can thus be taken as a mirror of natural environmental changes. Therefore, exploiting their microbial is of crucial importance. During the project we will analyze sediments of three lakes in Montenegro and Bosnia and Herzegovina. Lakes will be selected due to possible anthropogenic influences - ranging from lakes with minimal anthropogenic impact to that with significant anthropogenic impact. In order to get the full insight into these microbial communities, both microbiological and metagenomic approach will be used for analyses. Cultivable bacteria will be isolated from sediments and grown in aerobic and anaerobic conditions on various media. Cultivated bacteria will be then subjected to testing of antimicrobial compunds production. 16S rDNAmetagenomic analysis will be performed in order to determine the microbial diversity in lake sediments. Functional metagenomic libraries will be constructed in order to fully exploit the genomic potential of microbial communities from sediments. Functional metagenomic libraries will be expressed in E. coli and tested for the production of compounds active against carbapenem-resistant, multidrug-resistant Gram-negatives in order to avoid selection of antimicrobials that are already known, and those that can not overcome existing resistance mechanisms that are globally dispersed among Gram-negative pathogens. Also, functional metagenomic libraries will be screened for the presence of various antibiotic-resistance genetic determinants. After the selection of cosmid that encompasses potentially novel antimicrobial compound it will be subjected to sequencing in order to reveal gene(s) involved in biosynthesis of this compound. Also, we will determine biochemical properties of a novel antimicrobial compound, perform its chemical purification, as well as analyze the interactions between the compound and potential target molecules. However, since sampling of glacial lake sediments is possible only in the summer period due to high altitudes of lake localities we will, during first six months of the project, analyze laboratory collection of Lactic Acid Bacteria (LAB) for the producers of antimicrobial peptides active against multidrug-resistant Gram-negative bacteria. This unique collection includes (LAB) isolated from fermented milk products produced in households of Western Balkan according to traditional recipes as well as strains of human origin. Screening will be performed by means of agar well diffusion assay, and compounds active against tested bacteria will be subjected to further biochemical characterization.

Dss1’s roles in genome integrity and protein oxidative damage control

Principal Investigator: Dr Milorad Kojic

            This proposal is mainly about two pivotal cellular functions (genome integrity control and protein oxidative damage control) and it centrally concerns Dss1 protein. Elucidating the role of Dss1 in these processes is of fundamental scientific interest. Moreover, the knowledge will have important implications for understanding and possibly treatment of numerous human disorders and diseases.  In recent years the investigations of the Dss1 has largely been focused on its essential role in BRCA2-driven recombinational DNA repair. BRCA2 has emerged as the product of a breast cancer susceptibility gene in human and then came to be realized as a central component of the homologous recombination system. Although these studies clearly demonstrated the importance of the Dss1 protein for the number of BRCA2 functions, it remained mostly enigmatic how their interplay is coordinated with other cellular events to ensure the controlled maintenance of genetic integrity. Furthermore, the Dss1’s role in the detection and clearing of oxidatively damaged proteins was discovered fairly recently. This novel type of posttranslational protein modification (named as DSSylation) is conditionally induced by free radicals via an ATPase-mediated process. Like many other intriguing breakthrough study, this one too provided more questions than answers. Hence, this project seeks to shift emphasis from BRCA2-Dss1 interplay and to broaden our perspective of how homologous recombination is connected to other pivotal cellular events. The fundamental assumption is that Dss1 might well be the conduit that connects DNA repair to the protein oxidative damage control.  We plan to use the Ustilago maydis experimental system for this study because the facility it offers for performing rapid molecular genetic operations will provide a powerful means for attacking the problems. Specifically, we propose to: 1) perform an innovative screen to identify suppressors that could bypass cellular requirement for Dss1,  2) investigate the role of Dss1 in cellular response to oxidative insult,  3) identify new factors cooperating with Dss1 in its modifier activity, 4) identify the ATP-ase that catalyzes the binding of DSS1 to the oxidized proteins. The factors and processes studied in the project will significantly enhance our knowledge of the networks that govern cellular response to DNA damage and oxidative stress.  We therefore anticipate that our findings will greatly contribute towards further understanding of a number of human disorders (aging) and diseases (raging from inflammation to cancer). They should also help shape strategies for preventative diagnosis and therapy by providing insight into possible drug targets.

Structurally-guided identification of novel pharmacophores targeting Pseudomonas aeruginosa quorum sensing and biofilm formation

Principal Investigator: Dr Lidija Senerovic

Antibiotic resistance for both Gram-positive and Gram-negative pathogens is spreading worldwide at an increasing rate, presenting a challenging global health problem. The aim of this project is to discover and develop leads for new antimicrobial agents targeting bacterial biofilms, which are common sources of lower sensitivity or even resistance to antibiotics. Both historically and at the present time microbial natural products have been the major source of new drugs. Most clinical antibiotics are natural products or inspired by natural products. They usually came from screens of Actinomyces, soil-dwelling bacteria which are well-known for their production of valuable metabolites. During this project we will screen our untapped Streptomycetes culture collection accumulated for 20 years from variety of sources and ecological niches and search for the specific small molecules interfering with bacterial virulence rather than viability. Targeting bacterial virulence reduces selective pressure, which can decrease possibility to develop drug resistance and keeps the host endogenous microflora intact. Our screening will be based on bio-assays against P. aeruginosa quorum sensing-regulated biofilm formation.

Development of biopolymeric formulation of antifungal polyenes using medium chain length polyhydroxyalkanoate (PHA): validation against superficial mycoses

Principal Investigator: Dr Jasmina Nikodinovic-Runic

As the prevalence of superficial fungal infections in general population and more serious outcome of life-threatening invasive fungal infections are increasing there is a perpetual and unmet need for development of new and more advanced antifungal therapies. Therapeutic efficiency of so called ‘old drugs’ can be modified and tuned by designing appropriate drug delivery system. The major goal of this research project is controlled delivery of ‘old drugs’ such as polyenes to the specific site of action at the therapeutically optimal rate and dose using advanced biocompatible biopolymer (polyhydroxyalkanoate) as a carrier. During this project, for the first time biopolymeric formulation of two known antifungal polyenes nystatin and amphotericin B would be developed using biocompatible and biodegradable biopolymer medium chain length polyhydroxyalkanoate (mcl-PHA) and validated against a range of clinical fungal isolates. Two different biopolymeric preparations based on mcl-PHA would be developed, namely thin film and microparticles for encapsulation of polyene macrolides nystatin and amphotericin B. Resulting antifungal PHA would represent a novel material to achieve slow release and reduced toxicity of antifungals, and could further be developed into efficient sustained release antifungal bandages and desirable antifungal coatings.