Current Research Projects

Indranil Biswas

Indranil Biswas, Professor, Dept. of Microbiology, Molecular Genetics & Immunology, University of Kansas Medical Center

Project TitleDeveloping Assays to Identify Inhibitors of Hfq of Acinetobacter Baumannii

Project Summary

Acinetobacter baumannii, a gram-negative opportunistic pathogen, is becoming an important nosocomial causing a wide range of diseases and infections including ventilator-associated pneumonia and septicemia as well as urinary tract infections. The pathogen has emerged as one of the most highly antibiotic resistant in the US and elsewhere. Nearly, 70% of A. baumannii clinical isolates are now resistant to all drugs except collistin or tigecycline (known as extremely drug resistant or XDR). Furthermore, infections caused by A. baumannii that are resistant to all available antibiotics (known as pan-drug resistant or PDR) have already emerged and continue to increase since no new drug is in the pipeline that targets A. baumannii. The traditional antibiotics that target cell viability and growth perhaps are not the answer since they will drive the appearance of XDR or PDR further. The innovative approach would be the development of drugs that target the bacterial pathogenesis by inhibiting or controlling expressing of virulence factors. Hfq is a pleiotropic virulence regulator found in many pathogenic bacteria. It is a conserved protein that functions as a post-transcriptional regulator and displays RNA chaperone activity. Inactivation of hfq makes the cells sensitive to various environmental stresses, such as oxidative stress, displaying enhanced susceptibility to various antibiotics, and alteration of the synthesis of several proteins. Furthermore, pathogens lacking a functional Hfq protein are all attenuated for virulence. Therefore, Hfq is an ideal target for drug development to control a wide range of pathogens including A. baumannii.

The major goal of this study is to develop assays for a high-throughput screen (HTS) of small molecule inhibitors of Hfq using a heterologous reporter system and a native expression system. We expect that successful completion of this limited term study will establish an assay system than can be explored further for potential small molecule inhibitors of Hfq. Our long term goal is to stimulate new therapeutic strategies for A. baumannii infections by targeting Hfq and its regulatory mechanisms.

David Davido

David Davido, Associate Professor, Dept. of Molecular Biosciences, University of Kansas

Project Title: The Chemical Biology of HSV Gene Expression

Project Summary

The specific events that dictate herpes simplex virus type 1 (HSV-1)-cell interactions critically affect the outcome leading to either lytic or latent infection. An HSV-1 immediate-early (IE) regulatory protein that plays a key role in this process is infected cell protein 0 (ICP0). The ICP0 gene encodes a 775 amino acid (aa) protein that is a phosphorylated, nuclear E3 ubiquitin (Ub) ligase with the capacity to activate transcription of all classes of HSV-1 genes. ICP0 transactivates viral genes via its E3 ubiquitin ligase activity, ubiquitin being a post-translational modification typically associated with protein stability. As HSV-1 is an obligate intracellular pathogen that requires host factors to replicate, host cell factors are likely to play important roles in the ability of ICP0 to stimulate HSV gene expression. While insights as to how ICP0 and its interactions with cellular factors enhance HSV-1 replication has been primarily performed through cell biological and genetic based assays, a chemical biological approach using bioactives and natural compounds to understand ICP0 function and HSV-1 replication remain largely unstudied. Until potential inhibitors and pathways ICP0 interacts with are identified, it will be unclear as to the exact mechanisms ICP0 plays in the switch between the lytic and latent or quiescent phases of infection. Our long-term research goal is to elucidate the molecular interactions between HSV-1 and its host that modulate the HSV-1 life cycle and use this knowledge to ultimately develop therapeutic interventions for treating patients with HSV-1 diseases. The objective in this proposal is to initially identify novel mechanisms by which ICP0 stimulates viral gene expression to enhance HSV-1 productive infection using a chemical biology approach. Our central hypothesis is that inhibition of ICP0 function with small compounds will lead to the discovery of novel ICP0 interactions or pathways (with viral and/or cellular factors) that promote HSV-1 gene expression. Given the time frame of this pilot project grant, one specific aim is proposed: Specific Aim #1: Identify novel inhibitors of HSV-1 ICP0 and viral replication using chemical libraries that include compounds that recognize specific cellular targets/pathways.

Brandon DeKosky

Brandon DeKosky, Assistant Professor, Depts. of Pharmaceutical Chemistry and Chemical Engineering, University of Kansas

Project Title: A New Experimental Platform to Analyze anti-gB Antibodies in Human B Cells

Project Summary

This project will establish a new experimental pipeline for rapid analysis of antibody immune pressure against viral pathogens, which holds the potential to accelerate the discovery of therapeutic and vaccine candidates against persistent viruses. This project will develop new research tools to investigate adaptive immune pressure against human cytomegalovirus (HCMV), which is a highly prevalent pathogen infecting the majority of individuals in the world and causes significant morbidity and mortality in immunocompromised patients and in congenital infections with prevalence of around one in 200 births. Here we will develop a new antibody isolation assay to identify and express anti-HCMV glycoprotein B (gB) antibodies that have potential HCMV neutralization capacity among human antibody repertoires. We will also transfer established HCMV neutralization assays into a new high-throughput robotic format to rapidly screen our isolated antibodies for HCMV neutralization. This work will leverage established high-throughput immune profiling techniques recently invented by the PI to interrogate anti-HCMV antibody-based immunity and will greatly extend our capabilities in high-throughput sequencing and analysis of antiviral antibody repertoires. This pilot project will establish the protocols, research environment, and expertise to attract external funding and begin clinical research studies regarding the features of effective and ineffective adaptive immune pressure against HCMV infections. In particular, this project will establish a new research environment for follow-up collaborative studies investigating effective vs. ineffective adaptive immune pressure against HCMV in a prospective cohort of matched mother and infant pairs. In the long-term, the research catalyzed by this pilot project will accelerate growth of technologies for rapid analysis of adaptive immunity against persistent viruses. These new technologies will enable discovery of potent antibodies to prevent and treat viral infections in vulnerable populations, beginning with HCMV.

Revathi Govind

Revathi Govind, Assistant Professor of the Division of Biology, Kansas State University

Project TitleCurtailing Clostridium Difficile Virulence

Project Summary

Clostridium difficile is the leading cause of hospital-acquired diarrhea. Antibiotic use is the primary risk factor for the development of C. difficile-infections (CDI) because it disrupts normal protective gut flora and enables C. difficile to colonize the colon. The current treatment for CDI, administration of additional antibiotics, is increasingly ineffective and often results in relapse of the disease. New strategies to treat this important pathogen are urgently needed and one such approach is to target its virulence. Toxigenic C. difficile strains produce two toxins, toxin A and toxin B that are considered to be the major virulence factors. The toxins encoding genes, tcdA and tcdB are part of a pathogenicity locus, which also carry the gene encodes for the toxin genes positive regulator tcdR. TcdR is an alternate sigma factor that is specifically required for expression of tcdA and tcdB. In a preliminary study we found that tcdR in C. difficile to affect both toxin production and sporulation. It is hypothesized that a small molecule that inhibits TcdR would block toxin production along with sporulation and will serve as an anti-pathogenic agent against C. difficile. Different TcdR activated promoter-reporter fusions were developed and have been shown in E. coli to be appropriate for high-throughput screening. In the first specific aim, we propose to develop series of recombinanat TcdR activated promoter reporter fusions in E. coli and in B. subtilis for high throughput screening. Employing these reporter fusions, we will conduct a pilot screening of small molecule libraries from the KU-HTS core for compounds that inhibit TcdR activity. Second aim of the grant will focus on understanding the influence of TcdR on sporulation. We propose to study the influence on TcdR on sin (sporulation inhibition) locus transcription in C. difficile.

Alternate sigma factors are known to regulate virulence and virulence associated genes in many pathogenic bacteria. Including toxin genes, TcdR may regulate other virulence-associated genes in C. difficile. We have created and characterized, tcdR mutant in two different C. difficile strains. Mutation in tcdR affected both toxin production and sporulation in C. difficile. Microarray analysis revealed many differentially expressed sporulation-associated genes in tcdR mutant. In this project in our first aim, we propose to test the role of TcdR in C. difficile sporulation. In our second aim, we are proposing to monitor TcdR dependent promoter expression at cellular level, using a novel reporter system. During the current decade there has been a dramatic increase in the incidence and severity of C. difficile infections due to the emergence of hypertoxinogenic C. difficile strains. Our long- term goal is to unravel pathogenic mechanisms of C. difficile, thus new strategies to prevent, treat and manage C. difficile infection can be developed.


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