PI: Indranil Biswas, Ph.D., Associate Professor, Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center.
Streptococcus pyogenes, the group A streptococcus (GAS), is an important and common human pathogen which causes a wide variety of diseases including relatively mild, self-limiting infections of the throat and skin as well as severe invasive necrotizing fasciitis and streptococcal toxic shock syndrome. Bacterial pathogens encounter a number of stresses that are either due to attack by the immune system or result from bacterial entry into tissue sites that inhibit growth. Exposure of bacteria to these adverse environments can induce two kinds of stress responses: general stress response and specific stress response. The general stress response provides cross-protection against diverse environments by inducing a wide variety of genes regardless of initial stimulus and is controlled by a single or few master regulators. Specific stress responses facilitate a cells survival under a particular stress condition by inducing a subset of genes appropriate for that physiological condition and are regulated by unique groups of repressors. Very little is known about stress response genes and their regulation in GAS. Unlike other pathogens, GAS does not encode an alternate sigma factor that controls general stress response genes. In addition, general stress response genes have not been identified in GAS. However, Clp family of proteases that are involved in thermal and oxidative stress responses in other bacteria, are present in GAS. In this work, we propose to study stress response regulation in GAS. Specifically, we want to identify general stress response genes and study the role of two Clp proteases in general stress responses, with the following Specific Aims. In Aim 1, we will identify the genes that are required for survival and are induced by thermal stress, oxidative stress, and osmotic stress conditions. We will use transposon mutagenesis, DNA microarrays and proteomic approaches to identify differentially expressed genes/proteins under various stress inducing conditions. In Aim 2, we will study the role of ClpE and ClpP proteases in the regulation of stress responses and virulence expression. GAS encodes five Clp proteins that have close homologs in other bacteria. Among them, ClpE is uniquely present in gram-positive bacteria. The role and regulation of ClpE in general stress response and in pathogenesis has not been investigated in streptococci including GAS. In contrast, ClpP protease is ubiquitously present in all bacteria and is involved in general stress responses; however, its role in pathogenesis and in regulation varies significantly among bacteria. We will study the role of ClpE and ClpP in stress survival and identify various proteins and virulence factors that are regulated by ClpE and ClpP by proteomic analysis. We will use Pclp-reporter fusions and transposon mutagenesis to identify genes required for PclpE and PclpP expression (Aim 2C). In summary, this study will identify groups of stress response genes and virulence genes in this important human pathogen. In addition, the results can be extended to other pathogens like S. pneumoniae and may possibly identify new and potentially broad-spectrum drug targets.
Hepatitis B virus/hepatitis delta virus infection, its relation to pathogenesis
PI: Severin Gudima, Ph.D., Assistant Professor, Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center.
Human hepatitis B virus (HBV) infection is a major health risk. There are approximately 400 million carriers of persistent infection worldwide, of whom about 1 million die annually from HBV-induced liver cancer and chronic liver disease. Co- or super-infection with the sub-viral agent hepatitis delta virus (HDV) can lead to more severe pathology, increasing liver damage and risk of cirrhosis and hepatocellular carcinoma (HCC). This proposal is aimed to ascertain molecular mechanism of HBV/HDV infection with emphasis on pathogenesis.
Aim 1. The goal of this aim is to understand how the virus-induced pathogenesis is linked to a particular genotype of HBV. The project will compare aspects of replication, assembly, infectivity in primary human hepatocytes and host response for all known 8 HBV genotypes. The studies proposed will: (i) facilitate the understanding of the molecular mechanism of HBV genotype-specific pathogenesis; (ii) ascertain how HDV chronic infection is maintained; (iii) have a serious impact on the understanding of virus–host interactions. The finding of the most infectious HBV genotype will lead to three major applications: development of more potent peptide antivirals; performance of microarray studies using the system of in vitro infection of primary human hepatocytes; and establishment of HBV-susceptible cell lines.
Aim 2. To delineate the significance of the balance between HBV and HDV for outcome of infection, as will it become transient or chronic with possible risk of liver cancer, it is proposed to quantitatively examine what regulates competition or homeostasis between the two viruses during co-assembly, since both viruses share the same envelope. Also, using cultures of primary human hepatocytes, the patterns of co- and super-infection will be analyzed, addressing if the two viruses compete for susceptible cells, and if their replication rates are mutually affected. These studies will: (i) facilitate building the model of HBV-HDV homeostasis; (ii) enhance our understanding of virus-virus interactions in relation to pathogenesis; (iii) have significance in terms of the chronic infection mechanism by addressing the role of re-infection.
Modulation of NF-κB signaling by E. coli protein kinases
PI: Philip R. Hardwidge, Ph.D., Assistant Professor, Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center.
Enterohemorrhagic E. coli (EHEC) O157:H7 contributes greatly to the enormous economic and health
burden of food borne disease. EHEC causes severe diarrhea through contamination of beef and vegetable
products and also causes a fatal kidney disease for which there is no effective treatment or prophylaxis.
The central hypothesis for the proposed research is that the EHEC effector proteins NleH1-1 and NleH1-2
bind to the non-Rel NF-κB subunit Rps3 to disrupt specific host transcriptional responses to bacterial
infection. We have formulated this hypothesis based on our strong preliminary findings that demonstrate
NleH binding to Rps3 in vitro, alteration of NF-κB dependent transcription following NleH1-1/2 transfection,
and hypervirulence of EHEC strains lacking either nleH1-1 or nleH1-2 in a gnotobiotic piglet model of EHEC
infection. The specific aims are to:
1. Map the NleH-Rps3 binding domains. Our working hypothesis is that NleH1-1 and NleH1-2 bind the
mammalian non-Rel NF-kB subunit Rps3 to subvert its normal function.
2. Quantify the influence of NleH1-1/2 translocation on host transcription. Our working hypothesis is that
NleH1-1 represses NF-κB-dependent host transcription, whereas NleH1-2 stimulates NF-κB.
3. Measure the contribution of NleH1-1/2 to bacterial virulence in animal models of attaching/effacing
pathogens. Our working hypothesis is that NleH1-1/-2 promote bacterial transmission by maintaining an
optimal balance between bacterial colonization vs. host inflammatory responses.
The proposed research is innovative because it will test the hypothesis that translocated bacterial protein
kinases subvert the host innate response to infection by disrupting a novel molecular ‘specifier’ of selected
host transcriptional responses to external stimuli. These studies are expected to have a significant positive
impact on the design of new strategies to combat diarrheal pathogens, as they may identify host proteins
indispensible for bacterial infection as novel therapeutic targets for broad control of enteric pathogens.
Role of CNF1 in Escherichia coli meningitis
PI: Kee Jun Kim, Ph.D., Assistant Professor, Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center.
Despite advances in antimicrobial chemotherapy and supportive care, the mortality and morbidity associated with Escherichia coli meningitis remain significant due to incomplete understanding of the pathogenesis of this disease. E. coli K1 is the major cause of neonatal Gram-negative bacterial meningitis and its invasion of human brain microvascular endothelial cells (HBMEC) is a prerequisite for its penetration of blood-brain barrier (BBB) in vivo and in vitro. My colleagues and I have shown that cytotoxic necrotizing factor 1 (CNF1) is a major bacterial determinant contributing to E. coli K1 invasion of HBMEC and that laminin receptor (LR) is the cellular receptor for CNF1, which induces host cell actin cytoskeleton rearrangements through activation of RhoGTPases. Further characterization of CNF1-LR interaction suggests that LR plays essential role in CNF1-mediated E. coli K1 internalization into HBMEC, but it is incompletely understood how CNF1-LR interaction modulates actin cytoskeleton rearrangements in HBMEC, resulting in E. coli K1 invasion of HBMEC. Therefore, I hypothesize that E. coli K1 CNF1 interaction with its receptor (LR) triggers downstream signal transduction pathways responsible for actin cytoskeleton rearrangements and, in turn, E. coli K1 entry into HBMEC. To test my hypothesis, I propose to (1) determine whether activation of RhoGTPases is a proximal downstream of CNF1-laminin receptor (LR) interaction in HBMEC; (2) determine signaling molecules downstream of RhoGTPases activated through CNF1-LR interaction; (3) determine the role of ezrin in CNF1-mediated E. coli K1 invasion of HBMEC. The information derived from this study should enhance our understanding of the pathogenesis of E. coli meningitis, i.e., molecular mechanisms underlying CNF1-mediated E. coli K1 internalization into HBMEC and lead to the development of novel strategies to prevent E. coli meningitis.
Mechanics of hantavirus nucleocapsid protein mediated translation initiation of viral mRNA
PI: Mohammad A. Mir, Ph.D., Assistant Professor, Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center.
Eukaryotic mRNA translation initiation is a very complex process involving multiple translation initiation factors. Translation begins with recruitment of eIF4F complex at mRNA cap which engages 43S pre-initiation complex at mRNA 5 terminus. Another well characterized mechanism utilized by several viruses includes IRES translation initiation strategy that internally loads ribosomes on mRNA, independent of 5 cap. Hantaviruses, members of the Bunyaviridae family are emerging viruses that initiate mRNA translation by a different novel mechanism, using viral capsid protein (N) to engage the ribosome at mRNA cap, independent of eukaryotic eIF4F complex. We will further characterize N mediated translation initiation mechanism and illustrate possible benefits of this novel strategy that favor virus replication in infected cells. We will identify the components of 43S pre-initiation complex that interact with N. N specifically binds the viral mRNA 5 UTR with high affinity and preferentially facilitates the translation of viral mRNAs in vitro. We will identify and characterize the binding site for N on viral mRNA 5 UTR and will determine whether N preferentially facilitates the translation of viral mRNAs in host cells. N is also an RNA chaperone that unwinds RNA duplexes. However, this RNA chaperone activity is not involved in N mediated mRNA translation. Secondary structures in mRNA 5 UTR are removed by eIF4A (a component of eIF4F complex) during ribosome scanning and identification of AUG codon. Since N functionally supplants eIF4F complex, we hypothesize that N translocates the loaded ribosomes from 5 cap to the AUG codon, avoiding the regular scanning of 5 leader. Multifaceted experimental approaches have been designed to test this hypothesis. Host cells design various strategies to prevent virus replication, such as, interferon induced PKR over-expression leads to the phosphorylation of eIF2a, and transient shutdown of host mRNA translation. N significantly inhibits INF induced phosphorylation of eIF2a, suggesting that N likely blocks the virus induced host translation shutoff and prevents the cells from entering into an antiviral state. We will use multiple experimental approaches to check whether N mediated translation strategy is a viral counter measure against host cell antiviral response.
Post-transcriptional Regulation of Parvovirus B19 Capsid Gene Expression
PI: Jianming Qiu, Ph.D., Assistant Professor, Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center.
Parvovirus B19, the only parvovirus so far known to be pathogenic in humans, causes a variety of diseases, including erythema infectiosum in children, aplastic crisis in patients with chronic hemolytic anemia, persistent bone marrow failure in immunocompromised patients, and fetal hydrops in pregnant women. B19 is unique among animal DNA viruses in that it has a single promoter, and so its genetic diversity is controlled exclusively by post-transcriptional mechanisms. A single class of pre-mRNA molecules generated by B19 virus undergoes extensive alternative splicing and polyadenylation that generates sub-genomic mRNA molecules which program this diversity. Control of the synthesis of the capsid proteins is one of the key regulators of B19 tissue tropism. B19 permissive infection is characterized by a switch to increased capsid production. Other parvoviruses have an internal promoter that regulates production of capsid-encoding mRNA during the synthesis phase of viral replication, however, this mechanism is not available to B19. Control of expression of B19 capsid-coding genes by post-transcriptional mechanisms is the focus of this application. B19 has an efficient polyA site in the center of the genome. Use of this site precludes inclusion of the capsid coding ORFs into mRNA, and an understanding of how the choice is made to either polyadenylate, or read-through this site, is the top of Specific Aims. We propose to identify both the cis sequence and trans-factors that control these events, the mechanisms that govern their selective use, and exam how this choice is differently made in cells permissive or no-permissive for replication. How these post-transcriptional processes generate appropriate levels of the capsid proteins from the single pre-mRNA molecules encoded B19, is critical to our understanding of parvovirus gene expression, and the biology of parvovirus infection. However, in addition, these viral systems provide very tractable models with which to learn much about these basic cellular mechanisms in general.
This grant was made possible by NIH Grant Number P20 RR016443 from the COBRE program of the National Center for Research Resources.
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