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Student Experience

Career Opportunities for MMG Students: The career opportunities that are now available for Ph.D.s in microbiology are substantial and the goal of the MMG faculty is to guide the students in establishing a scientific foundation so that the students can be successful life-long learners. Graduates of the MMG program pursue many different career pathways and post-graduate training opportunities or employment. Some students continue their basic science training by conducting postdoctoral research at universities or in government laboratories. For instance, recent MMG Ph.D. graduates have undertaken postdoctoral research at Princeton University, Yale University, the University of Washington, Oxford University, the Centers for Disease Control and Prevention (CDC), the NIH and FDA. Other students continue their formal education by attending medical, law or public health schools while others gain employment in biotechnology firms. Indeed, our MMG Ph.D. graduates are presently faculty members in Microbiology and Immunology departments at universities, staff scientists at the CDC and NIH, scientists employed by biotechnology firms and large pharmaceutical companies, practicing physicians or lawyers or involved in scientific journalism enterprises.

A sampling of research projects undertaken by our current graduate students is described below.

Mark De Zalia (student in the Speck laboratory)

A hallmark of a herpesvirus infection is the establishment of a latent infection that persists for the life of the host. To this end, herpesviruses encode a variety of unique genes that act to establish and maintain the latent viral state and to periodically reactivate to a lytic viral state. Murine gammaherpesvirus 68 (γHV68) establishes a latent infection in mice, which provides a genetically tractable small animal model that can be utilized to identify host and viral factors that regulate gammaherpesvirus latency. M2 is a gene unique to γHV68 that appears to function as a signaling molecule. It is dispensable for lytic replication, but is required for the efficient establishment of latency as well as for reactivation from latency. My thesis research is focused on characterizing the transcriptional regulation of the γHV68 M2 gene. After identifying a promoter and a major transcript for the M2 gene, I constructed a number of viral mutants lacking critical portions of the promoter. These mutants were able to establish latency at frequencies similar to WT virus, but were crippled in their ability to reactivate from latency just as the M2 null virus, which, suggests either that a separate promoter drives M2 expression during the lytic phase or that the promoter is differentially regulated during latency establishment and reactivation. In addition, I am interested in the structure of the predominant M2 transcript. A mutant virus with a discrete mutation at the M2 splice acceptor sequence recapitulates the M2 null phenotype, however, a discrete mutation in the splice donor sequence only slightly decreases the establishment and reactivation frequencies. The remainder of my thesis research is focused on a thorough characterization of the M2 promoter, including identification of viral and host factors that act at the M2 promoter. This research will provide further insight into genetic events that lead to establishment of and reactivation from latency.

Shannon McNulty (student in the Kalman laboratory)

During morphogenesis, Poxviruses hijack eukaryotic vesicle trafficking pathways using kinesins to move along microtubules (MTs) to the cell periphery. Once at the cellular membrane, the virus is released from the MTs, and the viral membrane fuses with the cellular membrane, exposing a tethered virus to the extracelluar environment. Following membrane fusion, the extracellular particle stimulates nascent actin polymerization beneath the virion, which produces an actin-filled membrane protuberance ("actin tail"). The actin tail propels the particle towards an adjacent cell, initiating a new round of infection in a neighboring cell. Work from our lab has demonstrated that host tyrosine kinases of the Src- and Abl- families localize beneath the viral particle, and that both the Src- and Abl- family kinases redundantly mediate actin tail formation, whereas only the Abl-family kinases mediate virion release (Reeves, PM et al Nat. Med. 2005 11(7):731-9). We have been working to identify the targets of these kinases and their contribution to virion motility and release through complementary proteomic and genetic approaches. Candidate substrates were initially identified by quantitative Mass Spectrometry (iTraq). Cell lysates were generated from tyrosine kinase knockout cell lines, and cells treated with tyrosine kinase inhibitors (e.g. Gleevec, PD-166326, BMS-354825) in the presence or absence of vaccinia virus infection. These lysates were digested with trypsin, differentially labeled using iTraq reagents, and the experimental samples were combined. Phosphotyrosine peptides were immunoprecipitated using anti-phosphotyrosine antibodies and the eluate was analyzed with Q-TOF, which allows for protein sequence identification by tandem MS. The iTraq reporter groups also fragment in MS2; therefore, changes in the intensity of these reporter groups yield a relative quantification of phosphoprotein amounts from each experimental sample. Decreases in phosphorylation due to inactive or absent tyrosine kinases have allowed us to pinpoint substrates specifically phosphorylated by Src-family kinases or Abl-family kinases or both. To corroborate the MS results, in vitro kinase assays were performed with purified kinases and proteins from a VV Proteome Microarray. Using these approaches we have also identified phosphoproteins involved in aspects of virion maturation, viral membrane biogenesis, and virion entry and release. To elucidate the contribution of these proteins to aspects of the viral replication cycle, we are currently mutagenizing the phosphorylation sites within the viral and cellular proteins and assessing their contribution to viral replication, morphogenesis and dissemination. Novel processes identified by this approach may shed new light on not only aspects of Poxviral entry and release, but also identify new cellular mechanisms of vesicle transport, docking, fusion and fission.

Adrianne Nehrling Edwards (student in the Romeo laboratory)

A small RNA-binding protein, carbon storage regulator A (CsrA), is the key component of a global regulatory system in Escherichia coli K-12 and other bacteria. The Csr system activates certain processes associated with exponential phase growth while repressing several stationary phase functions. The Csr system activates glycolysis, motility, and acetate metabolism while repressing glycogen synthesis and catabolism, gluconeogensis, and biofilm formation by a post-transcriptional mechanism. Preliminary genomic array studies suggest that CsrA affects expression of approximately 400 genes, either directly or indirectly (Wu and Romeo, unpublished data). We hypothesize that a significant number of the messages of these genes are bound directly by CsrA. To determine the RNAs to which CsrA binds, a recombinant protein (CsrA-His6) was purified followed by extraction of bound RNA and subsequent cDNA synthesis. The identities of cDNAs are being determined using 454 Sequencing technology. Direct regulation of a select number of transcripts by CsrA will be confirmed through quantitative real-time PCR and RNA electrophoretic mobility shifts. Preliminary data indicate that CsrA binds to the messages of genes important for fundamental processes including cell division, metabolite uptake, and transcription and translation control. The transcripts identified will potentially uncover novel mechanisms of CsrA regulation, determine broad regulatory roles for the Csr system, and show direct CsrA regulation of important pathways and cellular functions in E. coli that may also be important in other nonpathogenic and pathogenic eubacteria.

Paul Johnson (student in the Shafer laboratory)

My work with the strict human pathogen Neisseria gonorrhoeae is focused on understanding how the AraC like transcriptional regulator MtrA impacts antimicrobial resistance and pathogenesis. MtrA is an important component of antimicrobial resistance in Neisseria gonorrhoeae as it regulates inducible resistance to select antimicrobial agents through enhancing transcription of the mtrCDE-encoded efflux pump. Recent work by traditional genetic/biochemical techniques indicates that the MtrA regulon extends beyond the MtrCDE efflux pump system and includes components of the Type IV pilus secretion system (pilMNOPQ) and a penicillin-binding protein (ponA). Expression of these genes appears to increase in the absence of MtrA indicating that MtrA acts as a repressor at these loci as opposed to the activator it is at the mtrCDE locus. In addition to classic techniques, our microarray analysis of an MtrA mutant strain has further identified genes positively or negatively regulated by MtrA. These MtrA-regulated genes encode components of amino acid biosynthesis pathways (e.g., glutamine), transport systems, antimicrobial resistance, and virulence. I am examining the mechanism by which MtrA regulates a subset of these genes and their involvement in antibiotic resistance and virulence.

Jeff Meisner (student in the Moran Laboratory)

Endospore formation in the bacterium Bacillus subtilis requires precise coordination of genetic and morphological events. This is accomplished by organizing the events into a series of dependent relationships whereby the initiation of late events is dependent on the completion of early events. At the onset of sporulation, an asymmetric cell division produces two progeny cells, the mother cell and the forespore. The mother cell engulfs the forespore, allowing maturation of the developing endospore. The genes necessary for forespore maturation are controlled by alternative RNA polymerase sigma factors that are activated following the completion of engulfment. In particular, sigma G activity in the forespore requires both the completion of engulfment and the products of the spoIIIA operon, expressed in the mother cell. Given that the spoIIIA locus is not necessary for engulfment, it may instead encode a checkpoint control mechanism that ensures sigma G activation occurs only after the completion of engulfment. However, relatively little is known about the organization and function of these gene products. The goal of my research is to understand how the products of the spoIIIA operon coordinate gene expression and morphogenesis and the consequences suffered by the cell in absence of these dependent relationships

M. Analise Zaunbrecher (student in the Shinnick laboratory)

Mycobacterium tuberculosis is the causative agent of Tuberculosis (TB) which annually claims over 2 million deaths worldwide. The emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains complicates treatment and control of the disease and escalates the risk to public health. Multidrug resistant strains of M. tuberculosis typically require the use of second line drugs for treatment. Although drug resistance mechanisms have been well defined for first line antibiotics, there exists only a limited understanding of resistance mechanisms to second line antibiotics. Kanamycin (KAN) is one second line drug that is becoming increasingly more significant in the treatment of MDR- and XDR-TB cases. Kanamycin resistance is observed in both clinical isolates and laboratory generated strains of M. tuberculosis. High level resistance has been attributed to mutations in the 16S rRNA gene, rrs. However, in 30-80% of kanamycin resistant clinical isolates, low level resistance is observed which can not be ascribed to any known mechanism of kanamycin resistance. These low level kanamycin resistant clinical isolates and laboratory generated strains suggest that another, uncharacterized mechanism of kanamycin resistance exists in M. tuberculosis. The focus my graduate research work is investigating the uncharacterized mechanism of kanamycin resistance in M. tuberculosis.

| GRADUATE DIVISION OF BIOLOGICAL AND BIOMEDICAL SCIENCES |