Faculty Bibliography

Staphylococcus aureus is a formidable human pathogen that uses secreted cytolytic factors to injure immune cells and promote infection of its host. Of these proteins, the bicomponent family of pore-forming leukocidins play critical roles in S. aureus pathogenesis. The regulatory mechanisms governing the expression of these toxins are incompletely defined. In this work, we performed a screen to identify transcriptional regulators involved in leukocidin expression in S. aureus strain USA300. We discovered that a metabolic sensor-regulator, RpiRc, is a potent and selective repressor of two leukocidins, LukED and LukSF-PV. Whole-genome transcriptomics, S. aureus exoprotein proteomics, and metabolomic analyses revealed that RpiRc influences the expression and production of disparate virulence factors. Additionally, RpiRc altered metabolic fluxes in the trichloroacetic acid cycle, glycolysis, and amino acid metabolism. Using mutational analyses, we confirmed and extended the observation that RpiRc signals through the accessory gene regulatory (Agr) quorum-sensing system in USA300. Specifically, RpiRc represses the rnaIII promoter, resulting in increased repressor of toxins (Rot) levels, which in turn negatively affect leukocidin expression. Inactivation of rpiRc phenocopied rot deletion and increased S. aureus killing of primary human polymorphonuclear leukocytes and the pathogenesis of bloodstream infection in vivo. Collectively, our results suggest that S. aureus senses metabolic shifts by RpiRc to differentially regulate the expression of leukocidins and to promote invasive disease. IMPORTANCE: The bicomponent pore-forming leukocidins play pivotal roles in the ability of S. aureus to kill multiple host immune cells, thus enabling this pathogen to have diverse tissue- and species-tropic effects. While the mechanisms of leukocidin-host receptor interactions have been studied in detail, the regulatory aspects of leukocidin expression are less well characterized. Moreover, the expression of the leukocidins is highly modular in vitro, suggesting the presence of regulators other than the known Agr, Rot, and S. aureus exoprotein pathways. Here, we describe how RpiRc, a metabolite-sensing transcription factor, mediates the repression of two specific leukocidin genes, lukED and pvl, which in turn has complex effects on the pathogenesis of S. aureus Our findings highlight the intricacies of leukocidin regulation by S. aureus and demonstrate the involvement of factors beyond traditional virulence factor regulators.

Monoclonal antibody (mAb) therapeutics targeting cancer, autoimmune diseases, inflammatory diseases, and infectious diseases are growing exponentially. Although numerous panels of mAbs targeting infectious disease agents have been developed, their progression into clinically useful mAbs is often hindered by the lack of sequence information and/or loss of hybridoma cells that produce them. Here we combine the power of crystallography and mass spectrometry to determine the amino acid sequence and glycosylation modification of the Fab fragment of a potent human astrovirus-neutralizing mAb. We used this information to engineer a recombinant antibody single-chain variable fragment that has the same specificity as the parent monoclonal antibody to bind to the astrovirus capsid protein. This antibody can now potentially be developed as a therapeutic and diagnostic agent.

Background: Mass Spectrometry has become an indispensable technique for studying proteins in biological systems. Thousands of proteins can be identified and quantified with state-of-the art instruments in a single experiment. Global changes in the proteome and certain post translational modifications can now be routinely characterized due to dramatic improvements in speed and sensitivity of mass spectrometric instruments and data analysis software. However, there are still areas of proteomics where analysis can be not as straight forward or need extra care in sample preparation. Examples are the characterization of disease specific circulating antibodies and the detection of virus-host protein protein interactions. Conclusions: Protein-Protein interactions are critical for all cellular processes. Understanding with which host proteins the virus interacts is crucial for understanding the mechanism of infection. Current strategies of affinity purifications of tagged viral proteins, chemical cross-linking of host-virus proteins as well as changes in post translational modifications (i.e. phosphorylation) upon infection will be presented. Common problems of affinity purifications and characterizing infection dependent post translational modifications will be discussed with special emphasis on sample preparation strategies. Establishing antibody repertoires of infected individuals by high throughput DNA sequencing has been rapidly advancing, yet the antibody composition in the blood of infected individuals remains largely unknown. Here, mass spectrometry is emerging as an enabling technology in combination with a match personal antibody database. Characterizing and quantifying the circulating antibodies in the blood of infected individuals is however not a trivial task. The majority of the antibody is structurally identical and it is therefore difficult to unambiguously identify which antibody is present in the sera of individuals. The majority of antibody derived peptides will be identical and shared between many different antibodies. Here, current strategies for identifying antibody specific peptides are described

Mycobacterium tuberculosis (Mtb) encodes five type VII secretion systems (T7SS), designated ESX-1-ESX-5, that are critical for growth and pathogenesis. The best characterized is ESX-1, which profoundly impacts host cell interactions. In contrast, the ESX-3 T7SS is implicated in metal homeostasis, but efforts to define its function have been limited by an inability to recover deletion mutants. We overcame this impediment using medium supplemented with various iron complexes to recover mutants with deletions encompassing select genes within esx-3 or the entire operon. The esx-3 mutants were defective in uptake of siderophore-bound iron and dramatically accumulated cell-associated mycobactin siderophores. Proteomic analyses of culture filtrate revealed that secretion of EsxG and EsxH was codependent and that EsxG-EsxH also facilitated secretion of several members of the proline-glutamic acid (PE) and proline-proline-glutamic acid (PPE) protein families (named for conserved PE and PPE N-terminal motifs). Substrates that depended on EsxG-EsxH for secretion included PE5, encoded within the esx-3 locus, and the evolutionarily related PE15-PPE20 encoded outside the esx-3 locus. In vivo characterization of the mutants unexpectedly showed that the ESX-3 secretion system plays both iron-dependent and -independent roles in Mtb pathogenesis. PE5-PPE4 was found to be critical for the siderophore-mediated iron-acquisition functions of ESX-3. The importance of this iron-acquisition function was dependent upon host genotype, suggesting a role for ESX-3 secretion in counteracting host defense mechanisms that restrict iron availability. Further, we demonstrate that the ESX-3 T7SS secretes certain effectors that are important for iron uptake while additional secreted effectors modulate virulence in an iron-independent fashion.

Hyaluronidases contribute to local and systemic damages after envenoming, since they act as spreading factors cleaving the hyaluronan presents in the connective tissues of the victim, facilitating the diffusion of venom components. Although hyaluronidases are ubiquitous in snake venoms, they still have not been detected in transcriptomic analysis of the Lachesis venom gland and neither in the proteome of its venom performed previously. This work purified a hyaluronidase from Lachesis muta rhombeata venom whose molecular mass was estimated by SDS-PAGE to be 60 kDa. The hyaluronidase was more active at pH 6 and 37 degrees C when salt concentration was kept constant and more active in the presence of 0.15 M monovalent ions when the pH was kept at 6. Venom was fractionated by reversed-phase liquid chromatography (RPLC). Edman sequencing after RPLC failed to detect hyaluronidase, but identified a new serine proteinase isoform. The hyaluronidase was identified by mass spectrometry analysis of the protein bands in SDS-PAGE. Additionally, phospholipase B was identified for the first time in Lachesis genus venom. The discovery of new bioactive molecules might contribute to the design of novel drugs and biotechnology products as well as to development of more effective treatments against the envenoming.

Despite minimal disparity at the sequence level, mammalian H3 variants bind to distinct sets of polypeptides. Although histone H3.1 predominates in cycling cells, our knowledge of the soluble complexes that it forms en route to deposition or following eviction from chromatin remains limited. Here, we provide a comprehensive analysis of the H3.1-binding proteome, with emphasis on its interactions with histone chaperones and components of the replication fork. Quantitative mass spectrometry revealed 170 protein interactions, whereas a large-scale biochemical fractionation of H3.1 and associated enzymatic activities uncovered over twenty stable protein complexes in dividing human cells. The sNASP and ASF1 chaperones play pivotal roles in the processing of soluble histones but do not associate with the active CDC45/MCM2-7/GINS (CMG) replicative helicase. We also find TONSL-MMS22L to function as a H3-H4 histone chaperone. It associates with the regulatory MCM5 subunit of the replicative helicase.

The vast majority of human tissue specimens are formalin-fixed, paraffin embedded (FFPE) archival samples, making this type of tissue a potential gold mine for medical research. It is now accepted that proteomics can be done using FFPE tissue and can generate similar results as snap-frozen tissue. However, the current methodology requires a large amount of starting protein, limiting the questions that can be answered in these types of proteomics studies and making cell-type specific proteomics studies difficult. Cell-type specific proteomics has the potential to greatly enhance understanding of cell functioning in both normal and disease states. Therefore, here we describe a new method that allows localized proteomics on individual cell populations isolated from FFPE tissue sections using laser capture microdissection. To demonstrate this technique we microdissected neurons from archived tissue blocks of the temporal cortex from patients with Alzheimer's disease. Using this method we identified over 400 proteins in microdissected neurons; on average 78% that were neuronal and 50% that were associated with Alzheimer's disease. Therefore, this technique is able to provide accurate and meaningful data and has great potential for any future study that wishes to perform localized proteomics using very small amounts of archived FFPE tissue.

PARP1 is the main sensor of single- and double-strand breaks in DNA and, in building chains of poly(ADP-ribose), promotes the recruitment of many downstream signaling and effector proteins involved in the DNA damage response (DDR). We show a robust physical interaction between PARP1 and the replication fork protein TIMELESS, distinct from the known TIMELESS-TIPIN complex, which activates the intra-S phase checkpoint. TIMELESS recruitment to laser-induced sites of DNA damage is dependent on its binding to PARP1, but not PARP1 activity. We also find that the PARP1-TIMELESS complex contains a number of established PARP1 substrates, and TIMELESS mutants unable to bind PARP1 are impaired in their ability to bind PARP1 substrates. Further, PARP1 binding to certain substrates and their recruitment to DNA damage lesions is impaired by TIMELESS knockdown, and TIMELESS silencing significantly impairs DNA double-strand break repair. We hypothesize that TIMELESS cooperates in the PARP1-mediated DDR.

HYPOTHESIS: Intrinsic differences in neurons of the vestibular ganglia result in the increased likelihood of superior vestibular ganglion involvement in vestibular neuritis. BACKGROUND: Vestibular neuritis is hypothesized to result from herpes simplex type I (HSV1) infection or reactivation in vestibular ganglia. Involvement of the inferior vestibular ganglion is extremely rare in patients with vestibular neuritis. METHODS: Primary cultures of rat superior and inferior vestibular ganglion neurons (VGNs) were cultivated separately. Neurons were lytically and latently infected with HSV1 with a US11-green fluorescent protein (GFP) chimera. Percentage lytic infection and baseline reactivation was assessed by microscopy for GFP fluorescence. Trichostatin-A (TSA) was used to stimulate HSV1 reactivation. Virion production was assessed by viral titers. Relative numbers of latency-associated (LAT) transcripts were determined by real-time reverse-transcription polymerase chain reaction (real-time RT-PCR). RESULTS: Lytic infection rates were equivalent between the two ganglia (p > 0.05). Lytic infections yielded similar amounts of plaque-forming units (p > 0.05). Relative amounts of LAT transcripts did not differ between latently infected superior and inferior VGNs. Latently infected cultures showed no differences in rates of baseline and TSA-induced HSV1 reactivation (p > 0.05). Production of virions was not significantly different between reactivated, latently infected superior versus inferior VGNs (p = 0.45). CONCLUSION: Differences in prevalence of superior and inferior vestibular neuritis do not result from intrinsic differences in HSV1 infection or virion production of these neurons. Other factors, such as the length and width of the bony canal containing the ganglia and nerves, account for the greater involvement of the superior vestibular ganglion in vestibular neuritis.