Helicobacter pylori is a bacterium that infects the stomach of about half the world’s population. While usually asymptomatic, H. pylori infection is the main cause of peptic ulceration, gastric lymphoma, and gastric adenocarcinoma. This suggests variability within populations of H. pylori in their ability to cause disease. I am researching the effect of a mutation in the gene cagY on the ability of H. pylori to cause disease. This gene produces CagY, a structural protein of the bacteria’s Type IV Secretion System, which is involved in causing disease. H. pylori transports different materials across the Type IV Secretion System, including the protein CagA. Host cells receiving this protein elongate and narrow (a change called the hummingbird phenotype) and produce interleukin-8, a chemical messenger of the immune system. In host cells, the protein CagA is phosphorylated (a process by which a phosphate group is added to a molecule). To test the effects of the cagY mutation on the bacteria’s Type IV Secretion System, I will infect human stomach cancer cells with the cagY mutant. To detect changes in these cells caused by the infection, I will use two techniques that detect specific proteins, Western Blot and ELISA. The Western Blot will detect the presence of phosphorylated CagA, while the ELISA will detect interleukin-8. The length of infected cells will be measured to detect the hummingbird phenotype. These tests will measure the function of the Type IV Secretion System in the cagY mutant. The absence of a functioning Type IV Secretion System in the cagY mutant will suggest a change in bacteria and host interaction and a potentially decreased ability for the bacterium to cause disease.

Chronic infections with Pseudomonas aeruginosa are a significant cause of deterioration of lung function in patients with cystic fibrosis (CF), and have substantial clinical importance due to their marked resistance to treatment with antibiotics. Furthermore, existing laboratory methods for testing bacterial susceptibility to antibiotics are poor indicators of clinical response to therapy, which further increase the difficulty associated with treating these infections. Current research shows that phenotypically diverse P. aeruginosa subpopulations evolve within a single patient during prolonged infection, and that clinical antibiotic therapy causes some of these subpopulations to change significantly in relative abundance. Observed changes in abundance following antibiotic treatment signify varying degrees of antibiotic susceptibility within each patient’s P. aeruginosa population as a whole, with an increase in a particular subpopulation abundance indicating resistance, and a decrease in a particular subpopulation abundance indicating sensitivity. As previous studies have only evaluated P. aeruginosa antibiotic sensitivity prior to antibiotic treatment, we attempted to demonstrate the correlation between an increase subpopulation abundance following clinical treatment and the degree of resistance to antibiotics in laboratory testing. This was done by evaluating the antibiotic minimum inhibitory concentration (MIC) of a range of isolates collected from CF patients who had undergone antibiotic therapy, using antibiotics which had been given to each patient in clinical treatment. Our results demonstrate a marked lack of correlation between relative subpopulation abundance and antibiotic MIC, with clinically resistant subpopulations showing the same or lower MIC than subpopulations which were clinically sensitive treatment. We conclude that isolates resisting treatment do not have a different MIC than isolates responding to treatment, suggesting that mechanisms other than classic antibiotic resistance are responsible for treatment resistance in the CF lung. Further studies to evaluate these mechanisms, such as biofilm formation and formation of persister cells, are currently under way.

The type VI secretion system (T6SS) is a large protein complex utilized by bacteria to transport proteins into other cells. Initially implicated in virulence, the T6SS also participates in interbacterial interactions. A T6SS found in the opportunistic pathogen Pseudomonas aeruginosa, the hemolysin coregulated protein secretion island I T6SS (H1-T6SS), has been found to deliver a set of toxins to other bacteria. This provides P. aeruginosa a fitness advantage over competing bacteria. The H1-T6SS can target neighboring P. aeruginosa, which protects itself with cognate immunity proteins. Deleting these immunity proteins leaves P. aeruginosa susceptible to self-attack. However, the H1-T6SS as well as other T6SSs can target other bacteria, specifically they only target certain species. One of the goals of this project is determine the nature of such specificity. These findings would implicate new functions for the H1-T6SS. To further this, I am seeking to extend the scope in which the T6SS is viewed, both mechanistically and genetically. In doing so I am searching for new genetic loci that participate in T6SS-dependent interactions. Taken together, this work provides insights into a new aspect of type VI secretion and may change the genetic composition of T6SSs.

 A small percentage of the HIV infected population remains asymptomatic for years to decades. Some of these persons, termed late progressors (LP), eventually progress to symptomatic infection, characterized by rapidly rising viral RNA levels and dramatic decreases in CD4+ cells, the main targets of HIV infection. This transition suggests significant changes in the virus-host relationship allowing viral replication to occur. To assess three possible hypotheses as to why the transition to progressive infection may occur, we examined blood plasma samples from four LPs, taken at multiple time points preceding the onset of disease progression. The gag and env genes of HIV isolates were amplified by the polymerase chain reaction (PCR), sequenced, and analyzed for viral population characteristics typical of either 1) superinfection with a second (less controllable) virus strain, 2) a change in the co-receptor used by the virus to infect CD4+ cells, or 3) viral escape, a phenomenon in which HIV-specific immune responses can no longer recognize, neutralize, or prevent replication of virus. Identification of superinfection consisted of locating subsets of virus exhibiting extreme levels of evolutionary divergence from the total virus population. Analysis for co-receptor usage focused on the env gene sequences, while viral escape was indicated by mutations at highly conserved amino acid sites and/or neighboring regions previously targetable by the host immune response. Any of these three hypotheses could potentially result in the sudden enhanced viral replication and disease progression observed in LPs. Examination of this data may clarify patterns of viral evolution in response to immune pressure, adding significantly to the understanding of late progression, viral escape and fitness, and overall HIV-host interactions. Thus, this study may potentially inform both timing of antiretroviral therapy for predicted late progressors and design of drugs and vaccines specific to identified critical regions of the HIV genome.

The lymphatic system is essential to fluid and cell transport, immune function, and tumor metastasis. Lymph drains from body extremities into lymph nodes, via lymphatic vessels, to present antigens to lymphocytes. This can result in an immune response. The existence of tumor cells in lymph nodes is a sign that cellular metastasis has occurred from the primary tumor. I am studying lymphangiogenesis, a process in which lymphatic vessels and sinuses grow and dilate in response to antigens or tumors, thus increasing the lymph flow and capacity to transport tumor cells in lymph nodes. Recently an antibody, 10.1.1, has been discovered to recognize an antigen that is selectively and highly expressed in lymphatic endothelium. The 10.1.1 antibody specifically induces lymph node lymphangiogenesis. This binding also induces proliferation of an unknown cell type in the lymph node. In comparison to the 10.1.1 antibody, injecting a nonspecific antibody control has no effect on lymphatic sinus growth. I am therefore studying how the 10.1.1 antibody rapidly induces lymphatic sinus growth, as well as what cell types contribute to making the new sinuses. I am using the technique of immunostaining to study the effects of the 10.1.1 antibody. By taking a frozen slice of lymphatic tissue and staining it with various antibodies to detect proliferating cells and their cell type, I can identify the specific kinds of cells that are forming into lymphatic sinuses. An understanding of how these lymphatic sinuses grow could help generate therapeutic strategies for manipulation of the lymphatic or immune responses. By inhibiting sinus growth and thus limiting lymph flow, it could be possible to prevent the metastasis of cancer.

 Mycobacterium tuberculosis infection is a leading cause of death worldwide, but the full details of how the human immune system responds to these bacteria are not known. Our lab studies CD1 proteins which allow T-cells to respond to bacterial lipids. We discovered that low expression of CD1A on dendritic cells is associated with an increased susceptibility to tuberculosis; however, the molecular details of CD1A-deficiency are not known. I cloned and analyzed the CD1A promoter from four CD1A-deficient and five control individuals. I identified twelve genetic variations in the CD1A promoter. Of these, eight were single nucleotide polymorphisms (SNPs), three were insertions or deletions, and one was a variable tandem repeat. One SNP (-199 T/C) and the variable tandem repeat showed an association with CD1A-deficiency. A T/T genotype at position -199 from the transcription start site correctly identified five out of five control subjects while a C/C genotype identified three out of four CD1A-deficient subjects. The variable tandem repeat was a string of 20 to 60 thymine bases. The CD1A-deficient subjects showed uniform repeat lengths, while the repeat lengths among control subjects were variable. These data identify two loci within the CD1A promoter that will be the target of future studies aimed at understanding the molecular mechanism of CD1A-deficiency.

Evolutionary conflicts involving mimicry are found throughout nature ranging in a multitude of levels from ecosystems to molecules. Moreover, structural mimicry is increasingly being recognized at the interface between viruses and hosts. The goal of my project is to provide structural and mechanistic insight into a newly discovered evolutionary ‘arms race’ between a viral protein and a host immunity protein. We have found that a poxviral protein IBP1 (IFIT Binding Protein 1) binds a host immunity protein, IFIT2, which represses viral mRNA translation. I hypothesize that IBP1 has convergently evolved to structurally mimic IFIT2’s binding partner, eIF3c, as a strategy to restore viral protein production. Because IBP1 has no known amino acid similarity to any other viral or host protein, the best way to test my hypothesis is to determine the molecular structure of IBP1 and compare it to eIF3c. I will biochemically engineer constructs to optimize the expression and amount of soluble IBP1 in E. coli and then utilize affinity columns to isolate IBP1, which will then be subjected to a myriad of crystallization conditions. Once optimized crystallization conditions have been found I will use X-ray diffraction techniques to solve the structure. Regardless of whether my hypothesis is correct, my work will provide atomic resolution detail for IBP1, which is a protein that is important for the fitness of the virus. If I find that IBP1 is indeed using molecular mimicry it will not only reveal the mechanism of IBP1 action, but it will also provide a case of molecular mimicry marked by the signature of convergent evolution. Furthermore, my work will make progress towards further understanding the interactions between hosts and viruses, which determine the results of potentially deadly infections in so many people.