Campylobacter jejuni is the principal cause of gastroenteritis worldwide. Contaminated chickens is the most important source of human infection by Campylobactor. In its hosts, C. jejuni colonizes the gastrointestinal tract despite the constitutive and induced secretion of cationic antimicrobial peptides (CAMPs). These peptides are a major component of the host's innate immune system and have been found to be effective against different bacterial species including Campylobacter . These observations suggest that Campylobacter has developed mechanisms to evade CAMPs. To test this hypothesis and identify genes presumably involved in adaptation and/or resistance to CAMPs, two approaches were pursued. In the first approach, the transcriptome profile of C. jejuni NCTC 11168 was analyzed upon exposure to increasing concentrations of protamine sulfate, which is a known antimicrobial peptide. In the second approach, a Campylobacter transposon mutant library was screened against two concentrations of protamine. The data obtained from both approaches revealed genes that could be important to overcome the effect of the studied antimicrobial agent and potentially other antimicrobial peptides. The transcriptome profile study unveiled the importance of two genes Cj1721c and Cj0355c, Cj1721c is an outer membrane protein with an unknown function, and Cj0355c, is an essential two-component system regulator. Phenotypic studies demonstrated that the Cj1721c knockout was more resistant to protamine, more motile, and had reduced biofilm-forming ability compared to the wild-type strain. In contrast, the strain that overexpresses Cj0355c was more sensitive to protamine and had greater biofilm-forming ability compared to the wild-type strain. Furthermore, it was attenuated in its ability to colonize the gastrointestinal tract of new born piglets. Moreover, transcriptome profiling of the strain that overexpresses Cj0335c in MH and MEM medium revealed potential pathways that could be regulated by this regulator. This observation suggests that membrane integrity could be sensed by a still unknown sensor and then the signal transferred to Cj0355c, which affects the expression of key genes that are involved in resistance to protamine sulfate. In summary, this study identifies previously uncharacterized genes (CJ1721c and Cj035c) that are involved in the resistance to protamine sulfate and potentially to other antimicrobial peptides. In addition, the performed transposon mutants library screening indicates that Campylobacter jejuni NCTC 11168 can intrinsically evade the effect of protamine sulfate. On the other hand, we showed that C. jeuni does not use the multidrug efflux pump (MATE) to resist the effect of protamine.

Contamination of soils with PAHs, substances that can pose serious environmental and human health risks, is a serious problem. Removal of risk requires effective and sustainable methods to decrease or eliminate toxicological hazard. Bioremediation is a sustainable option that can be accomplished by a variety of means. However, due to the limitations of classical culturing methods, there is a paucity of information regarding the composition of soil microbial communities, and moreover, the identity of organisms involved in contaminant catabolism. Newer, molecular techniques directly examine metagenomic DNA or RNA, and provide opportunities to investigate the genetic constitution of soil samples without the need for culturing. 16S rRNA has proven to be a valuable tool in the identification of soil microbes due to its highly conserved nature throughout the bacterial kingdom, in addition to inclusion of variable regions that are unique to a particular organism, or closely related group of organisms. This study used analysis of 16S rRNA gene sequences to examine the temporal changes in the soil microbial community in a PAH-contaminated soil during bioremediation. These results were compared with previously observed temporal changes in the chemical composition of the same soil during bioremediation. Interplay between the temporal changes of soil microbial community and the temporal changes of the PAH concentration was observed. The PAH concentration decreased an average of 77% across all treatments and reactors. Soil micro-organisms whose growth during treatment corresponded with a decline in PAH concentration included several known PAH-degraders such as Achromobacter, Acidovorax, Pseudomonas, Rhodanobacter and Stenotrophomonas. The metagenomic approach employed in this study can be used to evaluate innovative bioremediation methods, and moreover, identify members of the microbial community that are critical for the catabolism of PAHs.

Type-I interferons (IFN-I) play a key protective role during viral infections, however, their role during bacterial infections remains unclear. We evaluated the influence of IFN-I signalling during infection of mice with the facultative intracellular bacterium, Salmonella typhimurium (ST). Wild-type (WT) C57BL/6J mice succumbed to infection by day 7 and death was accelerated in mice deficient in key innate immune mediators (IFN-gamma, TNF-alpha, iNOS-2). Surprisingly, IFN-I receptor-deficient (IFN-I R-/-) mice survived ST infection up to 35 days. Despite enhanced inflammation, WT mice displayed uncontrolled ST burden and lower macrophage numbers in the spleen, compared with IFN-I R-/- mice. In vitro, IFN-I-deficient macrophages expressed reduced levels of TNF-alpha and NO2- in response to ST and displayed prolonged survival in comparison with WT macrophages. Since macrophages play key protective roles during intracellular bacterial infections, our results indicate that IFN-I signalling during ST infection promotes the elimination of macrophages resulting in poor pathogen control.

Vaccination is one of the major strategies available for combating viral infections in humans. The seasonal inactivated influenza virus vaccine elicits type-specific, protective neutralizing antibodies that are detectable in the serum of a vaccinated person; however, the very nature of the virus requires a reformulation of the vaccine to match the currently circulating strains in any given year. In the present study, a novel vaccination approach was developed to induce protective immunity against Influenza A. The approach consisted of delivering naked or protected mRNA encoding for Hemagglutinin as a vaccine. The vaccine was administrated to B6C3Fl mice and the overall immune response raised by an mRNA-based vaccine was assessed through the evaluation of the cellular and humoral responses induced by this type of vaccination and the in vitro protection it confers against Influenza A virus. Results from these studies suggest that rnRNA vaccination in the context of infectious diseases, specifically influenza, is feasible and that mRNA vaccines are capable of inducing a balanced immune response as characterized by the production of protective neutralizing antibodies and the induction of cellular immunity against influenza.

Haemophilus ducreyi, a Gram-negative and heme-dependent bacterium, is the causative agent of chancroid, a genital ulcer sexually transmitted infection. Although the precise molecular mechanism of heme acquisition in H. ducreyi is unclear, heme uptake likely proceeds via a receptor mediated process. The initial event involves binding to either of two outer membrane receptors, TdhA and HgbA. Once heme is deposited into the periplasmic space, a heme permease is postulated to transport heme across the periplasmic space to the inner membrane. In prior experiments, using protein expression profiling of the H. ducreyi periplasmic proteome, we identified a periplasmic-binding protein hHBP that we propose is a component of a heme trafficking operon. Biochemical and genetic approaches were used to functionally characterize hHBP. First, purified hHBP was incubated with increasing concentrations of heme and the mixtures were resolved by non-denaturing polyacylamide gel electrophoresis. Separated proteins were transferred onto PVDF membranes and heme-protein complexes were detected by enhanced chemiluminescence (ECL). Second, the hhbp gene was cloned in the E. coli recombinant mutant E. coli FB827 dppA::Km mppA::Cm (pAM238-HasR) which expresses the Serratia marcescens HasR heme receptor allowing heme translocaton into the periplasm, but denies heme entry into the cytoplasm because of the presence of the double mutation (dppA::Km mppA::Cm) resulting in a mutant lacking the two periplasmic proteins DppA and MppA. We found that heme binding to hHBP was saturable as determined by ECL. Genetic complementation in trans with hhbp repaired the growth defect of the mutant E. coli for heme utilization as an iron source. The growth restoration was comparable to that seen with the E. coli mutant complemented with the intact Dpp permease. Additionally, growth of the mutant was not rescued with the empty plasmid vector. We concluded that H. ducreyi hHBP functionally binds heme. Complementation of the E. coli mutant for heme competency supports the proposal that hHBP participates in the transit of heme in H. ducreyi.

Natural killer (NK) cells target virally infected cells and tumor cells through Ly49 receptors that recognize MHC-I molecules. Different inbred mouse strains possess disparate Ly49 receptor haplotypes, in addition to NK cell activity. C5781/6 mice express less Ly49 receptors than 129S1 mice, but have a more robust NK cell population. We intend to determine if the differences in NK cell activity between these inbred mice strains are due to the differences in Ly49 receptor haplotypes. C578116 mice with the Ly49 gene cluster of 129S1 origin were generated in order to compare 129S1 and 86 Ly49 contribution to NK cell function. These B6-Ly49129 congenic mice were confirmed to express the 129 Ly49 receptor pattern by flow cytometry. NK cell activity was assessed by cytotoxicity assays using a panel of NK-resistant and NK-susceptible tumor cell lines, the ability to clear infection with murine cytomegalovirus (MCMV) as well as the ability to reject MHC-I-deficient splenocytes in vivo. Susceptibility to MCMV infection by congenic mice was similar to 129 mice. However, there was a significant increase in rejection of MHC-I-deficient cells by the congenic mice in comparison to both the 86 and 129 mice. In addition, in vitro tumor cell killing by congenic NK cells was comparable to 86. The current results indicate that expression of an increased number of Ly49 receptors promotes better education of NK cells, which in turn leads to higher rejection of MHC deficient target cells.

HIV infection elicits defects in CD4 T-cell homeostasis in both a quantitative and qualitative manner. Interleukin-7 (IL-7) is essential to T-cell homeostasis and several groups have shown reduced levels of the IL-7 receptor alpha-chain (CD127) on both CD4 and CD8 T-cells in viremic HIV+ patients. Our lab has demonstrated that soluble HIV Tat protein specifically down regulates cell surface expression of CD127 on human CD8 T-cells. Once taken up by CD8 T-cells, Tat enters the cytoplasm and interacts directly with the cytosolic tail of CD 127 inducing receptor capping, endocytosis and degradation. The purpose of this study is to determine if, similar to CD8 T-cells, HIV Tat down regulates surface CD127 expression on CD4 T-cells and if this down regulation affects IL-7 signaling. To investigate this, primary CD4 T-cells isolated from healthy HIV-negative volunteers were incubated in medium alone or with Tat protein (10 ug/ml) for up to 72 hours and then analyzed by flow cytometry. As anticipated, soluble HIV Tat protein induces a significant decrease in surface CD127 expression on CD4 T-cells relative to cells cultured in medium alone. The effect was dose and time dependent, reversible, and could be blocked with anti-Tat antibodies or heparin. Tat down regulated CD127 equally on naive and memory cells and did not require new protein synthesis to have its effect. Tat's down regulation of CD127 was not associated with cell activation as there was no change in overall cell phenotype including expression of CD25 and CD56. Further, expression of CD132, the common gamma-chain which associates with CD127 to form the IL-7 receptor, was also unaffected by Tat. Notably, down regulation of CD127 by Tat resulted in lower induction of the anti-apoptotic protein Bd-2 following stimulation with IL-7. In view of the important role IL-7 plays in CD4 T-cell proliferation, homeostasis and survival, this down regulation of CD127 by Tat may well play a role in HIV-induced immune dysregulation and CD4 T-cell decline. Understanding this effect could lead to new therapeutic approaches to reverse the CD4 T-cell loss evident during HIV infection.

On a per capita basis, the incidence rate of type E botulism attributable to aged marine mammal meat in Nunavik is very high, with an average rate greater than 3 reported cases per 10,000 population for the period from 1996 to 2004. All cases reported for this period were clustered in Northern villages along the Ungava Bay. A survey for the presence of Clostridium botulinum type E along the coastline of Nunavik indicated a prevalence of spores along the southern coasts of Ungava Bay and Hudson Bay, indicating a possibility of contamination of seal meat from environmental sources during butchering. The highest concentration of spores was found in the Koksoak River (>5400 spores/kg), which flows into southern Ungava Bay. Spores were also found on seal skin and in seal intestinal contents, although at levels lower than found in environmental sources. Contamination of seal meat with C. botulinum type E spores may occur following contact with coastal rocks, tidal water, or shoreline soil, during the butchering process. Genomic analysis of coastal isolates of C. botulinum type E by PFGE indicated a heterogeneous population at the butchering sites, making it difficult to trace contamination sources. Epidemiologically related strains of C. botulinum type E could be identified by PFGE. Several distinct subtypes were involved in botulism incidents originating from Inuit villages of southern Ungava Bay, corroborating the existence of a high genetic diversity of C. botulinum type E in the environment. Some of these subtypes were detected inland and in large rivers flowing into southern Ungava Bay. The temperature used for igunaq preparation was the primary factor influencing the growth and toxigenesis of C. botulinum type E. All igunaq preparations incubated at 4°C were negative for BoNT/E. The use of traditional seal skin pouches or plastic containers did not influence the time to toxicity of seal igunaq preparations aged at abuse temperatures (10 and 20°C), regardless of inoculation dose. Changes in handling of seal meat during butchering to reduce contamination, combined with temperature control of the aging process to 4°C or less, should significantly reduce the risk of botulism from consumption of traditional Inuit foods.

CD44, a family of cell adhesion glycoproteins, is an important mediator in events such as tumorigenesis and metastasis, cell migration, and inflammatory responses. The molecular mechanisms in the regulation of CD44 expression are not well understood. In this study, I examined the regulation of CD44 expression in two models systems: human monocytic cells and Burkitt's lymphoma B cells. Lipopolysaccharide (LPS), a bacterial cell wall component, regulates CD44 expression, and modulates CD44-mediated biological effects in monocytic cells during inflammation and immune responses. In human monocytic cells, LPS and the proinflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha) are potent inducers of CD44 expression. To delineate the mechanism underlying LPS- and TNF-alpha-induced CD44 expression, the previous results in our laboratory showed the differential role of mitogen-activated protein kinase (MAPK) specifically c-Jun N-terminal kinase (JNK) in LPS-induced but not in TNF-alpha-induced CD44 expression. Therefore, we hypothesized that distinct signaling pathways are involved in the regulation of CD44 expression in a stimulus and cell-dependent manner. In this study, I investigated the signaling pathway involved in TNF-alpha-induced CD44 expression in human monocytic cells. I used human promonocytic THP-1 cells, transfected stably with CD14 receptor (THP-1/CD14) as a model system. My results showed the differential involvement of Ca2+ signaling molecules, in particular calmodulin (CaM) and CaM-dependent protein kinase-II (CaMK-II), in TNF-alpha-induced but not in LPS-induced CD44 expression. The CD44 promoter analysis suggests that the distinct transcription factors AP-1 and Egr-1 may be involved in TNF-alpha- and LPS-induced CD44 expression, respectively. In addition, I demonstrated the selective involvement of Ca 2+ signaling molecules, mainly CaM and CaMK-II, in TNF-alpha-induced CD44 expression through the activation of transcription factor AP-1. In contrast, LPS-induced CD44 expression was regulated by JNK MAPK through the activation of Egr-1. I have also demonstrated that phosphoinositide 3-kinase (PI3K) constitutes a key downstream component of both the signaling pathways involved in the regulation of LPS- and TNF-alpha-induced CD44 expression in human monocytic cells. My results suggest that the JNK-activated PI3K regulates LPS-induced CD44 expression through the activation of Egr-1, whereas TNF-alpha induces CD44 expression by a distinct CaM/CaMK-II-activated PI3K through the activation of AP-1 transcription factor. Further, to determine the involvement of the subunit of PI3K, I demonstrated that the regulatory subunit p85alpha is involved in both LPS- and TNF-alpha-induced CD44 expression, without involving the catalytic subunit, p110alpha. IL-4, a pleotropic cytokine, has been shown to enhance the survival and development of Burkitt's lymphoma (BL) B cells, and induces the expression of various costimulatory molecules including CD44. In addition, CD44 induction and its ability to bind hyaluronan (HA) have been suggested to play a vital role in in vivo BL tumor growth and dissemination. However, the role of IL-4 in the pathogenesis of BL and other B cell malignancies is not clear. In this study, I investigated the regulation of IL-4-induced CD44 expression in an Epstein-Barr virus (EBV)-transformed Burkitt's lymphoma B cell line, BL30/B95-8 cells. The results suggested that IL-4 did not induce the expression of the transcription factors Egr-1 and AP-1. However, IL-4 induced STAT-6 which plays a critical role in CD44 regulation in these cells. I demonstrated that STAT-6 is activated by two distinct signaling pathways namely Jak-1/3 and ERK MAPK and independent of the IRS-2/PI3K pathway. Therefore, STAT-6 and ERK MAPK may represent an important and novel target for regulation of CD44 expression and CD44-mediated immune responses in B cell proliferation- and maturation-related disease conditions and cancer malignancies. Further, my results also suggest a critical involvement of PI3K in the regulation of LPS- and TNF-alpha-induced CD44 expression in human monocytic cells, and hence, may represent a potential therapeutic target for inhibiting CD44 expression and consequent CD44-mediated cell migration, inflammation and autoimmune disorders.

Adenovirus vectors are undergoing evaluation in clinical trials for the treatment of a variety of human diseases. However, little is clearly understood regarding interactions between non-replicating adenovirus (Ad) vectors and the host nucleus. Here I have examined the role of chromatin in the regulation of helper-dependent Ad (hdAd) vector-encoded transgenes. Eviction of the Ad DNA packaging-protein VII, histone deposition, and transgene expression initiated within 2 hours of infection; hdAd assembled into physiologically-spaced nucleosomes within 6 hours. Histone deposition was transcription-independent, dependent on the histone chaperone HIRA, and essential for efficient viral gene expression. Having established that hdAd vectors assemble into chromatin, I examined the role of epigenetic factors in mediating the repressive effects of prokaryotic DNA attached to eukaryotic transgenes. To this end, I used helper-dependent adenovirus (hdAd) vectors with identical expression cassettes, but containing 22 kb of prokaryotic or eukaryotic stuffer DNA (hdAd-prok and hdAd-euk, respectively). I hypothesized that abundant CpG motifs in the hdAd-prok backbone are methylated, resulting in the formation of chromatin that is refractory to transcription. Within the host cell, the hdAd-prok transgene promoter was not associated with epigenetic markers of heterochromatin, although it did exhibit reduced association with acetylated histones H3 and H4. Inhibition of histone deacetylases or insertion of an insulator element between the transgene and stuffer DNA abrogated the inhibitory effects of prokaryotic DNA. However, I did not detect methylation of hdAd-prok DNA. Therefore, I examined the role of promyelocytic leukemia (PML) bodies and their constituents, Sp100 and Daxx - which have all been implicated in repression of DNA virus transcription - in repression of hdAd-prok. Intact PML bodies and the PML protein were not necessary for repression of hdAd-prok. Rather, Sp100 isoforms B and HMG, which bind to unmethylated CpGs, and Daxx, which interacts with histone deacetylases, repressed expression of hdAd-prok. Therefore, "foreign" prokaryotic DNA is recognized by Sp100B/HMG and Daxx, which repress associated genes via induction of histone deacetylation. I found that chromatin plays an important role in the regulation of Ad vector-encoded transgenes, suggesting that both genetic and epigenetic factors must be considered in the design of Ad vectors for gene therapy.