Progress 10/01/09 to 09/30/14
Outputs
Target Audience: We have demonstrated the essential role of anaerobiosis and redox potential as controlled by the global regulator FNR in the survival and virulence in Salmonella. The role of the redox state in Salmonella will be of great importance to understanding the physiology of this pathogenic organism and in developing vaccines. The development of safe, effective and economic mucosal vaccine is essential for protecting the health and safety of the consumer and the environment. Our new strain of Salmonella FNR-delete could be used as an oral vaccine against Salmonella spp. in Poultry, other farm animals and humans. In addition, this mutant strain could be engineered to serve as a vaccine vector for delivery of epitopes of other Disease Causing pathogens. Two US Patentswere issued: No. 8,101,168 B2 (January 24, 2012) & No. 8,435,506 B2 (May 7, 2013) covering this technology. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Trained Ph.D students (3) Trained many Undergraduate students (6) How have the results been disseminated to communities of interest? 1. Publication in the scintific literature 2. Oral presentations an National meetings 3. Review articles on the subject What do you plan to do during the next reporting period to accomplish the goals? This Project has been terminated. However, a soin-off project has started in October 2014.
Impacts
What was accomplished under these goals?
We reached a better understanding of the complex regulatory networks required for coordinating cellular metabolism and pathogenesis in response to changes in environmental stimuli (i.e., oxygen and iron in the host). The studies provided the next generation of microbiologists (Students and Professionals) with the training required to apply system biology approaches in solving biological and health related problems. Now we have a better appreciation and understanding of multi-layered, coordinately regulated circuits found in prokaryotes. Two US Patents were issued on the "Deficient FNR strain of Salmonella" that can be used as a vaccine against Salmonellosis
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Troxell, B. and H.M. Hassan (2013) Transcriptional regulation by Ferric Uptake Regulator (Fur) in pathogenic bacteria. Front. Cell. Infect. Microbiol. 3:59.
(doi:10.3389/fcimb.2013.00059)
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Husain, M., J. Jones-Carson, L. Liu, M. Song, J. R. Saah, B. Troxell, M. Mendoza, H. Hassan, and A. V�zquez-Torres (2014). Ferric uptake regulator-dependent antinitrosative defenses in Salmonella pathogenesis. Infect. & Immun. 82: 333-340
(doi:10.1128/IAI.01201-13)
|
Progress 10/01/12 to 09/30/13
Outputs
Target Audience: The target audience of our research program are the consumers, the poultry farmers and producers, and the scientific community. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? This project provided training for two undergraduate students and postdoctoral professional. We also provided summer training to 9 K-12 school teachers through the Kenan Institute. How have the results been disseminated to communities of interest? The results have been disseminated via scientific publications and presentations of Posters at scientific meetings. What do you plan to do during the next reporting period to accomplish the goals? : We will continue working on the regulation of pathogenesis in Salmonella in order to develop methods/vaccines for reducing the negative impact of this organism on food safety. Also, will continue working in the area of gut microbiome and its effect on Salmonellosis. In collaboration with scientists from NCSU and UNC-at Chapell Hill we will continue our work on Poultry microbiome and the effects of prebiotics and our Patented Salmonella vaccine on Salmonellosis.
Impacts
What was accomplished under these goals?
: We have demonstrated the essential role of anaerobiosis and redox potential as controlled by the global regulator FNR in the survival and virulence in Salmonella. The role of the redox state in Salmonella will be of great importance to understanding the physiology of this pathogenic organism and in developing vaccines. The development of safe, effective and economic mucosal vaccine is essential for protecting the health and safety of the consumer and the environment. Our new strain of Salmonella FNR-delete could be used as an oral vaccine against Salmonella spp. in Poultry, other farm animals and humans. In addition, this mutant strain could be engineered to serve as a vaccine vector for delivery of epitopes of other disease causing organisms. Two US Patents were issued: No. 8,101,168 B2 (January 24, 2012) & No. 8,435,506 B2 (May 7, 2013) covering this technology. We predict that our novel approach for developing new oral vaccines against food-borne pathogens will succeed in meeting the challenge.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Transcriptional regulation by Ferric Uptake Regulator (Fur) in pathogenic bacteria. Troxell, B and H.M. Hassan; Frontiers in Cellular and Infection Microbiology - Volume & Issue: 3:59. Page Number(s): 1-13
Publication Date: 10-13-2013 PubMed ID -- PMID: doi:10.3389/fcimb.2013.00059)
|
Progress 10/01/11 to 09/30/12
Outputs
OUTPUTS: Food-borne infections following the consumption of poultry meat and egg products contaminated with Salmonella enterica is a major public health concern. Salmonella enterica is a Gram-negative intracellular pathogen that causes gastroenteritis in humans. Although non-life-threatening in healthy adults, it can be fatal for children and immunocompromised individuals. The infection proceeds via two main stages: invasion and systemic infection. During the invasion stage, the pathogen adheres to and colonizes the intestines gaining access to the epithelial cells. Subsequently, Salmonella crosses the epithelial barrier, gets internalized by the macrophages, where it multiplies, spreads in the host, and causes systemic infection. Clearly, Salmonella must adapt quickly to the diverse environments it encounters in the host. In fact, from the gastrointestinal tract to the intracellular milieu, it is challenged with fluctuations in oxygen concentration and with numerous host-immune defenses that reduce its ability to colonize the host. PARTICIPANTS: Dr. Matt Koci - Associate Professor - Poultry Science Department - NC State University Mary Mendoza - Research Technician - Department of Microbiology, NC State University Dr. Bryan Troxell - Graduated Dr. Matt Evans - Graduated This program provided Graduate students (Ph.D.) with professional training in microbiological research TARGET AUDIENCES: The scientific community at large, and Farmers and producers of food and poultry products. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The ability of Salmonella to sense and adapt to sudden changes in its immediate environment is essential to its survival in the host and to its ability to cause disease. It is well known that the gut environment in the host is anoxic (i.e., anaerobic or microaerobic). Indeed, the gene required for the global regulation of anaerobic metabolism, Fumarate nitarte reduction (Fnr), is also important for the ability of Enterobacteria to adapt to the anaerobic environment in the host. For several years, we have been interested in the elucidation of the regulatory networks in S. Typhimurium that are involved in the coordinated regulation of cellular metabolism and pathogenesis in order to advance the development of novel strategies and therapeutics for the treatment and prevention of salmonellosis. We hypothesized that coordinated regulation of cellular redox and metabolic activity in S. Typhimurium plays an important role in its ability to survive and adapt to sudden environmental changes encountered in the host and cause illness. Indeed, we found that deleting this master global regulator, Fnr (d-fnr), of S. Typhimurium resulted in the loss of motility, flagella biosynthesis, and virulence in mice. We submitted a patent application on this technology and a US patent No. 8,101,168 B2 titled "Attenuated FNR Deficient Enterobacteria" was issued in January 2012. We also examined the roles of two other global regulators on Salmonella metabolism and virulence. Thus, we compared the transcriptional profiles of the virulent wild-type (WT) strain (ATCC 14028s) and its isogenic arcA mutant and fur mutant grown under anaerobic conditions. We found that ArcA directly or indirectly regulates 392 genes (8.5% of the genome); of these, 138 genes are poorly characterized. Additionally, we identified a set of 120 genes whose regulation was shared with the anaerobic redox regulator, Fnr. In addition, microarray analysis of the anaerobically grown delta fur S. Typhimurium identified 298 differentially expressed genes. Expression of several genes controlled by Fnr and NsrR appeared to be also dependent on Fur. Furthermore, Fur was required for the activity of the cytoplasmic superoxide disumutases (MnSOD and FeSOD). The regulation of FeSOD gene, sodB, occurred via small RNAs (i.e., the ryhB homologs, rfrA and rfrB) with the aid of the RNA chaperone Hfq. Additionally, in ∆fur, the expression of the gene coding for the ferritin-like protein (ftnB) was down-regulated, while the transcription of the gene coding for the nitric oxide detoxifying flavohemoglobin (hmpA) was up-regulated. In addition, the expression of the gene coding for the histone-like protein, H-NS (hns) was increased in delta fur.
Publications
- Qiu, R., J. Croom, R.A. Ali, C. Ashwell, H.M. Hassan, C.C. Chiang, and M.D. Koci.(2012). Direct fed microbial supplementation repartitions host energy to the immune system. J. Animal Sci. 90: 2639-2651.
|
Progress 10/01/09 to 09/30/10
Outputs
OUTPUTS: Iron is an essential element for the survival of living cells. However, excess iron is toxic and its uptake is exquisitely regulated by the ferric uptake regulator, Fur. In Salmonella, the Salmonella Pathogenicity Island-1 (SPI-1) encodes a type three secretion system (T3SS), which is required for invasion of host epithelial cells in the small intestine. A major activator of SPI-1 is HilA, which is encoded within SPI-1. One known regulator of hilA is Fur. The mechanism of hilA regulation by Fur is unknown. We found that Fur is required for virulence in Salmonella enterica serovar Typhimurium; and that Fur is required for the activation of hilA as well as of other HilA-dependent genes, invF and sipC. The Fur-dependent regulation of hilA was independent of PhoP, a known repressor of hilA. Instead, the expression of the gene coding for the histone-like protein, hns, was significantly derepressed in the fur mutant. Indeed, the activation of hilA by Fur was dependent on 28 nucleotides located upstream of hns. Moreover, we used chromatin immunoprecipitation (ChIP) to show that Fur bound, in vivo, to the upstream region of hns in a metal dependent fashion. Finally, deletion of fur in an hns mutant resulted in Fur-independent activation of hilA. In conclusion, Fur activates hilA by repressing the expression of hns. In an another study, we characterized the role of antioxidant enzymes (superoxide dismutases and hydroperoxidases) in bacterial (including Gram positive and Gram negative) resistance to organic acids. PARTICIPANTS: Bryan Troxell was a Ph.D. student in laboratory. He graduated in December 2009 and is currently a Postdoctoral/Research Associate in the department of Immunology and Microbiology at Indiana University School of Medicine. We also have collaborators from the department of Microbiology at University of Colorado School of Medicine and another collaborator at the department of Food Science and Nutrition at the University of Minnesota.The project provided training to several Ph.D. students and laboratory technician. TARGET AUDIENCES: Food Scientists, Vaccine companies, food producers, Basic Scientists, applied scientists, and the general public PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Salmonella Typhimurium is an intracellular pathogen and is a main cause of food-borne illness. The results from these studies and other studies from our laboratory demonstrated the essential role of low redox potential (i.e., anaerobiosis) controlled by "FNR" and iron homeostasis controlled by "Fur" in survival and virulence of Salmonella. Understanding the mechanism of pathogenesis in this organism will help in developing safe and effective vaccines against Salmonella and other related species. We have a patent application (Patent Pending) on a vaccine strain of Salmonella that could be used as an oral vaccine against Salmonella in farm animals and humans. We also discovered the role of antioxidants in protecting lactic acid probiotic bacteria and other organisms against acidic conditions in the stomach and in acid foods.
Publications
- Bruno-Barcena, J.M., M. A. Azcarate-Peril, and H. M. Hassan (2010). Role of antioxidant enzymes in bacterial resistance to organic acids. Appl. Environ. Microbiol. 76: 2747-2753.
- Dobrogosz, W. J., T. J. Peacock, and H. M. Hassan (2010). Evolution of the probiotic concept: from conception to validation and acceptance in medical science. Adv. Appl. Microbiol. 72:1-41.
- Troxell, B., M.L. Sikes, R.C. Fink, A. Vazquez-Torres, J. Jones-Carson, and H.M. Hassan. (2010). Fur negatively regulates hns and is required for the expression of HilA and virulence in Salmonella enterica serovar Typhimurium. J. Bacteriol. In press.
|
Progress 10/01/08 to 09/30/09
Outputs
OUTPUTS: Salmonella is one of the major causes of food-borne infections. Poultry/ meat products, and agriculture produce are the major source of Salmonella infections. Following ingestion of a contaminated food or water, the organism colonizes the host's intestinal epithelium and induces gastroenteritis (i.e., salmonellosis). The growth phase and growth conditions of the organism are important factors in attachment, invasion, and virulence. In general, Salmonella cells propagated under limited oxygen concentrations are more invasive than aerobically cultured cells. Recently, we hypothesized that the transition between aerobic and anaerobic environments in the host involves changes in a large number of genes that are regulated by the cooperative interactions between several global regulators (i.e., the regulators of redox and iron homeostasis). In Escherichia coli, FNR (Fumarate Nitrate Reductase) is one of the main regulatory proteins involved in oxygen sensing and in controlling the transcription of the genes required for the aerobic/anaerobic transition. However, the role of FNR in S. typhimurium is unknown. To assess its role in S. typhimurium, we constructed an FNR-minus (fnr) mutant in the pathogenic strain, ATCC14028S. The WT and the fnr mutant strains were grown under anaerobic conditions in a Coy Anaerobic Chamber. Total RNAs were isolated when the cultures reached an OD600 of 0.3 (mid-log). Microarray slides were used to compare the global expression patterns of the two strains (i.e., WT vs. FNR-). The data showed that 275 genes are controlled by FNR. These results demonstrated that FNR has an important role in S. typhimurium by serving as a transcriptional repressor/activator to coordinate cellular metabolism under anoxic conditions. we also found that FNR regulates many of the S. Typhimurium virulence genes (i.e., Salmonella pathogenicity island-1, SPI-1, and the virulence operon, srfAB). Furthermore, an FNR mutant was attenuated, both in vivo and ex vivo. The lethality of the mutant was completely abolished in the typhoid fever mouse model, and was quickly killed by mouse macrophages in culture. The data also suggested that this novel mutant strain of Salmonella could be useful as an oral vaccine in humans and animals against Salmonella. In addition, this mutant strain could be engineered to serve as a vaccine vector, Patent Pending. Furthermore, recent preliminary data from chicken macrophages indicated that the FNR-deficient strain of S. Typhimurium is also attenuated in chickens. We plan to demonstrate that this vaccine strain can provide protection against S. Typhimurium and other Salmonella serovars common in the poultry industry, and to reduce the shedding of Salmonella in the poultry foods and the environment. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Vaccine companies, Food Scientists, Food producers, Basic Scientists, Applied Scientists, and the General Public. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The results from these studies demonstrated the essential role of anaerobiosis and redox potential as controlled by the global regulator FNR in the survival and virulence in Salmonella. The role of the redox state in Salmonella will be of great importance to understanding the physiology of this pathogenic organism and in developing vaccines. The development of safe, effective and economic mucosal vaccine is essential for protecting the health and safety of the consumer and the environment. Our engineered strain of Salmonella FNR Mutant could be used as an oral vaccine against Salmonella spp. in humans and animals. In addition, this mutant strain could be engineered to serve as a vaccine vector for delivery of epitopes of other disease causing organisms. Currently, we have a Patent Pending (U.S. Patent Application # 11/780,358). We predict that our novel approach for developing new oral vaccines against food-borne pathogens will succeed in meeting the challenge.
Publications
- No publications reported this period
|
Progress 10/01/07 to 09/30/08
Outputs
OUTPUTS: Our research interests are focused on understanding the biochemical and molecular basis of the toxicity/mutagenicity of oxyradicals and the regulation of the biosynthesis of defense enzymes. We have established that the expression of the sodA gene which encodes the Mn-containing superoxide dismutase (MnSOD) in E. coli is tightly controlled by the level of superoxide radical (O2 ), the level of iron in the cells, and the redox state. These observations lead us to discover that the regulation of MnSOD biosynthesis is under negative control by an iron-containing regulatory protein where iron plays the role of a redox sensor. We isolated anaerobically derepressed sodA mutants using strains containing sodA-lacZ gene fusions. We were able to obtain and characterize cis- and trans-acting regulatory mutations. We also used site directed mutagenesis to identify the operator site of the sodA gene. We completed the identification of the trans-acting regulatory elements of sodA. The data, thus far, support our model (i.e., negative control via an iron-containing repressor), but it show that the regulatory circuit is much more complicated than originally thought. Thus, we found four trans-acting regulatory elements that act in concert to regulate the level of MnSOD in response to various environmental stimuli. The four regulatory elements are FUR (Ferric Uptake Regulation), ARC (Aerobic Regulatory Control), FNR (Fumarate-Nitrate Regulation) and IHF (Integration Host Factor). We would like to study the interactions of these repressors with the operator region of sodA and how the four repressors communicate with each other. We applied the same knowledge gained from our experiences with E. coli to a pathogenic strain of Salmonella. We used microarray technology to study the role(s) of the global regulators (FNR, ArcA, FUR) on Salmonella metabolism and virulence. A wealth of knowledge has been generated and the first manuscript on this new venture has been published in J. Bacteriology 2007. We discovered that these global regulators also control virulence. Based on our findings, we applied for a patent to use some of the mutants as oral vaccines (Patent Pending). We also collaborated with researchers at UNC-Chapel Hill in the genetic and Cancer research groups and demonstrated the ability of our engineered probiotic lactic acid bacteria that are capable of expressing MnSOD gene (sodA) from Streptococcus thermophilus to reduce IBD in a cancer mice model. The findings are significant and have been published. PARTICIPANTS: I have collaborative research efforts with Drs. Debbie Clare, Fred Breidt, and R. T. Klaenhammer of the Food Science Department (NCSU). Also, With Dr. Deborah Threadgill, Department of Genetic, University of North Carolina at Chapel Hill (Currently she is a member of our department). TARGET AUDIENCES: Our work identified a mutant strain of Salmonella that has the potential to be used as a vaccine for chiken and other farm animals for protection against salmonella infection and protacting the consumer against salmonellosis. We also discovered that antioxidant enzymes delivered in the intestinal tracts by probiotic lactobacilli can protects against inflammatory bowel diseases (IBD) and colen cancer. This information is targeted for the agriculture industry and the general public. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
1. Issue: The importance of oxygen derived free radicals in biological systems in both health and disease has become increasingly evident in the past ten years. Exciting discoveries have been made recently which have shown that these activated molecules are associated with a variety of biochemical reactions. The reactivity of free radicals confers upon them the potential for initiating structural alterations in final cellular macromolecules. Free radicals have been associated with the aging process, the initiation of cancer, ALS, Alzheimer's, Crohn's, and other diseases. 2. What has been done: An important cellular component which provides a protective function to living cells is the enzyme superoxide dismutase (SOD). The pioneering work of Irwin Fridovich's group at Duke University and Hosni Hassan's group at NC State University has led to the understanding of the biological function of SODS and other two enzymes, catalases and peroxidases, in providing protection against reactive oxygen species. Indeed, alteration/defects in these genes have been associated with cancer, aging, inflammation, and many diseases. Recent works from our laboratory have shown that Probiotics engineered to express these important antioxidant enzymes are useful in treating Inflammatory Bowel Diseases (IBD) and colon cancer in animal models. Future work is needed to include human studies. These antioxidant endowed probiotic organisms can also be applied to protect, ameliorate, and/or treat radiation induced gastrointestinal injuries. 3. Impact: Works in our laboratory provide the basic knowledge, at the molecular level, on the genetic regulation of these essential enzymes as well as the medical applications of these essential antioxidant enzymes. 4. Funding Sources: (1) Oxidative stress and the regulation of the antioxidant enzymes in prokaryotes. NC Agricultural Research Service, Project #NC006460 (2) Private gifts (3) Global Regulation of Pathogenesis in Salmonella typhimurium: Roles of Redox and Iron. Total cost $1,825,000/ Direct cost $1,250,000 (5 years) - to be resubmitted. 5. Contact: Hosni M. Hassan, Ph.D., Department of Microbiology, North Carolina State University, Raleigh, NC 27695-7615 - ph.: 919/515-7081 Email: hosni_hassan@ncsu.edu
Publications
- Clare, D.A., Z. Zheng, H.M. Hassan, H.E. Swaisgood, and G.L. Catignani. 2008. Antimicrobial properties of milkfat globule membrane fractions. J. Food Protection 71: 126-133.
- Kreske, A.C., K. Bjornsdottir, F. Dreidt, Jr., H.M. Hassan. 2008. Effects of pH, dissolved oxygen, and ionic strength on the survival of Escherichia coli O157:H7 in organic acid solutions. J. Food Protection 71: 2404-1409
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Progress 10/01/06 to 09/30/07
Outputs
OUTPUTS: In Escherichia coli, FNR is one of the main regulatory proteins involved in O2 - sensing. Since S. Typhimurium must successfully transition the broad fluctuations in [oxygen] encountered in the host. We assessed the role of FNR in S. Typhimurium. We constructed an isogenic fnr mutant in the virulent wild-type strain (ATCC 14028s) and compared their transcriptional profiles and pathogenicity in mice. Here, we report that, under anaerobic conditions, 311 genes (6.80% of the genome) are regulated directly or indirectly by FNR; of these, 87 genes (28%) are poorly characterized. Regulation by FNR in S. Typhimurium is similar, but distinct from that in E. coli. Thus, genes/operons involved in aerobic metabolism, NO.-detoxification, flagellar biosynthesis, motility, chemotaxis, and anaerobic carbon utilization are regulated by FNR in a similar fashion as in E. coli. However, genes/operons existing in E. coli, but regulated by FNR only in S. Typhimurium include those coding for
ethanolamine utilization, a universal stress protein, a ferritin-like protein, and a phosphotransacetylase. Interestingly, Salmonella-specific genes/operons regulated by FNR include numerous virulence genes within Salmonella pathogenicity island 1 (SPI-1), newly identified flagellar genes (mcpAC, cheV), and the virulence operon (srfABC). Furthermore, the role of FNR as a positive regulator of motility, flagella biosynthesis, and pathogenesis was confirmed by showing that the mutant is non-motile, lacks flagella, is attenuated in mice, and does not survive inside macrophages. The inability of the mutant to survive inside macrophages is likely due to its sensitivity to the reactive oxygen species generated by NADPH phagocyte oxidase. Recently, emerging evidence has implicated reactive oxygen species (ROS) in the pathogenesis of Inflammatory Bowel Diseases (IBD). Thus, we investigated the ability of MnSOD from Streptococcus thermophilus to reduce colitis symptoms in Interleukin (IL)
10-deficient mice using Lactobacillus gasseri as a delivery vehicle. Cohorts of 13-15 IL10-deficient mice were left untreated or supplemented with native L. gasseri or L. gasseri expressing MnSOD for four weeks. Colonic tissue was collected and inflammation was histologically scored. The presence of innate immune cells was investigated by immunohistochemistry and the host antioxidant response was determined by quantitative PCR. It was demonstrated that L. gasseri was stably maintained in mice for at least three days. L. gasseri producing MnSOD significantly reduced inflammation in IL10-deficient mice compared to untreated controls (P < 0.05), while the anti-inflammatory effects of both native and MnSOD producing L. gasseri were more pronounced in males. The anti-inflammatory effects of the L. gasseri were associated with a reduction in the infiltration of neutrophils and macrophages. This study demonstrates that L. gasseri producing MnSOD has significant anti-inflammatory activity
that reduces the severity of colitis in the IL10-deficient mouse.
PARTICIPANTS: 1- R. C. Fink - was a Postdoctoral Fellow - He is currently at the University of Florida as a faculty member 2- M. R. Evan - is a Ph.D. student that will graduate in May 2008 3- S. Porwollik and M. McClelland are collaborator from the University of California, San Diego 4- A. Vazquez-Torres and J. Jones-Carson are collaborators from the University of Colorado Medical ctr. 5- B. Troxell is a Ph.D. student in my laboratory - Planning to graduate in 2009. 6- D. S. Threadgill and I. M. Carroll are collaborators from the Univ. of NC Medical School at Chapel Hill, NC. 7- J. M. Andrus is a previous Ph.D. student from my Lab. He is currently an Assistant Professor at the University of Augusta, GA. 8- J.M. Bruno-Barcena is a previous Postdoctoral fellow in my Lab and is currently a Faculty member in the same department at NCSU. 9- M. A. Azcarate-Peril is a Research Associate and a collaborator from the Food Science Department at NCSU. 10- T.R. Klaenhammer is a collaborator from
the Food Science Department at NCSU
TARGET AUDIENCES: The Scientific Community at large and the general population.
PROJECT MODIFICATIONS: None
Impacts
The results from these studies demonstrated the essential and global role of FNR in regulating the virulence genes and anaerobic metabolism in Salmonella Typhimurium. These findings suggested that the FNR mutant could serve as a Salmonella Vaccine strain for immunizing Human and Farm Animals against Salmonella and other related pathogens. The vaccine strain could also be used to deliver foreign epitopes/antigens for immunization against other disease causing microorganisms. Indeed, a patent application has been filed and a Patent is pending. Previously, we have shown that the heterologous expression of MnSOD from Streptococcus thermophilus in probiotic lactic acid bacteria that do not contain this antioxidant enzyme resulted in better growth and resistance to hydrogen peroxide than the parent strains. Here, we showed that this MnSOD engineered organism can provide protection against Inflammatory Bowel Diseases. This finding has a great implication in medicine and in
the treated inflammatory and oxidative stress related disorders. Also, it shows that Probiotic Lactobacilli are excellent for delivering vaccines and epitopes to the intestinal tracts of humans and farm animals.
Publications
- Fink, R. C., M. R. Evans, S. Porwollik, A. Vazquez-Torres, J. Jones-Carson, B. Troxell, S. J. Libby, M. McClelland, and H. M. Hassan (2007). FNR is a global regulator of virulence and anaerobic metabolism in Salmonella enterica serovar Typhimurium (ATCC 14028s). J. Bacteriol. 189:2262-2273.
- Carroll, I.M., J.M. Andrus, J.M. Bruno-Barcena, T.R. Klaenhammer, H.M. Hassan, and D.S. Threadgill 2007. The anti-inflammatory properties of Lactobacillus gasseri expressing manganese superoxide dismutase (MnSOD) using the interleukin-10-deficient mouse model of colitis. Azcarate-Peril, M. A., J. M. Bruno-Barcena, H.M. Hassan, and T. R. Klaenhammer (2006). Transcriptional and functional analysis of oxalyl-coenzyme A (CoA) decarboxylase and formyl-CoA transferase genes from Lactobacillus acidophilus. Appl. Environ. Microbiol. 72: 1891-1899.(in press) Published electronically ahead of printed version (doi:10.1152/ajpgi.00132.2007).
- Azcarate-Peril, M. A., J. M. Bruno-Barcena, H.M. Hassan, and T. R. Klaenhammer (2006). Transcriptional and functional analysis of oxalyl-coenzyme A (CoA) decarboxylase and formyl-CoA transferase genes from Lactobacillus acidophilus. Appl. Environ. Microbiol. 72: 1891-1899.
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Progress 10/01/04 to 09/30/05
Outputs
A strategy for functional gene replacement in the chromosome of Lactobacillus gasseri is described. The phospho-beta-galactosidase II gene (lacII) was functionally replaced by the manganese superoxide dismutase (MnSOD) gene (sodA) from Streptococcus thermophilus, by adapting the insertional inactivation method described for lactobacilli (Russell and Klaenhammer. 2001, Appl. Environ. Microbiol. 67:4361-4364). L. gasseri carrying the heterologous sodA gene grew on lactose as efficiently as the wild type parent. An active MnSOD was expressed in the transgenic strain, and the enzyme migrated on PAGE-SOD activity gels to the same position as that of MnSOD from S. thermophilus. The expression of MnSOD from a single copy of sodA integrated in the chromosome of L. gasseri provided enhanced tolerance to hydrogen peroxide, and extended the viability of carbon/energy starved cultures stored at 25 degree Centigrade. This is the first report showing the successful utilization of
the pORI plasmids system to generate marker-free gene integration in L. gasseri strains.
Impacts
The integration and expression of S. thermophilus sodA gene in L. gasseri provided protection against hydrogen peroxide stress. The data show that MnSOD protects cells against hydrogen peroxide by removing O2- and preventing the redox cycling of iron. It also increased the survival and the shelf-life of the constructed strains. To our best knowledge, this is the first report of a sodA from S. thermophilus being integrated and expressed in other lactic acid bacteria.
Publications
- Bruno-Barcena, J.M., M.A. Azcarate-Peril, T.R. Klaenhammer, and H.M. Hassan (2005). Marker-free Chromosomal insertion of the manganese superoxide dismutase gene (sodA) from Streptococcus thermophilus into Lactobacillus gasseri. FEMS Microbiology Letters 246: 91-101.
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Progress 10/01/03 to 09/30/04
Outputs
Salmonella typhimurium is a gram-negative, facultative intracellular pathogen that infects the intestinal tracts of humans and animals. In the host, S. typhimurium encounters a wide range of oxygen concentrations going from oxic conditions in the stomach to near anoxic conditions in the distal sigmoid colon-rectal junction. Transitions from aerobic to anaerobic growth usually involve a large number of physiological changes in the cell. In Escherichia coli, FNR (Fumarate Nitrate Reductase) is one of the main regulatory proteins involved in oxygen sensing and in controlling the transcription of the genes required for the aerobic/anaerobic transition. However, the role of FNR in S. typhimurium is unknown. To assess its role in S. typhimurium, we constructed an FNR-minus (fnr) mutant in the pathogenic strain, ATCC14028S. The WT and the fnr mutant strains were grown under anaerobic conditions in a Coy Anaerobic Chamber. Total RNAs were isolated when the cultures reached an
OD600 of 0.3 (mid-log). Microarray slides were used to compare the global expression patterns of the two strains (i.e., WT vs. FNR-). The data showed that 275 genes are controlled by FNR. These results demonstrated that FNR has an important role in S. typhimurium by serving as a transcriptional repressor/activator to coordinate cellular metabolism under anoxic conditions. We also examined the potential advantages of providing a heterologous SOD to some of the intestinal lactobacilli. Thus, the gene encoding the manganese-containing superoxide dismutase (sodA) was cloned from Streptococcus thermophilus AO54 and expressed in four intestinal lactobacilli. A 1.2 kb PCR product containing the sodA was cloned into the shuttle vector pTRK563, to yield pSodA, which was functionally expressed and complemented an Escherichia coli strain deficient in Mn- and Fe-SODs. The plasmid, pSodA, was subsequently introduced and expressed in Lactobacillus gasseri NCK334, L. johnsonii NCK89, L. acidophilus
NCK56, and L. reuteri NCK932. Molecular and biochemical analyses confirmed the presence of the gene (sodA) and the expression of an active gene product (MnSOD) in these strains of lactobacilli. The specific activity of MnSOD was 6.7, 3.8, 5.8, and 60.7 (U/mg protein) for L. gasseri, L. johnsonii, L. acidophilus, and L. reuteri, respectively. The expression of S. thermophilus MnSOD in L. gasseri and L. acidophilus provided protection against hydrogen peroxide stress. The data show that MnSOD protects cells against hydrogen peroxide by removing O2- and preventing the redox cycling of iron. To our best knowledge, this is the first report of a sodA from S. thermophilus being expressed in other lactic acid bacteria.
Impacts
The results from these studies deminstrated the essential role of superoxide dismutase in aerobic survival. Thus, the heterologous expression of MnSOD from Streptococcus thermophilus in probiotic lactic acid bacteria that do not contain this antioxidant enzyme resulted in better growth and resistance to hydrogen peroxide than the parent strains. Also FNR is a global regulator in Salmonella typhimurium and unerstanding its regulatory function will be of great importance to understanding the physiology of this pathogenic organism.
Publications
- Kong, In-S., T.C. Bates, A. Huelsmann, H.M. Hassan, B.E. Smith, and J.D. Oliver (2004). Role of catalase and oxyR in the viable but nonculturable state of Vibrio vulnificus. FEMS Microbiology-Ecology 50: 133-142.
- Bruno-Barcena, J.M., J.M. Andrus, S.L. Libby, T.R. Klaenhammer, and H.M. Hassan. (2004). Expression of a heterlogous manganese superoxide dismutase gene in intestinal lactobacilli: protection against the toxicity of hydrogen peroxide. Appl. Environ. Microbiol. 70: 4702-4710.
- Ryan C. Fink, Matthew R. Evans, Michael McClelland, Stephen J. Libby, and Hosni M. Hassan. 2005. FNR is a global regulator in anaerobically grown Salmonella typhimurium. American Society for Microbiology- 105 General Meeting, Atlanta, GA (Abstract)
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