Citation Details

Journal Article

Animals; Bioware; Bone Marrow; Bone Marrow Cells; Disease Models, Animal; Female; Host-Pathogen Interactions; Humans; Knee Joint; Listeria monocytogenes; Listeriosis; Mice; Mice, Inbred BALB C; Mutation; pXen-5; Tibia

Murine listeriosis is one of the most comprehensive and well-studied models of infection, and Listeria monocytogenes has provided seminal information regarding bacterial pathogenesis. However, many aspects of the mouse model remain poorly understood, including carrier states and chronic colonization which represent important features of the spectrum of host-pathogen interaction. Bone marrow has recently been shown to harbor L. monocytogenes, which spreads from this location to the central nervous system. Bone could, therefore, be an important chronic reservoir, but this infection is difficult to study because it involves only a few bacteria and the extent of infection cannot be assessed until after the animal is sacrificed. We employed in vivo bioluminescence imaging to localize L. monocytogenes bone infections over time in live mice, revealing that the bacteria grow in discrete foci. These lesions can persist in many locations in the legs of mice and are not accompanied by a histological indication such as granuloma or a neutrophil infiltratate. We demonstrate that highly attenuated hly mutants, which have defective intracellular replication, are capable of prolonged focal infection of the bone marrow for periods of up to several weeks. These results support the recently proposed hypothesis that the bone marrow is a unique niche for L. monocytogenes.

Journal Article

Bioware; pXen-13

BACKGROUND There is no safe, effective human vaccine against brucellosis. Live attenuated Brucella strains are widely used to vaccinate animals. However these live Brucella vaccines can cause disease and are unsafe for humans. Killed Brucella or subunit vaccines are not effective in eliciting long term protection. In this study, we evaluate an approach using a live, non-pathogenic bacteria (E. coli) genetically engineered to mimic the brucellae pathway of infection and present antigens for an appropriate cytolitic T cell response. METHODS E. coli was modified to express invasin of Yersinia and listerialysin O (LLO) of Listeria to impart the necessary infectivity and antigen releasing traits of the intracellular pathogen, Brucella. This modified E. coli was considered our vaccine delivery system and was engineered to express Green Fluorescent Protein (GFP) or Brucella antigens for in vitro and in vivo immunological studies including cytokine profiling and cytotoxicity assays. RESULTS The E. coli vaccine vector was able to infect all cells tested and efficiently deliver therapeutics to the host cell. Using GFP as antigen, we demonstrate that the E. coli vaccine vector elicits a Th1 cytokine profile in both primary and secondary immune responses. Additionally, using this vector to deliver a Brucella antigen, we demonstrate the ability of the E. coli vaccine vector to induce specific Cytotoxic T Lymphocytes (CTLs). CONCLUSION Protection against most intracellular bacterial pathogens can be obtained mostly through cell mediated immunity. Data presented here suggest modified E. coli can be used as a vaccine vector for delivery of antigens and therapeutics mimicking the infection of the pathogen and inducing cell mediated immunity to that pathogen.

Journal Article

Administration, Intranasal; Animals; Bioware; Disease Models, Animal; Female; Humans; Immunohistochemistry; Intracellular Fluid; Lymphoid Tissue; Mice; Mice, Inbred BALB C; Nasal Mucosa; Nasopharynx; Palatine Tonsil; pXen-5; Streptococcal Infections; Streptococcus pyogenes

Human tonsils are suspected to be an antibiotic-impervious human reservoir for group A streptococcus. An intranasal infection model in mice and a bioluminescent-tagged strain were used to investigate this possibility. Viable streptococci were predominantly found both intra- and extracellularly in nasal-associated lymphoid tissue (NALT), a human tonsil homologue. Ulex europaeus-1, a membranous (M) cell-specific lectin, identified cells harboring streptococci at the epithelial surface of NALT and blocked bacterial colonization of this tissue. These results suggest that M cells in NALT transport this Gram-positive pathogen across the epithelial layers in a manner similar to those in Peyer's patches, which permit enteric pathogens to invade deeper tissues from the gastrointestinal tract.

Journal Article

Amino Acid Transport Systems, Neutral; Animals; Bacterial Proteins; Bioware; Blotting, Northern; Disease Models, Animal; Gene Expression Regulation, Bacterial; Humans; Lac Operon; Mice; Osmolar Concentration; Proline; pXen-5; Recombinant Fusion Proteins; Staphylococcal Infections; Staphylococcus aureus; Symporters; Transcriptional Activation

Staphylococcus aureus can grow virtually anywhere in the human body but needs to import proline through low- and high-affinity proline transporters to survive. This study examined the regulation of the S. aureus putP gene, which encodes a high-affinity proline permease. putP::lacZ and putP::lux transcriptional fusions were constructed and integrated into the genomes of several S. aureus strains. Enzyme activity was measured after growth in media with various osmolyte concentrations. As osmolarity rose, putP expression increased, with a plateau at 2 M for NaCl in strain LL3-1. Proline concentrations as low as 17.4 muM activated expression of the putP gene. The putP::lux fusion was also integrated into the genomes of S. aureus strains that were either SigB inactive (LL3-1, 8325-4, and SH1003) or SigB active (Newman and SH1000). SigB inactive strains showed increased putP gene expression as NaCl concentrations rose, whereas SigB active strains displayed a dramatic decrease in putP expression, suggesting that the alternative sigma factor B plays a negative role in putP regulation. Mice inoculated with S. aureus strains containing the putP::lux fusion exhibited up to a 715-fold increase in putP expression, although levels in the various murine organs differed. Moreover, urine from human patients infected with S. aureus showed elevated putP levels by use of a PCR procedure, whereas blood and some abscess material had no significant increase. Thus, putP is transcriptionally activated by a low-proline and high osmotic environment both in growth media and in murine or human clinical specimens.

Journal Article

Animals; Biofilms; Bioware; Colony-Forming Units Assay; Disease Susceptibility; Female; Luminescence; Mice; Mice, SCID; pXen-5; Staphylococcal Infections; Staphylococcus epidermidis; T-Lymphocytes; T-Lymphocytes, Cytotoxic

Staphylococcus epidermidis is the leading cause of hospital-acquired device-related infections, but there is a lack of suitable methods to investigate the pathogenesis of S. epidermidis infection. We created a bioluminescent strain of S. epidermidis and developed a subcutaneous catheter-related murine infection model for real-time monitoring of biofilm-associated infection. Additionally, we compared severely immunocompromised and immunocompetent mice, demonstrating the substantial effect of animal immune status on susceptibility to experimentally induced S. epidermidis disease. This study presents a novel approach for investigating the in vivo details of the pathogenesis of S. epidermidis infection.