Citation Details

Journal Article

Animals; Biofilms; Bioware; Bone Density Conservation Agents; Chronic Disease; Cytokines; Drug Evaluation, Preclinical; Humans; Immunity; Incidence; Jaw Diseases; Mice; Neovascularization, Physiologic; Osteoclasts; Osteomyelitis; Osteonecrosis; Staphylococcal Infections; Xen29

The effects of antiresorptive agents (e.g., alendronate [Aln], osteoprotegerin [OPG]) on bone infection are unknown. Thus, their effects on implant-associated osteomyelitis (OM) were investigated in mice using PBS (placebo), gentamycin, and etanercept (TNFR:Fc) controls. None of the drugs affected humoral immunity, angiogenesis, or chronic infection. However, the significant (P < 0.05 vs. PBS) inhibition of cortical osteolysis and decreased draining lymph node size in Aln- and OPG-treated mice was associated with a significant (P < 0.05) increase in the incidence of high-grade infections during the establishment of OM. In contrast, the high-grade infections in TNFR:Fc-treated mice were associated with immunosuppression, as evidenced by the absence of granulomas and presence of Gram(+) biofilm in the bone marrow. Collectively, these findings indicate that although antiresorptive agents do not exacerbate chronic OM, they can increase the bacterial load during early infection by decreasing lymphatic drainage and preventing the removal of necrotic bone that harbors the bacteria.

Journal Article

Adhesins, Bacterial; Animals; Antigens, CD46; Bacteremia; Bacterial Outer Membrane Proteins; Bacterial Proteins; Bacterial Translocation; Bioware; Blood-Brain Barrier; Central Nervous System; Disease Progression; Female; Luminescent Measurements; Luminescent Proteins; Male; Meningitis, Meningococcal; Meningococcal Infections; Mice; Mice, Transgenic; Nasal Cavity; pXen-13; Recombinant Fusion Proteins; Respiratory System; Sepsis; Thyroid Gland

Neisseria meningitidis is a human pathogen that causes septicemia and meningitis with high mortality. The disease progression is rapid and much remains unknown about the disease process. The understanding of disease development is crucial for development of novel therapeutic strategies and vaccines against meningococcal disease. The use of bioluminescent imaging combined with a mouse disease model allowed us to investigate the progression of meningococcal sepsis over time. Injection of bacteria in blood demonstrated waves of bacterial clearance and growth, which selected for Opa-expressing bacteria, indicating the importance of this bacterial protein. Further, N. meningitidis accumulated in the thyroid gland, while thyroid hormone T4 levels decreased. Bacteria reached the mucosal surfaces of the upper respiratory tract, which required expression of the meningococcal PilC1 adhesin. Surprisingly, PilC1 was dispensable for meningococcal growth in blood and for crossing of the blood-brain barrier, indicating that the major role of PilC1 is to interact with mucosal surfaces. This in vivo study reveals disease dynamics and organ targeting during meningococcal disease and presents a potent tool for further investigations of meningococcal pathogenesis and vaccines in vivo. This might lead to development of new strategies to improve the outcome of meningococcal disease in human patients.

Journal Article

Amino Acid Sequence; Animals; Bioware; Brucella melitensis; Brucellosis; Disease Models, Animal; Flagella; Gene Deletion; Gene Expression Regulation, Bacterial; Interferon Regulatory Factor-1; Macrophages; Mice; Mice, Mutant Strains; Molecular Sequence Data; Oligonucleotide Array Sequence Analysis; pXen-13; Quorum Sensing; Repressor Proteins; Trans-Activators; Virulence Factors

Brucella melitensis is an intracellular pathogen that establishes a replicative niche within macrophages. While the intracellular lifestyle of Brucella is poorly understood and few virulence factors have been identified, components of a quorum-sensing pathway in Brucella have recently been identified. The LuxR-type regulatory protein, VjbR, and an N-acylhomoserine lactone signaling molecule are both involved in regulating expression of the virB-encoded type IV secretion system. We have identified a second LuxR-type regulatory protein (BlxR) in Brucella. Microarray analysis of a blxR mutant suggests that BlxR regulates the expression of a number of genes, including those encoding the type IV secretion system and flagella. Confirming these results, deletion of blxR in B. melitensis reduced the transcriptional activities of promoters for the virB operon, flagellar genes, and another putative virulence factor gene, bopA. Furthermore, our data suggested that both BlxR and VjbR are positively autoregulated and cross-regulate the expression of each other. The blxR deletion strain exhibited reduced growth in macrophages, similar to that observed for a vjbR deletion strain. However, unlike the vjbR deletion, the blxR deletion did not fully attenuate virulence in mice. More strikingly, bioluminescent imaging revealed that dissemination of the blxR mutant was similar to that of wild-type B. melitensis, while the vjbR mutant was defective for systemic spread in IRF-1(-/-) mice, suggesting that these regulators are not functionally redundant but that they converge in a common pathway regulating bacterial processes.

Journal Article

Actinomycetales; Bioware; Genes, Reporter; Genetic Engineering; Luminescent Proteins; Lycopersicon esculentum; Mirabilis; Plant Diseases; pXen-13; Recombinant Proteins; Seeds; Staining and Labeling

Clavibacter michiganensis subsp. michiganensis is a Gram-positive bacterium that causes wilting and cankers, leading to severe economic losses in commercial tomato production worldwide. The disease is transmitted from infected seeds to seedlings and mechanically from plant to plant during seedling production, grafting, pruning, and harvesting. Because of the lack of tools for genetic manipulation, very little is known regarding the mechanisms of seed and seedling infection and movement of C. michiganensis subsp. michiganensis in grafted plants, two focal points for application of bacterial canker control measures in tomato. To facilitate studies on the C. michiganensis subsp. michiganensis movement in tomato seed and grafted plants, we isolated a bioluminescent C. michiganensis subsp. michiganensis strain using the modified Tn1409 containing a promoterless lux reporter. A total of 19 bioluminescent C. michiganensis subsp. michiganensis mutants were obtained. All mutants tested induced a hypersensitive response in Mirabilis jalapa and caused wilting of tomato plants. Real-time colonization studies of germinating seeds using a virulent, stable, constitutively bioluminescent strain, BL-Cmm17, showed that C. michiganensis subsp. michiganensis aggregated on hypocotyls and cotyledons at an early stage of germination. In grafted seedlings in which either the rootstock or scion was exposed to BL-Cmm17 via a contaminated grafting knife, bacteria were translocated in both directions from the graft union at higher inoculum doses. These results emphasize the use of bioluminescent C. michiganensis subsp. michiganensis to help better elucidate the C. michiganensis subsp. michiganensis-tomato plant interactions. Further, we demonstrated the broader applicability of this tool by successful transformation of C. michiganensis subsp. nebraskensis with Tn1409::lux. Thus, our approach would be highly useful to understand the pathogenesis of diseases caused by other subspecies of the agriculturally important C. michiganensis.

Journal Article

Animals; Bioware; Disease Models, Animal; Female; Lung; Mice; Mice, Inbred BALB C; Pneumococcal Infections; pXen-5; Serotyping; Streptococcus pneumoniae, Xen10, Xen7, Xen35

The variability of the course of infection by Streptococcus pneumoniae is well known but poorly understood. Most animal models of pneumonia, sepsis or meningitis have been forced to use site-specific bacterial inoculation to mimic localized human infection. This study examined the differences in the progression of disease-causing strains D39 (serotype 2), A66.1 (serotype 3) and TIGR4 (serotype 4) using isolates transformed with the Gram-positive lux transposon cassette, Tn4001 luxABCDE Km(r). Expression of the lux operon results in bioluminescence, permitting the detection of the bacteria within a living animal while using a CCD camera. Mice infected intranasally with A66.1 developed only pneumonia, those challenged with D39 experienced high-grade sepsis, while TIGR4 infection resulted in low-grade pneumonia and bacteremia ultimately progressing to meningitis. Quantitative analysis of bacterial titers confirmed these patterns, which were consistent across different lineages of mice. Mice anesthetized with ketamine and xylazine developed more severe forms of the disease compared with isoflurane. These studies unambiguously characterize 3 distinct models of the natural course of pneumococcal infection. Mapping these models provides a framework for detailed molecular modeling of pneumococcal virulence determinants at specific stages of disease.