2012 USF Health Research Day Virtual Book

Abstract #: 7 Presented by: Stella Kratzer, BS, Staff
Regulation of Mitosis in the AIDS Pathogen, Toxoplasma gondii
Stella Kratzer, Elena S. Suvorova, Josephine Fruehauf, and Michael White USF- College of Medicine,University of
South Florida, Morsani College of Medicine, Dept. of Molecular Medicine
Keywords: Apicomplexans, Toxoplasma gondii, Mitosis, Cell Cycle
Objective: The intracellular parasite, Toxoplasma gondii, causes widespread infections in humans and animals. In
the US the risk of permanent infection is 1:2 by age 50. Toxoplasma infections are dangerous in people with weak or
compromised immune systems where tissue destruction from rounds of parasite division go unchecked. New
treatments that are well tolerated are critically needed to combat toxoplasmosis in these patients.
Methods: To identify pathways required for parasite growth, we have used forward genetics to identify essential cell
cycle factors. Our study was focused on a class of conditional chromosomal mis-segregation mutants that show a
variety of defects in mitosis when shifted to the non-permissive temperature. We used cell biology methods to better
understand the mitotic defects of specific mutants and a cosmid genomic library was used to genetically rescue the
temperature restriction and identify the defective gene.
Results: The cell biology of these mutants demonstrated defects that led to irregular shapes and sizes of the
parasites, the formation of zoids absent their chromosomes, unequal amount of chromosome content in the
developing daughter buds, and retention of nuclear material in the mother cell. From a class of 18 mutants, we
complemented 12 mutants and in 8 mutants the candidate defective gene was identified. Mutant 11-51A1 carries a
defective Zn-finger protein that is weakly related to E3 ubiquitin ligases.
Conclusion: The study of conditional mutants gives us insight into the essential mechanisms required for parasite
division. Novel genes found only in the parasite family were identified with one gene encoding a unique Zn-finger
protein. We speculate this factor plays an important role in chromosome segregation in Toxoplasma.
Research supported by: NIH
Abstract #: 8 Presented by: Amorce Lima, Graduate Student
Attenuated Virulence of B. henselae Mutants in the Zebrafish Embryo Model
Amorce Lima 1 *, Lisa Smith 1 , Byeong Cha 2 and Burt Anderson 1 1 Department of Molecular Medicine, 2 Department
of Pathology and Cell Biology, School of Biomedical Sciences, Morsani College of Medicine, University of South
Florida, Tampa, Florida
Keywords: Zebrafish; Danio rerio; angiogenesis; bacteria; Bartonella henselae; virulence factors
Objectives: B. henselae (Bh) is an emerging zoonotic pathogen and the causative agent of cat scratch disease
and bacillary angiomatosis. Bacillary angiomatosis is a systemic disease that is characterized by tumor-like
lesions on the skin resulting from proliferation of the small blood vessels. VirB/VirD4 Type IV secretion system
and Bartonella adhesin A (BadA) are two important virulence factors shown to play a critical role in B. henselae
infection and angiogenesis in vitro. Although valuable knowledge has been acquired using in vitro models,
progress in Bartonella research has been hampered due to a lack of a practical in vivo model. The purpose of this
project is to develop a model of Bh infection using the Tg(fli1:EGFP) y1 transgenic line of zebrafish (Danio rerio)
embryos in order to test the role of Bh virulence factors in infection and in eliciting a pro-angiogenic host
response.
Methods: The embryos are microinjected at the early stage of development with wild type and mutant strains
derived from B. henselae Houston-1expressing red fluorescence protein. Quantitative PCR and confocal
microscopy analysis are performed to assess bacterial replication and bacterial load in the infected embryos;
qRT-PCR is used to quantitatively measure host response to Bh infection.
Results: Data from our experiments show that zebrafish embryos microinjected with WT Bh become infected and
display an angiogenic phenotype. Quantitative PCR results also confirm the expansion of the Bh in the zebrafish
embryo at different time-points after microinjection. Quantitative real-time PCR data show evidence of induction of
angiogenic response in the infected embryos compared to control embryos. Moreover, our data show that
zebrafish infected with Bh mutants for the virB and badA genes display an attenuated virulence compared to
those infected with WT Bh.
Conclusions: We conclude that the zebrafish embryo model will be invaluable in helping identify virulence factors
required for Bh infection and their role in eliciting a proangiogenic response.
Research Support by: National Institutes of Health grant AI038178 to Burt Anderson
50