Jon M. Davis
Emerging Infectious Diseases
Doctor of Philosophy
2006
Major Advisor: Alison D. O’Brien, Ph.D., Department of Microbiology and Immunology
Dissertation Title: The Modulation of Polymorphonuclear Neutrophil
Function by
Cytotoxic Necrotizing Factor Type 1 - Expressing Uropathogenic Escherichia
coli
ABSTRACT
Uropathogenic Escherichia coli (UPEC) cause more than 85% of all urinary tract infections (UTI). These infections primarily affect women, and over half of all women will experience at least one UTI in their lifetime. Animal models of UTI pathogenesis have provided some insight into the role of various UPEC virulence factors. In these animal studies, the toxin Cytotoxic Necrotizing Factor type 1 (CNF1) has been shown to have a significant role in the pathogenesis of UTI. One of the most striking features of CNF1-expressing UPEC infection in the in vivo models was magnitude of the acute inflammatory response. Compared to a cnf1 isogenic mutant, CNF1-expressing UPEC elicited an acute inflammatory response characterized by an extensive infiltration of polymorphonuclear leukocytes (PMN) into tissue infected with CNF1-expressing UPEC. In spite of the enhanced acute inflammatory response, the CNF1-expressing UPEC had a significant survival advantage compared to the cnf1 isogenic mutant. CNF1 is an AB toxin that deamidates the catalytically-active glutamine residue in the Rho family of small GTPases. The Rho family GTPases are intracellular signaling molecules responsible for the control of many cellular function in eukaryotic cells such as PMNs. In PMNs, Rho GTPases control the processes of phagocytosis and chemotaxis in addition to the generation of reactive oxygen species (ROS). These observations formed the foundation of the hypothesis that CNF1-expressing UPEC modulate PMN function. This hypothesis was tested in vitro with elicited mouse PMNs and showed that CNF1-expressing UPEC modulate the antimicrobial response of PMNs through several mechanisms. First, CNF1-expressing UPEC alter the capacity of PMNs to remodel their plasma membrane and cluster CD11b in response to serum-opsonized UPEC which leads to a diminished PMN phagocytic capacity. Second, CNF1 expressed by UPEC results in an increase in the amount of Rac2 in PMNs. Third, PMNs co-incubated with CNF1-expressing UPEC generate an enhanced intracellular ROS. Finally, CNF1-expressing UPEC release CNF1 via outer membrane vesicles that interact with the PMN membrane. These results support the hypothesis that CNF1 is a UPEC virulence factor and lead to a new molecular model of UPEC interaction with the host innate immune system.
Joseph C. Larsen
Emerging Infectious Diseases Graduate Program
Doctor of Philosophy
2004
Major Advisor: Patricia Guerry, Ph.D., Department of Enteric Diseases, Naval Medical Research Center
Dissertation Title: Characterization of a plasmid-encoded type IV
secretion system in
Campylobacter jejuni 81-176
ABSTRACT:
The Gram-negative bacterium Campylobacter jejuni is major cause of diarrheal
illness in both the United States and the world abroad. Although well established
as an etiological agent, little is understood regarding the molecular mechanisms
underlying its pathogenesis. Additionally, inter-strain variations in clinical
manifestations, in vitro epithelial cell invasion levels, and virulence in
animal models suggest that genetic elements account for these phenotypic differences.
One strain of C. jejuni, 81-176, is a particularly virulent strain that invades epithelial cells at levels higher than most C. jejuni isolates. This strain possesses two plasmids, designated pVir and pTet, both of which contain genes with homology to type IV secretion systems (TFSS). TFSS are systems capable of translocating DNA, protein, or nucleoprotein complexes across bacterial membranes. Such systems are found in a number of human pathogens, where they facilitate various pathogenic processes. A mutational analysis in some of the pVir TFSS genes reduced levels of intestinal epithelial cell invasion and natural competence. Additionally, a mutation in one of the TFSS genes led to an attenuation of virulence in the ferret diarrhea model. These data led us to hypothesize that the pVir TFSS contributes to the pathogenesis of C. jejuni 81-176 and accounts for the phenotypic differences observed in virulence models.
This work represents a characterization of the pVir TFSS found in C. jejuni 81-176. Following purification and the generation of antisera, it was demonstrated that VirB10, a structural component of the pVir TFSS is glycosylated. Lectin affinity, enzymatic cleavage, and a reconstitution of glycosylation in Escherichia coli suggested that VirB10 was glycosylated by the general N-linked glycosylation pathway (pgl) of C. jejuni. Site-specific mutagenesis of VirB10 revealed two glycosylation sites at N32 and N97. Previously, mutation of virB10 resulted in a modest reduction in levels of natural competence. Mutation of N97 but not N32 resulted in levels of natural competence consistent with the virB10 mutant, suggesting glycosylation is required for the function of the pVir TFSS.
Secondly, an initial biochemical characterization of a putative type IV secretion ATPase was undertaken. A VirB11 homolog from the pVir plasmid was expressed and purified. This protein was shown in both a time and concentration dependent manner to possess ATPase activity and mutation of the nucleotide-binding site of VirB11 resulted in a form of the protein that was devoid of enzymatic activity. Yeast 2-hybrid analysis and chemical cross-linking experiments suggested that VirB11 formed hexameric structures, consistent with other VirB11 family members.
Lastly, characterization of a pVir-encoded protein linked with the TFSS was performed. This protein, Cjp29, bears homology to eukaryotic cell proteins and its expression is dependent on the TFSS genes. The regulation with the TFSS suggests that Cjp29 may be a protein substrate of the pVir TFSS.
Taken together, the characterization of the pVir TFSS represented here has provided a substantial foundation upon which to launch future research to elucidate the specific contributions of this system to clinical illness.
Patricia Dantas Santos-Ciminera
Emerging Infectious Diseases Program
Doctor of Philosophy
2005
Major Advisor: Gerald V. Quinnan, Jr., M.D., Department of Preventive Medicine and Biometrics
Dissertation Title: Molecular Epidemiology of Epidemic Severe Malaria
Caused by
Plasmodium vivax in the State of Amazonas, Brazil.
ABSTRACT:
Malaria in South America is a major public health problem. In Brazil, most
of the cases occur in the Amazon Region, particularly in the State of Amazonas.
In Manaus, the capital of Amazonas, atypical cases of P. vivax infections,
including patients presenting with severe thrombocytopenia and bleeding, led
to the formulation of the hypothesis that severe disease could be related
to a particular, emergent and more pathogenic genotype of P. vivax. We described
the epidemiology of malaria for the Amazonas State and city of Manaus by comparing
patients admitted in the hospital to those treated as outpatients in the Fundação
de Medicina Tropical do Amazonas (FMT-AM). Admissions due to vivax malaria
increased significantly from 1997 through 2003 suggesting a change in clinical
presentation. The admitted group presented higher mean parasite counts, lower
platelet counts, and higher levels of liver enzymes, higher total and indirect
bilirubin, and higher blood urea nitrogen when compared to the outpatient
group. Clinical symptoms of severe disease, including hematuria, hemolytic
anemia, and thrombocytopenia were only noted in the admitted group. Furthermore,
the presence of a palpable liver was more frequent in admitted patients. Nucleic
acid sequences of three genes from P. vivax, the 18S SSUrRNA Type A gene,
CSP gene and MSP-1 gene, were determined. Strains from test samples were compared
to each other, to the reference strains Salvador I and Belém and to
sequences retrieved from the Gene Bank. There were two main polymorphisms
in the 18S SSUrRNA Type A gene, a cytosine/thymidine polymorphism at residue
100 of the alignment and a thymidine (T)/adenine (A) polymorphism at residue
117. Ten of eleven (10/11) admitted patients were 117:T compared to 13/21
outpatients. This frequency difference was statistically significant (P<0.05).
The Salvador I strain was T at this position and the Belém strain was
A. In the CSP gene, we identified 15 unique sequences of the VK210 strain;
one sample had a mixed infection with P. vivax-like. The sources of variation
in the CSP gene included the numbers of repeat segments, alanine/aspartic
acid polymorphism at position five of a common repeat, and sporadic mutations.
Frequent synonymous substitutions of the common repeat occurred in codons
1, 2 and 7, while the mutations at codon 5 were always non-synonymous. Among
MSP-1 gene sequences, four recombination sites were distinguished between
the interspecies conserved regions 5 and 6. Recombination among progenitor
strains, closely related to the Salvador I and Belém strains, was the
main source of diversity among the strains. The second most significant form
of variation was in the polyglutamine region of strains with Belém-like
sequences in the central part of this gene segment. There was no clustering
of MSP-1 sequences in patients with severe disease. It was not possible to
demonstrate the evolutionary relationship among our test samples by tests
of phylogeny that incorporated sequence data for all three genes tested. The
factors that may have limited the power of a combined analysis included small
sample size and differences in the mechanisms and extent of variation among
the genes. The retrospective study was unable to demonstrate that a particular
strain of P. vivax was responsible for severe disease requiring hospitalization.
This is the first detailed description of the genetic diversity among the
P. vivax population in the Amazonas State of Brazil.