Degree Not Listed / Department of Diagnostic Medicine/Pathobiology / Roman Reddy R. Ganta / Anaplasmataceae family rickettsial bacteria are mostly vector-transmitted pathogens causing important diseases in several vertebrates, including humans, canines, and ruminants. Ehrlichia chaffeensis, a tick-transmitted intraphagosomal rickettsial bacterium, is the causative agent of human monocytic ehrlichiosis (HME). Little is known about how this and other related rickettsial organisms are able to reside and replicate within an acidified phagosome environment. Similarly, it is unclear how the infectious form of the bacterium maintains pH homeostasis in the extracellular milieu where the pH is about 7.35-7.45, before its infection to a naïve host cell. Sodium/cation: proton antiporters are integral membrane proteins reported from a wide range of species. They exchange sodium or other monovalent cations against protons across a plasma membrane in maintaining the cytoplasmic pH of a cell. We recently described a mutation within the Ech_0379 gene of E. chaffeensis that is predicted to encode for a Na+/H+ antiporter protein. The mutation caused the attenuated growth of the organism in vertebrate hosts, resulting in a reduced level of the bacterial presence in the circulation. In this study, we evaluated several antiporter protein genes of E. chaffeensis. Its genome contains 10 coding sequences encoding for polypeptides which may form at least six functional proteins. To define their function, a sodium sensitive Escherichia coli strain having a mutation in two of its three antiporter protein genes (EP432) is used to carry out the functional complementation assay with E. chaffeensis genes from their respective promoters. The EP432 strain has a growth defect during its replication in the presence of NaCl that can be restored with functional complementation. All six E. chaffeensis genes could complement the growth defect of EP432 under acidic pH, while Ech_0379 and Ech_0179 also complemented at basic pH. Ech_0179 complemented at neutral pH as well. The complementation of all genes at neutral and basic pHs, except Ech_0179 and Ech_0379, made EP432 E. coli strain be more sensitive to the presence of 200 mM NaCl. The channeling activity is verified independently by constructing a proteoliposome in vitro with the recombinant protein Ech_0379. The recombinant protein showed antiporter activity at all three pHs in the presence of 100 or 200 mM NaCl when assessed using the recombinant proteoliposome. This research is the first description of antiporter proteins of E. chaffeensis.

La Ehrlichiosis es una enfermedad zoonótica emergente transmitida a los humanos a través de la picadura de garrapatas infectadas, de gran importancia en países tropicales y sub-tropicales. En el presente estudio se determinó la seropositividad contra Ehrlichia chaffeensis en propietarios de perros con antecedentes de ehrlichiosis mediante la prueba de inmunofluorescencia indirecta (IFI) y su asociación con el sexo, edad, exposición a garrapatas y nivel de contacto con los perros mediante la prueba de Chi cuadrado. Se evaluaron 95 personas sin distinción de sexo, edad o condición socio económica cuyos perros tenían historia de ehrlichiosis reciente, los cuales llenaron un cuestionario con datos clínicos y epidemiológicos de importancia. El estudio se realizo entre Enero del 2009 y Diciembre del 2010. Los resultados del total de personas consideradas en el presente estudio indicaron que el 31.6% (30/95) presentaron anticuerpos contra Ehrlichia chaffeensis. Las variables edad y exposición a garrapatas resultaron estadísticamente significantes (p<0.05), frente a la seropositividad contra Ehrlichia chaffeensis. De los pacientes seropositivos, el 80% son personas menores de 40 años (24/30), mientras que el 20% son personas de 40 años a más (6/30). Asimismo, de los pacientes seropositivos el 93.3% estuvieron expuestos a garrapatas (28/30) mientras que el 6.7% no estuvieron expuestos (2/30). Estos hallazgos confirman la exposición a Ehrlichia chaffeensis en propietarios de perros con antecedentes de ehrlichiosis en Lima Metropolitana, evidenciando asociación de los resultados con los factores de riesgo evaluados, tales como edad y exposición a garrapatas.

Doctor of Philosophy / Department of Biology / Stephen Keith Chapes / Ehrlichia chaffeensis is an obligate, intracellular bacterium that causes human monocytic ehrlichiosis (HME). The bacteria are vectored by the Lone Star tick (Amblyomma americanum), which is found primarily in the Midwestern and Southeastern United States E. chaffeensis was first reported in 1986 and HME was designated a nationally reportable disease by the United States Centers for Disease Control in 1999. Ehrlichia grows in several mammalian cell lines, but NO consensus model for pathogenesis exists for arthropods or vertebrates. Moreover, the host genes required for intracellular growth of this bacteria are unknown. We first established that the bacteria could infect and replicate both in vitro and in vivo in Drosophila melanogaster S2 cells and adult flies, respectively. We performed microarrays on S2 cells, comparing host gene expression between permissive or non-permissive conditions for E. chaffeensis growth. A total of 210 permissive, exclusive and 83 non-permissive, exclusive genes were up-regulated greater than 1.5-fold above uninfected cells. We screened flies mutant for genes identified in our microarrays for their ability to support Ehrlichia replication. Five mutant stocks were resistant to infection with Ehrlichia (genes CG6479, separation anxiety, CG3044, CG6364, and CG6543). qRT-PCR confirmed that bacterial load was decreased in mutant flies compared to wild-type controls. In particular, gene CG6364 is predicted to have uridine kinase activity. Thus, the in vivo mutation of this gene putatively disrupts the nucleotide salvage pathway, causing a decrease in bacterial replication. To further test the function of gene CG6364 in bacterial replication, we obtained cyclopentenyl cytosine (CPEC) from the National Cancer Institute. CPEC is a cytidine triphosphate (CTP) inhibitor known to deplete CTP pools in various cancers and to exhibit antiviral activity. Consequently, it inhibits de novo nucleotide synthesis, but doesn’t affect the nucleotide salvage pathway. When S2 cells were treated with CPEC and infected with Ehrlichia, an increase in bacterial replication was confirmed by qRT-PCR. Furthermore, addition of cytosine to S2 cells also resulted in increased bacterial replication. Therefore the nucleotide salvage pathway through cytidine appears necessary for bacterial replication. Our approach has successfully identified host genes that contribute to the pathogenicity of E. chaffeensis in Drosophila.

Master of Science / Department of Biology / S. K. Chapes / Ehrlichia chaffeensis is a Gram-negative, obligatorily intracytoplasmic bacterium and the causative agent of a tick-borne disease, human monocytic ehrlichiosis. In vertebrates, E. chaffeensis exhibits tropism for monocytes /macrophages. However, no clear requirements for cell tropism have been defined in ticks. Previously, our group identified two host genes that control E. chaffeensis replication in vivo in Drosophila. We used these two genes, CG6364 and separation anxiety (san) to test the hypothesis that E. chaffeensis replicates in arthropod hemocytes. Using the UAS/GAL4 RNAi system, we generated F1 flies (RNAi flies) and confirmed ubiquitous-or tissue-specific reduction in the transcript levels of the targeted genes. When RNAi flies were screened for Ehrlichia infections, we found that when either CG6364 or san were specifically suppressed in the hemocytes or in the fat body E. chaffeensis failed to replicate or cause infection. Deletion of these genes in the eyes, wings or the salivary glands did not impact fly susceptibility or bacterial replication within these organs. Our data demonstrate that in Drosophila, E. chaffeensis replicates within the hemocytes, the insect homolog of mammalian macrophages, and in the fat body, the liver homolog of mammals. This study provides insights about replication sites of E. chaffeensis in arthropods.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Roman Reddy Ganta / Ehrlichia chaffeensis a tick transmitted Anaplasmataceae family pathogen responsible for human monocytic ehrlichiosis. Differential gene expression appears to be an important pathogen adaptation mechanism for its survival in dual hosts. One of the ways to test this hypothesis is by performing mutational analysis that aids in altering the expression of genes. Mutagenesis is also a useful tool to study the effects of a gene function in an organism. Focus of my research has been to prepare several modified Himar transposon mutagenesis constructs for their value in introducing mutations in E. chaffeensis genome. While the work is in progress, research team from our group used existing Himar transposon mutagenesis plasmids and was able to create mutations in E. chaffeensis. Multiple mutations were identified by Southern blot analysis. I redirected my research efforts towards mapping the genomic insertion sites by performing the semi-random two step PCR (ST-PCR) method, followed by DNA sequence analysis. In this method, the first PCR is performed with genomic DNA as the template with a primer specific to the insertion segment and the second primer containing an anchored degenerate sequence segment. The product from the first PCR is used in the second PCR with nested transposon insertion primer and a primer designed to bind to the known sequence portion of degenerate primer segment. This method aided in identifying the genomic locations of four E. chaffeensis mutants and also was valuable in confirming four other sites mapped previously by the rescue cloning method. This is the first mutational analysis study in the genome of an Ehrlichia species. Mapping the genomic transposon insertion sites is the first critical step needed for the continued research to define the importance of the mutations in understanding the pathogenesis caused by the organism.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Roman Reddy R. Ganta / Ehrlichia chaffeensis is a Gram negative, rickettsial organism responsible for human monocytic ehrlichiosis, an emerging disease in people. E. chaffeensis infection to a vertebrate host occurs when the pathogen is inoculated by an infected tick, Amblyomma americanum. White-tailed deer is a reservoir host for this pathogen. The strategies employed by E. chaffeensis in support of its dual host adaptation and persistence are not clear. One of the possible mechanisms by which the pathogen adapts and persists, is by altering its gene expression in response to its host cell environments. Recently, we reported that E. chaffeensis protein expression including that from a 28 kDa outer membrane protein multigene locus (p28-Omp), is influenced by macrophage and tick cell environments. E. chaffeensis expresses p28-Omp gene 14 product predominantly when it is grown in tick cells and p28-Omp gene 19 protein in macrophages. We hypothesize that E. chaffeensis achieves its host-specific gene expression by employing transcriptional regulation by sensing the host cell signals. In support of this hypothesis, transcriptional analysis of 14 and 19 genes was performed utilizing several RNA analysis methods. The results supported our hypothesis that the gene regulation occurs at mRNA level in a host cell-specific manner. This analysis also identified transcription start sites and located putative promoters for the p28-Omp genes 14 and 19. Promoter regions of genes 14 and 19 were mapped to identify gene-specific differences, RNA polymerase binding sequences and the putative regulatory elements that may influence the promoter activities. Electrophoretic mobility shift assays revealed interaction of E. chaffeensis proteins with gene 14 and 19 promoters. Several E. chaffeensis putative regulatory proteins were expressed as recombinants and their effects on a p28-Omp gene promoter activity were evaluated. In summary, we demonstrated that the differences in the E. chaffeensis p28-Omp genes 14 and 19 are the result of their regulation at transcriptional level in response to the host cell environment. We also identified RNA polymerase binding regions and several DNA sequences that influenced promoter activity. This is the first description of a transcriptional machinery of E. chaffeensis. The data from these studies provide important insights about molecular mechanisms of gene regulation in E. chaffeensis.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Roman Reddy R. Ganta / Ehrlichia chaffeensis is an emerging tick-borne rickettsial pathogen that causes infection in people and several vertebrate animals. One of the striking features of E. chaffeensis infection is the prolonged persistence in its vertebrate and tick hosts. The mechanism of persistent infection and the reasons for the host immune system failure to clear the infection are not well understood. One hypothesis is that differential gene expression serves as an important adaptive mechanism used by E. chaffeensis in support of its continued survival in both tick and vertebrate hosts. One way to test this hypothesis is by performing mutational analysis. However, the methods for introducing mutations in this pathogen have not yet been documented and are challenging, possibly due to its obligate, intraphagosomal growth requirement. Recently, a novel gene mutation method called ‘TargeTron Gene Knockout System’ that is based on the modified group II intron insertion strategy has been developed. This method appears to be effective in creating mutations in a wide range of gram positive and gram negative bacterial organisms. The group II intron can be programmed for insertion into virtually any desired DNA target with possibly high frequency and specificity. In this study, I focus on creating mutations in E. chaffeensis using the TargeTron gene knockout system. I prepared modified group II intron constructs retargeting for insertion into three E. chaffeensis genes: Ech_0126 (a transcriptionally silent gene), macrophage-specific expressed gene (p28-Omp 19, Ech_1143) and tick cell-specific expressed gene (p28-Omp 14, Ech_1136). In support of driving the expression of the modified group II introns in E. chaffeensis, the pathogen- specific high-expressing gene promoter (tuf) was inserted upstream to the transcription start site. In addition, a chloramphenicol acetyltransferase gene with E. chaffeensis rpsl promoter was introduced for use as a selection marker. The constructs were then evaluated by transforming into E. chaffeensis. Transformants with mutations, introduced in two of the three genes (Ech_0126 and Ech_1143), were identified by PCR and Southern blot methods. Although the mutants are detectable for up to 48 hours, establishment of stable transformants remains to be challenging. The outcomes of this project will have important implications in defining the pathogenesis of E. chaffeensis, particularly to assess the differences in the organism in tick and vertebrate hosts.

Human Monocytic Ehrlichiosis (HME) is an emerging disease first described in 1987 and is transmitted by the bite of Amblyomma americanum. Over the past 10 years, the CDC has documented increasing ehrlichiosis case reports nationwide. Our study site is a golf-oriented retirement community located in the Cumberland Plateau of Tennessee. In 1993, four men at the study site had symptoms consistent with HME which prompted a CDC outbreak investigation and led community managers to mitigate ticks feeding on deer. The objectives of this study were to measure the efficacy of current tick mitigation attempts, to determine the level of infection and composition of tick-borne disease in the study area, and to assess which wildlife species are potentially acting as reservoirs for disease. Ticks were sampled in the community at eight sites of ‘4-poster’ acaricide applicator utilization and at seven untreated sites. Close to the ‘4-poster’ devices, larval, nymphal, and adult tick abundances were reduced by 90%, 68% and 49% respectively (larval p<0.001, nymphal p<0.001, adult p=0.005) relative to the untreated areas. We extracted DNA from A. americanum ticks collected at the treatment and non-treatment sites and tested for Ehrlichia spp. infections. Of 253 adult and nymphal A. americanum tested, we found 1.2% to be positive for Ehrlichia chaffeensis, 4.7% positive for Ehrlichia ewingii, and 1.6% positive for Panola Mountain Ehrlichia; in combination this prevalence is similar to that reported in other Ehrlichia-endemic areas of the eastern U.S.. We also performed blood meal analysis on DNA from A. americanum ticks and the results suggest that the most significant reservoir hosts for Ehrlichia spp. are white-tailed deer, turkeys, grey squirrels, and Passeriformes. We conclude that while the ‘4-poster’ acaricide applicators reduce the number of ticks close to treatment, at the density at which they are currently being used (8 applicators per 52.6 km2, average distance between applicators = 6.6km) they will have no large-scale effect on the community’s tick population. In order to accomplish area-wide reduction of A.americanum and Ehrlichia spp. in this locale, community managers should develop an integrated management strategy that utilizes other techniques in addition to ‘4-poster’ devices.

Amblyomma americanum is a hard tick species found in southern Indiana. Once a notorious pest to humans and livestock, A. americanum has now taken on a role as vector to pathogenic organisms. This study aimed to detect and quantify three microbes in A. americanum: Rickettsia amblyommii, Ehrlichia chaffeensis, and Borrelia lonestari. A primary objective of this study was to determine microbial interaction within a single A. americanum tick through quantification of each microbe within a co-infected tick. A second objective was to determine the density of R. amblyommii within the salivary glands of A. americanum ticks. Infection rates were 44%, 1%, and 0% for R. amblyommii, E. chaffeensis, and B. lonestari respectively. This study found no co-infected ticks, therefore no microbial interaction was determined. This study also found multiple drawbacks with utilizing quantitative real-time PCR to determine the density of R. amblyommii within the salivary glands of A. americanum ticks. / Department of Biology

Human monocytotropic ehrlichiosis (HME) is a tick-borne infectious disease caused by the bacterium Ehrlichia chaffeensis and transmitted by the lone star tick, Amblyomma americanum. The disease was recognized in Indiana for the first time in 1994. Since 1999, 11 cases have been confirmed in Indiana and two additional cases are under investigation. In the past five years, the cases have been reported from Crawford, Harrison, Warrick, Martin, Perry, Spencer, and Madison counties.A total of 2765 adult Amblyomma americanum ticks were collected from eight counties in southern Indiana during two field trips in May 2000. Ticks were pooled and examined for the presence of Ehrlichia chaffeensis using nested PCR with primers HE1 and HE3, specific for the 16S rRNA gene of the pathogen. Ninety-six pools of A. americanum specimens tested positive for E. chaffeensis DNA. This represented a minimum infection rate (MIR) of 3.5%.To identify different genetic forms (strains) of E. chaffeensis, the positive tick pools were probed for the Variable Length PCR Target (VLPT) gene of E. chaffeensis. The data were used to develop a geographic map of the distribution of the different strains of the pathogen. Overall, nine different genetic variants (91HE17, Arkansas, Jax, Liberty, Osceola, Sapulpa, St. Vincent, Wakulla, West Paces) of E. chaffeensis were identified from pools of ticks collected in four counties (Harrison, Perry, Pike, Warrick). All samples positive for the 16S rRNA were also positive for the VLPT gene.E. chaffeensis isolates are polymorphic in the number of repetitive sequences within the genes encoding the VLPT, and the isolates obtained illustrate this phenomenon. The high concordance rate between the 16S rRNA and the VLPT gene reveals that the VLPT gene is a very sensitive tool for detecting E. chaffeensis in the lone star ticks. We found no clear correlation between geographic distribution of different genetic variants of E. chaffeensis and the genetic polymorphism of the VLPT gene. Further study with a relatively larger sample size from a wider geographical area might be able to detect such a pattern. / Department of Physiology and Health Science