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The spirochete Borrelia hermsii avoids the immune response of its mammalian host through multiphasic antigenic variation. Serotype specificity is determined by Variable major proteins (Vmp), in the outer membrane. Through a non reciprocal recombination between linear plasmids, a formerly silent vmp gene replaces another vmp gene at a telomeric expression locus downstream from a common expression site. B. hermsii before and after the switch from serotype 7 to serotype 21, was examined in detail. The nucleotide sequence of the vmp7 and vmp21 genes and flanking regions was determined. The vmp7 and vmp21 are 77% identical in their coding sequence, and the deduced translation products are 63% identical. No antigenic cross reactivity is observed between Vmp7 and Vmp21. This suggests a folding of the proteins in which the similar regions are buried, and not exposed when it is presented at the bacterial surface. Vmp7 and Vmp21 have consensus sequences of prokaryotic lipoproteins and are processed as such when expressed in E. coli. The 5' regions of silent and expressed vmp7 and vmp21 were compared. Silent and active vmp7 and vmp21 genes shared a block of homologous sequence at their 5' ends. Sequences upstream of silent vmp7 and vmp21 genes lacked a promoter and differed substantially from each other. In this antigenic switch a vmp gene was activated by a recombination event which placed it downstream of a promoter. The vmp gene promoter is preceded by a poly(dT dA) ran and three imperfectlyrepeated elements of 2 kb. Each of the 2 kb repeats contains inverted repeats of approximately 0.2 kb at their termini. There is no evidence of the presence of similar elements elsewhere in the genome of B. hermsii. One or more of these elements may stimulate vmp gene switch or expression. The African relapsing fever species Borrelia crocidurae and the American species B. hermsii display many similarities. In both species the vmp genes are localised to linear plasmids, and the vmp genes are activated on the transcriptional level. The nucleotide sequence of their expression sites, however, are not related. Still, the possibility that the switch is mechanistically similar in B. crocidurae and B. hermsii, cannot be ruled out. The binding of B. crocidurae causes aggregation of erythrocytes around the spirochete. The aggregation is reminiscent of the erythrocyte rosetting seen in malarial infections. The erythrocytes at the B. crocidurae surface may protect them from clearance by the host. Thus, the rosetting may constitute an additional mechanism in B. crocidurae for the evasion of the immune reaction. / <p>Diss. (sammanfattning) Umeå : Umeå universitet, 1994, härtill 5 uppsatser.</p> / digitalisering@umu

This thesis investigates antigenic variation and its evolution in Plasmodium falciparum, the cause of the most deadly form of human malaria. Antigenic variation is a strategy for evading immunity by switching between antigenic variants during infection. In P. falciparum, such variable antigens confer different binding phenotypes that may affect parasite survival and have also been linked to pathology. Here, a new statistical method is described for determining the switching patterns that underlie antigenic variation. This method is then applied to experimental data to yield a full description of an antigenic switching network in P. falciparum. In light of the findings, theoretical modelling is used to show how immune selection and binding phenotypes may have contributed to the evolution of antigenic repertoire structure, expression order and virulence. Related models are also used to investigate parasite population diversity, providing possible explanations for observations reported here and elsewhere, with implications for vaccine design. Together, these chapters advance understanding of P. falciparum immune evasion and how it relates to pathology. This work further reinforces the role of host immunity in shaping pathogen population diversity at multiple levels.

A major virulence factor of the human malaria parasite Plasmodium falciparum is Plasmodium falciparum erythrocyte membrane protein 1(PfEMP-1). This protein is inserted into the erythrocyte membrane, giving cytoadherence properties. A family of genes called var, located sub-telomerically and in chromosome central clusters encode this protein. Var genes are expressed in a mutually exclusive manner, how this is controlled is unclear. A non-coding RNA (ncRNA) termed the GC-rich element (GRE) had been identified that is only located at the central clusters and is transcribed throughout the parasite lifecycle. A screen of the P. falciparum genome for novel ncRNAs identified ncRNAs from known classes. Novel transcripts were identified, but none in the proximity of var genes. We have investigated the role of the GRE in var gene regulation. A set of qRT-PCR primers have been designed and tested to follow var gene expression in the HB3 isolate, these are not cross-reactive with a published set for the 3D7 isolate. Alterations were made to the 3D7 set to remove cross-reactivity with HB3. Var gene expression was studied in 31 HB3 clones and progeny of the 3D7xHB3 genetic cross. Following var switching over five months in eleven HB3 clones showed that all of the clones ended up expressing var genes from the same central cluster on chromosome 4. GRE Transcription in these clones is linked to a specific class of var gene. Transcription from a single GRE locus occurs only when a var gene of the central UpsC class is expressed from the same cluster. Expression of other classes of var gene gives multiple transcripts from different GRE loci. Investigations into the in vitro binding properties of the GRE revealed an RNA:protein complex that can be resolved by electrophoresis. Proteomic analysis of the complex revealed predominantly ribosome proteins and translation factors.

Protozoan parasites of the genus Plasmodium are the causative agent of malaria. The most severe form of human malaria is caused by P. falciparum, responsible for approximately three quarters of a million deaths each year. One major problem in the treatment of malaria is resistance to existing chemotherapies. Consequently, there is an urgent need to identify and validate novel drug targets. A possible recently identified drug target is the PfNitA protein of P. falciparum which contains orthologues in other Plasmodium species but is absent from humans. The gene is annotated as a putative formate-nitrite transporter and orthologues are found in a range of prokaryotes as well as the lower eukaryotes algae and fungi. To determine the biological function of the protein, pfnita was expressed in Escherichia coli strains lacking the endogenous formate and nitrite transporters. In order to analyse the essentiality of the gene a reverse genetics approach was taken and the data discussed. Results indicate that the PfNitA protein is located in the plasma membrane and digestive vacuole of intraerythrocytic parasites suggesting a role in the uptake or excretion of metabolites. A second complexity with regard to treatment is the lack of a vaccine. A problem in crating a vaccine is antigenic variation. The PIR family of proteins contain a so-called hypervariable domain that has led to the suggestion that the family may play a role in antigenic variation. The objective of the work carried out in this thesis was to investigate the topology and structure of the PcCir2 protein of Plasmodium chabaudi, using E. coli as the expression host. The topology of Cir2 has been examined by means of reporter fusions and overexpression/purification studies undertaken towards crystallisation. As the PcCir2 amino acid sequence does not show significant homology to other proteins, structural data may provide insights into potential functional or binding domains.

vÃrus sincicial respiratÃrio humano (VSRh) à o agente viral mais freqÃentemente relacionado a infecÃÃes do trato respiratÃrio inferior em crianÃas menores de dois anos de idade. O VSRh à caracterizado antigenicamente em dois grupos: A e B, e cada grupo apresenta vÃrios subgrupos. A glicoproteÃna G à a principal responsÃvel pela a variaÃÃo antigÃnica inter e intragrupos desse vÃrus. Os objetivos desse estudo foram caracterizar os perÃodos epidÃmicos e a diversidade antigÃnica e genÃmica dos VSRh circulantes em Fortaleza, Cearà â Brasil, durante cinco perÃodos epidÃmicos consecutivos (2004-2008). A imunofluorescÃncia indireta (IFI) foi utilizada para a triagem de VSRh e de todos os vÃrus analisados e para a caracterizaÃÃo antigÃnica dos VSRh. A RT-nested-PCR seguida do seqÃenciamento parcial do gene G foi utilizada para a caracterizaÃÃo gÃnomica dos VSRh. O VSRh foi detectado em 456 das 2885 (15.8%) amostras. O pico dos perÃodos epidÃmicos de VSRh ocorreu nos meses de marÃo a maio relacionado à ocorrÃncia de chuvas. Um total de 282 VSRh (62,8%) foram caracterizados antigenicamente por IFI, sendo 170 VSRhA (60,3%) e 112 VSRhB (39,7%). Ambos os grupos circularam durante todo o perÃodo analisado sendo observado o predomÃnio de A em todos os anos. Um total de 250 VSRh (54,8%) foi submetido à RT-nested-PCR com amplificaÃÃo de 133 e seqÃenciamento de 86. A caracterizaÃÃo genÃmica dos VSRh identificou os subgrupos GA2 e GA5 para o VSRhA e os subgrupos GB3 e BA para o VSRhB. Esses quatro subgrupos co-circularam durante o ano de 2006. Nos anos de 2004, 2005 e 2007 verificou-se a presenÃa dos dois subgrupos de VSRhA. Em 2008 somente o GA2 circulou. Em 2004, 2007 e 2008 somente o subgrupo BA esteve presente. Em 2005 somente o GB3 circulou. Os VSRhA apresentaram uma maior variabilidade nas seqÃÃncias nucleotÃdicas, indicando uma possÃvel pressÃo seletiva positiva. Houve variaÃÃes no Ãnicio, fim e duraÃÃo de cada perÃodo epidÃmico de VSRh, assim como na circulaÃÃo de grupos e subgrupos. / The human respiratory syncytial virus (HRSV) is the major agent of lower respiratory tract in children under two years old. HRSV is characterized antigenically into two groups: A and B, and each group has several subgroups. Glycoprotein G is primarily responsible for the antigenic variation between and within groups of viruses. The aims of this study were to characterize the epidemic periods and the antigenic and genomic diversity of circulating HRSV in Fortaleza, Cearà - Brazil, for five consecutive epidemic periods (2004-2008). The screening of positive samples to HRSV and other viruses analyzed, as the antigenic characterization of HRSV was carried out by indirect immunofluorescence. RT-nested-PCR followed by partial sequencing of the gene G was used for genomic characterization of HRSV. The HRSV was detected in 456 of 2885 samples (15.8%). The peak of the epidemic periods of HRSV occurred from March to May related to rainfall. A total of 282 HRSV (62.8%) were characterized antigenically, with 170 HRSVA (60.3%) and 112 HRSVB (39.7%). Both groups circulated throughout the period analyzed with a predominance of HRSVA in all years of study. A total of 250 HRSV (54.8%) were submitted to RT-nested-PCR with amplification of 133 and sequencing of 86. The genomic characterization of HRSV identified subgroups GA2 and GA5 for HRSVA and subgroups GB3 and BA for HRSVB. In the years 2004, 2005 and 2007 both subgroups of HRSVA circulated. In 2008 only GA2 circulated. In 2004, 2007 and 2008 only the subgroup BA was present. In 2005 only the GB3 circulated. The HRSV A showed a higher variability in nucleotide sequences, indicating a possible positive selective pressure. There were variations in the beginning, end and duration of each epidemic period of HRSV, as well as in the occurrence of groups and subgroups.

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 89-111).

The diplomonads are a diverse group of eukaryotic flagellates found in microaerophilic and anaerobic environments. The most studied diplomonad is the intestinal parasite Giardia intestinalis, which infects a variety of mammals and cause diarrheal disease. Less is known about Spironucleus salmonicida, a parasite of salmonid fish, known to cause systemic infections with high mortality. We created a transfection system for S. salmonicida to study cellular functions and virulence in detail (Paper I). The system was applied to explore the mitochondrion-related organelle (MRO) in S. salmonicida. We showed that S. salmonicida possesses a hydrogenosome (Paper II) with a higher metabolic capacity than the corresponding MRO of Giardia, the mitosome. Evolutionary analysis of key hydrogenosomal proteins showed ancient origin, indicating their presence in the ancestral diplomonad and subsequent loss in Giardia. Annexins are of evolutionary interest since these proteins are found across all kingdoms. Annexin-like proteins are intriguingly expanded into multigene families in Giardia and Spironucleus. The annexins of S. salmonicida were characterized (Paper III) with distinct localizations to various cellular structures, including a putative adhesion structure anterior in the cell. The disease-causing Giardia trophozoites differentiate into infectious cysts, a process essential for transmission and virulence of the parasite. Cysts are often spread via contaminated water and exposed to environmental stressors, such as UV irradiation. We studied the survival and transcriptional response to this stress factor (Paper IV) and results showed the importance of active DNA replication machinery for parasite survival after DNA damage. In addition, we studied transcriptional changes along the trajectory of encystation (Paper V), which revealed a coordinated cascade of gene regulation. This was observed for the entire transcriptome as well as putative regulators. Large transcriptional changes appeared late in the process with the majority of differentially regulated genes encoding hypothetical proteins. We studied the localizations of several of these to gain information of their possible function. To conclude, the diplomonads are complex eukaryotic microbes with cellular processes adjusted to match their life styles. The work in this thesis has provided insight of their adaptations, differences and similarities, but also new interesting leads for future studies of diplomonad biology and virulence.

Thesis (M.S.)--University of Texas Health Science Center at Houston, School of Public Health, 2007. / Source: Masters Abstracts International, Volume: 45-05, page: 2360. Adviser: Cynthia L. Chappell.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 147-179).