The immunogenicity and protective efficacy of a DNA vaccine encoding a truncated groEL heat shock gene (pcDNA3/groEL) from Burkholderia pseudomallei was evaluated in vaccinated BALB/c mice infected with B. pseudomallei or B. cenocepacia. After vaccination, the levels of anti-GroEL total IgG and IgG2a were increased in mouse sera. The clonal expansion of the spleen cells increased, and the GroEL protein induced IFN-£^ production by spleen cells. The anti-GroEL antibody-mediated opsonic killing effect was not able to eliminate the growth of B. pseudomallei but was able to eliminate the growth of B. cenocepacia. After intravenous challenge of the vaccinated Balb/c mice with B. pseudomallei, the number of bacteria colonizing the in liver and/or spleen was not reduced. Over 50% of vaccinated mice infected with B. pseudomallei died within 7 days post-infection. By contrast, the bacterial loads in organs were significantly reduced if the vaccinated mice were infected with B. cenocepacia. All of vaccinated mice were alive 7 days post-infection. Liver damage, as assessed by histological observation, and abnormalities in the levels of liver enzymes rapidly resolved in vaccinated mice. We suggest that B. pseudomallei groEL plasmid DNA immunization of Balb/c mice induces a Th1-type immune response and provides cross-protection against B. cenocepacia but not against B. pseudomallei infection.

The causative agents of plague (Y. pestis), glanders (B. mallei) and melioidosis (B. pseudomallei) are included in critical agents of bioterrorisim. They belong to the most intensively studied agents during cold war, specially in the former Soviet Union (FSU). Mostly what is known about these agents, particularly (Y. pestis ) is not available in English language publications. Many of the studies are written in Russian language and published in Russian scientific journals. Thus, the work is designed to evaluate, published and unpublished Russian language written data obtained, in comparisions to the current state of global knowledge on the pathogens in concern.

O primeiro surto de melioidose no Brasil ocorreu quando quatro adolescentes apresentaram forma grave da doenÃa, com pneumonia e sepse, numa pequena comunidade rural do Nordeste do Brasil, em fevereiro de 2003. Burkolderia pseudomallei foi identificada, em cultura de sangue em um caso, inicialmente por mÃtodos convencionais e confirmado por amplificaÃÃo de Ãcido nuclÃico (PCR). O diagnÃstico foi presumÃvel nos outros trÃs casos. A investigaÃÃo epidemiolÃgica preliminar identificou os possÃveis fatores contribuintes: inÃcio dos sintomas durante perÃodo chuvoso,exposiÃÃo dos casos a solo e Ãgua em atividades de lazer, presenÃa de barragem de um rio prÃximo ao domicÃlio, Ãrea ao redor do domicÃlio exposta a grande chuva dois dias antes dos sintomas e casa com piso de terra. O fato das crianÃas adoecerem no mesmo dia aponta para uma possÃvel exposiÃÃo comum e simultÃnea. A barragem foi identificada como o local mais provÃvel da contaminaÃÃo e o mecanismo de transmissÃo possivelmente pode ter ocorrido por aspiraÃÃo, inalaÃÃo ou ingestÃo de Ãgua da barragem. O recente surto levanta o questionamento sobre a existÃncia da doenÃa no Brasil e mostra a necessidade de estudo ambiental detalhado para compreender sua ocorrÃncia. / The first melioidosis outbreak in Brazil happened when four adolescents presented a severe case of the disease, with pneumonia and sepsis, in a small rural community in the Northeast of Brazil, in February 2003. Burkolderia pseudomallei were identified through blood culture in one case, firstly using conventional methods and then confirmed by PCR. The diagnosis was presumptive in the other three cases. The preliminary epidemiological investigation identified the possible contributing factors: onset of the symptoms during the rainy season, exposure of the cases to the soil and water in leisure activities, presence of a river dam close to the residence, the area around the house had been exposed to heavy rain two days before the symptoms, and the house had dirt floor. The fact that the children got sick on the same day, points to a possible common and simultaneous exposure. The dam was identified as the most probable place of contamination, and the transmission mechanism might have happened through aspiration, inhalation or ingestion of water from the dam. The recent outbreak rises the important questioning about the existence of the disease in Brazil, and shows the need of a detailed environmental study to understand its occurrence.

Melioidosis, caused by the environmental Gram negative bacillus Burkholderia pseudomallei, is an emerging infectious disease affecting both animals and humans. B. pseudomallei has the ability to enter the host cell and escape from the phagosome. Once in the cytoplasm, the pathogen proliferates and expresses a virulence-associated protein known as BimA which polymerises cellular actin at the pole of the bacterium to promote its movement inter- and intracellularly, a process known as actin-based motility. This actin-based motility is also used as a strategy to evade host immune responses and survive intracellularly. In the first part of the thesis, we demonstrate that a B. pseudomallei ΔbimA mutant displays impaired intracellular survival compared to the isogenic parent strain in BALB/C bone-marrow derived macrophages (BMDMs), notably at later time points post-infection. Macrophages are the key innate immune cells that control B. pseudomallei in vivo and in vitro, and BALB/C mice provide an excellent model of acute human melioidosis. We also have determined that in BMDMs, the ΔbimA mutant is able to escape from the phagosome and enters the cytosol where it is unable to form actin tails. We used targeted, hypothesis-driven experiments to identify potential cell-autonomous innate mechanism/s of killing the mutant. First, we speculated that BimA mediates escape from autophagy. However our studies, including LC3-conversion assays, and bacterial co-localisation studies, failed to demonstrate a role for autophagy in clearance of the ΔbimA mutant from infected BMDMs. In the second part of this thesis, we investigated the role of Toll-like Receptors (TLR) in recognition and elimination of B. pseudomallei. MyD88 (Myeloid differentiation primary-response gene 88) and TRIF (TIR-domain-containing adaptor protein inducing IFNβ) are the main adaptor proteins involved in TLR signalling. We utilised the gene silencing technique using short interfering RNAs (siRNAs) to knockdown MyD88 transcript, and in a separate experiment used MyD88- or TRIF-blocking peptides. In addition, we investigated the involvement of canonical and non-canonical inflammasome pathways in cell-autonomous immunity of the BMDMs. However, none of these pathways were shown to be involved in clearance of the ΔbimA mutant from infected BMDMs. Finally we took an unbiased approach by microarray to characterise the global host transcriptome in BALB/C BMDMs upon B. pseudomallei infection, and to identify specific responses to the ΔbimA mutant. Analyses performed at the gene level revealed that several interferon signalling-related pathways are activated in cells infected with either the WT or ΔbimA mutant strains. A number of other pro-inflammatory mediators that are commonly seen in general inflammatory infections, such as IL-1α, IL-1β, IL-12β, and IL-6, were also upregulated. Interestingly, the cytoplasmic RNA sensors RIG-1 and MDA-5, thought primarily to be involved in the detection of RNA viruses, were also induced upon B. pseudomallei infection. Very few pathways were associated with a specific macrophage response to the ΔbimA mutant, indicating that an as yet undescribed pathway may play a role in sensing and eliminating the ΔbimA mutant. We conclude that actin-based motility mediates escape of B. pseudomallei from macrophage intracellular killing through a novel pathway which has yet to be unravelled.

Burkholderia pseudomallei (Bp) is a major public health concern in the endemic regions of southeast Asia and northern Australia, yet the organism has a worldwide distribution and cases are likely under-reported. In northeast Thailand the mortality rate associated with Bp infection is over 40%. The inherent resistance of Bp to multiple antibiotics impairs treatment, and relapse is seen in more than 25% of survivors. Beyond its public health significance, Bp is considered a potential biological warfare agent by the U.S. DHHS and was recently listed as a Tier 1 select agent. Despite enhanced research and vaccine efforts, traditional vaccine strategies employing attenuated bacterial strains, recombinant proteins, or purified polysaccharides have failed to elicit complete protection against aerosol challenge with Bp. We have previously shown that immunization with outer membrane vesicles (OMVs) derived from Bp can protect mice from lethal melioidosis. In this work we characterize the interactions of OMVs with antigen presenting cells in order to elucidate innate immune responses to the OMV vaccine. Vaccine-mediated antibody responses and protective efficacy were characterized in BALB/c mice. We also tested the safety and immunogenicity of the OMV vaccine in non-human primates (NHP). We show that Bp OMVs interact with dendritic cells and macrophages and are internalized by these antigen presenting cells (APCs).Internalization is dependent on actin polymerization and cholesterol present in APC membranes. OMVs also upregulate MHC class I and II on APCs, as well as promote the production of pro-inflammatory cytokines in a TLR2/4 dependent manner. Immunization of mice with Bp OMVs by the s.c. and i.m. routes induced the production of OMV-specific IgM and IgG and significantly protected mice against aerosol challenge. Addition of alum and MPL did not significantly change the antibody profiles of immunized mice and did not significantly enhance vaccine mediated protection. OMVs were well tolerated in a large animal NHP model. There were no adverse clinical reactions, and NHPs mounted significantly increased levels of OMV-specific IgG and OMV specific CD4+ T cell responses. These results suggest that Bp OMVs can stimulate innate and adaptive immune responses and may represent a safe and efficacious vaccine against melioidosis / acase@tulane.edu

Thematic Paper (M.Sc. (Clinical Tropical Medicine))--Mahidol University, 2006. / LICL has E-Thesis 0011 ; please contact computer services. LIRV has E-Thesis 0011 ; please contact circulation services.

Burkholderia pseudomallei is a Gram-negative intracellular bacterium that causes melioidosis, a serious disease of humans and animals in tropical countries. This pathogen can subvert the host cell actin machinery by a process known as actibased motility, for promoting its movement both within and between cells. The bacterial factor required for this process is known as BimA (Burkholderia intracellular motility A). Intracytoplasmic bacterial pathogens use distinct mechanisms for actin-based motility, hijacking host cytoskeletal proteins for their benefit. However, the molecular mechanism by which BimA subverts the cellular actin machinery is ill-defined. From an affinity approach coupled with mass spectrometry to identify cellular proteins recruited to BimA-expressing bacteria under conditions that promote actin polymerisation, a group of cellular proteins that are recruited to the B. pseudomallei surface in a BimA-dependent manner was identified. A subset of these proteins was independently validated with specific antisera including IQ motif containing GTPase activating protein 1 (IQGAP1). IQGAP1 is a ubiquitous scaffold protein that integrates several key cellular signalling pathways including those involved in actin dynamics. Previous studies demonstrated IQGAP1 was targeted by pathogens to regulate the actin cytoskeleton, for example promoting Salmonella invasion into epithelial cells or supporting cell attachment and pedestal formation of Enteropathogenic Escherichia coli. The aim of this study is to explore the roles of IQGAP1 in the intracellular life of B. pseudomallei. This present study revealed that IQGAP1 was recruited to B. pseudomallei actin tails in infected HeLa cells. This protein has not previously been associated with actin-based motility of other intracellular pathogens. To examine the effect on actibased motility of B. pseudomallei, siRNA was utilised to knockdown IQGAP1 in HeLa cells. After optimisation of siRNA transfection, IQGAP1 expression in HeLa cells was suppressed by approximately 70% as assessed by IQGAP1 immunoblotting. The siIQGAP1 knockdown cells were infected with B. pseudomallei. The bacteria could still form actin tails in the knockdown cells, however, the data showed a statistically significant increase in overall tail length with a concomitant decrease in actin density, compared with the tails formed by B. pseudomallei in control cells. Actin-based motility is essential in the life cycle of several cytoplasmic bacterial pathogens, particularly in cell-to- cell spread. After entry into the host cell cytosol, B. pseudomallei polymerises actin in a BimA-dependent manner and propels itself within and between cells. This is accompanied by cell fusion which generates multi-nucleated giant cells (MNGCs), a process mediated by a Type 6 Secretion System that is co-regulated with BimA. To gain an understanding of the impact of IQGAP1 on the intracellular life of B. pseudomallei, IQGAP1 was successfully knocked-out from HeLa cells using CRISPR-Cas9 technique. Interestingly, Burkholderia invasion was not affected in HeLa cells lacking IQGAP1. However, the bacteria showed a defect in intracellular survival in IQGAP1 knockout cells that was revealed after 6 hours post-infection. Moreover, there was no difference in the proportion of bacteria associated with actin in the control and knockout cells at 16 hours post-infection, although the bacteria formed longer actin tails in control cells with similar actin density. Consequently, the number of MNGCs decreased dramatically in the cells lacking IQGAP1, which was indicated by the absence of plaque formation. Another element of this study was to determine whether BimA and IQGAP1 are direct interacting partners. Using either an in vitro pulldown assay or in vivo yeast two-hybrid system, a direct interaction between these proteins could not be detected. It is, therefore, likely that IQGAP1 is recruited to B. pseudomallei actin tails through its intrinsic ability to interact with F-actin. Despite the lack of a direct interaction between these two proteins, an N-terminal IQGAP1 fragment significantly augmented BimA-mediated actin polymerisation in vitro. Taken together, this study provides the first evidence of the presence of IQGAP1 in B. pseudomallei actin tails and presents the importance of IQGAP1 in actin-based motility and intracellular life of this bacterium. Understanding the mechanism of B. pseudomallei actin-based motility is useful to gain insights into host cell actin dynamics and its role in pathogenesis. Targeting host cellular proteins that are required for the intracellular life of pathogens are a topical area of research, with the potential to be useful alternatives to classic antibiotic therapy. Indeed, IQGAP1 could be a potential novel therapeutic target to develop drugs for treating B. pseudomallei infection.

A thesis by Marc Ashley Bayliss entitled ‘Virus-like particles as a novel platform for delivery of protective Burkholderia antigens’ and submitted to the University of Exeter for the degree of Doctor of Philosophy. There is currently no licensed vaccine available for the global tropical pathogen Burkholderia pseudomallei which is the causative agent of melioidosis and a potential bio-threat agent. The capsule polysaccharide (CPS) expressed by B. pseudomallei has been shown to offer some protection against bacterial challenge. Polysaccharide immunogenicity can be enhanced by conjugation to a carrier protein and several licensed vaccines utilise this technology. Virus-like particles (VLPs) are non-infectious, non-replicating, viral proteins that self-assemble into viral structures and are in several licensed vaccines as primary antigens. VLPs are also effective delivery platforms for foreign antigens by genetic insertion or chemical conjugation. iQur, a collaborator on this project, has developed Tandem CoreTM that consists of two genetically linked hepatitis B core proteins that allow insertion of large proteins into each core whilst remaining assembly competent. The aim of this thesis was to assess the protective efficacy of Tandem CoreTM VLPs chemically conjugated to CPS and Tandem CoreTM Burkholderia protein fusion constructs. This involved three objectives; reduce the cost of CPS extraction; identify immunogenic Burkholderia proteins; and test candidate vaccine efficacy in an animal model of acute melioidosis against B. pseudomallei challenge. To reduce the cost of extraction, CPS was purified from B. thailandensis strain E555 and bacterial culture CPS concentration optimised which first required development of a quantitative ELISA. Immunogenic Burkholderia proteins were identified from the literature but Tandem CoreTM fusion constructs containing these proteins were not assembly competent. The Burkholderia proteins were added as co-antigens to the VLP CPS conjugate vaccine but did not improve efficacy. Tandem CoreTM VLPs conjugated to CPS were protective against B. pseudomallei challenge and were compared to CPS conjugated to Crm197: a commercially available carrier protein used in several licensed vaccines. At lower challenge doses, survival was greater in mice vaccinated with the VLP-CPS conjugate although at higher doses, Crm197-CPS efficacy was greater.

Many pathogenic microorganisms are believed to stochastically switch into low metabolic states that display resistance to supra-lethal levels of antibiotics. These so-called “persister” cells have been associated with recurrent infections and the development of antibiotic resistance. Whilst a compound that eliminates Staphylococcus aureus persister cells has been described, it is not active against Gram-negative bacteria. The aim of my PhD project was to develop a high-throughput assay for compounds that eradicate persister cells in the -proteobacterium Burkholderia thailandensis. Further to this, I aimed to develop “hit” compounds from screening into lead series through investigation of structure activity relationships and, use a chemical genetics approach to elucidate potential mechanisms of action. I developed a phenotypic assay to identify compounds that eradicate persister cells. The assay was based on the reduction of the resazurin based dye PrestoBlue. Optimization of the assay gave a Z’ prime of 0.41 when screened in high throughput at the DDU. Screening of the library of 61,250 compounds identified 2,127 compounds that gave a statistically significant reduction in persister cell numbers. Follow-up assays highlighted 29 compounds with a pIC50 greater than five. Detailed investigation allowed me to down select to six “best in class” compounds, which included the licensed drug chloroxine. A time dependent killing assay showed that chloroxine reduced levels of persister cells by three orders of magnitude over 72 hours (P = 0.01). Hit expansion around chloroxine using commercially available compounds did not identify any more potent compounds, but did highlight key features of the molecule for activity. Assay protocols were provided to collaborators at DSTL who were able to iv show that chloroxine is also active against persister cells formed by the tropical pathogen and Tier 1 biological agent Burkholderia pseudomallei. Investigations into the mechanism of action of chloroxine used Next Generation Sequencing of an over expression library, identifying two putative genes involved in inhibition of persister cells by chloroxine. My findings demonstrate a phenotypic assay against persister cells in Gram-negative bacteria, which has the power to identify potent anti-persister agents to assist in chemotherapy. Structural activity relationship and mechanism of action investigations have indicated lead series and genetic starting points for future development of this research. My PhD project has concluded with sufficient data for continuation of research following a number of leads and is at an ideal stage for instigation of a medicinal chemistry program for development of chloroxine as a clinical option for treatment of persistent melioidosis.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / A Burkholderia pseudomallei à um bacilo Gram-negativo nÃo-fermentador, saprÃfita ambiental capaz de causar melioidose em homens e animais. A doenÃa à considerada endÃmica em diversos paÃses, entre os quais destacam-se TailÃndia e AustrÃlia. Em fevereiro de 2003, ocorreu no Brasil, o primeiro isolamento e identificaÃÃo da bactÃria em quatro crianÃas da localidade de TejuÃuoca, CearÃ. Este trabalho consiste na caracterizaÃÃo fenotÃpica de 3 amostras de B. pseudomallei originÃrias dos pacientes do municÃpio de TejuÃuoca, com o propÃsito de comparar os dados obtidos de tais amostras com os dados da literatura. Foram avaliados: morfologia das colÃnias em diferentes meios de cultivo, assimilaÃÃo de L-arabinose, testes bioquÃmicos manuais e em sistema semi-automatizado API 20NE, teste de sensibilidade antibacteriano e diagnÃstico por PCR, a partir de cultivos bacterianos. Nos resultados obtidos, foi observado o padrÃo morfolÃgico caracterÃstico de B. pseudomallei em Ãgar sangue, chocolate, Ashdown, Mac Conkey, CLED e tripticase soja. As amostras 1 e 3 foram classificadas como mucÃides e a amostra 2 como rugosa. As trÃs amostras apresentaram os padrÃes fenotÃpicos caracterÃsticos de B. pseudomallei tanto nos testes bioquÃmicos manuais: motilidade, crescimento à 42ÂC, oxidase positiva e resistÃncia à polimixina B, como no Kit DiagnÃstico API 20NE. Neste Ãltimo, houve diferenÃa na esculina entre as amostras, o que nÃo interferiu no resultado final de identificaÃÃo, quando a leitura foi realizada com 48h. Todas as trÃs amostras foram incapazes de assimilar o carboidrato L-arabinose, quando testadas em meio sais mÃnimo e API 20NE, caracterÃstica de amostras virulentas de B. pseudomallei e utilizada tambÃm para diferenciar esta espÃcie da B. thailandensis, que à capaz de assimilar este carboidrato. O padrÃo de sensibilidade resultante do TSA em disco difusÃo apresentado pelas trÃs amostras foi o caracterÃstico da espÃcie B. pseudomallei. Os isolados foram resistentes à gentamicina, cefalotina, ciprofloxacina (1 amostra apresentou resistÃncia intermediÃria) e sulfa-trimetoprim; 2 amostras apresentaram sensibilidade intermediÃria à ceftriaxona. Todas as trÃs amostras foram sensÃveis à piperacilina-tazobactam, ticarcilina-Ãcido clavulÃnico, ceftazidima, imipenem, tetraciclina e cloranfenicol. Com o protocolo fenol-clorofÃrmio modificado de extraÃÃo de DNA, a PCR apresentou banda de 718 pb, o que confirmou o diagnÃstico da bactÃria tambÃm por mÃtodo molecular. O estudo confirma a presenÃa da B. pseudomallei em territÃrio brasileiro, com fenotipagem semelhante à descrita na literatura internacional. / Burkholderia pseudomallei is a Gram-negative non-fermentative bacilli, environmental saprophyte able to cause melioidosis on men and animals. The disease is considered endemic in several countries, especially in Thailand and Australia. The bacteria was isolated and identified for the first time in Brazil, february 2003, from four children that lived in a place called TejuÃuoca, CearÃ. This work consists of the phenotypic characterization of 3 strains of B. pseudomallei isolated from the patients from TejuÃuoca. The main aim of this study is to compare the data from these samples with the ones from the literature. It was assessed: the colonies morphology in different culture mediums, assimilation of L-arabinose, manual and semi-automatized biochemical tests in API 20NE, antibacterial sensitivity test and diagnosis by PCR, from bacterial cultures. In the obtained results, it was observed the morphological pattern of B. pseudomallei in blood agar, chocolate, Ashdown, Mac Conkey, CLED and trypticase soy agar. The strains 1 and 3 were classified as mucoid and the strain 2 as wrinkled. The three strains had shown the usual phenotypic patterns of B. pseudomallei as much in biochemical manual tests: motility, growth at 42ÂC, positive oxidase and resistance to polimixin B, as in the API 20NE Diagnosis Kit. In this last one, there was difference in the esculin test among the strains, when the reading was carried out with 48 hours, which did not change on the final identification. All of the three strains were unable to metabolize the carbohydrate L-arabinose, when tested in minimum medium salts and API 20NE, which is a characteristic of virulent strains of B. pseudomallei and is also used to differ this species from B. thailandensis, that is able to use the carbohydrate. The three isolates had shown a poor sensitivity pattern from disk diffusion on TSA, which were resistant to gentamicin, cefalotin, ciprofloxacin (one strain presented intermediate resistance) and sulfa-trimethoprim; two strains presented intermediate sensitivity to ceftriaxone. All of them were sensitive to piperacilin-tazobactam, ticarcilin-clavulanate, ceftazidime, imipenem, tetracycline and chloramphenicol. A modified extraction protocol based on phenol-chloroform was used to obtain DNA and later to test it by PCR, which had shown a 718 bp product, what also confirmed the diagnosis of the organisms by molecular method. The study confirms the presence of B. pseudomallei in Brazil with similar phenotype to that described in the international literature.