Burkholderia pseudomallei is an intracellular pathogen and the causative agent of melioidosis, a severe disease of humans and animals. One of the virulence factors critical for early stages of infection is the Burkholderia secretion apparatus (Bsa) Type 3 Secretion System (T3SS), a molecular syringe that injects bacterial proteins, called effectors, into eukaryotic cells where they subvert cellular functions to the benefit of the bacteria. Although the Bsa T3SS itself is known to be important for host cell invasion, intracellular replication, and virulence, only a few genuine effector proteins have been identified and the complete repertoire of proteins secreted by the system has not yet been fully characterized. The aims of this study are twofold. The first is to expand the repertoire of known effector proteins using modern proteomics techniques. The second is to explore the function of a subset of effector proteins to better understand their interaction with host cells. Isobaric Tags for Relative and Absolute Quantification (iTRAQ), a gel-free quantitative proteomics technique, was used to compare the secreted protein profiles of the Bsa T3SS hyper-secreting mutants of B. pseudomallei with the isogenic parent strain as well as a mutant incapable of effector protein secretion. This study provides one of the most comprehensive core secretomes of B. pseudomallei described to date and identified 26 putative Bsa-dependent secreted proteins that may be considered candidate effectors. Two of these proteins, BprD and BapA, were validated as novel effector proteins secreted by the Bsa T3SS of B. pseudomallei. To determine the possible function of two effector proteins, BipC and BapA, a yeast two-hybrid system was used to identify host cell proteins the effectors interact with. The proteins were screened against a library of human proteins for interactions. BapA interacted with 2 proteins while BipC interacted with 14. Both BapA and BipC were shown to interact with human C1QBP, a mitochondrial protein involved in inflammation, immunity and autophagy. Finally, the Bsa T3SS protein BipC was characterised in its ability to interact with actin. This study is the first evidence that BipC has the ability to bind to filamentous actin, but not monomeric actin. This binding is direct and no intermediate proteins are required for the interaction. Ectopic expression of BipC in eukaryotic cells caused cytoskeletal rearrangements consistent with an actin-binding protein. The core secretome represents a substantial resource of targets that will be mined for improved diagnostic assays and vaccines. Diagnostics that will detect early stages of disease to allow for more effective antimicrobial intervention are currently lacking. Furthermore, there is scope to design diagnostic assays with dual use such as to detect both melioidosis and infection of cystic fibrosis patients with the closely related opportunistic pathogen B. cepacia. The description of novel T3SS effector proteins is also of considerable value since T3SS proteins are often potent B- and T- cell antigens representing promising components of sub-unit vaccines. Such effector proteins commonly modulate cellular processes such as phagocytosis, inflammasome activation and cell cycle progression, hence the function of the predicted T3SS effectors will provide a series of future research opportunities.

MOREIRA, C. A. Estudo de sensibilidade do biofilme de Burkholderia pseudomallei a antibióticos de uso clínico e farnesol. 2013. 115 f. Tese (Doutorado em Microbiologia Médica) - Faculdade de Medicina, Universidade Federal do Ceará, Fortaleza, 2013. / Submitted by Erika Fernandes (erikaleitefernandes@gmail.com) on 2016-10-03T15:38:15Z No. of bitstreams: 1 2013_tese_camoreira.pdf: 2691368 bytes, checksum: 33ff8ee13ee8b744ebce1f67d391647c (MD5) / Approved for entry into archive by Erika Fernandes (erikaleitefernandes@gmail.com) on 2016-10-03T15:38:53Z (GMT) No. of bitstreams: 1 2013_tese_camoreira.pdf: 2691368 bytes, checksum: 33ff8ee13ee8b744ebce1f67d391647c (MD5) / Made available in DSpace on 2016-10-03T15:38:53Z (GMT). No. of bitstreams: 1 2013_tese_camoreira.pdf: 2691368 bytes, checksum: 33ff8ee13ee8b744ebce1f67d391647c (MD5) Previous issue date: 2013-09-27 / The melioidosis is an emerging infectious disease, especially in northeastern Brazil, with serious international implications, as well as a public health problem. Caused by the bacillus Burkholderia pseudomallei, this infection appears from tables asymptomatic to severe and often fatal frames. The high mortality rate of the disease (20-50%) make it a priority for global health agencies. In Brazil, cases of melioidosis were first reported in 2003 in Tejuçuoca municipality in Ceará. Since then, new cases of melioidosis were diagnosed in six other municipalities in Ceará. B. pseudomallei is intrinsically resistant to many antibiotics and recent studies have described cases of antimicrobial resistance used in the treatment of melioidosis. The investigation of the biofilm forming capacity of B. pseudomallei seems to be essential, since the biofilm allows the development of microcolonies within a protected environment, which relates to the environmental protection, adhesion, colonization, immune evasion and bonding the environmental cell. Therefore, the development of therapeutic alternatives antibiofilm, including new drugs is needed. Indeed, this study aimed to characterize the strains of B. pseudomallei of LAPERE collection as the biofilm production capacity and sensitivity in planktonic growth and biofilm. Selected drugs were ceftazidime (CAZ), doxycycline (DOX), imipenem (IPM), amoxicillin / clavulanate (AMC) and trimethoprim / sulfamethoxazole (SXT); and farnesol (FNS). All strains were classified as biofilm producers, being divided into: hard (5 strains), moderate (3 strains), and low (1 strain). Mean values ​​of minimum inhibitory concentration (MIC), minimum inhibitory concentration of biofilm (MBIC) and concentration minimum biofilm eradication in (MBEC) for these strains were determined for AMC (MIC 10.2 / 5.1 mg / L, 21 MBIC 3 / 10.6 mg / L and MBEC 27.6 / 13.8 mg / L) to CAZ (MIC 5.6 mg / L, MBIC 120.4 mg / L and MBEC 419.6 mg / L) to doxorubicin (MIC 0.28 mg / L, MBIC 1.3 mg / L and MBEC 3.8 mg / L) to IPM (MIC 0.597 mg / L, MBIC ≥ 256 mg / L and MBEC> 256 mg / G) to SXT (MIC 1.25 / 23.75 mg / L, MBIC ≤ 0.5 / 9.5 mg / L and MBEC <0.72 / 13.72 mg / L), and NSF (MIC and MBIC equal to 150 uM / L). The data obtained show especially for the inhibitory potential of three antibiotics studied - AMC, DOX and SXT and farnesol against strains of B. pseudomallei associated with biofilm. However, further studies are needed to investigate the mechanisms of action of these drugs on the biofilm, as well as the design of in vivo experiments to confirm the significance of these findings clinically. Moreover, the growth of these strains melanin formation conditions and biofilm showed that this combination makes them more resistant strains to the action of imipenem and farnesol. / A melioidose é uma doença infeciosa emergente, notadamente no nordeste do Brasil, com sérias implicações internacionais, assim como um problema de saúde pública. Causada pelo bacilo Burkholderia pseudomallei, esta infecção se apresenta desde quadros assintomáticos a quadros graves e frequentemente fatais. As altas taxas de mortalidade da doença (20 a 50%) a tornam uma prioridade para orgãos globais de saúde. No Brasil, casos de melioidose foram relatados pela primeira vez em 2003, no município de Tejuçuoca no Ceará. Desde então, novos casos de melioidose foram diagnosticados em outros seis municípios cearenses. B. pseudomallei é intrinsecamente resistente a muitos antibióticos e estudos recentes já descrevem casos resistência aos antimicrobianos utilizados no tratamento da melioidose. A investigação da capacidade de formação de biofilme por B. pseudomallei parece ser fundamental, já que o biofilme permite o desenvolvimento de microcolônias dentro de um ambiente protegido, o qual se relaciona com a proteção ambiental, adesão, colonização, evasão do sistema imune e ligação a células ambientais. Portanto, o desenvolvimento de alternativas terapêuticas antibiofilme, incluindo novas drogas, é necessário. Com efeito, este estudo teve como objetivo caracterizar as cepas de B. pseudomallei da coleção do LAPERE quanto à capacidade de produção de biofilme e sensibilidade em crescimento planctônico e em biofilme. As drogas selecionadas foram: ceftazidima (CAZ), doxiciclina (DOX), imipenem (IPM), amoxicilina/clavulanato (AMC) e sulfametoxazol/trimetoprim (SXT); e farnesol (FNS). Todas as cepas foram classificadas como produtoras de biofilme, sendo divididas em: forte (5 cepas), moderada (3 cepas), e fraca (1 cepa). Valores médios de concentração inibitória mínima (MIC), concentração inibitória mínima em biofilme (MBIC) e concentração de erradicação mínima em biofilme (MBEC) para estas cepas foram determinados para AMC (MIC 10,2/5,1 mg/L, MBIC 21,3/10,6 mg/L e MBEC 27,6/13,8 mg/L), para CAZ (MIC 5,6 mg/L, MBIC 120,4 mg/L e MBEC 419,6 mg/L), para DOX (MIC 0,28 mg/L, MBIC 1,3 mg/L e MBEC 3,8 mg/L), para IPM (MIC 0,597 mg/L, MBIC ≥ 256 mg/L e MBEC > 256 mg/L), para SXT (MIC 1,25/23,75 mg/L, MBIC ≤ 0,5/9,5 mg/L e MBEC < 0,72/13,72 mg/L), e para FNS (MIC e MBIC iguais a 150 µM/L). Os dados obtidos apontam especialmente para o potencial inibitório de três dos antibióticos estudados - AMC, DOX e SXT, e farnesol contra cepas de B. pseudomallei associadas a biofilme. Todavia, são necessários novos estudos para investigar os mecanismos de ação dessas drogas sobre o biofilme, bem como o delineamento de experimentos in vivo para confirmar a significância desses achados clinicamente. Ademais, o crescimento dessas cepas em condições de formação de melanina e biofilme evidenciou que esta associação torna as cepas mais resistentes à ação de imipenem e farnesol.

COUTO, Manuela Soares. Identificação molecular de cepas clínicas e ambientais de Burkholderia pseudomallei, oriundas do Estado do Ceará : análise baseada nas regiões 16S e 16S-23S do DNA ribossômico nuclear. 2099.0107 f. Dissertação (Mestrado em Ciências Médicas) - Universidade Federal do Ceará. Faculdade de Medicina, Fortaleza, 2009. / Submitted by denise santos (denise.santos@ufc.br) on 2013-12-05T14:02:29Z No. of bitstreams: 1 2009_dis_mscouto.pdf: 1672949 bytes, checksum: f161d01fb64b6c9b0e6980b0190093b1 (MD5) / Approved for entry into archive by denise santos(denise.santos@ufc.br) on 2013-12-05T14:03:42Z (GMT) No. of bitstreams: 1 2009_dis_mscouto.pdf: 1672949 bytes, checksum: f161d01fb64b6c9b0e6980b0190093b1 (MD5) / Made available in DSpace on 2013-12-05T14:03:43Z (GMT). No. of bitstreams: 1 2009_dis_mscouto.pdf: 1672949 bytes, checksum: f161d01fb64b6c9b0e6980b0190093b1 (MD5) Previous issue date: 2009 / Melioidosis is a potentially fatal disease caused by the bacterium Burkholderia pseudomallei, considered emerging in Brazil since the first cases were reported in 2003, on State of Ceará. This study aimed to perform the molecular identification of 31 isolates of B. pseudomallei (26 clinical and 5 environmental) maintained in the culture collection of CEMM (Specialized Center for Medical Mycology), based on sequences 16S and 16S-23S rRNA. The DNA of these samples was extracted with the kit Wizard ® Genomic DNA Purification (Promega), quantified by spectrophotometry and stored at 4°C. The amplification of a fragment of 302 bp of 16S-23S rRNA specific to B. pseudomallei was performed by PCR reaction with primers Bp1 and Bp4. The sequencing of 16S and 16S-23S rRNA was performed by using of the kit DYEnamicTM ET terminators cycle sequencing (GE Healthcare). The phylogenetic tree of 16S rRNA and the sequence identity matrix and sequence difference count matrix based on the 16S-23S rRNA were generated by the program MEGA4, version 4.1. The results confirmed the identification of 15 strains of B. pseudomallei (5 clinical and 10 environmental), which represents 48.4% of the isolates analyzed in this study. The phylogenetic tree based on 16S rRNA shows that the clinical and environmental isolates of B. pseudomallei of State of Ceará are evolutionarily clustered with the strains B. pseudomallei MSHR346 (Australia), B. pseudomallei 1106a (Thailand), B. pseudomallei K96243 (Thailand), B. pseudomallei 1710b (Thailand) and B. pseudomallei 668 (Australia). Using the same extraction kit was possible to extract DNA from B. pseudomallei directly from clinical specimen (bronchoalveolar lavage), confirming a new case of melioidosis in Ubajara/CE. In this study, the use of PCR for amplification of a fragment of 302 bp of 16S-23S rRNA identified correctly B. pseudomallei, and to confirm the discrimination between B. pseudomallei and B. mallei, the sequencing of the 16S and 16S-23S rRNA genes was performed. The technique of PCR coupled with sequencing of 16S and 16S-23S rRNA resulted in a high sensitivity and specificity of detection of B. pseudomallei in this study. / A melioidose é uma doença potencialmente fatal causada pela bactéria Burkholderia pseudomallei, sendo considerada emergente no Brasil desde que os primeiros casos foram reportados em 2003, no Estado do Ceará. Este estudo pretendeu realizar a identificação molecular de 31 isolados de B. pseudomallei (cinco clínicos e 26 ambientais) mantidos na coleção de culturas do CEMM (Centro Especializado em Micologia Médica), com base nas sequências 16S e 16S-23S DNAr. O DNA destas amostras foi extraído com o kit Wizard® Genomic DNA Purification (Promega), quantificado por espectrofotometria e armazenado a 4ºC. A amplificação de um fragmento de 302 pb da região espaçadora 16S-23S DNAr específico para B. pseudomallei foi realizada por meio de reação de PCR com os primers Bp1 e Bp4. O sequenciamento das regiões 16S e 16S-23S DNAr foi realizado pelo método da terminação da cadeia pelo didesoxinucleotídeo, usando-se o kit DYEnamicTM ET terminators cycle sequencing (GE Healthcare). A árvore filogenética da região 16S DNAr e as matrizes sequência identidade e contagem de diferenças baseadas na região 16S-23S DNAr foram geradas pelo programa MEGA4, versão 4.1. Os resultados confirmaram a identificação de 15 cepas de B. pseudomallei (cinco clínicas e dez ambientais), o que corresponde a 48.4% dos isolados em estudo. A árvore filogenética baseada na região 16S DNAr demonstra que os isolados clínicos e ambientais de B. pseudomallei do Estado do Ceará são evolutivamente agrupados com as cepas B. pseudomallei MSHR346 (Austrália), B. pseudomallei 1106a (Tailândia), B. pseudomallei K96243 (Tailândia), B. pseudomallei 1710b (Tailândia) e B. pseudomallei 668 (Austrália). Com a utilização do mesmo kit de extração também foi possível extrair DNA de B. pseudomallei diretamente de espécime clínico (lavado brônquico), confirmando um novo caso de melioidose no Município de Ubajara/CE. Em nosso estudo, o uso da PCR para a amplificação de um fragmento de 302 pb da região 16S-23S DNAr identificou corretamente B. pseudomallei, sendo que para confirmar a discriminação entre B. pseudomallei e B. mallei, o sequenciamento das regiões 16S e 16S-23S DNAr foi realizado. A técnica de PCR aliada ao sequenciamento das regiões 16S e 16S-23S do DNA ribossômico nuclear resultaram em uma elevada sensibilidade e especificidade de detecção de B. pseudomallei neste estudo.

The bacterium Burkholderia pseudomallei causes the disease melioidosis, a significant public health threat in endemic regions and is a potential biowarfare agent. Treatment of melioidosis is intensive and prolonged and there is no licensed vaccine to protect against it. The aim of this study was to characterise novel targets for antimicrobials to improve treatment of melioidosis. A holistic down selection process was undertaken in order to identify a range of possible novel and exploitable antimicrobial targets in Burkholderia pseudomallei. Four targets: FtsA, FtsZ, MraW and TonB were selected for characterisation by mutagenesis study. FtsA and FtsZ are early effectors of cell division and are considered potential antimicrobial drug targets in other pathogenic bacteria. Genes for both were shown likely to be essential for viability in Burkholderia pseudomallei, following attempted deletion of the genes, thus confirming their potential for drug targeting for treatment of melioidosis. MraW, a highly conserved methyltransferase, and TonB, the energiser for high affinity iron uptake in Gram negative bacteria, were also selected for characterisation as antimicrobial targets. In-frame deletions of the genes encoding these targets were constructed in B. pseudomallei K96243. In order to determine the roles played by MraW and TonB during infection, these mutants were characterised in several models of Burkholderia pseudomallei infection. Deletion of mraW rendered the bacteria non-motile and led to attenuation during infection of Balb/C mice. A small growth defect was seen early during infection of macrophages by this mutant, whilst no attenuation was seen on deletion of mraW in Galleria mellonella. Burkholderia pseudomallei ΔtonB required free iron supplementation for growth. This mutant had an improved ability to invade murine macrophages, though the mutant was attenuated in both Galleria mellonella and Balb/C mice. Attenuation of both mutants in a mammalian model of infection, support the strategy to target either of these proteins as novel targets for inhibition with small molecules during Burkholderia pseudomallei infection. However, an improved ability to infect macrophages by Burkholderia pseudomallei ΔtonB and non-complementation of this mutant by iron supplementation to Galleria mellonella suggests additional roles to iron uptake alone for TonB in Burkholderia pseudomallei, such as bacterial iron sensing and signalling.

Burkholderia pseudomallei is the aetiological agent of melioidosis and a potential bioterror threat. Infections are difficult to treat due to extensive antibiotic resistance and there is no prophylactic vaccine available. Studies have shown that the capsular polysaccharide (CPS) of B. pseudomallei is a virulence factor, immunogen and candidate antigen for a glycoconjugate vaccine. However, polysaccharides are complex to synthesise. One approach is to genetically engineer Escherichia coli to express the CPS; however, previous attempts at cloning the CPS coding locus from B. pseudomallei into E. coli were unsuccessful. This project proposes to clone only the essential genes from B. pseudomallei and to use native E. coli mechanisms to complete CPS synthesis. This would contribute to development of a new platform for the expression of any bespoke polysaccharide in E. coli. Six biosynthetic genes for the nucleotide sugar precursor were successfully expressed in E. coli. The structure of the precursor was verified by mass spectrometry. Precursor synthesis was also performed in an in vitro microfluidics system. This minimised the quantity of substrates and enzymes required, in preparation for the characterisation of glycosyltransferases required for CPS assembly. A novel assay for characterising glycosyltransferase activity was also developed, as current available options are prohibitively expensive and require significant quantities of glycosyltransferase which are difficult to purify. Finally, plasmids for the expression of additional glycosyltransferases to link the nascent B. pseudomallei CPS to truncated polysaccharides in E. coli were constructed. The aim of this project was to contribute to the development of a platform for the expression of bespoke polysaccharides in E. coli. The CPS of B. pseudomallei was chosen as the model polysaccharide as it has a simple structure and its manufacture is desirable for use in a vaccine against melioidosis.

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 and Burkholderia mallei cause the diseases melioidosis and glanders, respectively. These diseases are endemic mainly in southeastern Asia and northern Australia, but they also pose a bioterrorism threat in the developed world. These diseases have high mortality, partially due to the lack of vaccines and rapid, accurate diagnostic assays. The work discussed here represents a part of a larger project to develop a dependable diagnostic assay for use in both developing endemic areas and the developed world, as well as a subunit vaccine to protect against disease. In this study, several proteins from B. pseudomallei, B. mallei, and the closely related but less virulent B. thailandensis have been cloned, expressed and purified in order to develop highly sensitive and specific diagnostic reagents for the detection of B. pseudomallei and B. mallei in infected patient samples. Protein targets expressed in this study were also used in subunit vaccine development for melioidosis and glanders. / text

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.

Melioidosis is an important cause of community-acquired sepsis, endemic to Southeast Asia and Northern Australia. Melioidosis is caused by the soil saprophyte, Burkholderia pseudomallei, a motile Gram-negative bacillus, and is associated with a mortality rate that approaches 50% in Northeast Thailand. The most important risk factor for melioidosis is diabetes mellitus, and two-thirds of all adult patients with melioidosis have diabetes as a risk factor. It has been noted previously, however, that patients with diabetes have lower mortality than patients without diabetes. In this dissertation, we look at a cohort of 1160 consecutive adult melioidosis patients presenting to Sappasithiprasong Hospital in Ubon Ratchathani, Thailand, 410 (35%) of whom were diagnosed with diabetes prior to admission. We confirmed previous findings that diabetes protected from mortality in melioidosis, but also found that this protective effect was confined to a smaller subset of patients (208 patients) who were treated with glibenclamide prior to admission. Patients with hyperglycaemia (but no diagnosis of diabetes prior to admission) had the same mortality rate as patients without diabetes. In vitro experiments found no inhibitory effect of glibenclamide on bacterial growth, and we therefore looked for evidence of an effect of glibenclamide on the host. We conducted a gene expression study of circulating blood leukocytes in melioidosis patients and compared them to uninfected controls. In this study, we found that glibenclamide was associated with an anti-inflammatory effect on the host response to melioidosis. To further elucidate a mechanism for the action of glibenclamide, we studied the effect of glibenclamide therapy in a mouse model of melioidosis and found that the effect of glibenclamide was specific to interleukin-1β secretion. This reduction in interleukin-1β secretion was associated with reduced cellular influx into the lungs as well as lower bacterial loads in blood, liver and spleen.

Burkholderia pseudomallei is a gram-negative opportunistic soil pathogen that causes the life-threatening disease melioidosis. It is endemic in Northern Australia and Southeast Asia but can be found throughout many other regions in the world. Diabetes mellitus is a predisposing risk factor for infection with this organism and it has been demonstrated that diabetic males are particularly susceptible to severe infection. Previous research suggested that monocytes isolated from the whole blood of diabetic males demonstrated a decreased ability to produce the proinflammatory cytokines IL-1β and IL-8. We hypothesized that monocyte-derived macrophages from diabetic males would also secrete lower levels of pro-inflammatory cytokines and that this difference between gender and diabetic state would be more pronounced compared to those seen previously with monocytes. Twenty volunteer with type I diabetes mellitus (ten males and ten females), along with twenty healthy age- and gender-matched controls donated blood for this study. Monocytes were collected from whole blood and allowed to differentiate into macrophages with the use of human recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF). Macrophages were then divided into groups and infected with B. pseudomallei, B. thailandensis (a closely related by non-pathogenic bacterium that inhabits similar niches), and E. coli. An uninfected control was used as well. At six hours post-infection, mRNA was collected from all cells and qPCR was performed to determine cytokine expression levels. All mRNA values collected from cells which had been infected with bacterial agents were normalized against the corresponding concentrations of mRNA from mock-infected cells. Mean log fold increases in both IL-1β and IL-8 were computed and compared. Preliminary testing showed decreased levels of both IL-1β and IL-8 from B. pseudomallei-infected macrophages isolated from a diabetic male compared to the healthy, age-matched male control. Surprisingly, results from all forty donors demonstrated that gender and diabetic state were not significant factors in the proinflammatory responses of macrophages infected with B. pseudomallei, although further testing is needed to determine if these results were influenced by experimental parameters.