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 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.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Grupo DASA / Melioidose à uma doenÃa infecciosa grave causada por Burkholderia pseudomallei, um bacilo Gram-negativo encontrado no solo e na Ãgua. A doenÃa à endÃmica no sudeste asiÃtico e hiperendÃmica no norte da AustrÃlia, onde a letalidade permanece com uma taxa de 21%. No Brasil, à considerada uma doenÃa emergente desde marÃo de 2003. Nos Ãltimos oito anos, 12 casos ocorreram no Estado do Cearà e um notificado pelo Governo holandÃs, por se tratar de um turista que morreu de melioidose, apÃs visita ao CearÃ. Em razÃo da ocorrÃncia clÃnica de melioidose e do isolamento de B. pseudomallei no ambiente do Estado do CearÃ, este trabalho objetivou estudar as cepas clÃnicas e ambientais de B. pseudomallei isoladas no Estado no perÃodo de 2003 a 2011, visando a identificar as cepas por mÃtodos fenotÃpicos e moleculares, determinar o perfil de sensibilidade contra cinco agentes antimicrobianos (amoxicilina/clavulanato, ceftazidima, imipenem, doxicilina e sulfametoxazol/trimetoprim), realizar a genotipagem das cepas pela amplificaÃÃo aleatÃria de DNA polimÃrfico - Random Amplified Polymorphic DNA (RAPD), detectar o gene de virulÃncia Type Three Secretion System (TTSS), alÃm de avaliar os aspectos clÃnico-epidemiolÃgicos que caracterizaram a emergÃncia desta doenÃa no Brasil. Todas as 20 cepas (dez clÃnicas e dez ambientais) de B. pseudomallei foram precisamente identificadas tanto pela metodologia VITEK2 quanto pelo sequenciamento da regiÃo 16S do DNA, mostraram resultado negativo no teste de assimilaÃÃo de L-arabinose, e exibiram-se positivas para a detecÃÃo do gene de virulÃncia TTSS. As concentraÃÃes inibitÃrias mÃnimas (CIMs), obtidas por microdiluiÃÃo em caldo MÃeller-Hinton, demonstraram que todos os isolados (100%) foram sensÃveis ao imipenem, à doxicilina e ao sulfametoxazol- trimetoprim, no entanto, para amoxicilina/clavulanato e ceftazidima, a sensibilidade foi de 80 e 90%, respectivamente. A tÃcnica de RAPD evidenciou uma variabilidade genÃtica de 63% entre as cepas de B. pseudomallei oriundas do Estado do CearÃ, as quais foram agrupadas em trÃs clusters diferentes. Este trabalho decerto contribuirà para o conhecimento das caracterÃsticas fenotÃpicas e genotÃpicas das cepas de B. pseudomallei isoladas no Cearà e da atualizaÃÃo da vigilÃncia epidemiolÃgica dos casos de melioidose ocorridos no Estado, alÃm de contribuir para a conscientizaÃÃo dos ÃrgÃos de saÃde competentes para a inclusÃo do Cearà como zona endÃmica para esta enfermidade. / Melioidosis is a serious infectious disease caused by Burkholderia pseudomallei, a Gram negative rod, commonly found in soil and water. The disease is endemic in Southeastern Asia and hyperendemic in Northern Australia. Despite the initiation of empiric therapy, mortality remains at 21% in patients with melioidosis in Australia. In Brazil, it is considered an emerging disease, since April 2003, when it was first diagnosed in Ceara, Northeastern Brazil. In the last eight years, thirteen cases were reported, twelve local cases and one case reported by the Dutch government because of a tourist who died of melioidosis after a visit to Ceara. Considering the occurrence of melioidosis in CearÃ, this work aimed at studying these clinical and environmental strains of Burkholderia pseudomallei isolated from Cearà from 2003 to 2011, focusing on the bacterial and molecular identification; determining the susceptibility profile against five antimicrobial agents (amoxicillin/clavulanate, ceftazidime, imipenem, doxycycline and trimethoprim/sulfamethoxazole); genotyping through Random Amplified Polymorphic DNA (RAPD), detecting the virulence gene Type Three Secretion System (TTSS); and analyzing epidemiological and clinical aspects that characterized the emergence of this disease in Brazil. All 20 strains (10 clinical and 10 environment) from B. pseudomallei were accurately identified by both VITEK2  and sequencing of the 16S DNA, showed to be negative for the assimilation of L-arabinose and were positive results for the detection of the virulence gene TTSS. The minimum inhibitory concentrations (MICs) obtained through microdilution in MÃeller-Hinton broth, showed that all (100%) isolates were sensitive to imipenem, doxycycline and trimethoprim-sulfamethoxazole, however, the susceptibility rate to amoxicillin/clavulanate and ceftazidime was of 80 and 90%, respectively, with no differences between clinical and environmental strains. RAPD-PCR showed a genetic relatedness of 63% among the B. pseudomallei strains from the State of CearÃ, which were grouped in two different clusters. This work will contribute to the knowledge of phenotypic and genotypic characteristics of B. pseudomallei strains isolated in Cearà and the update of epidemiological surveillance of melioidosis cases in the state, also contribute to the awareness of agencies health authority for inclusion of Cearà State as an endemic area for this disease.

Methods for the rapid detection and differentiation of the Burkholderia pseudomallei complex comprising B. pseudomallei, B. mallei, and B. thailandensis, have been the topic of recent research due to the high degree of phenotypic and genotypic similarities of these species. B. pseudomallei and B. mallei are the causative agents of melioidosis and glanders, respectively. B. pseudomallei and B. mallei are recognized by the CDC as tier 1 select agents. Although B. thailandensis is generally avirulent in mammals, this species displays very similar phenotypic characteristics to that of B. pseudomallei. Optimal identification of these species remains problematic, due to the difficulty in developing a sensitive, selective, and accurate assay. To date, no real-time, multiplex PCR assay has been developed that can detect and differentiate between B. pseudomallei, B. mallei, and B. thailandensis in a single tube format. Here, we describe the development of such an assay that detects and differentiates the species of the B. pseudomallei complex. A real-time quadruplex qPCR assay, Bcom, was designed to target unique genomic regions of B. pseudomallei, B. mallei, B. thailandensis, and the B. pseudomallei complex that detects and differentiates the three species. A total of 299 isolates within the B. pseudomallei complex was evaluated in this study, as well as 15 near-neighbors and other bacterial species. The results showed that this quadruplex assay was capable of detecting the respective species in a given sample at a sensitivity between 288 fg and 277 pg of genomic DNA. The B. pseudomallei- and B. pseudomallei complex-specific assays tested negative on two presumed B. pseudomallei isolates. In addition, a third presumed B. pseudomallei isolate tested negative by the B. pseudomallei-specific test, but was detected by the B. thailandensis and B. pseudomallei complex-specific assays. After cultural and biochemical characterization, 16s rRNA sequencing, and multiple loci sequencing, it is proposed that B. pseudomallei 34 is B. thailandensis 82172 (Accession No. DQ388536), B. pseudomallei Darwin 175 is Elizabethkingia meningoseptica, and B. pseudomallei 135 is a new strain of B. ubonensis 135.