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Melioidosis, a human infectious disease with a high mortality rate in many tropical countries, is caused by the pathogen Burkholderia pseudomallei (B. pseudomallei). The function of the B. pseudomallei sigma S (RpoS) transcription factor in survival during the stationary growth phase and conditions of oxidative stress is well documented. Besides the rpoS, bioinformatics analysis of B. pseudomallei genome showed the existence of two rpoN genes, named rpoN1 and rpoN2. In this study, by using the mouse macrophage cell line RAW264.7 as a model of infection, the involvement of B. pseudomallei RpoS and RpoN2 in the invasion, intracellular survival leading to the reduction in multinucleated giant cell (MNGC) formation of RAW264.7 cell line were illustrated. We have demonstrated that the MNGC formation of RAW264.7 cell was dependent on a certain number of intracellular bacteria (at least 5 × 10 4 ). In addition, the same MNGC formation (15%) observed in RAW264.7 cells infected with either B. pseudomallei wild type with multiplicity of infection (MOI) 2 or RpoN2 mutant (∆rpoN2) with MOI 10 or RpoS mutant (∆rpoS) with MOI 100. The role of B. pseudomallei RpoS and RpoN2 in the regulation of type III secretion system on bipB-bipC gene expression was also illustrated in this study., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Burkholderia pseudomallei-a causative agent of melioidosis that is endemic in Southeast Asia and Northern Australia-is a Gram-negative bacterium transmitted to humans via inhalation, inoculation through skin abrasions, and ingestion. Melioidosis causes a range of clinical presentations including skin infection, pneumonia, and septicemia. Despite skin infection being one of the clinical symptoms of melioidosis, the pathogenesis of B. pseudomallei in skin fibroblasts has not yet been elucidated. In this study, we investigated B. pseudomallei pathogenesis in the HFF-1 human skin fibroblasts. On the basis of co-culture assays between different B. pseudomallei clinical strains and the HFF-1 human skin fibroblasts, we found that all B. pseudomallei strains have the ability to mediate invasion, intracellular replication, and multinucleated giant cell (MNGC) formation. Furthermore, all strains showed a significant increase in cytotoxicity in human fibroblasts, which coincides with the augmented expression of matrix metalloproteinase-2. Using B. pseudomallei mutants, we showed that the B. pseudomallei Bsa type III secretion system (T3SS) contributes to skin fibroblast pathogenesis, but O-polysaccharide, capsular polysaccharide, and short-chain dehydrogenase metabolism do not play a role in this process. Taken together, our findings reveal a probable connection for the Bsa T3SS in B. pseudomallei infection of skin fibroblasts, and this may be linked to the pathogenesis of cutaneous melioidosis., Competing Interests: The authors have declared that no competing interests exist.

Burkholderia pseudomallei is a Gram-negative bacterium and the causative agent of melioidosis. Despite advances in our understanding of the disease, B. pseudomallei poses a significant health risk, especially in regions of endemicity, where treatment requires prolonged antibiotic therapy. Even though the respiratory and percutaneous routes are well documented and considered the main ways to acquire the pathogen, the gastrointestinal tract is believed to be an underreported and underrecognized route of infection. In the present study, we describe the development of in vitro and in vivo models to study B. pseudomallei gastrointestinal infection. Further, we report that the type 6 secretion system (T6SS) and type 1 fimbriae are important virulence factors required for gastrointestinal infection. Using a human intestinal epithelial cell line and mouse primary intestinal epithelial cells (IECs), we demonstrated that B. pseudomallei adheres, invades, and forms multinucleated giant cells, ultimately leading to cell toxicity. We demonstrated that mannose-sensitive type 1 fimbria is involved in the initial adherence of B. pseudomallei to IECs, although the impact on full virulence was limited. Finally, we also showed that B. pseudomallei requires a functional T6SS for full virulence, bacterial dissemination, and lethality in mice infected by the intragastric route. Overall, we showed that B. pseudomallei is an enteric pathogen and that type 1 fimbria is important for B. pseudomallei intestinal adherence, and we identify a new role for T6SS as a key virulence factor in gastrointestinal infection. These studies highlight the importance of gastrointestinal melioidosis as an understudied route of infection and open a new avenue for the pathogenesis of B. pseudomallei ., (Copyright © 2020 American Society for Microbiology.)

Tetraspanins are four-span transmembrane proteins of host cells that facilitate infections by many pathogens. Burkholderia pseudomallei is an intracellular bacterium and the causative agent of melioidosis, a severe disease in tropical regions. This study investigated the role of tetraspanins in B. pseudomallei infection. We used flow cytometry to determine tetraspanins CD9, CD63, and CD81 expression on A549 and J774A.1 cells. Their roles in B. pseudomallei infection were investigated in vitro using monoclonal antibodies (MAbs) and recombinant large extracellular loop (EC2) proteins to pretreat cells before infection. Knockout of CD9 and CD81 in cells was performed using CRISPR Cas9 to confirm the role of tetraspanins. Pretreatment of A549 cells with MAb against CD9 and CD9-EC2 significantly enhanced B. pseudomallei internalization, but MAb against CD81 and CD81-EC2 inhibited MNGC formation. Reduction of MNGC formation was consistently observed in J774.A1 cells pretreated with MAbs specific to CD9 and CD81 and with CD9-EC2 and CD81-EC2. Data from knockout experiments confirmed that CD9 enhanced bacterial internalization and that CD81 inhibited MNGC formation. Our data indicate that tetraspanins are host cellular factors that mediated internalization and membrane fusion during B. pseudomallei infection. Tetraspanins may be the potential therapeutic targets for melioidosis.

Background: Burkholderia pseudomallei is an environmental bacterium that causes melioidosis. A facultative intracellular pathogen, B. pseudomallei can induce multinucleated giant cells (MNGCs) leading to plaque formation in vitro. B. pseudomallei can switch colony morphotypes under stress conditions. In addition, different isolates have been reported to have varying virulence in vivo, but genomic evolution and the relationship with plaque formation is poorly understood., Methodology/principle Findings: To gain insights into genetic underpinnings of virulence of B. pseudomallei, we screened plaque formation of 52 clinical isolates and 11 environmental isolates as well as 4 isogenic morphotype isolates of B. pseudomallei strains K96243 (types II and III) and 153 (types II and III) from Thailand in A549 and HeLa cells. All isolates except one environmental strain (A4) and K96243 morphotype II were able to induce plaque formation in both cell lines. Intracellular growth assay and confocal microscopy analyses demonstrated that the two plaque-forming-defective isolates were also impaired in intracellular replication, actin polymerization and MNGC formation in infected cells. Whole genome sequencing analysis and PCR revealed that both isolates had a large genomic loss on the same region in chromosome 2, which included Bim cluster, T3SS-3 and T6SS-5 genes., Conclusions/significance: Our plaque screening and genomic studies revealed evidence of impairment in plaque formation in environmental isolates of B. pseudomallei that is associated with large genomic loss of genes important for intracellular multiplication and MNGC formation. These findings suggest that the genomic and phenotypic differences of environmental isolates may be associated with clinical infection., Competing Interests: The authors have declared that no competing interests exist.

The human pathogen Burkholderia pseudomallei and the related species Burkholderia thailandensis are facultative intracellular bacteria characterized by the ability to escape into the cytosol of the host cell and to stimulate the formation of multinucleated giant cells (MNGCs). MNGC formation is induced via an unknown mechanism by bacterial type VI secretion system 5 (T6SS-5), which is an essential virulence factor in both species. Despite the vital role of the intracellular life cycle in the pathogenesis of the bacteria, the range of host cell types permissive for initiation and completion of the intracellular cycle is poorly defined. In the present study, we used several different types of human primary cells to evaluate bacterial entry, intracellular survival, and MNGC formation. We report the capacity of B. pseudomallei to enter, efficiently replicate in, and mediate MNGC formation of vein endothelial and bronchial epithelial cells, indicating that the T6SS-5 is important in the host-pathogen interaction in these cells. Furthermore, we show that B. pseudomallei invades fibroblasts and keratinocytes and survives inside these cells as well as in monocyte-derived macrophages and neutrophils for at least 17 h postinfection; however, MNGC formation is not induced in these cells. In contrast, infection of mixed neutrophils and RAW264.7 macrophages with B. thailandensis stimulated the formation of heterotypic MNGCs in a T6SS-5-dependent manner. In summary, the ability of the bacteria to enter and survive as well as induce MNGC formation in certain host cells may contribute to the pathogenesis observed in B. pseudomallei infection., (Copyright © 2017 American Society for Microbiology.)

The Burkholderia genus contains a group of soil-dwelling Gram-negative organisms that are prevalent in warm and humid climates. Two species in particular are able to cause disease in animals, B. mallei primarily infects Equus spp. and B. pseudomallei (BPS), that is able to cause potentially life-threatening disease in humans. BPS is naturally resistant to many antibiotics and there is no vaccine available. Although not a specialised human pathogen, BPS possesses a large genome and many virulence traits that allow it to adapt and survive very successfully in the human host. Key to this survival is the ability of BPS to replicate intracellularly. In this review we highlight recent advances in our understanding of the intracellular survival of BPS, including how it overcomes host immune defenses and other challenges to establish its niche and then spread the infection. Knowledge of these mechanisms increases our capacity for therapeutic interventions against a well-armed foe., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Burkholderia pseudomallei (Bp), a Gram-negative, motile, facultative intracellular bacterium is the causative agent of melioidosis in humans and animals. The Bp genome encodes a repertoire of virulence factors, including the cluster 3 type III secretion system (T3SS-3), the cluster 1 type VI secretion system (T6SS-1), and the intracellular motility protein BimA, that enable the pathogen to invade both phagocytic and non-phagocytic cells. A unique hallmark of Bp infection both in vitro and in vivo is its ability to induce cell-to-cell fusion of macrophages to form multinucleated giant cells (MNGCs), which to date are semi-quantitatively reported following visual inspection., Results: In this study we report the development of an automated high-content image acquisition and analysis assay to quantitate the Bp induced MNGC phenotype. Validation of the assay was performed using T6SS-1 (∆hcp1) and T3SS-3 (∆bsaZ) mutants of Bp that have been previously reported to exhibit defects in their ability to induce MNGCs. Finally, screening of a focused small molecule library identified several Histone Deacetylase (HDAC) inhibitors that inhibited Bp-induced MNGC formation of macrophages., Conclusions: We have successfully developed an automated HCI assay to quantitate MNGCs induced by Bp in macrophages. This assay was then used to characterize the phenotype of the Bp mutants for their ability to induce MNGC formation and identify small molecules that interfere with this process. Successful application of chemical genetics and functional reverse genetics siRNA approaches in the MNGC assay will help gain a better understanding of the molecular targets and cellular mechanisms responsible for the MNGC phenotype induced by Bp, by other bacteria such as Mycobacterium tuberculosis, or by exogenously added cytokines.

Burkholderia pseudomallei, the causative agent of melioidosis, contains a large pathogen genome (7.2 Mb) with ∼2,000 genes of putative or unknown function. Interactions with potential hosts and environmental factors may induce rapid adaptations in these B. pseudomallei genes, which can be discerned through evolutionary analysis of multiple B. pseudomallei genomes. Here we show that several previously uncharacterized B. pseudomallei genes bearing genetic signatures of rapid adaptation (positive selection) can induce diverse cellular phenotypes when expressed in mammalian cells. Notably, several of these phenotypes are plausibly related to virulence, including multinuclear giant cell formation, apoptosis, and autophagy induction. Specifically, we show that BPSS0180, a type VI cluster-associated gene, is capable of inducing autophagy in both phagocytic and nonphagocytic mammalian cells. Following infection of macrophages, a B. pseudomallei mutant disrupted in BPSS0180 exhibited significantly decreased colocalization with LC3 and impaired intracellular survival; these phenotypes were rescued by introduction of an intact BPSS0180 gene. The results suggest that BPSS0180 may be a novel inducer of host cell autophagy that contributes to B. pseudomallei intracellular growth. More generally, our study highlights the utility of applying evolutionary principles to microbial genomes to identify novel virulence genes.