Type VI Secretion System Accessory Protein TagAB-5 Promotes Burkholderia pseudomallei Pathogenicity in Human Microglia.

Central nervous system (CNS) melioidosis caused by Burkholderia pseudomallei is being increasingly reported. Because of the high mortality associated with CNS melioidosis, understanding the underlying mechanism of B. pseudomallei pathogenesis in the CNS needs to be intensively investigated to develop better therapeutic strategies against this deadly disease. The type VI secretion system (T6SS) is a multiprotein machine that uses a spring-like mechanism to inject effectors into target cells to benefit the infection process. In this study, the role of the T6SS accessory protein TagAB-5 in B. pseudomallei pathogenicity was examined using the human microglial cell line HCM3, a unique resident immune cell of the CNS acting as a primary mediator of inflammation. We constructed B. pseudomallei tagAB-5 mutant and complementary strains by the markerless allele replacement method. The effects of tagAB-5 deletion on the pathogenicity of B. pseudomallei were studied by bacterial infection assays of HCM3 cells. Compared with the wild type, the tagAB-5 mutant exhibited defective pathogenic abilities in intracellular replication, multinucleated giant cell formation, and induction of cell damage. Additionally, infection by the tagAB-5 mutant elicited a decreased production of interleukin 8 (IL-8) in HCM3, suggesting that efficient pathogenicity of B. pseudomallei is required for IL-8 production in microglia. However, no significant differences in virulence in the Galleria mellonella model were observed between the tagAB-5 mutant and the wild type. Taken together, this study indicated that microglia might be an important intracellular niche for B. pseudomallei, particularly in CNS infection, and TagAB-5 confers B. pseudomallei pathogenicity in these cells.

In Vitro Roles of Burkholderia Intracellular Motility A (BimA) in Infection of Human Neuroblastoma Cell Line.

The bacterial pathogen Burkholderia pseudomallei causes human melioidosis, which can infect the brain, leading to encephalitis and brain abscesses. Infection of the nervous system is a rare condition but is associated with an increased risk of mortality. Burkholderia intracellular motility A (BimA) was reported to play an important role in the invasion and infection of the central nervous system in a mouse model. Thus, to gain insight of the cellular mechanisms underlying the pathogenesis of neurological melioidosis, we explored the human neuronal proteomics to identify the host factors that are up- and downregulated during Burkholderia infection. When infected the SH-SY5Y cells with B. pseudomallei K96243 wild-type (WT), 194 host proteins showed a fold change of >2 compared with uninfected cells. Moreover, 123 proteins showed a fold change of >2 when infected with a knockout bimA mutant (ΔbimA) mutant compared with WT. The differentially expressed proteins were mainly associated with metabolic pathways and pathways linked to human diseases. Importantly, we observed the downregulation of proteins in the apoptosis and cytotoxicity pathway, and in vitro investigation with the ΔbimA mutant revealed the association of BimA with the induction of these pathways. Additionally, we disclosed that BimA was not required for invasion into the neuron cell line but was necessary for effective intracellular replication and multinucleated giant cell (MNGC) formation. These findings show the extraordinary capacity of B. pseudomallei in subverting and interfering with host cellular systems to establish infection and extend our understanding of B. pseudomallei BimA involvement in the pathogenesis of neurological melioidosis. IMPORTANCE Neurological melioidosis, caused by Burkholderia pseudomallei, can result in severe neurological damage and enhance the mortality rate of melioidosis patients. We investigate the involvement of the virulent factor BimA, which mediates actin-based motility, in the intracellular infection of neuroblastoma SH-SY5Y cells. Using proteomics-based analysis, we provide a list of host factors exploited by B. pseudomallei. The expression level of selected downregulated proteins in neuron cells infected with the ΔbimA mutant was determined by quantitative reverse transcription-PCR and was consistent with our proteomic data. The role of BimA in the apoptosis and cytotoxicity of SH-SY5Y cells infected by B. pseudomallei was uncovered in this study. Additionally, our research demonstrates that BimA is required for successful intracellular survival and cell fusion upon infection of neuron cells. Our findings have significant implications for understanding the pathogenesis of B. pseudomallei infections and developing novel therapeutic strategies to combat this deadly disease.

Cycle-Inhibiting Factor Is Associated with Burkholderia pseudomallei Invasion in Human Neuronal Cells.

Burkholderia pseudomallei is a pathogenic bacterium that causes human melioidosis, which is associated with a high mortality rate. However, the underlying mechanisms of B. pseudomallei pathogenesis are largely unknown. In this study, we examined the infection of human neuronal SH-Sy5y cells by several clinically relevant B. pseudomallei strains. We found that all tested B. pseudomallei strains can invade SH-Sy5y cells, undergo intracellular replication, cause actin-tail formation, and form multinucleated giant cells. Additionally, a deletion mutant of B. pseudomallei cycle-inhibiting factor (cif) was constructed that exhibited reduced invasion in SH-Sy5y cells. Complementation of cif restored invasion of the B. pseudomallei cif-deleted mutant. Our findings enhance understanding of B. pseudomallei pathogenicity in terms of the virulence factor Cif and demonstrate the function of Cif in neurological melioidosis. This may eventually lead to the discovery of novel targets for treatment and a strategy to control the disease.

Burkholderia pseudomallei pathogenesis in human skin fibroblasts: A Bsa type III secretion system is involved in the invasion, multinucleated giant cell formation, and cellular damage.

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.

Identification of Burkholderia pseudomallei Genes Induced During Infection of Macrophages by Differential Fluorescence Induction.

Burkholderia pseudomallei, the causative agent of melioidosis, can survive and replicate in macrophages. Little is known about B. pseudomallei genes that are induced during macrophage infection. We constructed a B. pseudomallei K96243 promoter trap library with genomic DNA fragments fused to the 5' end of a plasmid-borne gene encoding enhanced green fluorescent protein (eGFP). Microarray analysis showed that the library spanned 88% of the B. pseudomallei genome. The recombinant plasmids were introduced into Burkholderia thailandensis E264, and promoter fusions active during in vitro culture were removed. J774A.1 murine macrophages were infected with the promoter trap library, and J774A.1 cells containing fluorescent bacteria carrying plasmids with active promoters were isolated using flow cytometric-based cell sorting. Candidate macrophage-induced B. pseudomallei genes were identified from the location of the insertions containing an active promoter activity. A proportion of the 138 genes identified in this way have been previously reported to be involved in metabolism and transport, virulence, or adaptation. Novel macrophage-induced B. pseudomallei genes were also identified. Quantitative reverse-transcription PCR analysis of 13 selected genes confirmed gene induction during macrophage infection. Deletion mutants of two macrophage-induced genes from this study were attenuated in Galleria mellonella larvae, suggesting roles in virulence. B. pseudomallei genes activated during macrophage infection may contribute to intracellular life and pathogenesis and merit further investigation toward control strategies for melioidosis.

Inactivation of bpsl1039-1040 ATP-binding cassette transporter reduces intracellular survival in macrophages, biofilm formation and virulence in the murine model of Burkholderia pseudomallei infection.

Burkholderia pseudomallei, a gram-negative intracellular bacillus, is the causative agent of a tropical infectious disease called melioidosis. Bacterial ATP-binding cassette (ABC) transporters import and export a variety of molecules across bacterial cell membranes. At present, their significance in B. pseudomallei pathogenesis is poorly understood. We report here characterization of the BPSL1039-1040 ABC transporter. B. pseudomallei cultured in M9 medium supplemented with nitrate, demonstrated that BPSL1039-1040 is involved in nitrate transport for B. pseudomallei growth under anaerobic, but not aerobic conditions, suggesting that BPSL1039-1040 is functional under reduced oxygen tension. In addition, a nitrate reduction assay supported the function of BPSL1039-1040 as nitrate importer. A bpsl1039-1040 deficient mutant showed reduced biofilm formation as compared with the wild-type strain (P = 0.027) when cultured in LB medium supplemented with nitrate under anaerobic growth conditions. This reduction was not noticeable under aerobic conditions. This suggests that a gradient in oxygen levels could regulate the function of BPSL1039-1040 in B. pseudomallei nitrate metabolism. Furthermore, the B. pseudomallei bpsl1039-1040 mutant had a pronounced effect on plaque formation (P < 0.001), and was defective in intracellular survival in both non-phagocytic (HeLa) and phagocytic (J774A.1 macrophage) cells, suggesting reduced virulence in the mutant strain. The bpsl1039-1040 mutant was found to be attenuated in a BALB/c mouse intranasal infection model. Complementation of the bpsl1039-1040 deficient mutant with the plasmid-borne bpsl1039 gene could restore the phenotypes observed. We propose that the ability to acquire nitrate for survival under anaerobic conditions may, at least in part, be important for intracellular survival and has a contributory role in the pathogenesis of B. pseudomallei.

Identification of Candidate Host Cell Factors Required for Actin-Based Motility of Burkholderia pseudomallei.

Intracellular actin-based motility of the melioidosis pathogen Burkholderia pseudomallei requires the bacterial factor BimA. Located at one pole of the bacterium, BimA recruits and polymerizes cellular actin to promote bacterial motility within and between cells. Here, we describe an affinity approach coupled with mass spectrometry to identify cellular proteins recruited to BimA-expressing bacteria under conditions that promote actin polymerization. We identified a group of cellular proteins that are recruited to the B. pseudomallei surface in a BimA-dependent manner, a subset of which were independently validated with specific antisera including the ubiquitous scaffold protein Ras GTPase-activating-like protein (IQGAP1). IQGAP1 integrates several key cellular signaling pathways including those involved in actin dynamics and has been shown to be involved in the adhesion of attaching and effacing Escherichia coli to infected cells and invasion of host cells by Salmonella enterica serovar Typhimurium. Although a direct interaction between BimA and IQGAP1 could not be detected using either conventional pulldown or yeast two hybrid techniques, confocal microscopy revealed that IQGAP1 is recruited to B. pseudomallei actin tails in infected cells, and siRNA-mediated knockdown highlighted a role for this protein in controlling the length and actin density of B. pseudomallei actin tails.