Tetraspanins are involved in Burkholderia pseudomallei-induced cell-to-cell fusion of phagocytic and non-phagocytic cells

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

Burkholderia pseudomallei Is Genetically Diverse in Agricultural Land in Northeast Thailand

Burkholderia pseudomallei is the cause of melioidosis, a serious human infection most commonly diagnosed in southeast Asia and northern Australia. The organism lives in the soil in a specific geographical distribution and infection results from bacterial inoculation, inhalation or ingestion. The purpose of this study was to define the distribution and genetic diversity of B. pseudomallei in agricultural land where most human infections probably occur. We performed soil sampling and culture for the presence of B. pseudomallei in 100 equally spaced points within a rice paddy in northeast Thailand, and undertook genotyping of primary culture plate colonies from 11 sampling points. We identified 7 different genotypes, with relatively limited overlap between different sampling points. Two samples contained more than one B. pseudomallei genotype, in which a numerically dominant genotype coexisted with one or more additional genotypes present as a minority population. We conclude that genetic diversity and structuring of B. pseudomallei exists despite the effects of flooding and the physical and chemical processes associated with farming. These findings inform future efforts to define B. pseudomallei in the environment, and should be considered during the design stage of studies comparing B. pseudomallei isolated from the environment and from patients with invasive disease

Burkholderia pseudomallei Is Spatially Distributed in Soil in Northeast Thailand

Melioidosis is a severe infection caused by the environmental bacterium Burkholderia pseudomallei. Soil sampling is important to identify geographic regions where humans and animals are at risk of exposure. The purpose of this study was to examine a factor that has a major bearing on the accuracy of soil sampling: the spatial distribution of B. pseudomallei in soil of a specified sampling site. Soil sampling was performed using a fixed-interval grid of 100 sampling points in each of two sites (disused land and rice field) in northeast Thailand, and the presence and amount of B. pseudomallei determined using culture. Mapping of the presence and B. pseudomallei count demonstrated that samples taken from areas adjacent to sampling points that were culture positive (negative) for B. pseudomallei were also likely to be culture positive (negative), and samples taken from areas adjacent to sampling points with a high (low) B. pseudomallei count were also likely to yield a high (low) count (spatial autocorrelation). These data were used as the basis for highlighting several pitfalls in current approaches to soil sampling, together with a discussion of the suitability of a range of sampling strategies in different geographical locations and for different study objectives

Comparison of community-onset Staphylococcus argenteus and Staphylococcus aureus sepsis in Thailand: a prospective multicentre observational study.

Staphylococcus argenteus is a globally distributed cause of human infection, but diagnostic laboratories misidentify this as Staphylococcus aureus. We determined whether there is clinical utility in distinguishing between the two. A prospective cohort study of community-onset invasive staphylococcal sepsis was conducted in adults at four hospitals in northeast Thailand between 2010 and 2013. Of 311 patients analysed, 58 (19%) were infected with S. argenteus and 253 (81%) with S. aureus. Most S. argenteus (54/58) were multilocus sequence type 2250. Infection with S. argenteus was more common in males, but rates of bacteraemia and drainage procedures were similar in the two groups. S. argenteus precipitated significantly less respiratory failure than S. aureus (5.2% versus 20.2%, adjusted OR 0.21, 95% CI 0.06-0.74, p 0.015), with a similar but non-significant trend for shock (6.9% versus 12.3%, adjusted OR 0.46, 95% CI 0.15-1.44, p 0.18). This did not translate into a difference in death at 28 days (6.9% versus 8.7%, adjusted OR 0.80, 95% CI 0.24-2.65, p 0.72). S. argenteus was more susceptible to antimicrobial drugs compared with S. aureus, and contained fewer toxin genes although pvl was detected in 16% (9/58). We conclude that clinical differences exist in association with sepsis due to S. argenteus versus S. aureus

Abundance of ACVR1B transcript is elevated during septic conditions: perspectives obtained from a hands-on reductionist investigation

Sepsis is a complex heterogeneous condition, and the current lack of effective risk and outcome predictors hinders the improvement of its management. Using a reductionist approach leveraging publicly available transcriptomic data, we describe a knowledge gap for the role of ACVR1B (activin A receptor type 1B) in sepsis. ACVR1B, a member of the transforming growth factor-beta (TGF-beta) superfamily, was selected based on the following: 1) induction upon in vitro exposure of neutrophils from healthy subjects with the serum of septic patients (GSE49755), and 2) absence or minimal overlap between ACVR1B, sepsis, inflammation, or neutrophil in published literature. Moreover, ACVR1B expression is upregulated in septic melioidosis, a widespread cause of fatal sepsis in the tropics. Key biological concepts extracted from a series of PubMed queries established indirect links between ACVR1B and "cancer", "TGF-beta superfamily", "cell proliferation", "inhibitors of activin", and "apoptosis". We confirmed our observations by measuring ACVR1B transcript abundance in buffy coat samples obtained from healthy individuals (n=3) exposed to septic plasma (n = 26 melioidosis sepsis cases)ex vivo. Based on our re-investigation of publicly available transcriptomic data and newly generated ex vivo data, we provide perspective on the role of ACVR1B during sepsis. Additional experiments for addressing this knowledge gap are discussed

Plasma metabolomics reveals distinct biological and diagnostic signatures for melioidosis

Rationale: The global burden of sepsis is greatest in low-resource settings. Melioidosis, infection with the Gram-negative bacterium Burkholderia pseudomallei, is a frequent cause of fatal sepsis in endemic tropical regions such as Southeast Asia. Objectives: To investigate whether plasma metabolomics would identify biological pathways specific to melioidosis and yield clinically meaningful biomarkers. Methods: Using a comprehensive approach, differential enrichment of plasma metabolites and pathways were systematically evaluated in patients from a prospective cohort of individuals hospitalized in rural Thailand with infection. Statistical and bioinformatics methods were used to distinguish metabolomic features and processes specific to melioidosis patients, and between fatal and non-fatal cases. Measurements and Main Results: Metabolomic profiling and pathway enrichment analysis of plasma samples of melioidosis (n=175) and non-melioidosis infections (n=75) revealed a distinct immuno-metabolic state among patients with melioidosis, as suggested by excessive tryptophan catabolism in the kynurenine pathway and significantly increased lipid metabolism such as sphingomyelins and ceramide species. We derived a 12-metabolite classifier to distinguish melioidosis from other infections, with an area under the receiver operating characteristic curve of 0.87 in a second validation set of patients. Melioidosis non-survivors (n=94) had a significantly disturbed metabolome compared to survivors (n=81) with increased leucine, isoleucine and valine metabolism, and elevated circulating free fatty acids and acylcarnitines. A limited 8-metabolite panel shows promise as an early prognosticator of mortality in melioidosis. Conclusions: Melioidosis induces a distinct metabolomic state that can be leveraged to distinguish underlying pathophysiological mechanisms leading to increased risk of death. A twelve-metabolite signature accurately differentiates melioidosis from other infections and may have diagnostic applications. This article is open access and distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/

TLR4 genetic variation is associated with inflammatory responses in Gram-positive sepsis.

OBJECTIVES: To identify important pathogen recognition receptor (PRR) pathways regulating innate immune responses and outcome in Staphylococcus aureus sepsis. METHODS: We analysed whether candidate PRR pathway genetic variants were associated with killed S. aureus-induced cytokine responses ex vivo and performed follow-up in vitro studies. We tested the association of our top-ranked variant with cytokine responses and clinical outcomes in a prospective multicentre cohort of patients with staphylococcal sepsis. RESULTS: An intronic TLR4 polymorphism and expression quantitative trait locus, rs1927907, was highly associated with cytokine release induced by stimulation of blood from healthy Thai subjects with S. aureus ex vivo. S. aureus did not induce TLR4-dependent NF-κB activation in transfected HEK293 cells. In monocytes, tumor necrosis factor (TNF)-α release induced by S. aureus was not blunted by a TLR4/MD-2 neutralizing antibody, but in a monocyte cell line, TNF-α was reduced by knockdown of TLR4. In Thai patients with staphylococcal sepsis, rs1927907 was associated with higher interleukin (IL)-6 and IL-8 levels as well as with respiratory failure. S. aureus-induced responses in blood were most highly correlated with responses to Gram-negative stimulants whole blood. CONCLUSIONS: A genetic variant in TLR4 is associated with cytokine responses to S. aureus ex vivo and plasma cytokine levels and respiratory failure in staphylococcal sepsis. While S. aureus does not express lipopolysaccharide or activate TLR4 directly, the innate immune response to S. aureus does appear to be modulated by TLR4 and shares significant commonality with that induced by Gram-negative pathogens and lipopolysaccharide

Identification of Burkholderia cepacia strains that express a Burkholderia pseudomallei-like capsular polysaccharide

Burkholderia pseudomallei and Burkholderia cepacia are Gram-negative, soil-dwelling bacteria that are found in a wide variety of environmental niches. While B. pseudomallei is the causative agent of melioidosis in humans and animals, members of the B. cepacia complex typically only cause disease in immunocompromised hosts. In this study, we report the identification of B. cepacia strains isolated from either patients or soil in Laos and Thailand that express a B. pseudomallei-like 6-deoxyheptan capsular polysaccharide (CPS). These B. cepacia strains were initially identified based on their positive reactivity in a latex agglutination assay that uses the CPS-specific monoclonal antibody (mAb) 4B11. Mass spectrometry and recA sequencing confirmed the identity of these isolates as B. cepacia (formerly genomovar I). Total carbohydrates extracted from B. cepacia cell pellets reacted with B. pseudomallei CPS-specific mAbs MCA147, 3C5, and 4C4, but did not react with the B. pseudomallei lipopolysaccharide-specific mAb Pp-PS-W. Whole genome sequencing of the B. cepacia isolates revealed the presence of genes demonstrating significant homology to those comprising the B. pseudomallei CPS biosynthetic gene cluster. Collectively, our results provide compelling evidence that B. cepacia strains expressing the same CPS as B. pseudomallei co-exist in the environment alongside B. pseudomallei. Since CPS is a target that is often used for presumptive identification of B. pseudomallei, it is possible that the occurrence of these unique B. cepacia strains may complicate the diagnosis of melioidosis

Development of a prototype lateral flow immunoassay (LFI) for the rapid diagnosis of melioidosis.

Burkholderia pseudomallei is a soil-dwelling bacterium and the causative agent of melioidosis. Isolation of B. pseudomallei from clinical samples is the "gold standard" for the diagnosis of melioidosis; results can take 3-7 days to produce. Alternatively, antibody-based tests have low specificity due to a high percentage of seropositive individuals in endemic areas. There is a clear need to develop a rapid point-of-care antigen detection assay for the diagnosis of melioidosis. Previously, we employed In vivo Microbial Antigen Discovery (InMAD) to identify potential B. pseudomallei diagnostic biomarkers. The B. pseudomallei capsular polysaccharide (CPS) and numerous protein antigens were identified as potential candidates. Here, we describe the development of a diagnostic immunoassay based on the detection of CPS. Following production of a CPS-specific monoclonal antibody (mAb), an antigen-capture immunoassay was developed to determine the concentration of CPS within a panel of melioidosis patient serum and urine samples. The same mAb was used to produce a prototype Active Melioidosis Detect Lateral Flow Immunoassay (AMD LFI); the limit of detection of the LFI for CPS is comparable to the antigen-capture immunoassay (∼0.2 ng/ml). The analytical reactivity (inclusivity) of the AMD LFI was 98.7% (76/77) when tested against a large panel of B. pseudomallei isolates. Analytical specificity (cross-reactivity) testing determined that 97.2% of B. pseudomallei near neighbor species (35/36) were not reactive. The non-reactive B. pseudomallei strain and the reactive near neighbor strain can be explained through genetic sequence analysis. Importantly, we show the AMD LFI is capable of detecting CPS in a variety of patient samples. The LFI is currently being evaluated in Thailand and Australia; the focus is to optimize and validate testing procedures on melioidosis patient samples prior to initiation of a large, multisite pre-clinical evaluation

Genetic Diversity and Microevolution of Burkholderia pseudomallei in the Environment

The soil dwelling Gram-negative bacterium Burkholderia pseudomallei is the cause of melioidosis, a serious human infection that occurs in Southeast Asia and northern Australia. The purpose of this study was to evaluate the population genetic structure of B. pseudomallei in the environment. To achieve this, we undertook soil sampling and culture for the presence of B. pseudomallei in 100 equally spaced points within an area of disused land in northeast Thailand, and undertook detailed genotyping of primary plate colonies isolated from three independent sampling points. Our results demonstrated that multiple B. pseudomallei genotypes were present within a single soil sample, and that different genotypes were present at independent but nearby sampling points. The B. pseudomallei genetic population was unevenly distributed within a given sample, with a predominant genotype co-existing with several genotypes present as a minority population. We discuss the implications of this structuring of genotypic frequency in terms of micro-evolutionary dynamics and ecology, and how our results may inform future sampling strategies