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Mitophagy is critical for mitochondrial quality control and function to clear damaged mitochondria. Here, we found that Burkholderia pseudomallei maneuvered host mitophagy for its intracellular survival through the type III secretion system needle tip protein BipD. We identified BipD, interacting with BTB-containing proteins KLHL9 and KLHL13 by binding to the Back and Kelch domains, recruited NEDD8 family RING E3 ligase CUL3 in response to B. pseudomallei infection. Although evidently not involved in regulation of infectious diseases, KLHL9/KLHL13/CUL3 E3 ligase complex was essential for BipD-dependent ubiquitination of mitochondria in mouse macrophages. Mechanistically, we discovered the inner mitochondrial membrane IMMT via host ubiquitome profiling as a substrate of KLHL9/KLHL13/CUL3 complex. Notably, K63-linked ubiquitination of IMMT K211 was required for initiating host mitophagy, thereby reducing mitochondrial ROS production. Here, we show a unique mechanism used by bacterial pathogens that hijacks host mitophagy for their survival. The selective degradation of mitochondria by autophagy is essential for cell homeostasis. Here, Nan et al show that the effector protein BipD, secreted by Burkholderia pseudomallei , is able to modulate host mitophagy to promote its survival.

is a genus within the β that contains at least 90 validly named species which can be found in a diverse range of environments. A number of pathogenic species occur within the genus. These include and , opportunistic pathogens that can infect the lungs of patients with cystic fibrosis, and are members of the complex (Bcc). is also an opportunistic pathogen, but in contrast to Bcc species it causes the tropical human disease melioidosis, while its close relative is the causative agent of glanders in horses. For these pathogens to survive within a host and cause disease they must be able to acquire iron. This chemical element is essential for nearly all living organisms due to its important role in many enzymes and metabolic processes. In the mammalian host, the amount of accessible free iron is negligible due to the low solubility of the metal ion in its higher oxidation state and the tight binding of this element by host proteins such as ferritin and lactoferrin. As with other pathogenic bacteria, species have evolved an array of iron acquisition mechanisms with which to capture iron from the host environment. These mechanisms include the production and utilization of siderophores and the possession of a haem uptake system. Here, we summarize the known mechanisms of iron acquisition in pathogenic species and discuss the evidence for their importance in the context of virulence and the establishment of infection in the host. We have also carried out an extensive bioinformatic analysis to identify which siderophores are produced by each species that is pathogenic to humans.

Burkholderia pseudomallei is a saprophytic bacterium endemic throughout the tropics causing severe disease in humans and animals. Environmental signals such as the accumulation of inorganic ions mediates the biofilm forming capabilities and survival of B. pseudomallei . We have previously shown that B. pseudomallei responds to nitrate and nitrite by inhibiting biofilm formation and altering cyclic di-GMP signaling. To better understand the roles of nitrate-sensing in the biofilm inhibitory phenotype of B. pseudomallei , we created in-frame deletions of narX (Bp1026b_I1014) and narL (Bp1026b_I1013), which are adjacent components of a conserved nitrate-sensing two-component system. We observed transcriptional downregulation in key components of the biofilm matrix in response to nitrate and nitrite. Some of the most differentially expressed genes were nonribosomal peptide synthases (NRPS) and/or polyketide synthases (PKS) encoding the proteins for the biosynthesis of bactobolin, malleilactone, and syrbactin, and an uncharacterized cryptic NRPS biosynthetic cluster. RNA expression patterns were reversed in ∆ narX and ∆ narL mutants, suggesting that nitrate sensing is an important checkpoint for regulating the diverse metabolic changes occurring in the biofilm inhibitory phenotype. Moreover, in a macrophage model of infection, ∆ narX and ∆ narL mutants were attenuated in intracellular replication, suggesting that nitrate sensing contributes to survival in the host.

Melioidosis is a life-threatening tropical disease caused by an intracellular gram-negative bacterium Burkholderia pseudomallei . B. pseudomallei polymerizes the host cell actin through autotransporters, BimA, and BimC, to facilitate intracellular motility. Two variations of BimA in B. pseudomallei have been reported previously: BimA Bp and BimA B. mallei -like (BimA Bm ). However, little is known about genetic sequence variations within BimA and BimC, and their potential effect on the virulence of B. pseudomallei . This study analyzed 1,294 genomes from clinical isolates of patients admitted to nine hospitals in northeast Thailand between 2015 and 2018 and performed 3D structural analysis and plaque-forming efficiency assay. The genomic analysis identified 10 BimA Bp and 5 major BimC types, in the dominant and non-dominant lineages of the B. pseudomallei population structure. Our protein prediction analysis of all BimA Bp and major BimC variants revealed that their 3D structures were conserved compared to those of B. pseudomallei K96243. Sixteen representative strains of the most distant BimA Bp types were tested for plaque formation and the development of polar actin tails in A549 epithelial cells. We found that all isolates retained these functions. These findings enhance our understanding of the prevalence of BimA Bp and BimC variants and their implications for B. pseudomallei virulence.

Single-particle cryo-electron microscopy (cryo-EM) has become a powerful technique in the field of structural biology. However, the inability to reliably produce pure, homogeneous membrane protein samples hampers the progress of their structural determination. Here, we develop a bottom-up iterative method, Build and Retrieve (BaR), that enables the identification and determination of cryo-EM structures of a variety of inner and outer membrane proteins, including membrane protein complexes of different sizes and dimensions, from a heterogeneous, impure protein sample. We also use the BaR methodology to elucidate structural information from Escherichia coli K12 crude membrane and raw lysate. The findings demonstrate that it is possible to solve high-resolution structures of a number of relatively small (<100 kDa) and less abundant (<10%) unidentified membrane proteins within a single, heterogeneous sample. Importantly, these results highlight the potential of cryo-EM for systems structural proteomics.The iterative Build and Retrieve (BaR) methodology facilitates the solving of cryo-EM structures of multiple membrane (and soluble) proteins simultaneously, including small and low-abundance membrane proteins.

Abstract Bacterial secondary metabolites play important roles in promoting survival, though few have been carefully studied in their natural context. Numerous gene clusters code for secondary metabolites in the genomes of members of the Bptm group, made up of three closely related species with distinctly different lifestyles: the opportunistic pathogen Burkholderia pseudomallei, the non-pathogenic saprophyte Burkholderia thailandensis, and the host-adapted pathogen Burkholderia mallei. Several biosynthetic gene clusters are conserved across two or all three species, and this provides an opportunity to understand how the corresponding secondary metabolites contribute to survival in different contexts in nature. In this review, we discuss three secondary metabolites from the Bptm group: bactobolin, malleilactone (and malleicyprol), and the 4-hydroxy-3-methyl-2-alkylquinolines, providing an overview of each of their biosynthetic pathways and insight into their potential ecological roles. Results of studies on these secondary metabolites provide a window into how secondary metabolites contribute to bacterial survival in different environments, from host infections to polymicrobial soil communities.

Bacterial efflux pumps are an important pathogenicity trait because they extrude a variety of xenobiotics. Our laboratory previously identified in silico Burkholderia collagen-like protein 8 (Bucl8) in the hazardous pathogens Burkholderia pseudomallei and Burkholderia mallei . We hypothesize that Bucl8, which contains two predicted tandem outer membrane efflux pump domains, is a component of a putative efflux pump. Unique to Bucl8, as compared to other outer membrane proteins, is the presence of an extended extracellular region containing a collagen-like (CL) domain and a non-collagenous C-terminus (Ct). Molecular modeling and circular dichroism spectroscopy with a recombinant protein, corresponding to this extracellular CL-Ct portion of Bucl8, demonstrated that it adopts a collagen triple helix, whereas functional assays screening for Bucl8 ligands identified binding to fibrinogen. Bioinformatic analysis of the bucl8 gene locus revealed it resembles a classical efflux-pump operon. The bucl8 gene is co-localized with downstream fusCDE genes encoding fusaric acid (FA) resistance, and with an upstream gene, designated as fusR , encoding a LysR-type transcriptional regulator. Using reverse transcriptase (RT)-qPCR, we defined the boundaries and transcriptional organization of the fusR-bucl8-fusCDE operon. We found exogenous FA induced bucl8 transcription over 80-fold in B . pseudomallei , while deletion of the entire bucl8 locus decreased the minimum inhibitory concentration of FA 4-fold in its isogenic mutant. We furthermore showed that the putative Bucl8-associated pump expressed in the heterologous Escherichia coli host confers FA resistance. On the contrary, the Bucl8-associated pump did not confer resistance to a panel of clinically-relevant antimicrobials in Burkholderia and E . coli . We finally demonstrated that deletion of the bucl8 -locus drastically affects the growth of the mutant in L-broth. We determined that Bucl8 is a component of a novel tetrapartite efflux pump, which confers FA resistance, fibrinogen binding, and optimal growth.

A family of bacterial effectors including Cif homolog from Burkholderia pseudomallei (CHBP) and Cif from Enteropathogenic Escherichia coli (EPEC) adopt a functionally important papain-like hydrolytic fold. We show here that CHBP was a potent inhibitor of the eukaryotic ubiquitination pathway. CHBP acted as a deamidase that specifically and efficiently deamidated Gln⁴⁰ in ubiquitin and ubiquitin-like protein NEDD8 both in vitro and during Burkholderia infection. Deamidated ubiquitin was impaired in supporting ubiquitin-chain synthesis. Cif selectively deamidated NEDD8, which abolished the activity of neddylated Cullin-RING ubiquitin ligases (CRLs). Ubiquitination and ubiquitin-dependent degradation of multiple CRL substrates were impaired by Cif in EPEC-infected cells. Mutations of substrate-contacting residues in Cif abolished or attenuated EPEC-induced cytopathic phenotypes of cell cycle arrest and actin stress fiber formation.

Melioidosis, a febrile illness with disease states ranging from acute pneumonia or septicaemia to chronic abscesses, was first documented by Whitmore '' Krishnaswami (1912) . The causative agent, Burkholderia pseudomallei, was subsequently identified as a motile, gram-negative bacillus, which is principally an environmental saprophyte. Melioidosis has become an increasingly important disease in endemic areas such as northern Thailand and Australia ( Currie et al., 2000 ). This health burden, plus the classification of B. pseudomallei as a category B biological agent ( Rotz et al., 2002 ), has resulted in an escalation of research interest. This review focuses on the molecular and cellular basis of pathogenesis in melioidosis, with a comprehensive overview of the current knowledge on how B. pseudomallei can cause disease. The process of B. pseudomallei movement from the environmental reservoir to attachment and invasion of epithelial and macrophage cells and the subsequent intracellular survival and spread is outlined. Furthermore, the diverse assortment of virulence factors that allow B. pseudomallei to become an effective opportunistic pathogen, as well as to avoid or subvert the host immune response, is discussed. With the recent increase in genomic and molecular studies, the current understanding of the infection process of melioidosis has increased substantially, yet, much still remains to be elucidated.

Melioidosis is a fatal infection caused by the soil saprophyte Burkholderia pseudomallei. Early diagnosis and befitting medical management can significantly influence the clinical outcomes among patients with melioidosis. Witnessing an annual increment in the number of melioidosis cases, over the past few years, mainly from the developing tropical nations, the present study was undertaken to evaluate the diagnostic utility of Active Melioidosis DetectTMLateralFlow Assay (AMD-LFA), in comparison with enrichment culture and PCR. A total of 206clinical specimens obtained from 175 patients with clinical suspicion of melioidosis were considered for the evaluation. Positivity for B.pseudomallei using enrichment culture, PCR and AMD-LFA were observed among 63 (30.5%), 55 (26.6%) and 63 (30.5%) specimens respectively. The AMD-LFA failed to detect melioidosis from 9 culture-confirmed cases (6 whole blood specimens, 2 pus samples, and one synovial fluid). Further the test gave faint bands from 9 urine samples which were negative by culture and PCR. AMD-LFA demonstrated a sensitivity, specificity, of 85.71%(CI:74.61% to 93.25%) and 93.62% (CI:88.23% to 97.04%), with positive predictive value of 85.71% (CI: 75.98% to 91.92%) and negative predictive value of 93.62% (CI:88.89% to 96.42%). The test needs further evaluation in view of the faint bands from negative urine samples, for incorporating the test as a point of care assay.In view of its rapidity and ease of testing AMD-LFA might be useful in early diagnosis of melioidosis at resource constraint settings.