Burkholderia pseudomallei BipD modulates host mitophagy to evade killing.

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.

In vitro passage alters virulence, immune activation and proteomic profiles of Burkholderia pseudomallei.

Serial passage is a problem among many bacterial species, especially those where strains have been stored (banked) for several decades. Prior to banking with an organization such as ATCC, many bacterial strains were passaged for many years, so the characteristics of each strain may be extremely different. This is in addition to any differences in the original host environment. For Burkholderia pseudomallei, the number of serial passages should be carefully defined for each experiment because it undergoes adaptation during the course of serial passages. In the present study, we found that passaged B. pseudomallei fresh clinical isolates and reference strain in Luria-Bertani broth exhibited increased plaque formation, invasion, intracellular replication, Galleria mellonella killing abilities, and cytokine production of host cells. These bacteria also modulated proteomic profiles during in vitro passage. We presume that the modulation of protein expression during in vitro passage caused changes in virulence and immunogenicity phenotypes. Therefore, we emphasize the need for caution regarding the use of data from passaged B. pseudomallei. These findings of phenotypic adaptation during in vitro serial passage can help researchers working on B. pseudomallei and on other species to better understand disparate findings among strains that have been reported for many years.

Dysregulation of TNF-α and IFN-γ expression is a common host immune response in a chronically infected mouse model of melioidosis when comparing multiple human strains of Burkholderia pseudomallei.

BACKGROUND: Melioidosis is endemic in Southeast Asia and Northern Australia and is caused by the Gram-negative, facultative intracellular pathogen Burkholderia pseudomallei. Diagnosis of melioidosis is often difficult because of the protean clinical presentation of the disease, and it may mimic other diseases, such as tuberculosis. There are many different strains of B. pseudomallei that have been isolated from patients with melioidosis, but it was not clear if they could cause a similar disease in a chronic BALB/c murine model of melioidosis. Hence, we wanted to examine chronically infected mice exposed to different strains of B. pseudomallei to determine if there were differences in the host immune response to the pathogen. RESULTS: We identified common host immune responses exhibited in chronically infected BALB/c mice, although there was some heterogeneity in the host response in chronically infected mice after exposure to different strains of B. pseudomallei. They all displayed pyogranulomatous lesions in their spleens with a large influx of monocytes/macrophages, NK cells, and neutrophils identified by flow cytometry. Sera from chronically infected mice by ELISA exhibited elevated IgG titers to the pathogen, and we detected by Luminex micro-bead array technology a significant increase in the expression of inflammatory cytokines/chemokines, such as IFN-γ, IL-1α, IL-1ß, KC, and MIG. By immunohistochemical and in situ RNA hybridization analysis we found that the increased expression of proinflammatory cytokines (IL-1α, IL-1ß, TNF-α, IFN-γ) was confined primarily to the area with the pathogen within pyogranulomatous lesions. We also found that cultured splenocytes from chronically infected mice could express IFN-γ, TNF-α, and MIP-1α ex vivo without the need for additional exogenous stimulation. In addition by flow cytometry, we detected significant amounts of intracellular expression of TNF-α and IFN-γ without a protein transport blocker in monocytes/macrophages, NK cells, and neutrophils but not in CD4+ or CD8+ T cells in splenocytes from chronically infected mice. CONCLUSION: Taken together the common features we have identified in chronically infected mice when 10 different human clinical strains of B. pseudomallei were examined could serve as biomarkers when evaluating potential therapeutic agents in mice for the treatment of chronic melioidosis in humans.

D-LL-31 in combination with ceftazidime synergistically enhances bactericidal activity and biofilm destruction in Burkholderia pseudomallei.

Melioidosis is a severe disease caused by Burkholderia pseudomallei. The biofilm of B. pseudomallei acquires resistance to several antibiotics and may be related to relapse in melioidosis patients. Here, the killing activity of antimicrobial peptides (LL-37, LL-31) and the D-enantiomers (D-LL-37, D-LL-31) in combination with ceftazidime (CAZ) against B. pseudomallei 1026b, H777 and a biofilm mutant M10, derived from H777 grown under biofilm-stimulating conditions was observed. Using static conditions, D-LL-31 exhibited the strongest killing activity against the three isolates in a dose-dependent manner. IC50 values for D-LL-31 ranged from 1 to 6 µM, for isolates M10, H777, and 1026b, respectively. Moreover, D-LL-31 combined with CAZ synergistically decreased the IC50 values of the peptide and antibiotic and caused also disruption of biofilms of B. pseudomallei 1026b under flow conditions. Thus a combination of D-LL-31 and CAZ may enhance the efficacy of the currently used antibiotic treatments against B. pseudomallei.

Role of Toll-Like Receptor 5 (TLR5) in Experimental Melioidosis.

The Gram-negative intracellular pathogen Burkholderia pseudomallei is the causative agent of melioidosis, an important cause of sepsis in Southeast Asia. Recognition of pathogen-associated molecular patterns by Toll-like receptors (TLRs) is essential for an appropriate immune response during pathogen invasion. In patients with melioidosis, TLR5 is the most abundantly expressed TLR, and a hypofunctional TLR5 variant has been associated with improved survival. Here, we studied the functional role of TLR5 and its ligand flagellin in experimental melioidosis. First, we observed differential TLR5 expression in the pulmonary and hepatic compartments upon infection with B. pseudomallei Next, we found that B. pseudomallei-challenged TLR5-deficient (Tlr5-/- ) mice were more susceptible to infection than wild-type (WT) mice, as demonstrated by higher systemic bacterial loads, increased organ injury, and impaired survival. Lung bacterial loads were not different between the two groups. The phenotype was flagellin independent; no difference in in vivo virulence was observed for the flagellin-lacking mutant MM36 compared to the wild-type B. pseudomallei strain 1026b. Tlr5-/- mice showed a similar impaired antibacterial defense when infected with MM36 or 1026b. Ex vivo experiments showed that TLR5-deficient macrophages display markedly impaired phagocytosis of B. pseudomallei In conclusion, these data suggest that TLR5 deficiency has a detrimental flagellin-independent effect on the host response against pulmonary B. pseudomallei infection.

Modulation of bacterial virulence and fitness by host glutathione.

Glutathione is a low molecular weight thiol that is important for maintaining intracellular redox homeostasis. Some bacteria are able to import exogenous glutathione as a nutritional source and to counter oxidative stress. In cytosolic pathogens Burkholderia pseudomallei and Listeria monocytogenes, host glutathione regulates bacterial virulence. In B. pseudomallei, glutathione activates the membrane-bound histidine kinase sensor VirA that leads to activation of the Type VI Secretion System. In L. monocytogenes, host glutathione leads to the binding of bacterial glutathione to the master virulence regulator PrfA as an allosteric activator. Glutathione can also modulate virulence factors to control their activity by S-glutathionylation. Thus, host glutathione acts as a spacio-temporal cue for some pathogens to switch on their virulence programs at the right time and place.

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.

Adhesion and invasion attributes of Burkholderia pseudomallei are dependent on airway surface liquid and glucose concentrations in lung epithelial cells.

Physiological constituents in airway surface liquids (ASL) appear to impact the adherence and invasion potentials of Burkholderia pseudomallei contributing to recrudescent melioidosis. Here, we investigated the factors present in ASL that is likely to influence bacterial adhesion and invasion leading to improved understanding of bacterial pathogenesis. Six B. pseudomallei clinical isolates from different origins were used to investigate the ability of the bacteria to adhere and invade A549 human lung epithelial cells using a system that mimics the physiological ASL with different pH, NaCl, KCl, CaCl2 and glucose concentrations. These parameters resulted in markedly differential adherence and invasion abilities of B. pseudomallei to the lung epithelial cells. The concentration of 20 mM glucose dramatically increased adherence and invasion by increasing the rate of pili formation in depiliated bacteria. Glucose significantly increased adherence and invasion of B. pseudomallei to A549 cells, and presence of NaCl, KCl and CaCl2 markedly ablated the effect despite the presence of glucose. Our data established a link between glucose, enhanced adhesion and invasion potentials of B. pseudomallei, hinting increased susceptibility of individuals with diabetes mellitus to clinical melioidosis.

Burkholderia pseudomallei modulates host iron homeostasis to facilitate iron availability and intracellular survival.

BACKGROUND: The control over iron homeostasis is critical in host-pathogen-interaction. Iron plays not only multiple roles for bacterial growth and pathogenicity, but also for modulation of innate immune responses. Hepcidin is a key regulator of host iron metabolism triggering degradation of the iron exporter ferroportin. Although iron overload in humans is known to increase susceptibility to Burkholderia pseudomallei, it is unclear how the pathogen competes with the host for the metal during infection. This study aimed to investigate whether B. pseudomallei, the causative agent of melioidosis, modulates iron balance and how regulation of host cell iron content affects intracellular bacterial proliferation. PRINCIPAL FINDINGS: Upon infection of primary macrophages with B. pseudomallei, expression of ferroportin was downregulated resulting in higher iron availability within macrophages. Exogenous modification of iron export function by hepcidin or iron supplementation by ferric ammonium citrate led to increased intracellular iron pool stimulating B. pseudomallei growth, whereas the iron chelator deferoxamine reduced bacterial survival. Iron-loaded macrophages exhibited a lower expression of NADPH oxidase, iNOS, lipocalin 2, cytokines and activation of caspase-1. Infection of mice with the pathogen caused a diminished hepatic ferroportin expression, higher iron retention in the liver and lower iron levels in the serum (hypoferremia). In vivo administration of ferric ammonium citrate tended to promote the bacterial growth and inflammatory response, whereas limitation of iron availability significantly ameliorated bacterial clearance, attenuated serum cytokine levels and improved survival of infected mice. CONCLUSIONS: Our data indicate that modulation of the cellular iron balance is likely to be a strategy of B. pseudomallei to improve iron acquisition and to restrict antibacterial immune effector mechanisms and thereby to promote its intracellular growth. Moreover, we provide evidence that changes in host iron homeostasis can influence susceptibility to melioidosis, and suggest that iron chelating drugs might be an additional therapeutic option.

Entry, Intracellular Survival, and Multinucleated-Giant-Cell-Forming Activity of Burkholderia pseudomallei in Human Primary Phagocytic and Nonphagocytic Cells.

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.

Complement-activated vitronectin enhances the invasion of nonphagocytic cells by bacterial pathogens Burkholderia and Klebsiella.

Burkholderia pseudomallei is a serum-resistant Gram-negative bacterium capable of causing disseminated infections with metastatic complications. However, its interaction with nonphagocytic cells is poorly understood. We observed that exposure of B. pseudomallei and the closely related yet avirulent B. thailandensis to human plasma increased epithelial cell invasion by >20 fold. Enhanced invasion was primarily driven by a plasma factor, which required a functional complement cascade, but surprisingly, was downstream of C3 mediated opsonisation. Receptor blocking studies with RGD-domain containing peptide and αV ß3 blocking antibody identified complement-activated vitronectin as the factor facilitating this invasion. Plasma treatment led to the recruitment of vitronectin onto the bacterial surface, and its conversion into the active conformation. Activation of vitronectin, as well as increased invasion, required the complement pathway and was not observed in C3 or C5 depleted serum. The integrin inhibitor cilengitide, currently in clinical trials as an anti-angiogenesis agent, suppresses plasma-mediated Burkholderia invasion by ~95%, along with a downstream reduction in intracellular bacterial replication. We extend these findings to serum-resistant Klebsiella pneumoniae as well. Thus, the potential use of commercially available integrin inhibitors as anti-infective agents during selective bacterial infections should be explored.

Burkholderia pseudomallei-derived miR-3473 enhances NF-κB via targeting TRAF3 and is associated with different inflammatory responses compared to Burkholderia thailandensis in murine macrophages.

BACKGROUND: Burkholderia pseudomallei (Bp) is the causative agent of melioidosis, a kind of tropical disease. Burkholderia thailandensis (Bt), with a high sequence similarity to Bp, is thought to be an avirulent organism. Since there are numerous similarities between Bp and Bt, their differences in pathogenesis of host response and related mechanism are still undermined. In recent years, microRNAs have been researched in many diseases, but seldom involved in bacterial infection, bacteria-host interaction or explaining the differences between virulent and avirulent species. RESULTS: We found that Bp and Bt had similar phenotypes in terms of intracellular replication, dissemination (reflected by multinucleated giant cell formation), TNF-α release and apoptosis in RAW264.7 macrophages or TC-1 pulmonary cell but in different level. Especially, at the late infection phases (after 12 h post infection), Bp showed faster intracellular growth, stronger cytotoxicity, and higher TNF-α release. After microRNA array analysis, we found some microRNAs were significantly expressed in macrophages treated by Bp. miR-3473 was one of them specifically induced, but not significantly changed in Bt-treated macrophages. In addition, TargetScan suggested that miR-3473 possibly target TRAF3 (TNF receptor-associated factor 3), a well-known negative regulator of the NF-κB pathway, which was probably involved in the TNF-α induction and apoptosis in cells with Bp infection. In vivo, it was found that miR-3473 expression of total lungs cells from Bp-treated was higher than that from Bt-treated mice. And miR-3473 inhibitor was able to decrease the TNF-α release of mice and prolong the survival of mice with Bp infection. CONCLUSION: In sum, miR-3473 plays an important role in the differential pathogenicity of Bp and Bt via miR-3473-TRAF3-TNF-α network, and regulates TNF-α release, cell apoptosis and animal survival after Bp treatment. In this study, we have found a specific microRNA is related to bacterial virulence and provide insight into the mechanism for host-bacteria interaction, which suggests that potential oligonucleotides should be applied against bacterial infection.

Role of Canonical and Non-canonical Inflammasomes During Burkholderia Infection.

Burkholderia pseudomallei is a Gram-negative flagellate bacterium that causes melioidosis, a disease endemic to Southeast Asia and other tropical regions. Following infection of macrophages and other non-phagocytic cell types, B. pseudomallei or B. thailandensis (a related species that causes disease in mice but not humans) are able to escape the phagosome and replicate in the host cell cytoplasm. Resistance to infection with Burkholderia is dependent on the Nlrp3 and Nlrc4 inflammasomes and the non-canonical caspase-11 inflammasome. Nlrc4 mediates protection through induction of pyroptosis in the early phase of infection. As the infection progresses and as IL-18-dependent IFNγ production increases, caspase-11-dependent pyroptosis acquires a preponderant protective role. Production of IL-1ß and IL-18 during infection is primarily mediated by Nlrp3. IL-18 is essential for survival because of its ability to induce IFNγ production, which in turn activates macrophage microbicidal functions and primes for caspase-11 expression. In contrast, during melioidosis, IL-1ß has deleterious effects due to excessive recruitment of neutrophils to the lung and consequent tissue damage.

Burkholderia pseudomallei Differentially Regulates Host Innate Immune Response Genes for Intracellular Survival in Lung Epithelial Cells.

BACKGROUND: Burkholderia pseudomallei, the causative agent of melioidosis poses a serious threat to humankind. B. pseudomallei secretes numerous virulence proteins that alter host cell functions to escape from intracellular immune sensors. However, the events underlying disease pathogenesis are poorly understood. METHODS: We determined the ability of B. pseudomallei to invade and survive intracellularly in A549 human lung epithelial cells, and also investigated the early transcriptional responses using an Illumina HumanHT-12 v4 microarray platform, after three hours of exposure to live B. pseudomallei (BCMS) and its secreted proteins (CCMS). RESULTS: We found that the ability of B. pseudomallei to invade and survive intracellularly correlated with increase of multiplicity of infection and duration of contact. Activation of host carbohydrate metabolism and apoptosis as well as suppression of amino acid metabolism and innate immune responses both by live bacteria and its secreted proteins were evident. These early events might be linked to initial activation of host genes directed towards bacterial dissemination from lungs to target organs (via proposed in vivo mechanisms) or to escape potential sensing by macrophages. CONCLUSION: Understanding the early responses of A549 cells toward B. pseudomallei infection provide preliminary insights into the likely pathogenesis mechanisms underlying melioidosis, and could contribute to development of novel intervention strategies to combat B. pseudomallei infections.

Identification of CD4+ T-cell epitope and investigation of HLA distribution for the immunogenic proteins of Burkholderia pseudomallei using in silico approaches - A key vaccine development strategy for melioidosis.

Melioidosis is a serious infectious diseases affecting multi-organ system in humans with high mortality rate. The disease is caused by the bacterium, Burkholderia pseudomallei and it is intrinsically resistant to many antibiotics. Thus, there is an urgent need for protective vaccine against B. pseudomallei; which may reduce morbidity and mortality in endemic areas. The identification of peptides that bind to major histocompatibility complex II class helps in understanding the nature of immune response and identifying T-cell epitopes for the design of new vaccines. Previous studies indicate that, ompA, bipB, fliC and groEL proteins of B. pseudomallei stimulate CD4+ T-cell immune response and act as protective immunogens. However, the data for CD4+ T-cell epitopes of these immunogenic proteins are very limited. Hence, in this present study we attempted to identify CD4+ T-cell epitopes in B. pseudomallei immunogenic proteins using in silico approaches. We did population coverage analysis for these identified epitopic core sequences to identify individuals in endemic areas expected to respond to a given set of these epitopes on the basis of HLA genotype frequencies. We observed that eight epitopic core sequences, two from each immunogenic protein, were associated with the maximum number of HLA-DR binding alleles. These eight peptides are found to be immunogenic in more than 90% of population in endemic areas considered. Thus, these eight peptides containing epitopic core sequences may act as probable vaccine candidates and they may be considered for the development of epitope-based vaccines for melioidosis.

Novel engineered cationic antimicrobial peptides display broad-spectrum activity against Francisella tularensis, Yersinia pestis and Burkholderia pseudomallei.

Broad-spectrum antimicrobials are needed to effectively treat patients infected in the event of a pandemic or intentional release of a pathogen prior to confirmation of the pathogen's identity. Engineered cationic antimicrobial peptides (eCAPs) display activity against a number of bacterial pathogens including multi-drug-resistant strains. Two lead eCAPs, WLBU2 and WR12, were compared with human cathelicidin (LL-37) against three highly pathogenic bacteria: Francisella tularensis, Yersinia pestis and Burkholderia pseudomallei. Both WLBU2 and WR12 demonstrated bactericidal activity greater than that of LL-37, particularly against F. tularensis and Y. pestis. Only WLBU2 had bactericidal activity against B. pseudomallei. WLBU2, WR12 and LL-37 were all able to inhibit the growth of the three bacteria in vitro. Because these bacteria can be facultative intracellular pathogens, preferentially infecting macrophages and dendritic cells, we evaluated the activity of WLBU2 against F. tularensis in an ex vivo infection model with J774 cells, a mouse macrophage cell line. In that model WLBU2 was able to achieve greater than 50% killing of F. tularensis at a concentration of 12.5 µM. These data show the therapeutic potential of eCAPs, particularly WLBU2, as a broad-spectrum antimicrobial for treating highly pathogenic bacterial infections.

Mechanisms of Disease: Host-Pathogen Interactions between Burkholderia Species and Lung Epithelial Cells.

Members of the Burkholderia species can cause a range of severe, often fatal, respiratory diseases. A variety of in vitro models of infection have been developed in an attempt to elucidate the mechanism by which Burkholderia spp. gain entry to and interact with the body. The majority of studies have tended to focus on the interaction of bacteria with phagocytic cells with a paucity of information available with regard to the lung epithelium. However, the lung epithelium is becoming more widely recognized as an important player in innate immunity and the early response to infections. Here we review the complex relationship between Burkholderia species and epithelial cells with an emphasis on the most pathogenic species, Burkholderia pseudomallei and Burkholderia mallei. The current gaps in knowledge in our understanding are highlighted along with the epithelial host-pathogen interactions that offer potential opportunities for therapeutic intervention.

Membrane-Bound PenA ß-Lactamase of Burkholderia pseudomallei.

Burkholderia pseudomallei is the etiologic agent of melioidosis, a difficult-to-treat disease with diverse clinical manifestations. ß-Lactam antibiotics such as ceftazidime are crucial to the success of melioidosis therapy. Ceftazidime-resistant clinical isolates have been described, and the most common mechanism is point mutations affecting expression or critical amino acid residues of the chromosomally encoded class A PenA ß-lactamase. We previously showed that PenA was exported via the twin arginine translocase system and associated with the spheroplast fraction. We now show that PenA is a membrane-bound lipoprotein. The protein and accompanying ß-lactamase activity are found in the membrane fraction and can be extracted with Triton X-114. Treatment with globomycin of B. pseudomallei cells expressing PenA results in accumulation of the prolipoprotein. Mass spectrometric analysis of extracted membrane proteins reveals a protein peak whose mass is consistent with a triacylated PenA protein. Mutation of a crucial lipobox cysteine at position 23 to a serine residue results in loss of ß-lactamase activity and absence of detectable PenAC23S protein. A concomitant isoleucine-to-alanine change at position 20 in the signal peptide processing site in the PenAC23S mutant results in a nonlipidated protein (PenAI20A C23S) that is processed by signal peptidase I and exhibits ß-lactamase activity. The resistance profile of a B. pseudomallei strain expressing this protein is indistinguishable from the profile of the isogenic strain expressing wild-type PenA. The data show that PenA membrane association is not required for resistance and must serve another purpose.

Evaluating the role of phage-shock protein A in Burkholderia pseudomallei.

The phage-shock protein (Psp) response is an extracytoplasmic response system that is vital for maintenance of the cytoplasmic membrane when the cell encounters stressful conditions. The paradigm of the Psp response has been established in Escherichia coli. The response has been shown to be important for survival during the stationary phase, maintenance of the proton motive force across membranes and implicated in virulence. In this study, we identified a putative PspA homologue in Burkholderia pseudomallei, annotated as BPSL2105. Similar to the induction of PspA in E. coli, the expression of B. pseudomallei BPSL2105 was induced by heat shock. Deletion of BPSL2105 resulted in a survival defect in the late stationary phase coincident with dramatic changes in the pH of the culture medium. The B. pseudomallei BPSL2105 deletion mutant also displayed reduced survival in macrophage infection - the first indication that the Psp response plays a role during intracellular pathogenesis in this species. The purified protein formed large oligomeric structures similar to those observed for the PspA protein of E. coli, and PspA homologues in Bacillus, cyanobacteria and higher plants, providing further evidence to support the identification of BPSL2105 as a PspA-like protein in B. pseudomallei.

Bacterial Reductionism: Host Thiols Enhance Virulence.

Intracellular bacteria exploit host cytosolic signals to upregulate virulence genes. In this issue of Cell Host & Microbe, Wong et al. (2015) show that Burkholderia pseudomallei senses host cytosolic glutathione, a low-molecular-weight thiol, through the membrane-bound histidine sensor kinase VirA, highlighting the importance of inter-kingdom signaling in bacterial pathogenesis.