Outer Membrane Properties Research Articles

Characterising virulence in a nontoxigenic non-O1/non-O139 Vibrio cholerae isolate imported from Vietnam

Vibrio cholerae is a major human pathogen that can cause life-threatening acute diarrhea. V. cholerae are classified according to O-antigen polysaccharide outer membrane properties, where the serotypes O1 and O139 are strains that cause pandemics and epidemics while non-O1/non-O139 usually cause mild disease. The dynamic evolution of V. cholerae involves acquisition of new virulence factors through horizontal gene transfer and formerly nontoxigenic serogroups are increasingly being reported to cause severe forms of human disease.In this study we have serotyped one isolate (ST588-CPH) of imported V. cholerae from Vietnam to Denmark and performed whole genome sequencing to identify known virulence genes and furthermore studied the pattern of virulence in closely related pathogenic strains of V. cholerae.ST558-CPH was found to be a non-O1/non-O139 strain. Initial analysis from the whole genome sequencing gave a 96,6 % match to the O139-specific wbfZ gene, but in a second analysis with a higher identification threshold, the wbfZ gene was absent. We suggest a “de novo” display of a database misannotation, which explains the conflicting results. The MLST analysis revealed that the isolate belongs to the nontoxigenic non-O1/non-O139 sequence type ST558. ST558 has recently been reported as a sequence type forming a cluster of ST's that should be monitored, as it has shown to have virulence causing moderate to severe illness. Our analysis of virulence genes identified MakA, a recently discovered toxin, which seems to be generally present in both toxigenic and nontoxigenic strains.

A sensor histidine kinase from a plant-endosymbiont bacterium restores the virulence of a mammalian intracellular pathogen

Alphaproteobacteria include organisms living in close association with plants or animals. This interaction relies partly on orthologous two-component regulatory systems (TCS), with sensor and regulator proteins modulating the expression of conserved genes related to symbiosis/virulence. We assessed the ability of the exoS+Sm gene, encoding a sensor protein from the plant endosymbiont Sinorhizobium meliloti to substitute its orthologous bvrS in the related animal/human pathogen Brucella abortus. ExoS phosphorylated the B. abortus regulator BvrR in vitro and in cultured bacteria, showing conserved biological function. Production of ExoS in a B. abortus bvrS mutant reestablished replication in host cells and the capacity to infect mice. Bacterial outer membrane properties, the production of the type IV secretion system VirB, and its transcriptional regulators VjbR and BvrR were restored as compared to parental B. abortus. These results indicate that conserved traits of orthologous TCS from bacteria living in and sensing different environments are sufficient to achieve phenotypic plasticity and support bacterial survival. The knowledge of bacterial genetic networks regulating host interactions allows for an understanding of the subtle differences between symbiosis and parasitism. Rewiring these networks could provide new alternatives to control and prevent bacterial infection.

TamAB is regulated by PhoPQ and functions in outer membrane homeostasis during Salmonella pathogenesis.

Salmonella survive and replicate in macrophages, which normally kill bacteria by exposing them to a variety of harsh conditions and antimicrobial effectors, many of which target the bacterial cell envelope. The PhoPQ two-component system responds to the phagosome environment and induces factors that protect the outer membrane, allowing adaptation and growth in the macrophage. We show that PhoPQ induces the transcription of the tamAB operon both in vitro and in macrophages. The TamA protein is structurally similar to BamA, an essential protein in the Bam complex that assembles β-barrel proteins in the outer membrane, while TamB is an AsmA-family protein implicated in lipid transport between the inner and outer membranes. We show that the Bam machinery is stressed in vitro under low Mg2+, low pH conditions that mimic the phagosome. Not surprisingly, mutations affecting Bam function confer significant virulence defects. Although loss of TamAB alone confers no virulence defect, a tamAB deletion confers a synthetic phenotype in bam mutant backgrounds in animals and macrophages, and in vitro upon treatment with vancomycin or sodium dodecyl sulfate. Mutations affecting YhdP, which functions in partial redundancy with TamB, also confer synthetic phenotypes with bam mutations in the animal, but this interaction is not evident in vitro. Thus, in the harsh phagocytic environment of the macrophage, the outer membrane Bam machinery is compromised, and the TamAB system, and perhaps other PhoPQ-regulated factors, is induced to compensate. It is most likely that TamAB and other systems assist the Bam complex indirectly by affecting outer membrane properties. IMPORTANCE The TamAB system has been implicated in both outer membrane protein localization and phospholipid transport between the inner and outer membranes. We show that the β-barrel protein assembly complex, Bam, is stressed under conditions thought to mimic the macrophage phagosome. TamAB expression is controlled by the PhoPQ two-component system and induced in macrophages. This system somehow compensates for the Bam complex as evidenced by the fact that mutations affecting the two systems confer synthetic phenotypes in animals, macrophages, and in vitro in the presence of vancomycin or SDS. This study has implications concerning the role of TamAB in outer membrane homeostasis. It also contributes to our understanding of the systems necessary for Salmonella to adapt and reproduce within the macrophage phagosome.

Modeling and Simulation of Bacterial Outer Membranes with Lipopolysaccharides and Capsular Polysaccharides.

Capsule is one of the common virulence factors in Gram-negative bacteria protecting pathogens from host defenses and consists of long-chain capsular polysaccharides (CPS) anchored in the outer membrane (OM). Elucidating structural properties of CPS is important to understand its biological functions as well as the OM properties. However, the outer leaflet of the OM in current simulation studies is represented exclusively by LPS due to the complexity and diversity of CPS. In this work, representative Escherichia coli CPS, KLPS (a lipid A-linked form) and KPG (a phosphatidylglycerol-linked form), are modeled and incorporated into various symmetric bilayers with co-existing LPS in different ratios. All-atom molecular dynamics simulations of these systems have been conducted to characterize various bilayer properties. Incorporation of KLPS makes the acyl chains of LPS more rigid and ordered, while incorporation of KPG makes them less ordered and flexible. These results are consistent with the calculated area per lipid (APL) of LPS, in which the APL of LPS becomes smaller when KLPS is incorporated, whereas it gets larger when KPG is included. Torsional analysis reveals that the influence of the CPS presence on the conformational distributions of the glycosidic linkages of LPS is small, and minor differences are also detected for the inner and outer regions of the CPS. Combined with previously modeled enterobacterial common antigens (ECAs) in the form of mixed bilayers, this work provides more realistic OM models as well as the basis for characterization of interactions between the OM and OM proteins.

Type III Secretion System Repressor RhpR Induces GrlP, a Glycine-Rich Outer Membrane Lipoprotein with Functions in Regulating the Periplasmic Space and Pleiotropic Responses.

The two-component system RhpRS was initially identified as a regulator of genes encoding the type III secretion system (T3SS) in Pseudomonas syringae. Phosphorylated RhpR (P-RhpR) negatively regulates the T3SS genes by repressing the hrpR promoter, but directly activates the expression of a small gene named here as grlp. Here, we show that grlp is expressed higher in rich medium than in minimal medium in P. s. pv. tomato DC3000 and encodes a glycine rich lipoprotein (GrlP) located in the outer membrane (OM). The grlp gene has a pleiotropic effect on bacterial behaviors such as reductions in pathogenicity, swimming motility, biofilm formation, tolerance to various stresses and antibiotics, and long-term survival when overexpressed, but induces these responses when it is deleted in P. s. pv. tomato DC3000. Overexpression of grlp increases the size of periplasm while deletion of grlp decreases the periplasmic space. Further, GrlP interacts with OprI, the ortholog of E. coli OM lipoprotein Lpp, a key player in determining the size of periplasm and mechanic stiffness of the OM by tethering the OM to peptidoglycan (PG) in periplasm. As periplasmic space and OM mechanics play central roles in regulating bacterial physiology, we speculate that GrlP probably imposes its functions on bacterial physiology by regulating the periplasmic space and OM mechanics. These findings suggest that the T3SS gene regulation is closely coordinated with bacterial cell envelope properties by RhpRS in P. syringe. IMPORTANCE The OM of Gram-negative bacteria is the most front line in contact with extracellular milieu. OM is not only a protective layer, but also a structure that determines the envelope stiffness. Recent evidence indicated that components determining the periplasmic space and cross-links of lipopolysaccharide on the OM play key roles in regulating the mechanical properties of the OM. However, whether the OM composition and mechanical properties are coordinated with the expression of the T3SS genes is unknown. Here, we found that the two-component system (TCS) regulator P-RhpR, a direct repressor of the T3SS regulator hrpRS operon, directly activates the expression of the OM lipoprotein gene grlp bearing a function in regulating the periplasmic space. This finding suggests a coordination between the OM properties and the T3SS gene regulation and reveals a new target for control of the T3SS gene expression and bacterial pathogenicity.

Clinically Relevant Bacterial Outer Membrane Models for Antibiotic Screening Applications.

Antibiotic resistance is a growing global health concern that has been increasing in prevalence over the past few decades. In Gram-negative bacteria, the outer membrane is an additional barrier through which antibiotics must traverse to kill the bacterium. In addition, outer membrane features and properties, like membrane surface charge, lipopolysaccharide (LPS) length, and membrane porins, can be altered in response to antibiotics and therefore, further mediate resistance. Model membranes have been used to mimic bacterial membranes to study antibiotic-induced membrane changes but often lack the compositional complexity of the actual outer membrane. Here, we developed a surface-supported membrane platform using outer membrane vesicles (OMVs) from clinically relevant Gram-negative bacteria and use it to characterize membrane biophysical properties and investigate its interaction with antibacterial compounds. We demonstrate that this platform maintains critical features of outer membranes, like fluidity, while retaining complex membrane components, like OMPs and LPS, which are central to membrane-mediated antibiotic resistance. This platform offers a non-pathogenic, cell-free surface to study such phenomena that is compatible with advanced microscopy and surface characterization tools like quartz crystal microbalance. We confirm these OMV bilayers recapitulate membrane interactions (or lack thereof) with the antibiotic compounds polymyxin B, bacitracin, and vancomycin, validating their use as representative models for the bacterial surface. By forming OMV bilayers from different strains, we envision that this platform could be used to investigate underlying biophysical differences in outer membranes leading to resistance, to screen and identify membrane-active antibiotics, or for the development of phage technologies targeting a particular membrane surface component.

Characterizing Escherichia coli's transcriptional response to different styrene exposure modes reveals novel toxicity and tolerance insights.

The global transcriptional response of Escherichia coli to styrene and potential influence of exposure source was determined by performing RNA sequencing (RNA-seq) analysis on both styrene-producing and styrene-exposed cells. In both cases, styrene exposure appears to cause both cell envelope and DNA damage, to which cells respond by down-regulating key genes/pathways involved in DNA replication, protein production, and cell wall biogenesis. Among the most significantly up-regulated genes were those involved with phage shock protein response (e.g. pspABCDE/G), general stress regulators (e.g. marA, rpoH), and membrane-altering genes (notably, bhsA, ompR, ldtC), whereas efflux transporters were, surprisingly, unaffected. Subsequent studies with styrene addition demonstrate how strains lacking ompR [involved in controlling outer membrane (OM) composition/osmoregulation] or any of tolQ, tolA, or tolR (involved in OM constriction) each displayed over 40% reduced growth relative to wild-type. Conversely, despite reducing basal fitness, overexpression of plsX (involved in phospholipid biosynthesis) led to 70% greater growth when styrene exposed. These collective differences point to the likely importance of OM properties in controlling native styrene tolerance. Overall, the collective behaviours suggest that, regardless of source, prolonged exposure to inhibitory styrene levels causes cells to shift from‘growth mode’ to ‘survival mode’, redistributing cellular resources to fuel native tolerance mechanisms.

The essential inner membrane protein YejM is a metalloenzyme

Recent recurrent outbreaks of Gram-negative bacteria show the critical need to target essential bacterial mechanisms to fight the increase of antibiotic resistance. Pathogenic Gram-negative bacteria have developed several strategies to protect themselves against the host immune response and antibiotics. One such strategy is to remodel the outer membrane where several genes are involved. yejM was discovered as an essential gene in E. coli and S. typhimurium that plays a critical role in their virulence by changing the outer membrane permeability. How the inner membrane protein YejM with its periplasmic domain changes membrane properties remains unknown. Despite overwhelming structural similarity between the periplasmic domains of two YejM homologues with hydrolases like arylsulfatases, no enzymatic activity has been previously reported for YejM. Our studies reveal an intact active site with bound metal ions in the structure of YejM periplasmic domain. Furthermore, we show that YejM has a phosphatase activity that is dependent on the presence of magnesium ions and is linked to its function of regulating outer membrane properties. Understanding the molecular mechanism by which YejM is involved in outer membrane remodeling will help to identify a new drug target in the fight against the increased antibiotic resistance.

A How-To Guide for Mode of Action Analysis of Antimicrobial Peptides.

Antimicrobial peptides (AMPs) are a promising alternative to classical antibiotics in the fight against multi-resistant bacteria. They are produced by organisms from all domains of life and constitute a nearly universal defense mechanism against infectious agents. No drug can be approved without information about its mechanism of action. In order to use them in a clinical setting, it is pivotal to understand how AMPs work. While many pore-forming AMPs are well-characterized in model membrane systems, non-pore-forming peptides are often poorly understood. Moreover, there is evidence that pore formation may not happen or not play a role in vivo. It is therefore imperative to study how AMPs interact with their targets in vivo and consequently kill microorganisms. This has been difficult in the past, since established methods did not provide much mechanistic detail. Especially, methods to study membrane-active compounds have been scarce. Recent advances, in particular in microscopy technology and cell biological labeling techniques, now allow studying mechanisms of AMPs in unprecedented detail. This review gives an overview of available in vivo methods to investigate the antibacterial mechanisms of AMPs. In addition to classical mode of action classification assays, we discuss global profiling techniques, such as genomic and proteomic approaches, as well as bacterial cytological profiling and other cell biological assays. We cover approaches to determine the effects of AMPs on cell morphology, outer membrane, cell wall, and inner membrane properties, cellular macromolecules, and protein targets. We particularly expand on methods to examine cytoplasmic membrane parameters, such as composition, thickness, organization, fluidity, potential, and the functionality of membrane-associated processes. This review aims to provide a guide for researchers, who seek a broad overview of the available methodology to study the mechanisms of AMPs in living bacteria.

The protection effect of trehalose on the multinuclear microcapsules based on gelatin and high methyl pectin coacervate during freeze-drying

Drying of coacervates can cause various stresses which may lead to the damage of the outer layer membrane, aggregation, and poor rehydration properties. Gelatin/pectin coacervates prepared with addition of trehalose as cryoprotectant were subjected to freeze-drying. The addition of trehalose (15%) improved wettability, bulk density and encapsulation efficiency. Fourier transform infrared (FTIR) results revealed the formation of a hydrogen bond between coacervates and trehalose. Trehalose increased the degradation temperature of coacervates from 200-250 °C to 300–350 °C. Scanning electron microscopy analysis showed that samples with (10% and 15%) trehalose had a spherical shape. The trehalose added microcapsules improved the rehydration state and maintained the integrity of multinuclear wall and spherical shape. The retention and stability of cinnamaldehyde stored at 45 °C were enhanced by trehalose addition. The amount of trehalose, the interaction between coacervates and trehalose are the main reasons to affect thermal stability, rehydration properties, particle size and the protection degree of core material.

Molecular characterization of the outer membrane of Pseudomonas aeruginosa

Discovery of new antibiotics effective against Gram-negative bacteria is hampered by the low permeability of the bacterial two-membrane cell envelopes. The outer membrane (OM) is an asymmetric bilayer of lipopolysaccharides (LPS) and phospholipids and is a significant permeability barrier to noxious compounds. Molecular characterization of the OM is essential for understanding how antibiotics penetrate this barrier, and for the development of new therapeutic strategies and rational drug design. We carry out all-atom (AA) and coarse-grained (CG) molecular dynamics (MD) simulations to characterize the fully assembled OM bilayer of Pseudomonas aeruginosa, a human pathogen resistant to most clinical antibiotics. These simulations were run using AA and CG parameters that we developed to model the most abundant LPS of P. aeruginosa, specifically the A band O-antigen. Our simulations reproduce several experimentally determined physical properties such as area per lipid, membrane stiffness and increased stability due to the interaction with divalent cations. These tested parameters allowed us to study various OM properties including the effect of divalent cations and OM electroporation. In addition, the massive and extended carbohydrate head groups increase water permeation, which propagates towards the aliphatic tails of the inner leaflet. This effect contributes to the low ordering of the lipids in the inner leaflet. Further, evaluation is needed to understand whether the increased solvation of outer membrane could modulate permeation of highly polar drugs.

Outer Membrane Permeability of Cyanobacterium Synechocystis sp. Strain PCC 6803: Studies of Passive Diffusion of Small Organic Nutrients Reveal the Absence of Classical Porins and Intrinsically Low Permeability.

The outer membrane of heterotrophic Gram-negative bacteria plays the role of a selective permeability barrier that prevents the influx of toxic compounds while allowing the nonspecific passage of small hydrophilic nutrients through porin channels. Compared with heterotrophic Gram-negative bacteria, the outer membrane properties of cyanobacteria, which are Gram-negative photoautotrophs, are not clearly understood. In this study, using small carbohydrates, amino acids, and inorganic ions as permeation probes, we determined the outer membrane permeability of Synechocystis sp. strain PCC 6803 in intact cells and in proteoliposomes reconstituted with outer membrane proteins. The permeability of this cyanobacterium was >20-fold lower than that of Escherichia coli The predominant outer membrane proteins Slr1841, Slr1908, and Slr0042 were not permeable to organic nutrients and allowed only the passage of inorganic ions. Only the less abundant outer membrane protein Slr1270, a homolog of the E. coli export channel TolC, was permeable to organic solutes. The activity of Slr1270 as a channel was verified in a recombinant Slr1270-producing E. coli outer membrane. The lack of putative porins and the low outer membrane permeability appear to suit the cyanobacterial autotrophic lifestyle; the highly impermeable outer membrane would be advantageous to cellular survival by protecting the cell from toxic compounds, especially when the cellular physiology is not dependent on the uptake of organic nutrients.IMPORTANCE Because the outer membrane of Gram-negative bacteria affects the flux rates for various substances into and out of the cell, its permeability is closely associated with cellular physiology. The outer membrane properties of cyanobacteria, which are photoautotrophic Gram-negative bacteria, are not clearly understood. Here, we examined the outer membrane of Synechocystis sp. strain PCC 6803. We revealed that it is relatively permeable to inorganic ions but is markedly less permeable to organic nutrients, with >20-fold lower permeability than the outer membrane of Escherichia coli Such permeability appears to fit the cyanobacterial lifestyle, in which the diffusion pathway for inorganic solutes may suffice to sustain the autotrophic physiology, illustrating a link between outer membrane permeability and the cellular lifestyle.

Omp31 plays an important role on outer membrane properties and intracellular survival of Brucella melitensis in murine macrophages and HeLa cells.

Brucellosis is an infectious disease that affects practically all species of mammals, including human, and is a major zoonosis worldwide. Brucella spp. are facultative intracellular pathogens that have the ability to survive and multiply in phagocytic and nonphagocytic cells such as trophoblast and epithelial cells. Among the six recognized species of the genus Brucella, Brucella melitensis is the main etiological agent involved in goat brucellosis and is also the most pathogenic for human. It causes significant losses in livestock production as a result of abortions, metritis, infertility, and birth of weak animals. Outer membrane proteins (OMPs) are exposed on the bacterial surface and are in contact with cells and effectors of the host immune response, whereby they could be important virulence factors of Brucella species. To evaluate this hypothesis, the gene encoding for the major outer membrane protein Omp31 was amplified, cloned into pUC18 plasmid, and inactivated by inserting a kanamycin cassette, rendering pLVM31 plasmid which was transformed into B. melitensis wild-type strain to obtain LVM31 mutant strain. The Outer membrane (OM) properties of the mutant strain were compared with B. melitensis Bm133 wild-type and B. melitensis Rev1 vaccine strains, in assessing its susceptibility to polymyxin B, sodium deoxycholate, and nonimmune serum. The mutant strain was assessed in vitro with survival assays in murine macrophages J774.A1 and HeLa cells. Our results demonstrate that LVM31 mutant is more susceptible to polymyxin B, sodium deoxycholate, and nonimmune serum than control strains; moreover, Omp31 mutation caused a decrease in the internalization and a significant decrease in the intracellular survival compared with the reference strains in both cell lines. These results allow us to conclude that Omp31 is important for maintaining OM integrity, but also it is necessary for bacterial internalization, establishment and development of an optimal replication niche, and essential for survival and intracellular multiplication.

Elevated cholesterol levels have a poor prognosis in a cholestasis scenario.

Cholestasis results from defective bile flow through the biliary ducts leading to the accumulation of bile acids (BAs) in hepatocytes and serum. It has been seen that cholestasis is associated with hypercholesterolemia, which is a prerequisite for gallstone formation and primary biliary cirrhosis, being some of the most common gastrointestinal disorders in Western societies. Cytotoxic BAs induce proinflammatory mediators, oxidative stress, and apoptosis in hepatocytes, whereas cytoprotective BAs prevent them; they can also modify the plasmatic membrane structure of cells or mitochondrial outer membrane properties as well as the distribution of cholesterol, altering various proteins involved in BAs homeostasis.

Trait changes induced by species interactions in two phenotypically distinct strains of a marine dinoflagellate.

Populations of the toxigenic marine dinoflagellate Alexandrium are composed of multiple genotypes that display phenotypic variation for traits known to influence top-down processes, such as the ability to lyse co-occurring competitors and prospective grazers. We performed a detailed molecular analysis of species interactions to determine how different genotypes perceive and respond to other species. In a controlled laboratory culture study, we exposed two A. fundyense strains that differ in their capacity to produce lytic compounds to the dinoflagellate grazer Polykrikos kofoidii, and analyzed transcriptomic changes during this interaction. Approximately 5% of all analyzed genes were differentially expressed between the two Alexandrium strains under control conditions (without grazer presence) with fold-change differences that were proportionally higher than those observed in grazer treatments. Species interactions led to the genotype-specific expression of genes involved in endocytotic processes, cell cycle control and outer membrane properties, and signal transduction and gene expression regulatory processes followed similar patterns for both genotypes. The genotype-specific trait changes observed in this study exemplify the complex responses to chemically mediated species interactions within the plankton and their regulation at the gene level.

Brucella ovis PA mutants for outer membrane proteins Omp10, Omp19, SP41, and BepC are not altered in their virulence and outer membrane properties

Mutants in several genes have been obtained on the genetic background of virulent rough (lacking O-polysaccharide) Brucella ovis PA. The target genes encode outer membrane proteins previously associated with the virulence of smooth (bearing O-polysaccharide chains in the lipopolysaccharide) Brucella strains. Multiple attempts to delete omp16, coding for a homologue to peptidoglycan-associated lipoproteins, were unsuccessful, which suggests that Omp16 is probably essential for in vitro survival of B. ovis PA. Single deletion of omp10 or omp19 – that encode two other outer membrane lipoproteins – was achieved, but the simultaneous removal of both genes failed, suggesting an essential complementary function between both proteins. Two other deletion mutants, defective in the Tol-C-homologue BepC or in the SP41 adhesin, were also obtained. Surprisingly when compared to previous results obtained with smooth Brucella, none of the B. ovis mutants showed attenuation in the virulence, either in the mouse model or in cellular models of professional and non-professional phagocytes. Additionally, and in contrast to the observations reported with smooth Brucella strains, several properties related to the outer membrane remained almost unaltered. These results evidence new distinctive traits between naturally rough B. ovis and smooth brucellae.

Deoxycholic acid modulates cell death signaling through changes in mitochondrial membrane properties

Cytotoxic bile acids, such as deoxycholic acid (DCA), are responsible for hepatocyte cell death during intrahepatic cholestasis. The mechanisms responsible for this effect are unclear, and recent studies conflict, pointing to either a modulation of plasma membrane structure or mitochondrial-mediated toxicity through perturbation of mitochondrial outer membrane (MOM) properties. We conducted a comprehensive comparative study of the impact of cytotoxic and cytoprotective bile acids on the membrane structure of different cellular compartments. We show that DCA increases the plasma membrane fluidity of hepatocytes to a minor extent, and that this effect is not correlated with the incidence of apoptosis. Additionally, plasma membrane fluidity recovers to normal values over time suggesting the presence of cellular compensatory mechanisms for this perturbation. Colocalization experiments in living cells confirmed the presence of bile acids within mitochondrial membranes. Experiments with active isolated mitochondria revealed that physiologically active concentrations of DCA change MOM order in a concentration- and time-dependent manner, and that these changes preceded the mitochondrial permeability transition. Importantly, these effects are not observed on liposomes mimicking MOM lipid composition, suggesting that DCA apoptotic activity depends on features of mitochondrial membranes that are absent in protein-free mimetic liposomes, such as the double-membrane structure, lipid asymmetry, or mitochondrial protein environment. In contrast, the mechanism of action of cytoprotective bile acids is likely not associated with changes in cellular membrane structure.

Role of Burkholderia cenocepacia afcE and afcF genes in determining lipid-metabolism-associated phenotypes

Burkholderia cenocepacia is an opportunistic pathogen that primarily infects cystic fibrosis patients. Previously we have reported that mutations in shvR, a LysR-type transcriptional regulator, and ShvR-regulated genes BCAS0208 and BCAS0201 (designated afcE and afcF, respectively) affect colony morphotype, biofilm and pellicle formation and virulence in B. cenocepacia. In this study we investigated the role of afcE and afcF in influencing lipid-metabolism-associated phenotypes. As previously reported for K56-2ΔshvR, the Δ2afcE and afcF : : lux mutants had no antifungal activity against Fusarium and Rhizoctonia solani, suggesting that these genes are involved in synthesis of a membrane-associated antifungal lipopeptide. Strains Δ2afcE and afcF : : lux had reduced swarming motility and altered cell membrane morphology, both of which were restored to wild-type levels upon providing these genes in trans. Both K56-2ΔshvR and Δ2afcE showed increased uptake of the hydrophobic fluorescent probe N-phenylnaphthylamine (NPN), indicating altered outer membrane properties. Total lipid profiles determined by TLC revealed distinct differences in cellular lipid compositions of K56-2ΔshvR, Δ2afcE and afcF : : lux compared with K56-2. Taken together, these results indicate that afcE and afcF are involved in metabolic pathway(s) influencing lipid profiles and affect both cell surface and antifungal properties of B. cenocepacia.

When it comes to drug discovery not all Gram‐negative bacterial biodefence pathogens are created equal: Burkholderia pseudomallei is different

When it comes to drug discovery not all Gram‐negative bacterial biodefence pathogens are created equal: <i>Burkholderia pseudomallei</i> is different

Metaxin deficiency alters mitochondrialmembrane permeability and leads to resistance to TNF-induced cell killing

Metaxin, a mitochondrial outer membrane protein, is critical for TNF-induced cell death in L929 cells. Its deficiency, caused by retroviral insertion-mediated mutagenesis, renders L929 cells resistance to TNF killing. In this study, we further characterized metaxin deficiency-caused TNF resistance in parallel with Bcl-X(L) overexpression-mediated death resistance. We did not find obvious change in mitochondria membrane potential in metaxin-deficient (Met(mut)) and Bcl-X(L)-overexpressing cells, but we did find an increase in the release rate of the mitochondrial membrane potential probe rhodamine 123 (Rh123) that was preloaded into mitochondria. In addition, overexpression of a function-interfering mutant of metaxin (MetaΔTM/C) or Bcl-X(L) in MCF-7.3.28 cells also resulted in an acquired resistance to TNF killing and a faster rate of Rh123 release, indicating a close correlation between TNF resistance and higher rates of the dye release from the mitochondria. The release of Rh123 can be controlled by the mitochondrial membrane permeability transition (PT) pore, as targeting an inner membrane component of the PT pore by cyclosporin A (CsA) inhibited Rh123 release. However, metaxin deficiency and Bcl-X(L) overexpression apparently affect Rh123 release from a site(s) different from that of CsA, as CsA can overcome their effect. Though both metaxin and Bcl-X(L) appear to function on the outer mitochondrial membrane, they do not interact with each other. They may use different mechanisms to increase the permeability of Rh123, since previous studies have suggested that metaxin may influence certain outer membrane porins while Bcl-X(L) may form pores on the outer membrane. The alteration of the mitochondrial outer membrane properties by metaxin deficiency and Bcl-X(L) overexpression, as indicated by a quicker Rh123 release, may be helpful in maintaining mitochondrial integrity.