Genetic Screen for Cell Fitness in High or Low Oxygen Highlights Mitochondrial and Lipid Metabolism.

Human cells are able to sense and adapt to variations in oxygen levels. Historically, much research in this field has focused on hypoxia-inducible factor (HIF) signaling and reactive oxygen species (ROS). Here, we perform genome-wide CRISPR growth screens at 21%, 5%, and 1% oxygen to systematically identify gene knockouts with relative fitness defects in high oxygen (213 genes) or low oxygen (109 genes), most without known connection to HIF or ROS. Knockouts of many mitochondrial pathways thought to be essential, including complex I and enzymes in Fe-S biosynthesis, grow relatively well at low oxygen and thus are buffered by hypoxia. In contrast, in certain cell types, knockout of lipid biosynthetic and peroxisomal genes causes fitness defects only in low oxygen. Our resource nominates genetic diseases whose severity may be modulated by oxygen and links hundreds of genes to oxygen homeostasis.

Membrane homeostasis beyond fluidity: control of membrane compressibility.

Biomembranes are complex materials composed of lipids and proteins that compartmentalize biochemistry. They are actively remodeled in response to physical and metabolic cues, as well as during cell differentiation and stress. The concept of homeoviscous adaptation has become a textbook example of membrane responsiveness. Here, we discuss limitations and common misconceptions revolving around it. By highlighting key moments in the life cycle of a transmembrane protein, we illustrate that membrane thickness and a finely regulated membrane compressibility are crucial to facilitate proper membrane protein insertion, function, sorting, and inheritance. We propose that the unfolded protein response (UPR) provides a mechanism for endoplasmic reticulum (ER) membrane homeostasis by sensing aberrant transverse membrane stiffening and triggering adaptive responses that re-establish membrane compressibility.

Low membrane fluidity triggers lipid phase separation and protein segregation in living bacteria.

All living organisms adapt their membrane lipid composition in response to changes in their environment or diet. These conserved membrane-adaptive processes have been studied extensively. However, key concepts of membrane biology linked to regulation of lipid composition including homeoviscous adaptation maintaining stable levels of membrane fluidity, and gel-fluid phase separation resulting in domain formation, heavily rely upon in vitro studies with model membranes or lipid extracts. Using the bacterial model organisms Escherichia coli and Bacillus subtilis, we now show that inadequate in vivo membrane fluidity interferes with essential complex cellular processes including cytokinesis, envelope expansion, chromosome replication/segregation and maintenance of membrane potential. Furthermore, we demonstrate that very low membrane fluidity is indeed capable of triggering large-scale lipid phase separation and protein segregation in intact, protein-crowded membranes of living cells; a process that coincides with the minimal level of fluidity capable of supporting growth. Importantly, the in vivo lipid phase separation is not associated with a breakdown of the membrane diffusion barrier function, thus explaining why the phase separation process induced by low fluidity is biologically reversible.

Cholesterol in the cargo membrane amplifies tau inhibition of kinesin-1-based transport.

Intracellular cargos are often membrane-enclosed and transported by microtubule-based motors in the presence of microtubule-associated proteins (MAPs). Whereas increasing evidence reveals how MAPs impact the interactions between motors and microtubules, critical questions remain about the impact of the cargo membrane on transport. Here we combined in vitro optical trapping with theoretical approaches to determine the effect of a lipid cargo membrane on kinesin-based transport in the presence of MAP tau. Our results demonstrate that attaching kinesin to a fluid lipid membrane reduces the inhibitory effect of tau on kinesin. Moreover, adding cholesterol, which reduces kinesin diffusion in the cargo membrane, amplifies the inhibitory effect of tau on kinesin binding in a dosage-dependent manner. We propose that reduction of kinesin diffusion in the cargo membrane underlies the effect of cholesterol on kinesin binding in the presence of tau, and we provide a simple model for this proposed mechanism. Our study establishes a direct link between cargo membrane cholesterol and MAP-based regulation of kinesin-1. The cholesterol effects uncovered here may more broadly extend to other lipid alterations that impact motor diffusion in the cargo membrane, including those associated with aging and neurological diseases.

The mechanistic basis of the membrane-permeabilizing activities of the virulence-associated protein A (VapA) from Rhodococcus equi.

Pathogenic Rhodococcus equi release the virulence-associated protein A (VapA) within macrophage phagosomes. VapA permeabilizes phagosome and lysosome membranes and reduces acidification of both compartments. Using biophysical techniques, we found that VapA interacts with model membranes in four steps: (i) binding, change of mechanical properties, (ii) formation of specific membrane domains, (iii) permeabilization within the domains, and (iv) pH-specific transformation of domains. Biosensor data revealed that VapA binds to membranes in one step at pH 6.5 and in two steps at pH 4.5 and decreases membrane fluidity. The integration of VapA into lipid monolayers was only significant at lateral pressures <20 mN m-1 indicating preferential incorporation into membrane regions with reduced integrity. Atomic force microscopy of lipid mono- and bilayers showed that VapA increased the surface heterogeneity of liquid disordered domains. Furthermore, VapA led to the formation of a new microstructured domain type and, at pH 4.5, to the formation of 5 nm high domains. VapA binding, its integration and lipid domain formation depended on lipid composition, pH, protein concentration and lateral membrane pressure. VapA-mediated permeabilization is clearly distinct from that caused by classical microbial pore formers and is a key contribution to the multiplication of Rhodococcus equi in phagosomes.

Detergent-induced quantitatively limited formation of diacyl phosphatidylinositol dimannoside in Mycobacterium smegmatis.

Mycobacterial plasma membrane, together with the peptidoglycan-arabinogalactan cell wall and waxy outer membrane, creates a robust permeability barrier against xenobiotics. The fact that several antituberculosis drugs target plasma membrane-embedded enzymes underscores the importance of the plasma membrane in bacterial physiology and pathogenesis. Nevertheless, its accurate phospholipid composition remains undefined, with conflicting reports on the abundance of phosphatidylinositol mannosides (PIMs), physiologically important glycolipids evolutionarily conserved among mycobacteria and related bacteria. Some studies indicate cardiolipin, phosphatidylethanolamine, and phosphatidylinositol as dominant structural phospholipids. Conversely, some suggest PIMs dominate the plasma membrane. A striking example of the latter is the use of reverse micelle extraction, showing diacyl phosphatidylinositol dimannoside (Ac2PIM2) as the most abundant phospholipid in a model organism, Mycobacterium smegmatis. Our recent work reveals a rapid response mechanism to membrane-fluidizing stress in mycobacterial plasma membrane: monoacyl phosphatidylinositol dimannoside and hexamannoside (AcPIM2 and AcPIM6) are converted to diacyl forms (Ac2PIM2 and Ac2PIM6). Given the dynamic nature of PIMs, we aimed to resolve the conflicting data in the literature. We show that unstressed M. smegmatis lacks an Ac2PIM2-dominated plasma membrane. Ac2PIM2 accumulation is induced by experimental conditions involving sodium docusate, a component of the reverse micellar solution. Using chemically synthesized PIMs as standards, we accurately quantified phospholipid ratio in M. smegmatis through liquid chromatography-mass spectrometry, revealing that mycobacterial plasma membrane is dominated by cardiolipin, phosphatidylethanolamine, and phosphatidylinositol. PIMs are quantitatively minor but responsive to environmental stresses in M. smegmatis. Our study paves the way for accurate modeling of mycobacterial plasma membrane.

AdipoR2 recruits protein interactors to promote fatty acid elongation and membrane fluidity.

The human AdipoR2 and its Caenorhabditis elegans homolog PAQR-2 are multipass plasma membrane proteins that protect cells against membrane rigidification. However, how AdipoR2 promotes membrane fluidity mechanistically is not clear. Using 13C-labeled fatty acids, we show that AdipoR2 can promote the elongation and incorporation of membrane-fluidizing polyunsaturated fatty acids into phospholipids. To elucidate the molecular basis of these activities, we performed immunoprecipitations of tagged AdipoR2 and PAQR-2 expressed in HEK293 cells or whole C. elegans, respectively, and identified coimmunoprecipitated proteins using mass spectrometry. We found that several of the evolutionarily conserved AdipoR2/PAQR-2 interactors are important for fatty acid elongation and incorporation into phospholipids. We experimentally verified some of these interactions, namely, with the dehydratase HACD3 that is essential for the third of four steps in long-chain fatty acid elongation and ACSL4 that is important for activation of unsaturated fatty acids and their channeling into phospholipids. We conclude that AdipoR2 and PAQR-2 can recruit protein interactors to promote the production and incorporation of unsaturated fatty acids into phospholipids.

Bacterial membrane dynamics: Compartmentalization and repair.

In every bacterial cell, the plasma membrane plays a key role in viability as it forms a selective barrier between the inside of the cell and its environment. This barrier function depends on the physical state of the lipid bilayer and the proteins embedded or associated with the bilayer. Over the past decade or so, it has become apparent that many membrane-organizing proteins and principles, which were described in eukaryote systems, are ubiquitous and play important roles in bacterial cells. In this minireview, we focus on the enigmatic roles of bacterial flotillins in membrane compartmentalization and bacterial dynamins and ESCRT-like systems in membrane repair and remodeling.

High Myristic Acid in Glycerolipids Enhances the Repair of Photodamaged Photosystem II under Strong Light.

Cyanobacteria inhabit areas with a broad range of light, temperature and nutrient conditions. The robustness of cyanobacterial cells, which can survive under different conditions, may depend on the resilience of photosynthetic activity. Cyanothece sp. PCC 8801 (Cyanothece), a freshwater cyanobacterium isolated from a Taiwanese rice field, had a higher repair activity of photodamaged photosystem II (PSII) under intense light than Synechocystis sp. PCC 6803 (Synechocystis), another freshwater cyanobacterium. Cyanothece contains myristic acid (14:0) as the major fatty acid at the sn-2 position of the glycerolipids. To investigate the role of 14:0 in the repair of photodamaged PSII, we used a Synechocystis transformant expressing a T-1274 encoding a lysophosphatidic acid acyltransferase (LPAAT) from Cyanothece. The wild-type and transformant cells contained 0.2 and 20.1 mol% of 14:0 in glycerolipids, respectively. The higher content of 14:0 in the transformants increased the fluidity of the thylakoid membrane. In the transformants, PSII repair was accelerated due to an enhancement in the de novo synthesis of D1 protein, and the production of singlet oxygen (1O2), which inhibited protein synthesis, was suppressed. The high content of 14:0 increased transfer of light energy received by phycobilisomes to PSI and CP47 in PSII and the content of carotenoids. These results indicated that an increase in 14:0 reduced 1O2 formation and enhanced PSII repair. The higher content of 14:0 in the glycerolipids may be required as a survival strategy for Cyanothece inhabiting a rice field under direct sunlight.

Dictyostelium discoideum cells retain nutrients when the cells are about to outgrow their food source.

Dictyostelium discoideum is a unicellular eukaryote that eats bacteria, and eventually outgrows the bacteria. D. discoideum cells accumulate extracellular polyphosphate (polyP), and the polyP concentration increases as the local cell density increases. At high cell densities, the correspondingly high extracellular polyP concentrations allow cells to sense that they are about to outgrow their food supply and starve, causing the D. discoideum cells to inhibit their proliferation. In this report, we show that high extracellular polyP inhibits exocytosis of undigested or partially digested nutrients. PolyP decreases plasma membrane recycling and apparent cell membrane fluidity, and this requires the G protein-coupled polyP receptor GrlD, the polyphosphate kinase Ppk1 and the inositol hexakisphosphate kinase I6kA. PolyP alters protein contents in detergent-insoluble crude cytoskeletons, but does not significantly affect random cell motility, cell speed or F-actin levels. Together, these data suggest that D. discoideum cells use polyP as a signal to sense their local cell density and reduce cell membrane fluidity and membrane recycling, perhaps as a mechanism to retain ingested food when the cells are about to starve. This article has an associated First Person interview with the first author of the paper.

Feasibility Study for the Use of Gene Electrotransfer and Cell Electrofusion as a Single-Step Technique for the Generation of Activated Cancer Cell Vaccines.

Cell-based therapies hold great potential for cancer immunotherapy. This approach is based on manipulation of dendritic cells to activate immune system against specific cancer antigens. For the development of an effective cell vaccine platform, gene transfer, and cell fusion have been used for modification of dendritic or tumor cells to express immune (co)stimulatory signals and to load dendritic cells with tumor antigens. Both, gene transfer and cell fusion can be achieved by single technique, a cell membrane electroporation. The cell membrane exposed to external electric field becomes temporarily permeable, enabling introduction of genetic material, and also fusogenic, enabling the fusion of cells in the close contact. We tested the feasability of combining gene electrotransfer and electrofusion into a single-step technique and evaluated the effects of electroporation buffer, pulse parameters, and cell membrane fluidity for single or combined method of gene delivery or cell fusdion. We determined the percentage of fused cells expressing green fluorescence protein (GFP) in a murine cell model of melanoma B16F1, cell line used in our previous studies. Our results suggest that gene electrotransfer and cell electrofusion can be applied in a single step. The percentage of viable hybrid cells expressing GFP depends on electric pulse parameters and the composition of the electroporation buffer. Furthermore, our results suggest that cell membrane fluidity is not related to the efficiency of the gene electrotransfer and electrofusion. The protocol is compatible with microfluidic devices, however further optimization of electric pulse parameters and buffers is still needed.

Amino acid substitutions in norovirus VP1 dictate host dissemination via variations in cellular attachment.

IMPORTANCE: All viruses initiate infection by utilizing receptors to attach to target host cells. These virus-receptor interactions can therefore dictate viral replication and pathogenesis. Understanding the nature of virus-receptor interactions could also be important for the development of novel therapies. Noroviruses are non-enveloped icosahedral viruses of medical importance. They are a common cause of acute gastroenteritis with no approved vaccine or therapy and are a tractable model for studying fundamental virus biology. In this study, we utilized the murine norovirus model system to show that variation in a single amino acid of the major capsid protein alone can affect viral infectivity through improved attachment to suspension cells. Modulating plasma membrane mobility reduced infectivity, suggesting an importance of membrane mobility for receptor recruitment and/or receptor conformation. Furthermore, different substitutions at this site altered viral tissue distribution in a murine model, illustrating how in-host capsid evolution could influence viral infectivity and/or immune evasion.

Erythrocyte membrane fluidity: A novel biomarker of residual cardiovascular risk in type 2 diabetes.

AIMS: Improving the composition of circulating fatty acids (FA) leads to a reduction in cardiovascular diseases (CVD) in high-risk individuals. The membrane fluidity of red blood cells (RBC), which reflects circulating FA status, may be a valid biomarker of cardiovascular (CV) risk in type 2 diabetes (T2D). METHODS: Red blood cell membrane fluidity, quantified as general polarization (GP), was assessed in 234 subjects with T2D, 86 with prior major CVD. Based on GP distribution, a cut-off of .445 was used to divide the study cohort into two groups: the first with higher GP, called GEL, and the second, defined as lower GP (LGP). Lipidomic analysis was performed to evaluate FA composition of RBC membranes. RESULTS: Although with comparable CV risk factors, the LGP group had a greater percentage of patients with major CVD than the GEL group (40% vs 24%, respectively, p < .05). Moreover, in a logistic regression analysis, a lower GP value was independently associated with the presence of macrovascular complications. Lipidomic analysis showed a clear shift of LGP membranes towards a pro-inflammatory condition due to higher content of arachidonic acid and increased omega 6/omega 3 index. CONCLUSIONS: Increased membrane fluidity is associated with a higher CV risk in subjects with T2D. If confirmed in prospective studies, membrane fluidity could be a new biomarker for residual CV risk assessment in T2D.

Exposure of Legionella pneumophila to low-shear modeled microgravity: impact on stress response, membrane lipid composition, pathogenicity to macrophages and interrelated genes expression.

Here, we studied the effect of low-shear modeled microgravity (LSMMG) on cross stress resistance (heat, acid, and oxidative), fatty acid content, and pathogenicity along with alteration in expression of stress-/virulence-associated genes in Legionella pneumophila. The stress resistance analysis result indicated that bacteria cultivated under LSMMG environments showed higher resistance with elevated D-values at 55 °C and in 1 mM of hydrogen peroxide (H2O2) conditions compared to normal gravity (NG)-grown bacteria. On the other hand, there was no significant difference in tolerance (p < 0.05) toward simulated gastric fluid (pH-2.5) acid conditions. In fatty acid analysis, our result showed that a total amount of saturated and cyclic fatty acids was increased in LSMMG-grown cells; as a consequence, they might possess low membrane fluidity. An upregulated expression level was noticed for stress-related genes (hslV, htrA, grpE, groL, htpG, clpB, clpX, dnaJ, dnaK, rpoH, rpoE, rpoS, kaiB, kaiC, lpp1114, ahpC1, ahpC2, ahpD, grlA, and gst) under LSMMG conditions. The reduced virulence (less intracellular bacteria and less % of induce apoptosis in RAW 264.7 macrophages) of L. pneumophila under LSMMG conditions may be because of downregulation related genes (dotA, dotB, dotC, dotD, dotG, dotH, dotL, dotM, dotN, icmK, icmB, icmS, icmT, icmW, ladC, rtxA, letA, rpoN, fleQ, fleR, and fliA). In the LSMMG group, the expression of inflammation-related factors, such as IL-1α, TNF-α, IL-6, and IL-8, was observed to be reduced in infected macrophages. Also, scanning electron microscopy (SEM) analysis showed less number of LSMMG-cultivated bacteria attached to the host macrophages compared to NG. Thus, our study provides understandings about the changes in lipid composition and different genes expression due to LSMMG conditions, which apparently influence the alterations of L. pneumophila' stress/virulence response.

Differential interactions of essential and toxic metal ions with biologically relevant phosphatidic acid and phosphatidylserine membranes.

Metal pollutants are a growing concern due to increased use in mining and other industrial processes. Moreover, the use of metals in daily life is becoming increasingly prevalent. Metals such as manganese (Mn), cobalt (Co), and nickel (Ni) are toxic in high amounts whereas lead (Pb) and cadmium (Cd) are acutely toxic at low µM concentrations. These metals are associated with system dysfunction in humans including cancer, neurodegenerative diseases, Alzheimer's disease, Parkinson's disease, and other cellular process'. One known but lesser studied target of these metals are lipids that are key membrane building blocks or serve signalling functions. It was shown that Mn, Co, Ni, Pb, and Cd cause rigidification of liposomes and increase the phase transition in membranes composed of both saturated or partly unsaturated phosphatidic acid (PA) and phosphatidylserine (PS). The selected metals showed differential effects that were more pronounced on saturated lipids. In addition, more rigidity was induced in the biologically relevant liquid-crystalline phase. Moreover, metal affinity, induced rigidification and liposome size increases also varied with the headgroup architecture, whereby the carboxyl group of PS appeared to play an important role. Thus, it can be inferred that Mn, Co, Ni, Cd, and Pb may have preferred binding coordination with the lipid headgroup, degree of acyl chain unsaturation, and membrane phase.

Addition of low concentration of cholesterol-loaded cyclodextrin (CLC) has a positive effect on cryopreserved canine spermatozoa evaluated by andrological and biophysical methods.

BACKGROUND: This study was conducted to find the best concentration of cholesterol-loaded cyclodextrin (CLC) which has a positive impact on canine post thaw semen quality. Three different concentrations of CLC (0.83 mg/ml; 1.66 mg/ml; 3.32 mg/ml) and 2-hydroxylpropyl-beta-cyclodextrin (HBCD) (1.66 mg/ml) were used in addition to cryopreservation extender and compared with the control after thawing. Samples were assessed using computer-assisted semen analyzer (CASA), flow cytometry, fluorimeter by measuring the fluorescence anisotropy (ANISO) and determining the generalized membrane polarization (GP). RESULTS: An addition of 0.83 mg/ml CLC significantly increased the percentage of progressive motile (PROG) and rapid spermatozoa (RAP) (P < 0.05). 1.66 mg/ml HBCD decreased progressive motility of spermatozoa and population with rapid movement relative to the control (P < 0.05). Furthermore, the groups with an addition of 1.66 mg/ml and 3.32 mg/ml of CLC, as well as the group with only cyclodextrin, increased percentage of dead spermatozoa without lipid peroxidation and decreased percentage of viable spermatozoa without LPO which was lower in these groups than in the control (P < 0.05). Other sperm parameters assessed on flow cytometer were not significantly different. The addition of CLC at 0.83 mg/ml and 3.32 mg/ml concentrations and 1.66 mg/ml of HBCD caused an increase in ANISO measured at 23 ºC (P < 0.05). CONCLUSIONS: In conclusion, the results suggest that increasing cholesterol in the plasma membrane of canine spermatozoa can improve their freezability. However, only low concentrations of CLC may improve semen quality after thawing without adversely affecting other parameters.

Effect of Lipid Composition on the Interaction of Liposomes with THP-1-Derived Macrophages.

Recently, liposomal formulations that target macrophages have been used to treat lung diseases. However, the detailed mechanism of the cellular uptake must be elucidated to identify a formulation with excellent cellular uptake efficiency to treat non-tuberculous mycobacterial lung disease. We studied the effect of lipid composition on the cellular uptake of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/cholesterol (Chol) liposomes with a size of approximately 200 nm into THP-1-derived macrophages. The amount of DPPC/Chol liposomes (80/20 mol%) was greater than that of DPPC/Chol (60/40 mol%) and DPPC/Chol (67/33 mol%) liposomes. The anisotropy of 1,6-diphenyl-1,3,5-hexatriene indicated that the membrane fluidity of the DPPC/Chol (80/20 mol%) liposomes was higher than that of the other two liposomes. DPPC/Chol (80/20 mol%) and DPPC/Chol (67/33 mol%) liposomes were taken up via clathrin- and caveolae-mediated endocytosis and phagocytosis. However, proteins involved in cellular uptake through ligand-receptor interactions were adsorbed to a greater extent on DPPC/Chol (80/20 mol%) liposomes than on DPPC/Chol (67/33 mol%) liposomes. Pretreatment of cells with antibodies against the low-density lipoprotein receptor and scavenger receptor type B1 largely inhibited the uptake efficiency of DPPC/Chol (80/20 mol%) liposomes. Our results indicate that the membrane fluidity of DPPC/Chol liposomes, which is controlled by the Chol ratio, is an important factor in controlling protein adsorption and the subsequent uptake efficiency of liposomes.

Lipid Giant Vesicles Engulf Living Bacteria Triggered by Minor Enhancement in Membrane Fluidity.

Antibacterial amphiphiles normally kill bacteria by destroying the bacterial membrane. Whether and how antibacterial amphiphiles alter normal cell membrane and lead to subsequent effects on pathogen invasion into cells have been scarcely promulgated. Herein, by taking four antibacterial gemini amphiphiles with different spacer groups to modulate cell-mimic phospholipid giant unilamellar vesicles (GUVs), bacteria adhesion on the modified GUVs surface and bacteria engulfment process by the GUVs are clearly captured by confocal laser scanning microscopy. Further characterization shows that the enhanced cationic surface charge of GUVs by the amphiphiles determines the bacteria adhesion amount, while the involvement of amphiphile in GUVs results in looser molecular arrangement and concomitant higher fluidity in the bilayer membranes, facilitating the bacteria intruding into GUVs. This study sheds new light on the effect of amphiphiles on membrane bilayer and the concurrent effect on pathogen invasion into cell mimics and broadens the nonprotein-mediated endocytosis pathway for live bacteria.

Zinc Cations Uniquely Stabilize Cell Membrane for Cell Cryopreservation.

We report, for the first time, merely using a small amount of (0.039% w/w) Zn(II) instead of very high concentration (25%-50% w/w) of conventional cryoprotective agents (CPAs), i.e., glycerol, during the cryopreservation of red blood cells (RBCs) can lead to a comparable post-thaw recovery rate of ∼95% while avoiding the tedious gradient washout process for the removal of CPA afterward. The result is remarkable, since Zn(II) does not have the ice-controlling ability reported to be critical for CPA. It benefits from its moderate interaction with lipid molecules, facilitating the formation of small and dynamic lipid clusters. Consequently, the membrane fluidity is maintained, and the cells are resilient to osmotic and mechanical stresses during cryopreservation. This study first reports the ion-specific effect on stabilizing the cell membrane; meanwhile, reversibly tuning the structure of biological samples against injuries during the cooling and rewarming provides a new strategy for cryopreservation.

Rapid Bacterial Detection and Gram-Identification Using Bacterially Activated, Macrophage-Membrane-Coated Nanowired-Si Surfaces in a Microfluidic Device.

Bacterially induced sepsis requires rapid bacterial detection and identification. Hours count for critically ill septic patients, while current culture-based detection requires at least 10 h up to several days. Here, we apply a microfluidic device equipped with a bacterially activated, macrophage-membrane-coating on nanowired-Si adsorbent surfaces for rapid, bacterial detection and Gram-identification in bacterially contaminated blood. Perfusion of suspensions of Gram-negative or Gram-positive bacteria through a microfluidic device equipped with membrane-coated adsorbent surfaces detected low (<10 CFU/mL) bacterial levels. Subsequent, in situ fluorescence-staining yielded Gram-identification for guiding antibiotic selection. In mixed Escherichia coli and Staphylococcus aureus suspensions, Gram-negative and Gram-positive bacteria were detected in the same ratios as those fixed in suspension. Results were validated with a 100% correct score by blinded evaluation (two observers) of 15 human blood samples, spiked with widely different bacterial strains or combinations of strains, demonstrating the potential of the platform for rapid (1.5 h in total) diagnosis of bacterial sepsis.