Bacterial Lipoproteins Research Articles

Identification and characterization of a novel stress-responsive outer membrane protein Lip40 from Actinobacillus pleuropneumoniae

BackgroundActinobacillus pleuropneumoniae, a Gram-negative bacterium, is the causative agent of porcine pleuropneumonia, a highly contagious and often fatal disease. Because current vaccines confer limited protection against A. pleuropneumoniae infection, the development of more effective vaccines is urgently required. The identification of immunogenic and protective antigens, such as an outer-membrane lipoprotein, will advance this purpose.ResultsSixty putative lipoproteins were predicted from the genomic sequence of A. pleuropneumoniae using multiple algorithms. Here, we focused on the characteristics of the putative lipoprotein Lip40 from A. pleuropneumoniae strain SLW01 (serovar 1). Lip40 shares sequence similarity with many bacterial lipoproteins, and the structural prediction of Lip40 suggests that it is similar to A. pleuropneumoniae TbpB. The N-terminus of Lip40 contains an interesting tandemly repeated sequence, Q(E/D/P)QPK. Real-time RT–PCR indicated that the expression of lip40 was significantly upregulated at 42 °C, at 16 °C, and under anaerobic conditions. Recombinant Lip40 (rLip40) produced in Escherichia coli BL21(DE3) was specifically recognized by porcine convalescent serum directed against A. pleuropneumoniae. Lip40 was confirmed to localize at the bacterial outer membrane, and its expression was significantly stimulated when A. pleuropneumoniae was cultured under various stress conditions. Lip40 also protected 75 % of mice from fatal virulent A. pleuropneumoniae infection.ConclusionsThe immunogenic outer-membrane protein Lip40 is stress responsive, protects mice against infection, and might be a virulence determinant. Further investigation of Lip40 should expedite vaccine development and provide insight into the pathogenesis of A. pleuropneumoniae.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-015-0199-8) contains supplementary material, which is available to authorized users.

Quantitative Lipoproteomics in Clostridium difficile Reveals a Role for Lipoproteins in Sporulation

Bacterial lipoproteins are surface exposed, anchored to the membrane by S-diacylglyceryl modification of the N-terminal cysteine thiol. They play important roles in many essential cellular processes and in bacterial pathogenesis. For example, Clostridium difficile is a Gram-positive anaerobe that causes severe gastrointestinal disease; however, its lipoproteome remains poorly characterized. Here we describe the application of metabolic tagging with alkyne-tagged lipid analogs, in combination with quantitative proteomics, to profile protein lipidation across diverse C.difficile strains and on inactivation of specific components of the lipoprotein biogenesis pathway. These studies provide the first comprehensive map of the C.difficile lipoproteome, demonstrate the existence of two active lipoprotein signal peptidases, and provide insights into lipoprotein function, implicating the lipoproteome in transmission of this pathogen.

Analyzing the molecular mechanism of lipoprotein localization in Brucella.

Bacterial lipoproteins possess diverse structure and functionality, ranging from bacterial physiology to pathogenic processes. As such many lipoproteins, originating from Brucella are exploited as potential vaccines to countermeasure brucellosis infection in the host. These membrane proteins are translocated from the cytoplasm to the cell membrane where they are anchored peripherally by a multifaceted targeting mechanism. Although much research has focused on the identification and classification of Brucella lipoproteins and their potential use as vaccine candidates for the treatment of Brucellosis, the underlying route for the translocation of these lipoproteins to the outer surface of the Brucella (and other pathogens) outer membrane (OM) remains mostly unknown. This is partly due to the complexity of the organism and evasive tactics used to escape the host immune system, the variation in biological structure and activity of lipoproteins, combined with the complex nature of the translocation machinery. The biosynthetic pathway of Brucella lipoproteins involves a distinct secretion system aiding translocation from the cytoplasm, where they are modified by lipidation, sorted by the lipoprotein localization machinery pathway and thereafter equipped for export to the OM. Surface localized lipoproteins in Brucella may employ a lipoprotein flippase or the β-barrel assembly complex for translocation. This review provides an overview of the characterized Brucella OM proteins that form part of the OM, including a handful of other characterized bacterial lipoproteins and their mechanisms of translocation. Lipoprotein localization pathways in gram negative bacteria will be used as a model to identify gaps in Brucella lipoprotein localization and infer a potential pathway. Of particular interest are the dual topology lipoproteins identified in Escherichia coli and Haemophilus influenza. The localization and topology of these lipoproteins from other gram negative bacteria are well characterized and may be useful to infer a solution to better understand the translocation process in Brucella.

Outer membrane lipoprotein biogenesis: Lol is not the end.

Bacterial lipoproteins are lipid-anchored proteins that contain acyl groups covalently attached to the N-terminal cysteine residue of the mature protein. Lipoproteins are synthesized in precursor form with an N-terminal signal sequence (SS) that targets translocation across the cytoplasmic or inner membrane (IM). Lipid modification and SS processing take place at the periplasmic face of the IM. Outer membrane (OM) lipoproteins take the localization of lipoproteins (Lol) export pathway, which ends with the insertion of the N-terminal lipid moiety into the inner leaflet of the OM. For many lipoproteins, the biogenesis pathway ends here. We provide examples of lipoproteins that adopt complex topologies in the OM that include transmembrane and surface-exposed domains. Biogenesis of such lipoproteins requires additional steps beyond the Lol pathway. In at least one case, lipoprotein sequences reach the cell surface by being threaded through the lumen of a beta-barrel protein in an assembly reaction that requires the heteropentomeric Bam complex. The inability to predict surface exposure reinforces the importance of experimental verification of lipoprotein topology and we will discuss some of the methods used to study OM protein topology.

Myeloid-related protein 8 induces self-tolerance and cross-tolerance to bacterial infection via TLR4- and TLR2-mediated signal pathways.

Myeloid-related protein 8 (Mrp8) is the active component of Mrp8/14 protein complex released by phagocytes at the site of infection and stimulates inflammatory responses. However, it is unclear whether Mrp8 could induce self-tolerance and cross-tolerance to bacterial infection. Here we report that Mrp8 triggered TNF-α and IL-6 release via a Toll-like receptor 4 (TLR4)-dependent manner. Pre-stimulation of murine macrophages and human monocytes with Mrp8 induced self-tolerance to Mrp8 re-stimulation and cross-tolerance to lipopolysaccharide (LPS), bacterial lipoprotein (BLP), gram-negative and gram-positive bacterial challenges, with substantially attenuated TNF-α and IL-6 release. Moreover, Mrp8 tolerisation significantly reduced serum TNF-α and IL-6, increased polymorphonuclear neutrophil (PMN) recruitment and accelerated bacterial clearance, thus protecting mice against LPS-induced lethality and cecal ligation and puncture (CLP)-induced polymicrobial sepsis. In addition to TLR4, TLR2 also contributed to Mrp8-induced inflammatory response and tolerance. Down-regulation of phosphorylated p38 by Mrp8 pre-stimulation was predominantly responsible for the intracellular mechanism of Mrp8-induced tolerance. Thus, our findings of Mrp8-induced self-tolerance and cross-tolerance may provide a potential strategy for attenuating an overwhelming proinflammatory cascade and enhancing antimicrobial responses during microbial sepsis.

The Human Pathogen Streptococcus pyogenes Releases Lipoproteins as Lipoprotein-rich Membrane Vesicles

Bacterial lipoproteins are attractive vaccine candidates because they represent a major class of cell surface-exposed proteins in many bacteria and are considered as potential pathogen-associated molecular patterns sensed by Toll-like receptors with built-in adjuvanticity. Although Gram-negative lipoproteins have been extensively characterized, little is known about Gram-positive lipoproteins. We isolated from Streptococcus pyogenes a large amount of lipoproteins organized in vesicles. These vesicles were obtained by weakening the bacterial cell wall with a sublethal concentration of penicillin. Lipid and proteomic analysis of the vesicles revealed that they were enriched in phosphatidylglycerol and almost exclusively composed of lipoproteins. In association with lipoproteins, a few hypothetical proteins, penicillin-binding proteins, and several members of the ExPortal, a membrane microdomain responsible for the maturation of secreted proteins, were identified. The typical lipidic moiety was apparently not necessary for lipoprotein insertion in the vesicle bilayer because they were also recovered from the isogenic diacylglyceryl transferase deletion mutant. The vesicles were not able to activate specific Toll-like receptor 2, indicating that lipoproteins organized in these vesicular structures do not act as pathogen-associated molecular patterns. In light of these findings, we propose to name these new structures Lipoprotein-rich Membrane Vesicles.

Immunogenicity of a novel enhanced consensus DNA vaccine encoding the leptospiral protein LipL45

Leptospirosis is a bacterial zoonotic disease caused by an infection with a spirochete belonging to the genus Leptospira. In animals, leptospirosis displays a wide range of pathologies, including fever, abortion, icterus, and uveitis. Conversely, infection in humans is associated with multi-organ injury, resulting in an increased rate of fatalities. Pathogenic leptospires are able to translocate through cell monolayers at a rate significantly greater than that of non-pathogenic leptospires. Thus, vaccine approaches have been focused on targeting bacterial motility, lipopolysaccharides (LPSs), lipoproteins, outer-membrane proteins (OMPs) and other potential virulence factors. Previous studies have indicated that leptospiral proteins elicit long-lasting immunological memory in infected humans. In the study reported here, the efficacy of a synthetic consensus DNA vaccine developed against the Leptospira membrane lipoprotein LipL45 was tested. After in vivo electroporation (EP) mediated intramuscular immunization with a synthetic LipL45 DNA vaccine (pLipL45) immunized mice developed a significant cellular response along with the development of anti-LipL45-specific antibodies. Specifically, the pLipL45 vaccine induced a significant Th1 type immune response, indicated by the higher production of IL-12 and IFN-γ cytokines. The results presented here are the first demonstration that a LipL45 based DNA immunogen has potential as a anti-Leptospira vaccine.

Staphylococcus aureus induces IL-8 expression through its lipoproteins in the human intestinal epithelial cell, Caco-2

Staphylococcus aureus can cause the intestinal inflammatory diseases. However, little is known about the molecular mechanism of S. aureus infection in the intestine. In the present study, we investigated whether S. aureus could stimulate human intestinal epithelial cells triggering inflammation. When the human intestinal epithelial cell-line, Caco-2, and the primary colon cells were stimulated with ethanol-inactivated S. aureus, IL-8 expression was induced in a dose-dependent manner. The inactivated S. aureus preferentially stimulated Toll-like receptor (TLR) 2 rather than TLR4. Lipoproteins, lipoteichoic acid (LTA), and peptidoglycan (PGN) are considered as potential TLR2 ligands of S. aureus. Interestingly, S aureus lipoproteins and Pam2CSK4 mimicking Gram-positive bacterial lipoproteins, but not LTA and PGN of S. aureus, significantly induced IL-8 expression in Caco-2 cells. Furthermore, lipoprotein-deficient S. aureus mutant strain failed to induce IL-8 production. Collectively, these results suggest that S. aureus stimulates the human intestinal epithelial cells to induce the chemokine IL-8 production through its lipoproteins, potentially contributing the development of intestinal inflammation.

Lipoteichoic acid of Lactobacillus plantarum inhibits Staphylococcus aureus-induced IL-8 expression in human intestinal epithelial cells (IRM11P.641)

Abstract Lactobacilli as probiotics are well known to alleviate intestinal inflammation. However, the molecular basis for the mechanism has not been well understood. Here, we investigated the inhibitory effect of Lactobacillus plantarum lipoteichoic acid (Lp.LTA) on the induction of interleukin-8 (IL-8), which plays an important role in inflammatory responses by recruiting various immune cells, in human intestinal epithelial cells, Caco-2. Staphylococcus aureus but not the lipoprotein-deficient S. aureus mutant, induced IL-8 expression, implying that the lipoproteins are crucial in the induction of IL-8 by S. aureus. Pam2CSK4, a synthetic lipopeptide that is known as a mimic of Gram-positive bacterial lipoproteins, also induced IL-8 expression in Caco-2 cells. While heat-inactivated L. plantarum or peptidoglycan of L. plantarum did not inhibit Pam2CSK4-induced IL-8 expression, Lp.LTA significantly inhibited its expression. Deacylated or dealanylated Lp.LTA failed to inhibit Pam2CSK4-induced IL-8 expression, suggesting that both lipid and D-alanine moieties are responsible for the inhibitory potency of Lp.LTA. Furthermore, Lp.LTA inhibited the activation of Toll-like receptor 2 sensing bacterial lipoproteins and the intracellular signal transduction involving p38 kinase, JNK and NF-κB in Pam2CSK4-stimulated Caco-2 cells. Taken together, these results suggest that Lp.LTA alleviates S. aureus-induced intestinal inflammatory responses by inhibiting IL-8 expression.

Dual orientation of the outer membrane lipoprotein Pal in Escherichia coli.

Peptidoglycan associated lipoprotein (Pal) of Escherichia coli (E. coli) is a characteristic bacterial lipoprotein, with an N-terminal lipid moiety anchoring it to the outer membrane. Since its discovery over three decades ago, Pal has been well studied for its participation in the Tol-Pal complex which spans the periplasm and has been proposed to play important roles in bacterial survival, pathogenesis and virulence. Previous studies of Pal place the lipoprotein in the periplasm of E. coli, allowing it to interact with Tol proteins and the peptidoglycan layer. Here, we describe for the first time, a subpopulation of Pal which is present on the cell surface of E. coli. Flow cytometry and confocal microscopy detect anti-Pal antibodies on the surface of intact E. coli cells. Interestingly, Pal is surface exposed in an 'all or nothing' manner, such that most of the cells contain only internal Pal, with fewer cells ( < 20 %) exhibiting surface Pal.

The molecular mechanism of bacterial lipoprotein modification--how, when and why?

Posttranslational modification of proteins by lipidation is a common process in biological systems. Lipids provide protein stability, interaction with other membrane components, and in some cases, due to reversibility of the process, a mechanism for regulating protein localization and function. Bacterial lipoproteins possess fatty acids at their amino-termini that are derived from phospholipids, and this lipid moiety anchors the proteins into the membrane. These lipids, as is the case for lipopolysaccharides and lipoteichoic acids, play an important role in signaling of the innate immune system through the interaction with Toll-like receptors. Over the past three years, tremendous progress has been made in understanding the mechanism by which lipoproteins become lipidated. Advanced methodology in mass spectrometry, proteomics and genome-wide analyses allowed precise characterization of lipoprotein modifications and the identification of the enzymes catalyzing the reactions in diverse bacterial species. This review will highlight new findings on bacterial lipoprotein modification with focus on the reaction mechanisms and the role of lipoproteins in cell envelope homeostasis.

Residues located on membrane-embedded flexible loops are essential for the second step of the apolipoprotein N-acyltransferase reaction.

Apolipoprotein N-acyltransferase (Lnt) is an essential membrane-bound enzyme that catalyzes the third and last step in the post-translational modification of bacterial lipoproteins. In order to identify essential residues implicated in substrate recognition and/or binding we screened for non-functional variants of Lnt obtained by error-prone polymerase chain reaction in a complementation assay using a lnt depletion strain. Mutations included amino acid substitutions in the active site and of residues located on flexible loops in the catalytic periplasmic domain. All, but one mutation, led to the formation of the thioester acyl-enzyme intermediate and to the accumulation of apo-Lpp, suggesting that these residues are involved in the second step of the reaction. A large cytoplasmic loop contains a highly conserved region and two hydrophobic segments. Accessibility analysis to alkylating reagents of substituted cysteine residues introduced in this region demonstrated that the hydrophobic segments do not completely span the membrane. Two residues in the highly conserved cytoplasmic region were shown to be essential for Lnt function. Together, our data suggest that amino acids located on flexible cytoplasmic and periplasmic loops, predicted to be membrane embedded, are required for efficient N-acylation of lipoproteins.

Multi-cellular 3D human primary liver cell culture elevates metabolic activity under fluidic flow.

We have developed a low-cost liver cell culture device that creates fluidic flow over a 3D primary liver cell culture that consists of multiple liver cell types, including hepatocytes and non-parenchymal cells (fibroblasts, stellate cells, and Kupffer cells). We tested the performance of the cell culture under fluidic flow for 14 days, finding that hepatocytes produced albumin and urea at elevated levels compared to static cultures. Hepatocytes also responded with induction of P450 (CYP1A1 and CYP3A4) enzyme activity when challenged with P450 inducers, although we did not find significant differences between static and fluidic cultures. Non-parenchymal cells were similarly responsive, producing interleukin 8 (IL-8) when challenged with 10 μM bacterial lipoprotein (LPS). To create the fluidic flow in an inexpensive manner, we used a rocking platform that tilts the cell culture devices at angles between ±12°, resulting in a periodically changing hydrostatic pressure drop between reservoirs and the accompanying periodically changing fluidic flow (average flow rate of 650 μL min(-1), and a maximum shear stress of 0.64 dyne cm(-2)). The increase in metabolic activity is consistent with the hypothesis that, similar to unidirectional fluidic flow, primary liver cell cultures increase their metabolic activity in response to fluidic flow periodically changes direction. Since fluidic flow that changes direction periodically drastically changes the behavior of other cells types that are shear sensitive, our findings support the theory that the increase in hepatic metabolic activity associated with fluidic flow is either activated by mechanisms other than shear sensing (for example increased opportunities for gas and metabolite exchange), or that it follows a shear sensing mechanism that does not depend on the direction of shear. Our mode of device operation allows us to evaluate drugs under fluidic cell culture conditions and at low device manufacturing and operation costs.

Oral administration of a Spirulina extract enriched for Braun-type lipoproteins protects mice against influenza A (H1N1) virus infection

A growing body of research indicates that oral administration of bacteria (such as probiotics) can exhibit a protective effect against influenza A (H1N1) viral infection in mice. In the present study, we used a mouse model to examine whether oral administration of Immulina®, a commercial extract from the cyanobacteria Arthrospira (Spirulina) platensis, can reduce the severity of illness resulting from influenza A (H1N1) viral infection. The main active compounds within Immulina® are bacterial Braun-type lipoproteins that activate innate immune cells through a toll-like receptor (TLR) 2-dependent pathway. Mice that were fed Immulina® for 30 days before and 21 days after infection with influenza A (H1N1) virus exhibited a statistically significant reduction in the severity of infection. Compared to the control group, Immulina®-fed mice exhibited less weight loss, increased appetite, decreased clinical signs of disease, and lower lung histopathology scores. The results from the present study adds to the increasing evidence that oral administration of bacterial components that activate innate immune cells, whether derived from a bacterial preparation (probiotics or cyanobacteria) or from plant material containing endophytic bacteria, can exhibit a protective effect against influenza A (H1N1) viral infection.

Lipoteichoic acid from Lactobacillus plantarum inhibits Pam2CSK4-induced IL-8 production in human intestinal epithelial cells

Lactobacilli are probiotic bacteria that are considered to be beneficial in the gastrointestinal tract of humans. Although lactobacilli are well known to alleviate intestinal inflammation, the molecular basis of this phenomenon is poorly understood. In this study, we investigated the effect of Lactobacillus plantarum lipoteichoic acid (Lp.LTA), which is a major cell wall component of this species, on the production of interleukin (IL)-8 in human intestinal epithelial Caco-2 cells. Treatment with Pam2CSK4, a synthetic lipopeptide that is known to mimic Gram-positive bacterial lipoproteins as an important virulence factor, significantly induced IL-8 expression in Caco-2 cells. However, neither heat-inactivated L. plantarum nor L. plantarum peptidoglycan inhibited Pam2CSK4-induced IL-8 mRNA expression. In addition, both a deacylated form and a dealanylated form of Lp.LTA failed to inhibit Pam2CSK4-induced IL-8 expression, indicating that the lipid and D-alanine moieties are critical for Lp.LTA-mediated inhibition. Moreover, Lp.LTA inhibited Pam2CSK4-induced activation of p38 kinase, JNK, and NF-κB transcription factor by suppressing toll-like receptor 2 activation. Collectively, these results suggest that Lp.LTA exerts anti-inflammatory effects on human intestinal epithelial cells by blocking IL-8 production.

Immune response profile elicited by the model antigen ovalbumin expressed in fusion with the bacterial OprI lipoprotein

The use of immunogenic formulations targeting pattern recognition receptors towards modulation of immune responses is a promising strategy to develop better vaccines against infectious and malignant diseases. Molecules targeting TLR2 offer interesting properties that are relevant for vaccine development, including the possibility to covalently attach the lipidic ligands to the antigens. However, the type of immune response elicited by these formulations is still controversial. In this work, we used the model antigen ovalbumin (OVA) expressed in fusion with the bacterial lipoprotein OprI in order to characterize the immunomodulatory properties of this TLR ligand. Murine bone marrow-derived dendritic cells stimulated with OprI-OVA fusion lipoprotein produced high levels of the pro-inflammatory cytokines TNF-α and IL-6 and also IL-10, IL-12(p70) and IL-27, while TGF-β and IL-23 were not detected. Using OT-II and OT-I mice, an enhancement of MHC class II and class I antigen presentation was observed for the OVA antigen in fusion with OprI. Mice immunized by intraperitoneal route with this fusion lipoprotein in prime-boost protocols developed strong specific antibody responses including IgG1, IgG2c, IgG2b, IgG3 and IgE. These results, together with data obtained by restimulation of splenocytes from the immunized mice, point to an immune response profile that does not correspond to a strict Th1 or Th2 polarization. Finally, in a challenge experiment using a melanoma syngeneic mouse model (B16-OVA), prophylactic inoculation with OprI fused with the unrelated antigen eGFP increased the tumor growth, while the fusion with the tumor-associated antigen OVA delayed the tumor growth and increased mice survival.

The structure of rice weevil pectin methylesterase.

Rice weevils (Sitophilus oryzae) use a pectin methylesterase (EC 3.1.1.11), along with other enzymes, to digest cell walls in cereal grains. The enzyme is a right-handed β-helix protein, but is circularly permuted relative to plant and bacterial pectin methylesterases, as shown by the crystal structure determination reported here. This is the first structure of an animal pectin methylesterase. Diffraction data were collected to 1.8 Å resolution some time ago for this crystal form, but structure solution required the use of molecular-replacement techniques that have been developed and similar structures that have been deposited in the last 15 years. Comparison of the structure of the rice weevil pectin methylesterase with that from Dickeya dandantii (formerly Erwinia chrysanthemi) indicates that the reaction mechanisms are the same for the insect, plant and bacterial pectin methylesterases. The similarity of the structure of the rice weevil enzyme to the Escherichia coli lipoprotein YbhC suggests that the evolutionary origin of the rice weevil enzyme was a bacterial lipoprotein, the gene for which was transferred to a primitive ancestor of modern weevils and other Curculionidae. Structural comparison of the rice weevil pectin methylesterase with plant and bacterial enzymes demonstrates that the rice weevil protein is circularly permuted relative to the plant and bacterial molecules.

Structural Features of a Highly Conserved Omp16 Protein of Pasteurella multocida Strains and Comparison with Related Peptidoglycan-associated Lipoproteins (PAL)

Bacterial lipoproteins of varying size and functional role in physiology/pathogenesis are widely prevalent among Gram-negative bacterial species. Among virulent bacteria, Pasteurella multocida is known to be associated with diverse infectious diseases of livestock worldwide and possess several outer membrane proteins (OMPs) as virulence factors. In the present study, we cloned and sequenced omp16 gene of different P. multocida strains (n = 9) of Indian origin and compared with representative related bacterial species from Pasteurellaceae (n = 28) as well as others (n = 11). Phylogenetic and multiple sequence alignment revealed high degree conservation of Omp16 among P. multocida strains. However, it significantly differed from similar peptidoglycans-associated lipoproteins of other Gram-negative bacterial species. Notably, a conversed lipobox at N-terminus of proteins was noticed among all bacterial species. Further, three dimensional homology model of Omp16 was predicted and its structural features were analyzed. The study indicated the potential possibilities to use either native or recombinant Omp16 protein in developing species-specific diagnostic assay or broadly active subunit vaccine along with suitable adjuvant for pasteurellosis in livestock.

Crystal Structure of TIR Domain of TLR6 Reveals Novel Dimeric Interface of TIR–TIR Interaction for Toll-Like Receptor Signaling Pathway

Toll-like receptors (TLRs) are responsible for recognition of particular pathogens during the innate immune response and cytoplasmic Toll/interleukin-1 receptor (TIR) domain responsible for downstream signaling. TLR6 working with TLR2 can detect bacterial lipoprotein leading signal for nuclear factor-kappaB activation for immune response. To better understand TLR-mediated signaling event in the innate immune system, in this study, we report the first crystal structure of the TIR domain of TLR6 at 2.2Å resolution. Our structure reveals novel homo-dimerization interfaces, which might be a critical for the interaction with TIR-containing adaptor proteins and itself. We also report structural similarities and differences of TLR6 with those of other TIR domains, which may be functionally relevant.

A CRISPR-Cas system enhances envelope integrity mediating antibiotic resistance and inflammasome evasion.

Clustered, regularly interspaced, short palindromic repeats-CRISPR associated (CRISPR-Cas) systems defend bacteria against foreign nucleic acids, such as during bacteriophage infection and transformation, processes which cause envelope stress. It is unclear if these machineries enhance membrane integrity to combat this stress. Here, we show that the Cas9-dependent CRISPR-Cas system of the intracellular bacterial pathogen Francisella novicida is involved in enhancing envelope integrity through the regulation of a bacterial lipoprotein. This action ultimately provides increased resistance to numerous membrane stressors, including antibiotics. We further find that this previously unappreciated function of Cas9 is critical during infection, as it promotes evasion of the host innate immune absent in melanoma 2/apoptosis associated speck-like protein containing a CARD (AIM2/ASC) inflammasome. Interestingly, the attenuation of the cas9 mutant is complemented only in mice lacking both the AIM2/ASC inflammasome and the bacterial lipoprotein sensor Toll-like receptor 2, but not in single knockout mice, demonstrating that Cas9 is essential for evasion of both pathways. These data represent a paradigm shift in our understanding of the function of CRISPR-Cas systems as regulators of bacterial physiology and provide a framework with which to investigate the roles of these systems in myriad bacteria, including pathogens and commensals.