Membrane Lipoprotein Research Articles

TamL is a key player of the outer membrane homeostasis in Bacteroidota.

TamL is a key player of the outer membrane homeostasis in Bacteroidota.

NlpD as a crucial factor in desiccation resistance and biofilm formation in Cronobacter sakazakii

Cronobacter sakazakii is a Gram-negative bacterium known for causing severe infections in neonates, particularly through contaminated infant formula. This study investigated the role of the outer membrane lipoprotein NlpD in the environmental tolerance of C. sakazakii. A nlpD knockout mutant was constructed, and its impact on desiccation resistance, biofilm formation, motility, and proteomic profiles was evaluated and compared with that of the wild-type strain. The nlpD mutant presented reduced desiccation tolerance, reduced ability to form a biofilm, and altered surface hydrophobicity and motility patterns. The complemented strain restored these phenotypic changes, confirming that the observed effects were specifically due to the deletion of nlpD. Proteomic analysis revealed significant differential expression of proteins involved in metabolic and biosynthetic pathways upon nlpD deletion. These findings emphasize the multifaceted role of NlpD in enhancing the environmental tolerance of C. sakazakii, suggesting its importance in the resilience and survival of the bacterium in adverse conditions.

Outer membrane lipoproteins: late to the party, but the center of attention.

An outer membrane (OM) is the hallmark feature that is often used to distinguish "Gram-negative" bacteria. Our understanding of how the OM is built rests largely on studies of Escherichia coli. In that organism-and seemingly in all species of the Proteobacterial phyla-the essential pathways that assemble the OM each rely on one or more lipoproteins that have been trafficked to the OM. Hence, the lipoprotein trafficking pathway appeared to be foundational for the ability of these bacteria to build their OM. However, such a notion now appears to be misguided. New phylogenetic analyses now show us that lipoprotein trafficking was likely the very last of the essential OM assembly systems to have evolved. The emergence of lipoprotein trafficking must have been a powerful innovation for the ancestors of Proteobacteria, given how it assumed such a central place in OM biogenesis. In this minireview, we broadly discuss the biosynthesis and trafficking of lipoproteins and ponder why the newest OM assembly system (lipoprotein trafficking) has become so key to building the Proteobacterial OM. We examine the diversity among lipoprotein trafficking systems, noting uniting commonalities and highlighting key differences. Current novel antibiotic development is targeted against a small subset of Proteobacterial species that cause severe human diseases; several inhibitors of lipoprotein biosynthesis and OM trafficking have been recently reported that may become new antibiotics. Understanding the diversity in lipoprotein trafficking may yield selective new antibiotics that preferentially kill important human pathogens while sparing species of normal healthy flora.

The effect of clinically relevant antibiotics on bacterial extracellular vesicle release from E. coli

The effect of clinically relevant antibiotics on bacterial extracellular vesicle release from E. coli

Deciphering the molecular basis of lipoprotein recognition and transport by LolCDE

Outer membrane (OM) lipoproteins serve vital roles in Gram-negative bacteria, contributing to their pathogenicity and drug resistance. For these lipoproteins to function, they must be transported from the inner membrane (IM), where they are assembled, to the OM by the ABC transporter LolCDE. We have previously captured structural snapshots of LolCDE in multiple states, revealing its dynamic conformational changes. However, the exact mechanism by which LolCDE recognizes and transfers lipoprotein between domains remains unclear. Here, we characterized the E. coli LolCDE complex bound with endogenous lipoprotein or ATP to explore the molecular features governing its substrate binding and transport functions. We found that the N-terminal unstructured linker of lipoprotein is critical for efficient binding by LolCDE; it must be sufficiently long to keep the lipoprotein’s main body outside the complex while allowing the triacyl chains to bind within the central cavity. Mutagenic assays identified key residues that mediate allosteric communication between the cytoplasmic and transmembrane domains and in the periplasmic domain to form a lipoprotein transport pathway at the LolC–LolE interface. This study provides insights into the OM lipoprotein relocation process mediated by LolCDE, with significant implications for antimicrobial drug development.

RcsF-independent mechanisms of signaling within the Rcs phosphorelay.

The Rcs (regulator of capsule synthesis) phosphorelay is a conserved cell envelope stress response mechanism in enterobacteria. It responds to perturbations at the cell surface and the peptidoglycan layer from a variety of sources, including antimicrobial peptides, beta-lactams, and changes in osmolarity. RcsF, an outer membrane lipoprotein, is the sensor for this pathway and activates the phosphorelay by interacting with an inner membrane protein IgaA. IgaA is essential; it negatively regulates the signaling by interacting with the phosphotransferase RcsD. We previously showed that RcsF-dependent signaling does not require the periplasmic domain of the histidine kinase RcsC and identified a dominant negative mutant of RcsD that can block signaling via increased interactions with IgaA. However, how the inducing signals are sensed and how signal is transduced to activate the transcription of the Rcs regulon remains unclear. In this study, we investigated how the Rcs cascade functions without its only known sensor, RcsF, and characterized the underlying mechanisms for three distinct RcsF-independent inducers. Previous reports showed that Rcs activity can be induced in the absence of RcsF by a loss of function mutation in the periplasmic oxidoreductase DsbA or by overexpression of the DnaK cochaperone DjlA. We identified an inner membrane protein, DrpB, as a multicopy RcsF-independent Rcs activator in E. coli. The loss of the periplasmic oxidoreductase DsbA and the overexpression of the DnaK cochaperone DjlA each trigger the Rcs cascade in the absence of RcsF by weakening IgaA-RcsD interactions in different ways. In contrast, the cell-division associated protein DrpB uniquely requires the RcsC periplasmic domain for activation; this domain is not needed for RcsF-dependent signaling. This suggests the possibility that the RcsC periplasmic domain acts as a sensor for some Rcs signals. Overall, the results add new understanding to how this complex phosphorelay can be activated by diverse mechanisms.

The BfmRS stress response protects Acinetobacter baumannii against defects in outer membrane lipoprotein biogenesis.

As the cell's surface, the outer membrane (OM) of bacteria, such as Acinetobacter baumannii, is continuously under assault from the environment or host. OM integrity is needed for cell survival, and envelope stress responses (ESRs) act to detect and repair any defects. ESRs are well-defined in Escherichia coli but are poorly conserved. We sought to identify an ESR for the essential process of OM lipoprotein biogenesis in A. baumannii. We found that the BfmRS two-component system performs this function and does so without relying on its NlpE sensor homolog, suggesting a novel mechanism of stress sensing is involved in A. baumannii. Our work identifies a key cellular role for BfmRS.

Delta-9 desaturase reduction in gastrointestinal cells induced to senescence by doxorubicin.

The condition of cellular senescence has specific features, including an altered lipid metabolism. Delta-9 desaturase (Δ9) catalyzes the conversion of saturated fatty acids, such as palmitic acid and stearic acid, into their monounsaturated forms, palmitoleic and oleic acid, respectively. Δ9 activity is important for most lipid functions, such as membrane fluidity, lipoprotein metabolism and energy storage. The present study aimed to investigate differences in the expression of Δ9 in senescence-induced pancreatic (MIA-PaCa-2 and PANC-1) and hepatic (Hepa-RG and HLF) cancer cell lines. Cellular senescence was induced by growing cells in the presence of the chemotherapic drug doxorubicin. Senescence status was determined by the senescence-associated beta-galactosidase activity assay kit combined with the p21 and senescence associated secretory phenotype protein assay. Δ9 was downregulated in all senescence-induced cell lines compared to control cells, in both the lipidomic analysis and when measuring protein levels via western blotting. Hence, our findings demonstrate that the study of membrane lipid composition and the expression levels of Δ9 could potentially form the basis for future applications investigating the state of cellular senescence.

Cell division cycle fluctuation of Pal concentration in Escherichia coli.

The Tol-Pal proteins stabilize the outer membrane during cell division in many Gram-negative bacteria, including Escherichia coli. Pal is an outer membrane lipoprotein that can bind peptidoglycan. It accumulates at the septum during division by a mobilization-and-capture mechanism. This work further substantiates and extends knowledge of Pal's localization in E. coli using immunolabelling; this method enables the detection of endogenous proteins. The midcell localization of Pal and TolB, as seen with fluorescent protein fusions, during cell division, was confirmed. The retention of Pal in newly formed cell poles seemed to persist longer than observed with fluorescent Pal fusions. The concentration of endogenous Pal during the cell division cycle fluctuated: it decreased initially (to half the fluorescence concentration (32.1 au µm-3) of the maximum (64.1 au µm-3) reached during the cell cycle) and then increased during the second half of the cell division cycle. We probed for possible regulators and proposed two new putative regulators of Pal. By deleting the periplasmic protease, Prc decreased the total Pal abundance (to ~65% of the fluorescence concentration in WT cells) and affected its concentration fluctuation during the cell cycle. This suggests that Prc controls a cell division stage-specific regulator of Pal. Immunolabelling also supported the prediction that the small RNA MicA suppresses Pal expression (the fluorescence concentration of Pal in cells without MicA is double that of Pal in WT cells). However, the regulation by MicA occurred in a cell cycle-independent manner. All these findings urge further research on the tight regulation of the dividing cell envelope stability.

The Salmonella enterica EnvE is an Outer Membrane Lipoprotein and Its Gene Expression Leads to Transcriptional Repression of the Virulence Gene msgA.

The envE gene of Salmonella enterica serovar Typhimurium is encoded within Salmonella Pathogenicity Island-11 (SPI-11) and is located immediately upstream of the virulence gene msgA (macrophage survival gene A) in the same transcriptional orientation. To date, the characteristics and roles of envE remain largely unexplored. In this study, we show that EnvE, a predicted lipoprotein, is localized on the outer membrane using sucrose gradient ultracentrifugation. Under oxidative stress conditions, envE transcription is suppressed, while msgA transcription is induced, indicating an inverse correlation between the mRNA levels of the two neighboring genes. Importantly, inactivation of envE leads to constitutive transcription of msgA regardless of the presence of oxidative stress. Moreover, trans-complementation of the envE mutant with a plasmid-borne envE fails to prevent the induction of msgA transcription, suggesting that envE functions as a cis-regulatory element rather than a trans-acting factor. We further show that both inactivation and complementation of envE confer wild-type levels of resistance to oxidative stress by ensuring the expression of msgA. Our data suggest that the S. enterica envE gene encodes an outer membrane lipoprotein, and its transcription represses msgA expression in a cis-acting manner, probably by transcriptional interference, although the exact molecular details are yet unclear.

Identification of immunogenic antigens and evaluation of vaccine candidates against Clostridium perfringens

Identification of immunogenic antigens and evaluation of vaccine candidates against Clostridium perfringens

Mycoplasma genitalium membrane lipoprotein induces GAPDH malonylation in urethral epithelial cells to regulate cytokine response

Mycoplasma genitalium membrane lipoprotein induces GAPDH malonylation in urethral epithelial cells to regulate cytokine response

Intra-strain colony biofilm heterogeneity in uropathogenic Escherichia coli and the effect of the NlpI lipoprotein

Intra-strain colony biofilm heterogeneity in uropathogenic Escherichia coli and the effect of the NlpI lipoprotein

Computational Design of Cyclic Peptide Inhibitors of a Bacterial Membrane Lipoprotein Peptidase.

There remains a critical need for new antibiotics against multi-drug-resistant Gram-negative bacteria, a major global threat that continues to impact mortality rates. Lipoprotein signal peptidase II is an essential enzyme in the lipoprotein biosynthetic pathway of Gram-negative bacteria, making it an attractive target for antibacterial drug discovery. Although natural inhibitors of LspA have been identified, such as the cyclic depsipeptide globomycin, poor stability and production difficulties limit their use in a clinical setting. We harness computational design to generate stable de novo cyclic peptide analogues of globomycin. Only 12 peptides needed to be synthesized and tested to yield potent inhibitors, avoiding costly preparation of large libraries and screening campaigns. The most potent analogues showed comparable or better antimicrobial activity than globomycin in microdilution assays against ESKAPE-E pathogens. This work highlights computational design as a general strategy to combat antibiotic resistance.

Leptospiral LipL45 lipoprotein undergoes processing and shares structural similarities with bacterial sigma regulators.

Leptospiral LipL45 lipoprotein undergoes processing and shares structural similarities with bacterial sigma regulators.

Comprehensive Proteomics Analysis of Polyhydroxyalkanoate (PHA) Biology in Pseudomonas putida KT2440: The Outer Membrane Lipoprotein OprL is a Newly Identified Phasin

Pseudomonas putida KT2440 is an important bioplastic-producing industrial microorganism capable of synthesizing the polymeric carbon-rich storage material, polyhydroxyalkanoate (PHA). PHA is sequestered in discrete PHA granules, or carbonosomes, and accumulates under conditions of stress, for example, low levels of available nitrogen. The pha locus responsible for PHA metabolism encodes both anabolic and catabolic enzymes, a transcription factor, and carbonosome-localized proteins termed phasins. The functions of phasins are incompletely understood but genetic disruption of their function causes PHA-related phenotypes. To improve our understanding of these proteins, we investigated the PHA pathways of P.putida KT2440 using three types of experiments. First, we profiled cells grown in nitrogen-limited and nitrogen-excess media using global expression proteomics, identifying sets of proteins found to coordinately increase or decrease within clustered pathways. Next, we analyzed the protein composition of isolated carbonosomes, identifying two new putative components. We carried out physical interaction screens focused on PHA-related proteins, generating a protein-protein network comprising 434 connected proteins. Finally, we confirmed that the outer membrane protein OprL (the Pal component of the Pal-Tol system) localizes to the carbonosome and shows a PHA-related phenotype and therefore is a novel phasin. The combined datasets represent a valuable overview of the protein components of the PHA system in P.putida highlighting the complex nature of regulatory interactions responsive to nutrient stress.

The Trimeric Autotransporter Adhesin SadA from Salmonella spp. as a Novel Bacterial Surface Display System.

Bacterial surface display platforms have been developed for applications such as vaccine delivery and peptide library screening. The type V secretion system is an attractive anchoring motif for the surface expression of foreign proteins in gram-negative bacteria. SadA belongs to subtype C of the type V secretion system derived from Salmonella spp. and promotes biofilm formation and host cell adherence. The inner membrane lipoprotein SadB is important for SadA translocation. In this study, SadA was used as an anchoring motif to expose heterologous proteins in Salmonella typhimurium using SadB. The ability of SadA to display heterologous proteins on the S. typhimurium surface in the presence of SadB was approximately three-fold higher than that in its absence of SadB. Compared to full-length SadA, truncated SadAs (SadA877 and SadA269) showed similar display capacities when exposing the B-cell epitopes of urease B from Helicobacter pylori (UreB158-172aa and UreB349-363aa). We grafted different protein domains, including mScarlet (red fluorescent protein), the urease B fragment (UreBm) from H. pylori SS1, and/or protective antigen domain 4 from Bacillus anthracis A16R (PAD4), onto SadA877 or SadA1292. Whole-cell dot blotting, immunofluorescence, and flow cytometric analyses confirmed the localization of Flag×3-mScarlet (~30 kDa) and Flag×3-UreBm-mScarlet (~58 kDa) to the S. typhimurium surface using truncated SadA877 or SadA1292 as an anchoring motif. However, Flag×3-UreBm-PAD4-mScarlet (~75 kDa) was displayed on S. typhimurium using SadA1292. The oral administrated pSadBA1292-FUM/StmΔygeAΔmurI and pSadBA877-FUM/StmΔygeAΔmurI could elicit a significant mucosal and humoral immunity response. SadA could thus be used as an anchoring motif for the surface expression of large heterologous proteins as a potential strategy for attenuated bacterial vaccine development.

Fatty acids of Helicobacter pylori lipoproteins CagT and Lpp20.

Bacterial lipoproteins are post-translationally modified by the addition of acyl chains that anchor the protein to bacterial membranes. This modification includes two ester-linked and one amide-linked acyl chain on lipoproteins from Gram-negative bacteria. Helicobacter pylori lipoproteins have important functions in pathogenesis (including delivering the CagA oncoprotein to mammalian cells) and are recognized by host innate and adaptive immune systems. The number and variety of acyl chains on lipoproteins impact the innate immune response through Toll-like receptor 2. The acyl chains added to lipoproteins are derived from membrane phospholipids. H. pylori membrane phospholipids have previously been shown to consist primarily of C14:0 and C19:0 cyclopropane-containing acyl chains. However, the acyl composition of H. pylori lipoproteins has not been determined. In this study, we characterized the acyl composition of two representative H. pylori lipoproteins, Lpp20 and CagT. Fatty acid methyl esters were prepared from both purified lipoproteins and analyzed by gas chromatography-mass spectrometry. For comparison, we also analyzed H. pylori phospholipids. Consistent with previous studies, we observed that the H. pylori phospholipids contain primarily C14:0 and C19:0 cyclopropane-containing fatty acids. In contrast, both the ester-linked and amide-linked fatty acids found in H. pylori lipoproteins were observed to be almost exclusively C16:0 and C18:0. A discrepancy between the acyl composition of membrane phospholipids and lipoproteins as reported here for H. pylori has been previously reported in other bacteria including Borrelia and Brucella. We discuss possible mechanisms.IMPORTANCEColonization of the stomach by Helicobacter pylori is an important risk factor in the development of gastric cancer, the third leading cause of cancer-related death worldwide. H. pylori persists in the stomach despite an immune response against the bacteria. Recognition of lipoproteins by TLR2 contributes to the innate immune response to H. pylori. However, the role of H. pylori lipoproteins in bacterial persistence is poorly understood. As the host response to lipoproteins depends on the acyl chain content, defining the acyl composition of H. pylori lipoproteins is an important step in characterizing how lipoproteins contribute to persistence.

Identification and Characterization of Surface Adhesive Proteins of Prevotella Intermedia that Bind to Saliva and Extracellular Matrix Proteins

Adhesin proteins Prevotella intermedia mediate the attachment of bacteria to host cells and thereby induce the development of periodontal disease. These adhesin proteins are important targets for developing vaccines for treating periodontal disease. This study used two groups of host proteins to capture potential adhesins: salivary proteins and host extracellular matrix proteins (fibrinogen, fibronectin, laminin, collagen I, and collagen IV). We identified 17 adhesin candidates using this approach, including outer membrane proteins, proteases, hemagglutinins, lipoproteins, and porins. Two adhesins (PIN_A0102 and PIN_A1455) were expressed as recombinant proteins and purified, and their binding to ECM proteins was analyzed by the ELISA method. The outer membrane protein (PIN_A0102) was shown to bind to ECM proteins (fibrinogen and fibronectin). Another protein, the protease/hemagglutinin PrT (PIN_A1373), binds to saliva proteins, fibrinogen, and lectin. Thus, P. intermedia 17 adhesin proteins have been identified that effectively bind to host proteins and are potential candidates for developing vaccines against periodontal disease.

An octameric PqiC toroid stabilises the outer-membrane interaction of the PqiABC transport system

The E. coli Paraquat Inducible (Pqi) Pathway is a putative Gram-negative phospholipid transport system. The pathway comprises three components: an integral inner membrane protein (PqiA), a periplasmic spanning MCE family protein (PqiB) and an outer membrane lipoprotein (PqiC). Interactions between all complex components, including stoichiometry, remain uncharacterised; nevertheless, once assembled into their quaternary complex, the trio of Pqi proteins are anticipated to provide a continuous channel between the inner and outer membranes of diderms. Here, we present X-ray structures of both the native and a truncated, soluble construct of the PqiC lipoprotein, providing insight into its biological assembly, and utilise neutron reflectometry to characterise the nature of the PqiB-PqiC-membrane interaction. Finally, we employ phenotypic complementation assays to probe specific PqiC residues, which imply the interaction between PqiB and PqiC is less intimate than previously anticipated.