Lipoprotein Signal Peptidase Research Articles

Cosmid based mutagenesis causes genetic instability in Streptomyces coelicolor, as shown by targeting of the lipoprotein signal peptidase gene.

Bacterial lipoproteins are extracellular proteins tethered to cell membranes by covalently attached lipids. Deleting the lipoprotein signal peptidase (lsp) gene in Streptomyces coelicolor results in growth and developmental defects that cannot be restored by reintroducing lsp. This led us to hypothesise that lsp is essential and that the lsp mutant we isolated previously had acquired compensatory secondary mutations. Here we report resequencing of the genomes of wild-type M145 and the cis-complemented ∆lsp mutant (BJT1004) to map and identify these secondary mutations but we show that they do not increase the efficiency of disrupting lsp and are not lsp suppressors. We provide evidence that they are induced by introducing the cosmid St4A10∆lsp, as part of ReDirect PCR mutagenesis protocol, which transiently duplicates a number of important cell division genes. Disruption of lsp using a suicide vector (which does not result in gene duplication) still results in growth and developmental delays and we conclude that loss of Lsp function results in developmental defects due to the loss of all lipoproteins from the cell membrane. Significantly, our results also indicate the use of cosmid libraries for the genetic manipulation of bacteria can lead to phenotypes not necessarily linked to the gene(s) of interest.

Structural basis of lipoprotein signal peptidase II action and inhibition by the antibiotic globomycin.

With functions that range from cell envelope structure to signal transduction and transport, lipoproteins constitute 2 to 3% of bacterial genomes and play critical roles in bacterial physiology, pathogenicity, and antibiotic resistance. Lipoproteins are synthesized with a signal peptide securing them to the cytoplasmic membrane with the lipoprotein domain in the periplasm or outside the cell. Posttranslational processing requires a signal peptidase II (LspA) that removes the signal peptide. Here, we report the crystal structure of LspA from Pseudomonas aeruginosa complexed with the antimicrobial globomycin at 2.8 angstrom resolution. Mutagenesis studies identify LspA as an aspartyl peptidase. In an example of molecular mimicry, globomycin appears to inhibit by acting as a noncleavable peptide that sterically blocks the active site. This structure should inform rational antibiotic drug discovery.

TLR2-Modulating Lipoproteins of the Mycobacterium tuberculosis Complex Enhance the HIV Infectivity of CD4+ T Cells.

Co-infection with Mycobacterium tuberculosis accelerates progression from HIV to AIDS. Our previous studies showed that M. tuberculosis complex, unlike M. smegmatis, enhances TLR2-dependent susceptibility of CD4+ T cells to HIV. The M. tuberculosis complex produces multiple TLR2-stimulating lipoproteins, which are absent in M. smegmatis. M. tuberculosis production of mature lipoproteins and TLR2 stimulation is dependent on cleavage by lipoprotein signal peptidase A (LspA). In order to determine the role of potential TLR2-stimulating lipoproteins on mycobacterial-mediated HIV infectivity of CD4+ T cells, we generated M. smegmatis recombinant strains overexpressing genes encoding various M. bovis BCG lipoproteins, as well as a Mycobacterium bovis BCG strain deficient in LspA (ΔlspA). Exposure of human peripheral blood mononuclear cells (PBMC) to M. smegmatis strains overexpressing the BCG lipoproteins, LprF (p<0.01), LprH (p<0.05), LprI (p<0.05), LprP (p<0.001), LprQ (p<0.005), MPT83 (p<0.005), or PhoS1 (p<0.05), resulted in increased HIV infectivity of CD4+ T cells isolated from these PBMC. Conversely, infection of PBMC with ΔlspA reduced HIV infectivity of CD4+ T cells by 40% relative to BCG-infected cells (p<0.05). These results may have important implications for TB vaccination programs in areas with high mother-to-child HIV transmission.

LspA gene of Mycobacterium tuberculosis co-transcribes with Rv1540 and induced by surface and acidic stress

Lipoprotein signal peptidase, lspA (Rv1539), is the only known gene in mycobacterial genome for cleaving the signal sequence from prolipoprotein to form mature lipoprotein. It has been implicated in maintaining the virulence of Mycobacterium tuberculosis. The regulation of lspA had not been studied so far. Here, we identify a novel operon lspA–Rv1540 in M. tuberculosis. We detected co-transcription of the open reading frames of lspA–Rv1540 in in-vitro as well as in ex-vivo conditions. Analysis of the sequence upstream to lspA revealed a strong promoter activity that was shown to be induced significantly by surface stress and acidic environment.

Structural Insights of LspA, from Mycobacterium Tuberculosis, using Solid State NMR Spectroscopy

Lipoprotein Signal Peptidase A (LspA) is a type II signal peptidase that is an essential enzyme in one of the three distinct steps in lipoprotein maturation. After targeting the prolipoprotein, LspA cleaves the signal peptide of lipoprotein to produce the precursor for the mature lipoprotein.In Mycobacterium tuberculosis (Mtb), gene Rv1539 encodes LspA, an inner membrane protein of 21 kDa (202 amino acid residues). In vivo and in vitro experimental evidence confirms that LspA is essential for Mtb and its contribution for the antibiotic resistance. Small molecules have access to the active site of this enzyme which is located extracellular to the cytoplasmic membrane. All type II signal peptidases have five conserved domains but the absence of those domains in eukaryotic cells makes LspA an appealing drug target for the treatment of tuberculosis. Globomycin, a cyclic-peptide can inhibit LspA in a non-competitive manner.The structure of small helical membrane proteins is very sensitive to their environment and therefore it is vital to characterize the structure them in a native-like environment, such as a synthetic lipid bilayer or a bacterial cell membrane. Solid state NMR spectroscopy is able to make such structural characterizations in such environments. Here, the challenging, sample preparation steps are described and initial Magic Angle Spinning solid state NMR spectra are reported from 13C isotopically labeled LspA in native E.coli and synthetic lipid bilayers. In addition, uniformly oriented samples for solid state NMR spectra of 15N isotopically labeled LspA, have been prepared in synthetic lipid bilayers providing initial structural insights.

Influence of Detergent Properties on the Solubilization and Function of Membrane Proteins

Detergent micelles are often used as a bilayer mimic for membrane proteins for functional assays and high-resolution structural studies, but selecting the appropriate detergent for the protein requires extensive time and resources. The long-term goal of this research is to rationally select a membrane mimic for a given membrane protein that stabilizes fold and native function based on matching physical properties of the detergent and protein. To determine the important micelle and membrane properties, lipoprotein signal peptidase A (LspA) from Chlamydia trachomatis is used as a model system. To screen detergents that support the native function, LspA was purified in over 30 detergent micelles with varying physical properties. LspA is soluble in zwitterionic detergents and solubility remains independent of detergent alkyl chain length, solubilizing in detergents with wide range of hydrophobic radius, 27-37A. A detergent may maintain protein solubility without maintaining function or fold; therefore, the function of LspA in these detergents is currently being studied. To assess the activity of native membrane and solubilized LspA, a signal peptide containing the LspA cleavage consensus sequence and donor-quencher pair on each termini was designed. Preliminary HPLC and fluorescence data demonstrate a cleavage of the signal peptide upon addition of LspA in the native membrane and a comparison to different protein-detergent complexes will be presented. The results presented will identify solubilizing detergents, monitor enzymatic activity, and ultimately correlate the physical properties of the solubilizing detergent and protein structure and function.

Secondary Structure and Topology of the Transmembrane Domain of LspA, from Mycobacterium Tuberculosis, using Solid State NMR Spectroscopy: Initial Study

Lipoprotein Signal Peptidase A (LspA) belongs to type II signal peptidases and it is an essential enzyme in one of the three distinct steps in lipoprotein maturation. After targeting the prolipoprotein, LspA cleaves the signal peptide to result in the signal peptide cleaved lipoprotein, which is the precursor for the mature lipoprotein.In Mycobacterium tuberculosis (Mtb), LspA is encoded by the gene Rv1539 resulting in a 202 amino acid residue peptide with a molecular weight of 21 kDa that is predicted to possess four transmembrane helices. It has been recognized in vivo that LspA is an essential protein for Mtb virulence and that it may also contribute to antibiotic resistance. As a result LspA has been identified as a potential drug target for the treatment of tuberculosis. Globomycin, a cyclic-peptide is known to inhibit LspA in a non-competitive manner.Solid-state NMR enables studies of membrane proteins in a lipid bilayer environment. Since the native-like environment is important for studying the structure and function of membrane proteins, ssNMR is increasingly useful. In Oriented Sample ssNMR used here, the protein is uniformly aligned with respect to the magnetic field and 15N spectra of isotopically labeled protein have provided initial structural insights.Here, all sample preparation steps and initial spectroscopy will be emphasized. Expression of LspA in E.coli, purification using IMAC, reconstitution of LspA into proteoliposomes via dialysis and the preparation of bicelles as an alignment media with different types of lipids and detergents will be presented. 1D NMR 31P and 15N spectra and 2D separated local field NMR spectra of uniformly and selectively labeled LspA will be shown.

The ppm Operon Is Essential for Acylation and Glycosylation of Lipoproteins in Corynebacterium glutamicum

BackgroundDue to their contribution to bacterial virulence, lipoproteins and members of the lipoprotein biogenesis pathway represent potent drug targets. Following translocation across the inner membrane, lipoprotein precursors are acylated by lipoprotein diacylglycerol transferase (Lgt), cleaved off their signal peptides by lipoprotein signal peptidase (Lsp) and, in Gram-negative bacteria, further triacylated by lipoprotein N-acyl transferase (Lnt). The existence of an active apolipoprotein N-acyltransferase (Ms-Ppm2) involved in the N-acylation of LppX was recently reported in M. smegmatis. Ms-Ppm2 is part of the ppm operon in which Ppm1, a polyprenol-monophosphomannose synthase, has been shown to be essential in lipoglycans synthesis but whose function in lipoprotein biosynthesis is completely unknown.ResultsIn order to clarify the role of the ppm operon in lipoprotein biosynthesis, we investigated the post-translational modifications of two model lipoproteins (AmyE and LppX) in C. glutamicum Δppm1 and Δppm2 mutants. Our results show that both proteins are anchored into the membrane and that their N-termini are N-acylated by Cg-Ppm2. The acylated N-terminal peptide of LppX was also found to be modified by hexose moieties. This O-glycosylation is localized in the N-terminal peptide of LppX and disappeared in the Δppm1 mutant. While compromised in the absence of Cg-Ppm2, LppX O-glycosylation could be restored when Cg-Ppm1, Cg-Ppm2 or the homologous Mt-Ppm1 of M. tuberculosis was overexpressed.ConclusionTogether, these results show for the first time that Cg-Ppm1 (Ppm synthase) and Cg-Ppm2 (Lnt) operate in a common biosynthetic pathway in which lipoprotein N-acylation and glycosylation are tightly coupled.

Towards the Backbone Structure Determination of the Membrane Protein, LspA, from Mycobacterium Tuberculosis using Solid State NMR

Lipoprotein Signal Peptidase A (LspA) is an essential enzyme in one of the three distinct steps in lipoprotein maturation. After targeting, the signal peptide of a prolipoprotein, the peptide is cleaved by LspA to result in the mature lipoprotein.In Mycobacterium tuberculosis (Mtb), LspA is encoded by the gene RV1539 resulting in a 202 amino acid residue peptide with a molecular weight is 21 kDa that is predicted to possess four transmembrane helices. It has been recognized in vivo that LspA is an essential protein for Mtb virulence and that it may also contribute to antibiotic resistance, as a result LspA has been identified as a potential drug target for the treatment of tuberculosis. Globomycin, a cyclic-peptide is known to inhibit LspA in a non-competitive manner.Solid-state NMR uniformly aligned samples is being used to determine the structure of the protein backbone. Anisotropic 15N-1H dipolar couplings and 15N chemical shifts are obtained from separated local field NMR spectra of the protein in aligned bilayer preparations. The resulting orientational restraints can be used to generate a high resolution structure as well as a determination of how the helices are oriented with respect to their environment.In this study, LspA is reconstituted into synthetic lipids (DMPC/DMPG and POPC/POPG) to mimic the membrane environment and it is aligned uniaxially either mechanically (using glass slides) or magnetically (using bicelles).

The Acylation State of Surface Lipoproteins of Mollicute Acholeplasma laidlawii

Acylation of the N-terminal Cys residue is an essential, ubiquitous, and uniquely bacterial posttranslational modification that allows anchoring of proteins to the lipid membrane. In gram-negative bacteria, acylation proceeds through three sequential steps requiring lipoprotein diacylglyceryltransferase, lipoprotein signal peptidase, and finally lipoprotein N-acyltransferase. The apparent lack of genes coding for recognizable homologs of lipoprotein N-acyltransferase in gram-positive bacteria and Mollicutes suggests that the final step of the protein acylation process may be absent in these organisms. In this work, we monitored the acylation state of eight major lipoproteins of the mollicute Acholeplasma laidlawii using a combination of standard two-dimensional gel electrophoresis protein separation, blotting to nitrocellulose membranes, and MALDI-MS identification of modified N-terminal tryptic peptides. We show that for each A. laidlawii lipoprotein studied a third fatty acid in an amide linkage on the N-terminal Cys residue is present, whereas diacylated species were not detected. The result thus proves that A. laidlawii encodes a lipoprotein N-acyltransferase activity. We hypothesize that N-acyltransferases encoded by genes non-homologous to N-acyltransferases of gram-negative bacteria are also present in other mollicutes and gram-positive bacteria.

Dissecting the complete lipoprotein biogenesis pathway in Streptomyces scabies

Following translocation, bacterial lipoproteins are lipidated by lipoprotein diacylglycerol transferase (Lgt) and cleaved of their signal peptides by lipoprotein signal peptidase (Lsp). In Gram-negative bacteria and mycobacteria, lipoproteins are further lipidated by lipoprotein N-acyl transferase (Lnt), to give triacylated lipoproteins. Streptomyces are unusual amongst Gram-positive bacteria because they export large numbers of lipoproteins via the twin arginine protein transport (Tat) pathway. Furthermore, some Streptomyces species encode two Lgt homologues and all Streptomyces species encode two homologues of Lnt. Here we characterize lipoprotein biogenesis in the plant pathogen Streptomyces scabies and report that lgt and lsp mutants are defective in growth and development while only moderately affected in virulence. Lipoproteins are lost from the membrane in an S. scabies lgt mutant but restored by expression of Streptomyces coelicolor lgt1 or lgt2 confirming that both encode functional Lgt enzymes. Furthermore, lipoproteins are N-acylated in Streptomyces with efficient N-acylation dependent on Lnt1 and Lnt2. However, deletion of lnt1 and lnt2 has no effect on growth, development or virulence. We thus present a detailed study of lipoprotein biogenesis in Streptomyces, the first study of Lnt function in a monoderm bacterium and the first study of bacterial lipoproteins as virulence factors in a plant pathogen.

Biodegradation of malachite green by strain Pseudomonas sp. K9 and cloning of the tmr2 gene associated with an ISPpu12.

A bacterial strain K9 capable of degrading malachite green was isolated from the sludge of the wastewater treatment system of a chemical plant. It was identified preliminarily as Pseudomonas sp. Strain K9 was also able to degrade other triphenylmethane dyes, such as Crystal Violet and Basic Fuchsin. The gene tmr2, encoding the triphenylmethane reductase, was cloned from strain K9, and functionally expressed in E. coli. A 5946-bp DNA fragment including the tmr2 gene was cloned from the genomic DNA of strain K9 by chromosome walking. Its sequence analysis showed that tmr2 was associated with a typical mobile element ISPpu12 consisting of tnpA (encoding a transposase), lspA (encoding a lipoprotein signal peptidase) and orf1 (encoding a putative MerR family regulator), orf2 (encoding a CDF family heavy metal/H(+) antiporter). This association was also found in another malachite green-degrading strain Pseudomonas sp. MDB-1, which indicated that the tmr2 gene might be a horizontally transferable gene.

Investigating lipoprotein biogenesis and function in the model Gram‐positive bacterium Streptomyces coelicolor

Lipoproteins are a distinct class of bacterial membrane proteins that are translocated across the cytoplasmic membrane primarily by the Sec general secretory pathway and then lipidated on a conserved cysteine by the enzyme lipoprotein diacylglycerol transferase (Lgt). The signal peptide is cleaved by lipoprotein signal peptidase (Lsp) to leave the lipid-modified cysteine at the N-terminus of the mature lipoprotein. In all Gram-positive bacteria tested to date this pathway is non-essential and the lipid attaches the protein to the outer leaflet of the cytoplasmic membrane. Here we identify lipoproteins in the model Gram-positive bacterium Streptomyces coelicolor using bioinformatics coupled with proteomic and downstream analysis. We report that Streptomyces species translocate large numbers of lipoproteins out via the Tat (twin arginine translocase) pathway and we present evidence that lipoprotein biogenesis might be an essential pathway in S. coelicolor. This is the first analysis of lipoproteins and lipoprotein biogenesis in Streptomyces and provides the first evidence that lipoprotein biogenesis could be essential in a Gram-positive bacterium. This report also provides the first experimental evidence that Tat plays a major role in the translocation of lipoproteins in a specific bacterium.

Isolation of a methyl parathion-degrading strain Stenotrophomonas sp. SMSP-1 and cloning of the ophc2 gene

A rod-shaped, gram-negative bacterium Stenotrophomonas sp. SMSP-1 was isolated from the sludge of a wastewater treating system of a pesticide manufacturer. Strain SMSP-1 could hydrolyze methyl parathion to p-nitrophenol (PNP) and dimethyl phosphorothioate but could not degrade PNP further. Strain SMSP-1 was able to hydrolyze other organophosphate pesticides, including fenitrothion, ethyl parathion, fenthion, and phoxim, but not chlorpyrifos. A 4395-bp DNA fragment, including an organophosphorus hydrolase encoding gene ophc2, was cloned from the chromosome of strain SMSP-1 using the shotgun technique. Its sequence analysis showed that ophc2 was associated with a typical mobile element ISPpu12 consisting of tnpA (encoding a transposase), lspA (encoding a lipoprotein signal peptidase), and orf1 (encoding a CDF family heavy metal/H(+) antiporter). The ophc2 gene was effectively expressed in E. coli. This is the second report of cloning the ophc2 gene and the first report of this gene from the genus of Stenotrophomonas.

Calcium Binds to LipL32, a Lipoprotein from Pathogenic Leptospira, and Modulates Fibronectin Binding

Tubulointerstitial nephritis is a cardinal renal manifestation of leptospirosis. LipL32, a major lipoprotein and a virulence factor, locates on the outer membrane of the pathogen Leptospira. It evades immune response by recognizing and adhering to extracellular matrix components of the host cell. The crystal structure of Ca(2+)-bound LipL32 was determined at 2.3 A resolution. LipL32 has a novel polyD sequence of seven aspartates that forms a continuous acidic surface patch for Ca(2+) binding. A significant conformational change was observed for the Ca(2+)-bound form of LipL32. Calcium binding to LipL32 was determined by isothermal titration calorimetry. The binding of fibronectin to LipL32 was observed by Stains-all CD and enzyme-linked immunosorbent assay experiments. The interaction between LipL32 and fibronectin might be associated with Ca(2+) binding. Based on the crystal structure of Ca(2+)-bound LipL32 and the Stains-all results, fibronectin probably binds near the polyD region on LipL32. Ca(2+) binding to LipL32 might be important for Leptospira to interact with the extracellular matrix of the host cell.

Making Membrane Protein LspA Samples, and Its Uniformly Aligned Full Length Investigated by Solid State NMR

Here is a novel procedure for making a membrane protein sample, Lipoprotein Signal Peptidase (LspA) for structural studies by solid state NMR. LspA is expressed in E.Coli, purified and refolded with detergent on a Ni-NTA affinitive column and uniformly aligned in lipid bilayer on glass slides. Final concentrations of reconstituted LspA of up to 35mg/mL have been achieved. Reconstitution of LspA in detergent micelles was monitored by CD and solution NMR HSQC. The aligned LspA in lipid bilayer was monitored by solid state NMR to indicate the degree of alignment. Based on these results, ssNMR spectrum will be obtained to show resonance patterns known as PISA wheel for the transmembrane domains of LspA.

In the absence of Lgt, lipoproteins are shed from Streptococcus uberis independently of Lsp

The role of lipoprotein diacylglyceryl transferase (Lgt) and lipoprotein signal peptidase (Lsp) responsible for processing lipoproteins was investigated in Streptococcus uberis, a common cause of bovine mastitis. In the absence of Lgt, three lipoproteins [MtuA (SUB0473), Hap (SUB1625) and an extracellular solute-binding protein (SUB0365)] were detected in extracellular locations. All were shown by Edman degradation analysis to be cleaved on the carboxy side of the LXXC lipobox. Detection of MtuA, a lipoprotein shown previously to be essential for infectivity and virulence, was used as a surrogate lipoprotein marker to locate and assess processing of lipoproteins. The absence of Lgt did not prevent location of MtuA to the cell membrane, its location in the wild-type strain but, in contrast to the situation with wild-type, did result in a widespread location of this protein. In the absence of both Lgt and Lsp, MtuA was similarly released from the bacterial cell. In such strains, however, the cell-associated MtuA represented the full-length gene product, indicating that Lsp was able to cleave non-lipidated (lipo)proteins but was not responsible for their release from this bacterium.

RP105 Facilitates Macrophage Activation by Mycobacterium tuberculosis Lipoproteins

RP105, phylogenetically related to Toll-like receptor (TLR)-4, is reported to facilitate B cell activation by the TLR4-agonist lipopolysaccharide (LPS)--but to limit LPS-induced cytokine production by antigen-presenting cells. Here, we show that the role of RP105 extends beyond LPS recognition and that RP105 positively regulates macrophage responses to Mycobacterium tuberculosis (Mtb) lipoproteins. Mtb-infected RP105(-/-) mice exhibited impaired proinflammatory cytokine responses associated with enhanced bacterial burden and increased lung pathology. The Mtb 19 kDa lipoprotein induced release of tumor necrosis factor in a manner dependent on both TLR2 and RP105, and macrophage activation by Mtb lacking mature lipoproteins was not RP105 dependent. Thus, mycobacterial lipoproteins are RP105 agonists. RP105 physically interacted with TLR2, and both RP105 and TLR2 were required for optimal macrophage activation by Mtb. Our data identify RP105 as an accessory molecule for TLR2, forming part of the receptor complex for innate immune recognition of mycobacterial lipoproteins.

LspA inactivation in Mycobacterium tuberculosis results in attenuation without affecting phagosome maturation arrest

The success of Mycobacterium tuberculosis depends on its ability to survive within host macrophages. Here, M. tuberculosis avoids the acidic, hydrolytically competent environment of the phagolysosome by arresting phagosome maturation. Having shown previously that a M. tuberculosis mutant deficient in lipoprotein signal peptidase (LspA) is strongly attenuated in vivo in a mouse model of infection, we now studied putative mechanisms involved in attenuation of the lspA : : aph mutant at a cellular level. In this work we investigated the ability of the mutant to interfere with two host defence mechanisms, i.e. Toll-like receptor (TLR)2-dependent immune response and phagosome maturation. While mycobacterial lipoproteins have been reported to trigger a TLR2 signalling pathway critical for innate immune responses, we found that growth control of the lspA : : aph mutant was independent of TLR2. In addition, the lspA : : aph mutant arrested phagosome maturation to an extent similar to that of the wild-type, as measured by lysosomal-associated membrane protein 1 (LAMP1) co-localization and intraphagosomal pH. These observations demonstrate severe attenuation even in the presence of arrested phagosome maturation, and point to a role for the early phagosome in growth restriction of the M. tuberculosis lspA mutant.

Recovery of a Functional Class 2 Integron from an Escherichia coli Strain Mediating a Urinary Tract Infection

A class 2 integron was found in an Escherichia coli isolate mediating a urinary tract infection. Unlike other class 2 integrons from pathogens, the encoded IntI2 protein was functional. The integron possessed a dfrA14 cassette, and a second novel cassette in which a lipoprotein signal peptidase gene is predicted.