Serratia marcescens secretes several proteins, such as the lipase LipA, the metalloprotease PrtA, and the heme-binding protein HasA, which is required for heme acquisition, through two N-terminal signal peptide-independent systems that are classified as bacterial ATP-binding cassette (ABC) exporters. One is the ABC exporter for HasA, consisting of the ABC protein HasD, the membrane fusion protein (MFP) HasE, and the outer membrane protein (OMP) HasF. The second, composed of LipB (an ABC protein), LipC (an MFP), and LipD (an OMP), promotes secretion of LipA and PrtA in Escherichia coli recombinant clones. PrtA, which shows homology to the Erwinia chrysanthemi metalloproteases, is efficiently secreted by E. coli cells carrying the E. chrysanthemi ABC exporter PrtD (ABC protein)-PrtE (MFP)-PrtF (OMP). The existence of distinct systems in this bacterium and of various substrates for these systems allowed the study of protein secretion by heterologous Has, Lip, and Prt systems and by Has-Lip and Lip-Prt hybrid exporters in the genuine host as well as in E. coli. For that purpose, lipB-, lipC-, and lipD-deficient mutants were isolated from S. marcescens 8000 and their secretion of LipA and PrtA was analyzed. This demonstrated that a unique exporter, the Lip apparatus, in S. marcescens secretes both LipA and PrtA. Hybrid exporters were tested for secretion of HasA and LipA. The LipB-HasE-HasF exporter allowed secretion of LipA but not HasA, showing that the ABC protein LipB is responsible for the substrate specificity. LipA, HasA, and E. chrysanthemi PrtC were secreted via heterologous exporters and via some hybrid exporters. Analysis of secretion via hybrid exporters showed that specific interactions occur between MFPs and OMPs in these systems. These genetic experiments demonstrated that specific interactions between the ABC protein and the MFP are required for the formation of active exporters.
The extracellular lipase of Serratia marcescens Sr41, lacking a typical N-terminal signal sequence, is secreted via a signal peptide-independent pathway. The 20-kb SacI DNA fragment which allowed the extracellular lipase secretion was cloned from S. marcescens by selection of a phenotype conferring the extracellular lipase activity on the Escherichia coli cells. The subcloned 6.5-kb EcoRV fragment was revealed to contain three open reading frames which are composed of 588, 443, and 437 amino acid residues constituting an operon (lipBCD). Comparisons of the deduced amino acid sequences of the lipB, lipC, and lipD genes with those of the Erwinia chrysanthemi prtDEC, prtEEC, and prtFEC genes encoding the secretion apparatus of the E. chrysanthemi protease showed 55, 46, and 42% identity, respectively. The products of the lipB and lipC genes were 54 and 45% identical to the S. marcescens hasD and hasE gene products, respectively, which were secretory components for the S. marcescens heme-binding protein and metalloprotease. In the E. coli DH5 cells, all three lipBCD genes were essential for the extracellular secretion of both S. marcescens lipase and metalloprotease proteins, both of which lack an N-terminal signal sequence and are secreted via a signal-independent pathway. Although the function of the lipD gene seemed to be analogous to those of the prtFEC and tolC genes encoding third secretory components of ABC transporters, the E. coli TolC protein, which was functional for the S. marcescens Has system, could not replace LipD in the LipB-LipC-LipD transporter reconstituted in E. coli. These results indicated that these three proteins are components of the device which allows extracellular secretion of the extracellular proteins of S. marcescens and that their style is similar to that of the PrtDEF(EC) system.
The lipase subfamilies I.1 and I.2 show more than 33% homology in the amino acid sequences and most members share another common property that their genes are clustered with the secondary genes whose protein products are required for folding the lipase into an active conformation and secretion into the culture medium. In previous studies, the lipase (LipA) and its chaperone (LipB) from Ralstonia sp. M1 were overexpressed in E. coli and the lipase was successfully refolded in vitro. The purpose of this study was to enhance the production of the active lipase LipA from Ralstonia sp. M1 in the heterologous host E. coli without in vitro refolding process, using two-plasmid co-expression systems and dual expression cassette plasmid systems.
To produce more active lipase from Ralstonia sp. M1 in E. coli without in vitro refolding process but with the help of overexpression of the chaperone (LipB1 and LipB3 corresponding to 56-aa truncated and 26-aa truncated chaperone LipB), six different expression systems including 2 two-plasmid co-expression systems (E. coli BL21/pELipABa + pELipB1k and BL21/pELipABa + pELipB3k) and 4 dual expression cassette plasmid systems (BL21/pELipAB-LipB1a, BL21/pELipAB-LipB3a, BL21/pELipA-LipB1a, and BL21/pELipA-LipB3a) were constructed. The two-plasmid co-expression systems (E. coli BL21/pELipABa + pELipB1k and BL21/pELipABa + pELipB3k) produced the active lipase at a level of 4 times as high as the single expression cassette plasmid system E. coli BL21/pELipABa did. For the first time, the dual expression cassette plasmid systems BL21/pELipAB-LipB1a and BL21/pELipAB-LipB3a yielded 29- and 19-fold production of the active lipase in comparison with the single expression cassette plasmid system E. coli BL21/pELipABa, respectively. Although the lipase amount was equally expressed in all these expression systems (40% of total cellular protein) and only a small fraction of the overexpressed lipase was folded in vivo into the functional lipase in soluble form whereas the main fraction was still inactive in the form of inclusion bodies. Another controversial finding was that the dual expression cassette plasmid systems E. coli BL21/pELipAB-LipB1a and E. coli/pELipAB-LipB3a secreted the active lipase into the culture medium of 51 and 29 times as high as the single expression cassette plasmid system E. coli pELipABa did, respectively, which has never been reported before. Another interesting finding was that the lipase form LipA6xHis (mature lipase fused with 6× histidine tag) expressed in the dual expression cassette plasmid systems (BL21/pELipA-LipB1a and BL21/pELipA-LipB3a) showed no lipase activity although the expression level of the lipase and two chaperone forms LipB1 and LipB3 in these systems remained as high as that in E. coli BL21/pELipABa + pELipB1k, BL21/pELipABa + pELipB3k, BL21/pELipAB-LipB1a, and BL21/pELipAB-LipB3a. The addition of Neptune oil or detergents into the LB medium increased the lipase production and secretion by up to 94%.
Our findings demonstrated that a dual expression cassette plasmid system E. coli could overproduce and secrete the active chaperone-dependent lipase (subfamilies I.1 and I.2) in vivo and an improved dual expression cassette plasmid system E. coli could be potentially applied for industrial-scale production of subfamily I.1 and I.2 lipases.
Ralstonia sp. M1; Lipase; Chaperone; Functional expression; Secretion
Type VI secretion systems (T6SS) are trans-envelope machines dedicated to the secretion of virulence factors into eukaryotic or prokaryotic cells, therefore required for pathogenesis and/or for competition towards neighboring bacteria. The T6SS apparatus resembles the injection device of bacteriophage T4, and is anchored to the cell envelope through a membrane complex. This membrane complex is composed of the TssL, TssM and TagL inner membrane anchored proteins and of the TssJ outer membrane lipoprotein. Here, we report the crystal structure of the enteroaggregative Escherichia coli Sci1 TssJ lipoprotein, a two four-stranded β-sheets protein that exhibits a transthyretin fold with an additional α-helical domain and a protruding loop. We showed that TssJ contacts TssM through this loop since a loop depleted mutant failed to interact with TssM in vitro or in vivo. Biophysical analysis of TssM and TssJ-TssM interaction suggest a structural model of the membrane-anchored outer shell of T6SS. Collectively, our results provide an improved understanding of T6SS assembly and encourage structure-aided drug design of novel antimicrobials targeting T6SS.
Type VI secretion systems (T6SS) are specialized secretion machines responsible for the transport of virulence factors. T6SS are versatile as they are able to target both eukaryotic and prokaryotic cells. They therefore play an important role in pathogenesis by acting directly on the host, as well as eliminating competing bacteria from the niche. At a molecular level, T6SS are composed of a minimum of 13 proteins called core-components, all required for the activity of the secretion system. These core-components can be divided in two groups: soluble proteins having a common evolution history with bacteriophage T4 subunits, and membrane or membrane-associated proteins required for anchoring the bacteriophage-like structure to the envelope. Here, we report the crystal structure of one of the membrane-associated core component, the TssJ lipoprotein. The structure exhibits a transthyretin fold supplemented with additional structural elements. One of these, a loop connecting two beta-strands, is responsible for the interaction of the TssJ lipoprotein with the C-terminal domain of the inner membrane protein TssM. We propose that these two proteins link the two membranes and form a channel accommodating the bacteriophage-like structure. These results provide important new insights to understand the biogenesis of these secretion apparati.
Triacylglycerol lipases (EC 126.96.36.199) catalyze both hydrolysis and synthesis reactions with a broad spectrum of substrates rendering them especially suitable for many biotechnological applications. Most lipases used today originate from mesophilic organisms and are susceptible to thermal denaturation whereas only few possess high thermotolerance. Here, we report on the identification and characterization of two novel thermostable bacterial lipases identified by functional metagenomic screenings. Metagenomic libraries were constructed from enrichment cultures maintained at 65 to 75°C and screened resulting in the identification of initially 10 clones with lipolytic activities. Subsequently, two ORFs were identified encoding lipases, LipS and LipT. Comparative sequence analyses suggested that both enzymes are members of novel lipase families. LipS is a 30.2 kDa protein and revealed a half-life of 48 h at 70°C. The lipT gene encoded for a multimeric enzyme with a half-life of 3 h at 70°C. LipS had an optimum temperature at 70°C and LipT at 75°C. Both enzymes catalyzed hydrolysis of long-chain (C12 and C14) fatty acid esters and additionally hydrolyzed a number of industry-relevant substrates. LipS was highly specific for (R)-ibuprofen-phenyl ester with an enantiomeric excess (ee) of 99%. Furthermore, LipS was able to synthesize 1-propyl laurate and 1-tetradecyl myristate at 70°C with rates similar to those of the lipase CalB from Candida antarctica. LipS represents the first example of a thermostable metagenome-derived lipase with significant synthesis activities. Its X-ray structure was solved with a resolution of 1.99 Å revealing an unusually compact lid structure.
We report the cloning of the gene encoding the 32-kDa lipoprotein, designated LipL32, the most prominent protein in the leptospiral protein profile. We obtained the N-terminal amino acid sequence of a staphylococcal V8 proteolytic-digest fragment to design an oligonucleotide probe. A Lambda-Zap II library containing EcoRI fragments of Leptospira kirschneri DNA was screened, and a 5.0-kb DNA fragment which contained the entire structural lipL32 gene was identified. Several lines of evidence indicate that LipL32 is lipid modified in a manner similar to that of other procaryotic lipoproteins. The deduced amino acid sequence of LipL32 would encode a 272-amino-acid polypeptide with a 19-amino-acid signal peptide, followed by a lipoprotein signal peptidase cleavage site. LipL32 is intrinsically labeled during incubation of L. kirschneri in media containing [3H]palmitate. The linkage of palmitate and the amino-terminal cysteine of LipL32 is acid labile. LipL32 is completely solubilized by Triton X-114 extraction of L. kirschneri; phase separation results in partitioning of LipL32 exclusively into the hydrophobic, detergent phase, indicating that it is a component of the leptospiral outer membrane. CaCl2 (20 mM) must be present during phase separation for recovery of LipL32. LipL32 is expressed not only during cultivation but also during mammalian infection. Immunohistochemistry demonstrated intense LipL32 reactivity with L. kirschneri infecting proximal tubules of hamster kidneys. LipL32 is also a prominent immunogen during human leptospirosis. The sequence and expression of LipL32 is highly conserved among pathogenic Leptospira species. These findings indicate that LipL32 may be important in the pathogenesis, diagnosis, and prevention of leptospirosis.
Lipases have been implicated to be of importance in the life cycle development, virulence, and transmission of a variety of parasitic organisms. Potential functions include the acquisition of host resources for energy metabolism and as simple building blocks for the synthesis of complex parasite lipids important for membrane remodeling and structural purposes. Using a molecular approach, we identified and characterized the structure of an LdLip3-lipase gene from the primitive trypanosomatid pathogen of humans, Leishmania donovani. The LdLip3 encodes a ~ 33 kDa protein, with a well-conserved sub-strate-binding and catalytic domains characteristic of members of the serine lipase-protein family. Further, we showed that LdLip3 mRNA is constitutively expressed by both the insect vector (i.e., promastigote) and mammalian (i.e., amastigote) life cycle developmental forms of this protozoan parasite. Moreover, a homologous episomal expression system was used to express an HA epitope-tagged LdLip3 chimeric construct (LdLip3∷HA) in these parasites. Expression of the LdLip3 chimera was verified in these transfectants by Western blots and indirect immunofluorescence analyses. Results of coupled immuno-affinity purification and enzyme activity experiments demonstrated that the LdLip3∷HA chimeric protein was secreted/released by transfected L. donovani parasites and that it possessed functional lipase enzyme activity. Taken together these observations suggest that this novel secretory lipase might play essential role(s) in the survival, growth, and development of this important group of human pathogens.
Leishmania; Human parasite; Gene structure; Trypanosomatid; Kinetoplastid protozoan; Lipase
Acinetobacter calcoaceticus BD413 produces an extracellular lipase, which is encoded by the lipA gene. Five lipase-deficient mutants have been generated via random insertion mutagenesis. Phenotypic characterization of these mutants revealed the presence of as many as four lipolytic enzymes in A. calcoaceticus. Biochemical evidence classified four of the mutants as export mutants, which presumably are defective in translocation of the lipase across the outer membrane. The additional mutant, designated AAC302, displays a LipA- phenotype, and yet the mutation in this strain was localized 0.84 kbp upstream of lipA. Sequence analysis of this region revealed an open reading frame, designated lipB, that is disrupted in AAC302. The protein encoded by this open reading frame shows extensive similarity to a chaperone-like helper protein of several pseudomonads, required for the production of extracellular lipase. Via complementation of AAC302 with a functional extrachromosomal copy of lipA, it could be determined that LipB is essential for lipase production. As shown by the use of a translational LipB-PhoA fusion construct, the C-terminal part of LipB of A. calcoaceticus BD413 is located outside the cytoplasm. Sequence analysis further strongly suggests that A. calcoaceticus LipB is N terminally anchored in the cytoplasmic membrane. Therefore, analogous to the situation in Pseudomonas species, however, lipB in A. calcoaceticus is located upstream of the structural lipase gene. lipB and lipA form a bicistronic operon, and the two genes are cotranscribed from an Escherichia coli sigma 70-type promoter. The reversed order of genes, in comparison with the situation in Pseudomonas species, suggests that LipA and LipB are produced in equimolar amounts. Therefore, the helper protein presumably does not only have a catalytic function, e.g., in folding of the lipase, but is also likely to act as a lipase-specific chaperone. A detailed model of the export route of the lipase of A. calcoaceticus BD413 is proposed.
We report the cloning of the gene encoding a 36-kDa leptospiral outer membrane lipoprotein, designated LipL36. We obtained the N-terminal amino acid sequence of a staphylococcal V8 proteolytic-digest fragment in order to design an oligonucleotide probe. A Lambda-Zap II library containing EcoRI fragments of Leptospira kirschneri DNA was screened, and a 2.3-kb DNA fragment which contained the entire structural lipL36 gene was identified. Several lines of evidence indicate that LipL36 is lipid modified in a manner similar to that of LipL41, a leptospiral outer membrane lipoprotein we described in a previous study (E. S. Shang, T. A. Summers, and D. A. Haake, Infect. Immun. 64:2322–2330, 1996). The deduced amino acid sequence of LipL36 would constitute a 364-amino-acid polypeptide with a 20-amino-acid signal peptide, followed by an L-X-Y-C lipoprotein signal peptidase cleavage site. LipL36 is solubilized by Triton X-114 extraction of L. kirschneri; phase separation results in partitioning of LipL36 exclusively into the hydrophobic, detergent phase. LipL36 is intrinsically labeled during incubation of L. kirschneri in media containing [3H]palmitate. Processing of LipL36 is inhibited by globomycin, a selective inhibitor of lipoprotein signal peptidase. After processing, LipL36 is exported to the outer membrane along with LipL41 and lipopolysaccharide. Unlike LipL41, there appears to be differential expression of LipL36. In early-log-phase cultures, LipL36 is one of the most abundant L. kirschneri proteins. However, LipL36 levels drop considerably beginning in mid-log phase. LipL36 expression in vivo was evaluated by examining the humoral immune response to leptospiral antigens in the hamster model of leptospirosis. Hamsters surviving challenge with culture-adapted virulent L. kirschneri generate a strong antibody response to LipL36. In contrast, sera from hamsters surviving challenge with host-adapted L. kirschneri do not recognize LipL36. These findings suggest that LipL36 expression is downregulated during mammalian infection, providing a marker for studying the mechanisms by which pathogenic Leptospira species adapt to the host environment.
LipL32 is the major leptospiral outer membrane lipoprotein expressed during infection and is the immunodominant antigen recognized during the humoral immune response to leptospirosis in humans. In this study, we investigated novel aspects of LipL32. In order to define the immunodominant domains(s) of the molecule, subfragments corresponding to the N-terminal, intermediate, and C-terminal portions of the LipL32 gene were cloned and the proteins were expressed and purified by metal affinity chromatography. Our immunoblot results indicate that the C-terminal and intermediate domains of LipL32 are recognized by sera of patients with laboratory-confirmed leptospirosis. An immunoglobulin M response was detected exclusively against the LipL32 C-terminal fragment in both the acute and convalescent phases of illness. We also evaluated the capacity of LipL32 to interact with extracellular matrix (ECM) components. Dose-dependent, specific binding of LipL32 to collagen type IV and plasma fibronectin was observed, and the binding capacity could be attributed to the C-terminal portion of this molecule. Both heparin and gelatin could inhibit LipL32 binding to fibronectin in a concentration-dependent manner, indicating that the 30-kDa heparin-binding and 45-kDa gelatin-binding domains of fibronectin are involved in this interaction. Taken together, our results provide evidence that the LipL32 C terminus is recognized early in the course of infection and is the domain responsible for mediating interaction with ECM proteins.
The capsule of N. meningitidis serogroup B, (α2→8)-linked polysialic acid and the capsules of other meningococcal serogroups and of other gram-negative bacterial pathogens are anchored in the outer membrane through a 1,2-diacylglycerol moiety. Previous work on the meningococcal cps complex in Escherichia coli K-12 indicated that deletion of genes designated lipA and lipB caused intracellular accumulation of hyperelongated capsule polymers lacking the phospholipid substitution. To better understand the role of lip and lipB in capsule expression in a meningococcal background, the location, sequence, and relationship to related bacterial capsule genes were defined and specific mutations in lipA and lipB were generated in the serogroup B meningococcal strain NMB. The lipA and lipB genes are located on the 3′ end of the ctr operon and are most likely transcribed independently. Inactivation of lipA, lipB, and both resulted in the same total levels of capsular polymer production as in the parental controls; however, these mutants were as sensitive as an unencapsulated mutant to killing by normal human serum. Immunogold electron microscopy and flow cytometric analyses revealed intracellular inclusions of capsular polymers in lipA, lipB, and lipA lipB mutants. Capsular polymers purified from lipA, lipB, and lipA lipB mutants were lipidated. The phospholipid anchor was shown by gas chromatography-mass spectroscopy analysis to be a phosphodiester-linked 1,2-dipalmitoyl (C16:0) glycerol moiety and was identical in structure to that found on the wild-type meningococcal capsule polymers. Thus, lipA and lipB do not encode proteins responsible for diacylglycerophosphatidic acid substitution of the meningococcal capsule polymer; rather, they are required for proper translocation and surface expression of the lipidated polymer.
In the companion paper (Martin et al., 2011) we reported that Bacillus subtilis requires three proteins for lipoic acid metabolism, all of which are members of the lipoate protein ligase family. Two of the proteins, LipM and LplJ, have been shown to be an octanoyltransferase and a lipoate:protein ligase, respectively. The third protein, LipL, is essential for lipoic acid synthesis, but had no detectable octanoyltransferase or ligase activity either in vitro or in vivo. We report that LipM specifically modifies the glycine cleavage system protein, GcvH, and therefore another mechanism must exist for modification of other lipoic acid requiring enzymes (e.g., pyruvate dehydrogenase). We show that this function is provided by LipL which catalyzes the amidotransfer (transamidation) of the octanoyl moiety from octanoyl-GcvH to the E2 subunit of pyruvate dehydrogenase. LipL activity was demonstrated in vitro with purified components and proceeds via a thioester-linked acyl-enzyme intermediate. As predicted, ΔgcvH strains are lipoate auxotrophs. LipL represents a new enzyme activity. It is a GcvH:[lipoyl domain] amidotransferase that probably employs a Cys-Lys catalytic dyad. Although the active site cysteine residues of LipL and LipB are located in different positions within the polypeptide chains, alignment of their structures show these residues occupy similar positions. Thus, these two homologous enzymes have convergent architectures.
Post-Translational Modification; Lipoic Acid; Bacillus subtilis
β-amyloid (Aβ) is the main protein component of the amyloid plaques associated with Alzheimer’s disease. Transthyretin (TTR) is a homotetramer that circulates in both blood and cerebrospinal fluid. Wild-type transthyretin (TTR) amyloid deposits are linked to senile systemic amyloidosis, a common disease of aging, while several TTR mutants are linked to familial amyloid polyneuropathy. Several recent studies provide support for the hypothesis that these two amyloidogenic proteins interact, and that this interaction is biologically relevant. For example, upregulation of TTR expression in Tg2576 mice was linked to protection from toxic effects of Aβ deposition [Stein, T.D. and Johnson, J.A. (2002) J. Neurosci. 22: 7380–7388]. We examined the interaction of Aβ with wt TTR as well as two mutants: F87M/L110M, engineered to be a stable monomer, and T119M, a naturally occurring mutant with higher tetrameric stability than wildtype. Based on enzyme-linked immunoassays as well as crosslinking experiments, we conclude that Aβ monomers bind more strongly to TTR monomers than to TTR tetramers. The data further suggest that TTR tetramers interact preferably with Aβ aggregates rather than Aβ monomers. Through tandem mass spectrometry analysis of crosslinked TTR-Aβ fragments, we identified the A strand, in the inner β-sheet of TTR, as well as the EF helix, as regions of TTR that are involved with Aβ association. Light scattering and electron microscopy studies demonstrate that the outcome of the TTR-Aβ interaction strongly depends on TTR quaternary structure. While TTR tetramers may modestly enhance aggregation, TTR monomers decidedly arrest Aβ aggregate growth. These data provide important new insights into the nature of TTR-Aβ interactions. Such interactions may regulate TTR-mediated protection against Aβ toxicity.
Two ATP-binding cassette (ABC) exporters are present in Pseudomonas fluorescens no. 33; one is the recently reported AprDEF system and the other is HasDEF, which exports a heme acquisition protein, HasA. The hasDEF genes were cloned by DNA hybridization with a DNA probe coding for the LipB protein, one of the components of the Serratia marcescens ABC exporter Lip system. P. fluorescens HasA showed sequence identity of 40 to 49% with HasA proteins from Pseudomonas aeruginosa and Serratia marcescens. The P. fluorescens Has exporter secreted HasA proteins from P. fluorescens and P. aeruginosa but not S. marcescens HasA in Escherichia coli, whereas the Has exporter from S. marcescens allowed secretion of all three HasA proteins. The P. fluorescens HasDEF system also promoted the secretion of the lipase and alkaline protease of P. fluorescens. Hybrid exporter analysis demonstrated that the HasD proteins, which are ABC proteins, are involved in the discrimination of export substrates. Chimeric HasA proteins containing both P. fluorescens and S. marcescens sequences were produced and tested for secretion through the Has exporters. The C-terminal region of HasA was shown to be involved in the secretion specificity of the P. fluorescens Has exporter.
Metagenomic analyses have advanced our understanding of ecological microbial diversity, but to what extent can metagenomic data be used to predict the metabolic capacity of difficult-to-study organisms and their abiotic environmental interactions? We tackle this question, using a comparative genomic approach, by considering the molecular basis of aerobiosis within archaea. Lipoylation, the covalent attachment of lipoic acid to 2-oxoacid dehydrogenase multienzyme complexes (OADHCs), is essential for metabolism in aerobic bacteria and eukarya. Lipoylation is catalysed either by lipoate protein ligase (LplA), which in archaea is typically encoded by two genes (LplA-N and LplA-C), or by a lipoyl(octanoyl) transferase (LipB or LipM) plus a lipoic acid synthetase (LipA). Does the genomic presence of lipoylation and OADHC genes across archaea from diverse habitats correlate with aerobiosis? First, analyses of 11,826 biotin protein ligase (BPL)-LplA-LipB transferase family members and 147 archaeal genomes identified 85 species with lipoylation capabilities and provided support for multiple ancestral acquisitions of lipoylation pathways during archaeal evolution. Second, with the exception of the Sulfolobales order, the majority of species possessing lipoylation systems exclusively retain LplA, or either LipB or LipM, consistent with archaeal genome streamlining. Third, obligate anaerobic archaea display widespread loss of lipoylation and OADHC genes. Conversely, a high level of correspondence is observed between aerobiosis and the presence of LplA/LipB/LipM, LipA and OADHC E2, consistent with the role of lipoylation in aerobic metabolism. This correspondence between OADHC lipoylation capacity and aerobiosis indicates that genomic pathway profiling in archaea is informative and that well characterized pathways may be predictive in relation to abiotic conditions in difficult-to-study extremophiles. Given the highly variable retention of gene repertoires across the archaea, the extension of comparative genomic pathway profiling to broader metabolic and homeostasis networks should be useful in revealing characteristics from metagenomic datasets related to adaptations to diverse environments.
Leptospira interrogans are bacterial pathogens of animal that cause zoonotic infections in human. Outer membrane proteins of leptospire are among the most effective antigens which can stimulate remarkable immune responses during the infection processes, and thus are currently considered leading candidate vaccine antigens. The objective of the present study is to predict and confirm major combined B and T cell epitopes of leptospiral outer membrane proteins OmpL1 and LipL41, as well as to evaluate their capacity in the induction of immune responses in BALB/c mice.
In this study, four epitopes from OmpL1 and four from LipL41 conserved regions were evaluated for their potential utilization in leptospire vaccines. Firstly, combined B and T cell epitopes were predicted by softwares and expressed using a phage display system. OmpL1 residues 87-98 and 173-191 (OmpL187-98 and OmpL1173-191) and LipL4130-48, LipL41233-256 of LipL41 were identified as immunodominant B cell epitopes by Western blot. Epitopes OmpL1173-191, OmpL1297-320 of OmpL1 and LipL41233-256, LipL41263-282 of LipL41 were identified as immunodominant CD4+ T cell epitopes through proliferation analysis of splenocytes from recombinant OmpL1 (rOmpL1) or recombinant LipL41 (rLipL41)-immunized BALB/c (H-2d) mice. These epitopes induced responses of CD4+ T cells and Th1 (T helper cells) type cytokine responses during the infection.
This work identified combined T and B cell immunodominant epitopes in outer membrane proteins OmpL1 and LipL41 of Leptospira interrogans. OmpL1173-191 of OmpL1 and LipL41233-256 of LipL41 could be useful in a vaccine against Leptospira. The findings could also contribute to the development of effective cross-protective vaccine strategies for leptospirosis.
Leptospirosis is one of the most widespread zoonotic diseases in the world. It is caused by the pathogen Leptospira that results in multiple-organ failure, in particular of the kidney. Outer membrane lipoprotein is the suspected virulence factor of Leptospira. In Leptospira spp LipL41 is one major lipoprotein and is highly conserved. Previous study suggests that LipL41 bears hemin-binding ability and might play a possible role in iron regulation and storage. However, the characterization of hemin-binding ability of LipL41 is still unclear. Here the hemin-binding ability of LipL41 was examined, yielding a Kd = 0.59 ± 0.14 μM. Two possible heme regulatory motifs (HRMs), C[P/S], were found in LipL41 at 140Cys-Ser and 220Cys-Pro. The mutation study indicates that Cys140 and Cys220 might be cooperatively involved in hemin binding. A supramolecular assembly of LipL41 was determined by transmission electron microscopy. The LipL41 oligomer consists of 36 molecules and folds as a double-layered particle. At the C-terminus of LipL41, there are two tetratricopeptide repeats (TPRs), which might be involved in the protein-protein interaction of the supramolecular assembly.
We isolated the LIP2 gene from the lipolytic yeast Yarrowia lipolytica. It was found to encode a 334-amino-acid precursor protein. The secreted lipase is a 301-amino-acid glycosylated polypeptide which is a member of the triacylglycerol hydrolase family (EC 188.8.131.52). The Lip2p precursor protein is processed by the KEX2-like endoprotease encoded by XPR6. Deletion of the XPR6 gene resulted in the secretion of an active but less stable proenzyme. Thus, the pro region does not inhibit lipase secretion and activity. However, it does play an essential role in the production of a stable enzyme. Processing was found to be correct in LIP2A (multiple LIP2 copy integrant)-overexpressing strains, which secreted 100 times more activity than the wild type, demonstrating that XPR6 maturation was not limiting. No extracellular lipase activity was detected with the lip2 knockout (KO) strain, strongly suggesting that extracellular lipase activity results from expression of the LIP2 gene. Nevertheless, the lip2 KO strain is still able to grow on triglycerides, suggesting an alternative pathway for triglyceride utilization in Y. lipolytica.
Plasmids containing a Pseudomonas sp. strain 109 extracellular lipase gene (lipL) lacking NH2-terminal sequence and a lipase modulator gene (limL) lacking the NH2-terminal hydrophobic region were constructed and expressed independently in Escherichia coli by using the T7 promoter expression vector system. Recombinant LipL (rLipL) was produced as inclusion bodies, whereas recombinant LimL (rLimL) was present as a soluble protein. During in vitro renaturation of the purified rLipL inclusion bodies after they had been dissolved in 8 M urea, addition of rLimL was essential to solubilize and modulate rLipL. The solubility and activity of rLipL were influenced by the rLimL/rLipL molar ratio; the highest level of solubility was obtained at an rLimL/rLipL ratio of 4:5, whereas the highest activity level was obtained at an rLimL/rLipL ratio of 4:1. After renaturation, rLipL and rLimL were coprecipitated with anti-rLipL antibody, indicating the formation of an rLipL-rLimL complex. Activity of the native lipase purified from Pseudomonas sp. strain 109 was also inhibited by rLimL. By Western blotting (immunoblotting) with anti-rLimL antibody, native LimL was detected in Pseudomonas cells solubilized by sarcosyl treatment. LimL was purified from Pseudomonas sp. strain 109, and the NH2-terminal amino acid sequence was determined to be NH2-Leu-Glu-Pro-Ser-Pro-Ala-Pro-. We propose that to prevent membrane degradation, LimL weakens lipase activity inside the cell, especially in the periplasm, in addition to modulating lipase folding.
Saccharomyces cerevisiae cannot produce extracellular lipase and utilize low‐cost lipid substrates. This study aimed to express extracellular lipase from Yarrowia lipolytica in S. cerevisiae, construct recombinant oily substrate consumer strains, and compare the roles of native and mutant Y. lipolytica extracellular lipases in S. cerevisiae. The LIP2 gene of Y. lipolytica DSM3286 and its mutant Y. lipolytica U6 were isolated and cloned by expression vector in S. cerevisiae. New recombinant S. cerevisiae strains FDS100 containing the native LIP2 gene, and FDS101 containing the mutant LIP2 gene were produced 10 and 15 U ml
−1 extracellular lipase respectively, on a production medium containing olive oil. New recombinant S. cerevisiae strains produce acceptable amount of extracellular lipase in comparison with Y. lipolytica wild‐type strains. These strains can utilize olive oil and lipids as low‐cost substrates to produce bioethanol, single cell protein and other biotechnologically valuable products. The recombinant S. cerevisiae strain with mutant LIP2 produced lipase with 1.5‐fold higher activity. The LIP2 gene of Y. lipolytica was expressed in S. cerevisiae as a heterologous protein without any modifications. Strong components of the Y. lipolytica expression/secretion system could be used for high‐level production of recombinant proteins in S. cerevisiae.
Leptospiral protein antigens are of interest as potential virulence factors and as candidate serodiagnostic and immunoprotective reagents. We identified leptospiral protein antigens by screening a genomic expression library with serum from a rabbit hyperimmunized with formalin-killed, virulent Leptospira kirschneri serovar grippotyphosa. Genes expressing known outer membrane lipoproteins LipL32 and LipL41, the heat shock protein GroEL, and the α, β, and β′ subunits of RNA polymerase were isolated from the library. In addition, a new leptospiral gene that in Escherichia coli expressed a 45-kDa antigen with an amino-terminal signal peptide followed by the spirochetal lipobox Val−4-Phe−3-Asn−2-Ala−1↓Cys+1 was isolated. We designated this putative lipoprotein LipL45. Immunoblot analysis of a panel of Leptospira strains probed with LipL45 antiserum demonstrated that many low-passage strains expressed LipL45. In contrast, LipL45 was not detected in high-passage, culture-attenuated strains, suggesting that LipL45 is a virulence-associated protein. In addition, all leptospiral strains tested, irrespective of culture passage, expressed a 31-kDa antigen that was recognized by LipL45 antiserum. Southern blot and peptide mapping studies indicated that this 31-kDa antigen was derived from the carboxy terminus of LipL45; therefore, it was designated P31LipL45. Membrane fractionation studies demonstrated that P31LipL45 is a peripheral membrane protein. Finally, we found that P31LipL45 levels increased as Leptospira entered the stationary phase, indicating that P31LipL45 levels were regulated. Hamsters infected with L. kirschneri formed an antibody response to LipL45, indicating that LipL45 was expressed during infection. Furthermore, the immunohistochemistry of kidneys from infected hamsters indicated that LipL45 was expressed by L. kirschneri that colonized the renal tubule. These observations suggest that expression of LipL45 responds to environmental cues, including those encountered during infection of a mammalian host.
Protein secretion systems are critical to bacterial virulence and interactions with other organisms. The Type VI secretion system (T6SS) is found in many bacterial species and is used to target either eukaryotic cells or competitor bacteria. However, T6SS-secreted proteins have proven surprisingly elusive. Here, we identified two secreted substrates of the antibacterial T6SS from the opportunistic human pathogen, Serratia marcescens. Ssp1 and Ssp2, both encoded within the T6SS gene cluster, were confirmed as antibacterial toxins delivered by the T6SS. Four related proteins encoded around the Ssp proteins (‘Rap’ proteins) included two specifically conferring self-resistance (‘immunity’) against T6SS-dependent Ssp1 or Ssp2 toxicity. Biochemical characterization revealed specific, tight binding between cognate Ssp–Rap pairs, forming complexes of 2:2 stoichiometry. The atomic structures of two Rap proteins were solved, revealing a novel helical fold, dependent on a structural disulphide bond, a structural feature consistent with their functional localization. Homologues of the Serratia Ssp and Rap proteins are found encoded together within other T6SS gene clusters, thus they represent founder members of new families of T6SS-secreted and cognate immunity proteins. We suggest that Ssp proteins are the original substrates of the S. marcescens T6SS, before horizontal acquisition of other T6SS-secreted toxins. Molecular insight has been provided into how pathogens utilize antibacterial T6SSs to overcome competitors and succeed in polymicrobial niches.
We previously identified a Serratia marcescens extracellular protein, HasA, able to bind heme and required for iron acquisition from heme and hemoglobin by the bacterium. This novel type of extracellular protein does not have a signal peptide and does not show sequence similarities to other proteins. HasA secretion was reconstituted in Escherichia coli, and we show here that like many proteins lacking a signal peptide, HasA has a C-terminal targeting sequence and is secreted by a specific ATP binding cassette (ABC) transporter consisting of three proteins, one inner membrane protein with a conserved ATP binding domain, called the ABC; a second inner membrane protein; and a third, outer membrane component. Since the three S. marcescens components of the HasA transporter have not yet been identified, the reconstituted HasA secretion system is a hybrid. It consists of the two S. marcescens inner membrane-specific components, HasD and HasE, associated with an outer membrane component coming from another bacterial ABC transporter, such as the E. coli TolC protein, the outer membrane component of the hemolysin transporter, or the Erwinia chrysanthemi PrtF protein, the outer membrane component of the protease transporter. This hybrid transporter was first shown to allow the secretion of the S. marcescens metalloprotease and the E. chrysanthemi metalloproteases B and C. On account of that, the two S. marcescens components HasD and HasE were previously named PrtDSM and PrtESM, respectively. However, HasA is secreted neither by the PrtD-PrtE-PrtF transporter (the genuine E. chrysanthemi protease transporter) nor by the HlyB-HlhD-TolC transporter (the hemolysin transporter). Moreover, HasA, coexpressed in the same cell, strongly inhibits the secretion of proteases B and C by their own transporter, indicating that the E. chrysanthemi transporter recognizes HasA. Since PrtF could replace TolC in the constitution of the HasA transporter, this indicates that the secretion block does not take place at the level of the outer membrane component but rather at an earlier step of interaction between HasA and the inner membrane components.
The lipA gene encoding an extracellular lipase was cloned from the wild-type strain of Serratia marcescens Sr41. Nucleotide sequencing showed a major open reading frame encoding a 64.9-kDa protein of 613 amino acid residues; the deduced amino acid sequence contains a lipase consensus sequence, GXSXG. The lipase had 66 and 56% homologies with the lipases of Pseudomonas fluorescens B52 and P. fluorescens SIK W1, respectively, but did not show any overall homology with lipases from other origins. The Escherichia coli cells carrying the S. marcescens lipA gene did not secrete the lipase into the medium. The S. marcescens lipase had no conventional N-terminal signal sequence but was also not subjected to any processing at both the N-terminal and C-terminal regions. A specific short region similar to the regions of secretory proteins having no N-terminal signal peptide was observed in the amino acid sequence. Expression of the lipA gene in S. marcescens was affected by the carbon source and the addition of Tween 80.
Leptospira interrogans is responsible for leptospirosis, a zoonosis of worldwide distribution. LipL32 is the major outer membrane protein of pathogenic leptospires, accounting for up to 75% of total outer membrane protein. In recent times LipL32 has become the focus of intense study because of its surface location, dominance in the host immune response, and conservation among pathogenic species. In this study, an lipL32 mutant was constructed in L. interrogans using transposon mutagenesis. The lipL32 mutant had normal morphology and growth rate compared to the wild type and was equally adherent to extracellular matrix. Protein composition of the cell membranes was found to be largely unaffected by the loss of LipL32, with no obvious compensatory increase in other proteins. Microarray studies found no obvious stress response or upregulation of genes that may compensate for the loss of LipL32 but did suggest an association between LipL32 and the synthesis of heme and vitamin B12. When hamsters were inoculated by systemic and mucosal routes, the mutant caused acute severe disease manifestations that were indistinguishable from wild-type L. interrogans infection. In the rat model of chronic infection, the LipL32 mutant colonized the renal tubules as efficiently as the wild-type strain. In conclusion, this study showed that LipL32 does not play a role in either the acute or chronic models of infection. Considering the abundance and conservation of LipL32 among all pathogenic Leptospira spp. and its absence in saprophytic Leptospira, this finding is remarkable. The role of this protein in leptospiral biology and pathogenesis thus remains elusive.