Christophe Gilbert

Heterologous Expression of an Immunogenic Pneumococcal Type 3 Capsular Polysaccharide in Lactococcus lactis

ABSTRACT In order to develop a new system for the analysis of capsular biosynthetic pathways we have explored the possibility of expressing type 3 capsular polysaccharide (CPS) from the pathogenStreptococcus pneumoniae in Lactococcus lactis, an unencapsulated lactic acid bacterium being developed as a vaccine delivery vehicle for mucosal immunization. Only three of the four type 3 CPS biosynthesis genes were found to be necessary for the abundant formation (120 mg liter−1) of an extracellular type 3 CPS in L. lactis, implying a role for the type 3-specific synthase in the extracellular transport of the CPS or implying the existence of an alternative export system in L. lactis. The authenticity of the expressed heterologous polysaccharide was established by chemical and immunological analyses. Proton and carbon nuclear magnetic resonance spectroscopy of CPSs purified from L. lactis and S. pneumoniae showed that the two CPS structures were identical. When mice were immunized intraperitoneally with 3.5 × 106 CFU of live recombinant lactococci expressing a total of approximately 0.5 μg of type 3 CPS, the immune responses elicited appeared identical to those observed in mice inoculated with 0.5 μg of type 3 CPS purified from S. pneumoniae. These findings show that L. lactis is a useful host in which to study the role and function of genes involved in the production of bacterial capsules. Additionally, L. lactis shows potential as a host for the safe production of capsule antigens and as a vaccine delivery vehicle for polysaccharide antigens.

Cloning, sequencing and characterization of the pepIP gene encoding a proline iminopeptidase from Lactobacillus delbrueckii subsp. bulgaricus CNRZ 397.

The proline iminopeptidase (PepIP) of Lactobacillus delbrueckii subsp. bulgaricus is a major peptidase located in the cell envelope. Its structural gene (pepIP) has been cloned into pUC18 and expressed at a very high level in Escherichia coli to give a PepIP activity 15,000-fold higher than that found in L. delbrueckii subsp. bulgaricus. The nucleotide sequence of the pepIP gene revealed an open reading frame of 295 codons encoding a protein with a predicted M(r) of 33,006, which is consistent with the apparent size of the gene product. The amino acid sequence of PepIP shows significant homology with those of other hydrolases involved in the degradation of cyclic compounds. In particular, there is a region which includes an identified catalytic site containing a serine residue and a motif specific for the active sites of prolyloligopeptidases (Gly-X-Ser-X-Gly-Gly). The PepIP opens a new way for supplying cells with proline using the peptides resulting from the proteolytic degradation of caseins.

Proline iminopeptidase from Lactobacillus delbrueckii subsp. bulgaricus CNRZ 397 : purification and characterization

Proline iminopeptidase (PepIP) is a major peptidase in Lactobacillus delbrueckii subsp. bularicus CNRZ397, encoded by the pepIP gene. Amplification and expression of this gene in Escherichia coli K12 resulted in a very high level of enzyme production. Moreover, export into the E. coli periplasm of 45% of PepIP activity allowed us to purify the enzyme easily by a single ion-exchange chromatography step. PepIP is a trimer of Mr 100000 , composed of three identical subunits. In the presence of 0.1% BSA, PepIP activity was optimal at pH 6-7 and stable at temperatures below 40 degrees C. The enzyme was strongly inhibited by 3,4-dichloroisocoumarin, a serine protease inhibitor, by bestatin and by heavy metal ions. It was also inactivated by p-chloromercuribenzoate, but was reactivated by adding dithiothreitol. PepIP is characterized by a high specificity towards di- or tripeptides with proline at the NH2-terminal position, but is not able to hydrolyse longer peptides, or peptides with hydroxyproline at the NH2-end. The NH2-terminal amino acid sequence of the purified PepIP corresponds to the amino acid sequence deduced from the nucleotide sequence of the pepIP gene.

Comparison of cell surface proteinase activities within the Lactobacillus genus

Whole cells of Lactobacillus delbrueckii subsp. bulgaricus CNRZ 397 ( Lb. bulgaricus CNRZ 397) are able to hydrolyse α- and β-caseins. We have isolated a mutant of Lb. bulgaricus altered for growth in milk and unable to hydrolyse α- or β-casein. Normal growth was restored by adding amino acids or tryptone to milk. No significant difference between the peptidase activities of parent and mutant strains was observed. The cell surface caseinolytic activities of three lactobacilli species and Lactococcus lactis subsp. lactis ( Lc. lactis ) were compared. As expected, the characteristics of the cell surface proteinase activity of Lb. casei were similar to those of Lc. lactis . We showed that the cleavage specificities of the cell surface proteinase activities from lactobacilli were species-dependent and at least three types of activity were distinguished. The regulation of the biosynthesis of cell surface proteinase activities was medium-dependent and different within the Lactobacillus genus and even within the Lb. delbrueckii species. In contrast to Lb. bulgaricus , the cell surface proteinase activity of Lb. lactis was totally inhibited in a medium rich in peptides or amino acids. In contrast, the cell surface of Lb. helveticus probably displayed two proteinases with different cleavage specificities and with a biosynthesis regulation sensitive to different medium components.

The TolC Protein of Legionella pneumophila Plays a Major Role in Multi-Drug Resistance and the Early Steps of Host Invasion

Pneumonia associated with Iegionnairess disease is initiated in humans after inhalation of contaminated aerosols. In the environment, Legionella pneumophila is thought to survive and multiply as an intracellular parasite within free-living amoeba. In the genome of L. pneumophila Lens, we identified a unique gene, tolC, encoding a protein that is highly homologous to the outer membrane protein TolC of Escherichia coli. Deletion of tolC by allelic exchange in L. pneumophila caused increased sensitivity to various drugs. The complementation of the tolC mutation in trans restored drug resistance, indicating that TolC is involved in multi-drug efflux machinery. In addition, deletion of tolC caused a significant attenuation of virulence towards both amoebae and macrophages. Thus, the TolC protein appears to play a crucial role in virulence which could be mediated by its involvement in efflux pump mechanisms. These findings will be helpful in unraveling the pathogenic mechanisms of L. pneumophila as well as in developing new therapeutic agents affecting the efflux of toxic compounds.

Two cell-wall-associated aminopeptidases from Lactobacillus helveticus and the purification and characterization of APII from strain ITGL1

Lactobacillus helveticus ITGL1 is able to hydrolyse many amino-acyl and dipeptidyl-p-nitroanilides. Analysis of heat inactivation kinetics, metal ion and protease inhibitor effects, and the subcellular location of aminopeptidase activities in both the parental strain and mutants deficient in lysyl-p-nitroanilide hydrolysis, led to the characterization of two cell-wall-associated aminopeptidases, APII and APIV. APII, which catalysed L-lysine p-nitroanilide hydrolysis, was purified about 28-fold to homogeneity from cell-wall extracts of L. helveticus ITGL1 and characterized. The purified enzyme appeared to be monomeric, with a molecular mass of 97 kDa. Aminopeptidase activity was greatest at pH 6.5 and 50 degrees C. APII was completely inhibited by bestatin, chelating agents such as EDTA or 1,10-phenanthroline and the divalent cations Zn2+ and Cu2+. The activity of the EDTA-treated enzyme was restored by Co2+, Ca2+ or Mn2+. Although APII was able to degrade several dipeptides and tripeptides with hydrophobic N-terminal amino acid (Leu, Ala), it was inactive on peptides containing Pro or Gly, and may thus contribute to the development of cheese flavour by processing bitter peptides.

Determination of the Domain of the Lactobacillus delbrueckii subsp. bulgaricus Cell Surface Proteinase PrtB Involved in Attachment to the Cell Wall after Heterologous Expression of the prtB Gene in Lactococcus lactis

ABSTRACT Belonging to the subtilase family, the cell surface proteinase (CSP) PrtB of Lactobacillus delbrueckii subsp. bulgaricus differs from other CSPs synthesized by lactic acid bacteria. Expression of the prtB gene under its own promoter was shown to complement the proteinase-deficient strain MG1363 (PrtP− PrtM−) of Lactococcus lactis subsp. cremoris. Surprisingly, the maturation process of PrtB, unlike that of lactococcal CSP PrtPs, does not require a specific PrtM-like chaperone. The carboxy end of PrtB was previously shown to be different from the consensus anchoring region of other CSPs and exhibits an imperfect duplication of 59 amino acids with a high lysine content. By using a deletion strategy, the removal of the last 99 amino acids, including the degenerated anchoring signal (LPKKT), was found to be sufficient to release a part of the truncated PrtB into the culture medium and led to an increase in PrtB activity. This truncated PrtB is still active and enables L. lactis MG1363 to grow in milk supplemented with glucose. By contrast, deletion of the last 806 amino acids of PrtB led to the secretion of an inactive proteinase. Thus, the utmost carboxy end of PrtB is involved in attachment to the bacterial cell wall. Proteinase PrtB constitutes a powerful tool for cell surface display of heterologous proteins like antigens.

The Legionella Kinase LegK2 Targets the ARP2/3 Complex To Inhibit Actin Nucleation on Phagosomes and Allow Bacterial Evasion of the Late Endocytic Pathway

ABSTRACT Legionella pneumophila, the etiological agent of legionellosis, replicates within phagocytic cells. Crucial to biogenesis of the replicative vacuole is the Dot/Icm type 4 secretion system, which translocates a large number of effectors into the host cell cytosol. Among them is LegK2, a protein kinase that plays a key role in Legionella infection. Here, we identified the actin nucleator ARP2/3 complex as a target of LegK2. LegK2 phosphorylates the ARPC1B and ARP3 subunits of the ARP2/3 complex. LegK2-dependent ARP2/3 phosphorylation triggers global actin cytoskeleton remodeling in cells, and it impairs actin tail formation by Listeria monocytogenes, a well-known ARP2/3-dependent process. During infection, LegK2 is addressed to the Legionella-containing vacuole surface and inhibits actin polymerization on the phagosome, as revealed by legK2 gene inactivation. Consequently, LegK2 prevents late endosome/lysosome association with the phagosome and finally contributes to remodeling of the bacterium-containing phagosome into a replicative niche. The inhibition of actin polymerization by LegK2 and its effect on endosome trafficking are ARP2/3 dependent since it can be phenocopied by a specific chemical inhibitor of the ARP2/3 complex. Thus, LegK2-ARP2/3 interplay highlights an original mechanism of bacterial virulence with an unexpected role in local actin remodeling that allows bacteria to control vesicle trafficking in order to escape host defenses. IMPORTANCE Deciphering the individual contribution of each Dot/Icm type 4 secretion system substrate to the intracellular life-style of L. pneumophila remains the principal challenge in understanding the molecular basis of Legionella virulence. Our finding that LegK2 is a Dot/Icm effector that inhibits actin polymerization on the Legionella-containing vacuole importantly contributes to the deciphering of the molecular mechanisms evolved by Legionella to counteract the endocytic pathway. Indeed, our results highlight the essential role of LegK2 in preventing late endosomes from fusing with the phagosome. More generally, this work is the first demonstration of local actin remodeling as a mechanism used by bacteria to control organelle trafficking. Further, by characterizing the role of the bacterial protein kinase LegK2, we reinforce the concept that posttranslational modifications are key strategies used by pathogens to evade host cell defenses. Deciphering the individual contribution of each Dot/Icm type 4 secretion system substrate to the intracellular life-style of L. pneumophila remains the principal challenge in understanding the molecular basis of Legionella virulence. Our finding that LegK2 is a Dot/Icm effector that inhibits actin polymerization on the Legionella-containing vacuole importantly contributes to the deciphering of the molecular mechanisms evolved by Legionella to counteract the endocytic pathway. Indeed, our results highlight the essential role of LegK2 in preventing late endosomes from fusing with the phagosome. More generally, this work is the first demonstration of local actin remodeling as a mechanism used by bacteria to control organelle trafficking. Further, by characterizing the role of the bacterial protein kinase LegK2, we reinforce the concept that posttranslational modifications are key strategies used by pathogens to evade host cell defenses.

The atypical two-component sensor kinase Lpl0330 from Legionella pneumophila controls the bifunctional diguanylate cyclase-phosphodiesterase Lpl0329 to modulate bis-(3'-5')-cyclic dimeric GMP synthesis.

A significant part of bacterial two-component system response regulators contains effector domains predicted to be involved in metabolism of bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP), a second messenger that plays a key role in many physiological processes. The intracellular level of c-di-GMP is controlled by diguanylate cyclase and phosphodiesterases activities associated with GGDEF and EAL domains, respectively. The Legionella pneumophila Lens genome displays 22 GGDEF/EAL domain-encoding genes. One of them, lpl0329, encodes a protein containing a two-component system receiver domain and both GGDEF and EAL domains. Here, we demonstrated that the GGDEF and EAL domains of Lpl0329 are both functional and lead to simultaneous synthesis and hydrolysis of c-di-GMP. Moreover, these two opposite activities are finely regulated by Lpl0329 phosphorylation due to the atypical histidine kinase Lpl0330. Indeed, Lpl0330 was found to autophosphorylate on a histidine residue in an atypical H box, which is conserved in various bacteria species and thus defines a new histidine kinase subfamily. Lpl0330 also catalyzes the phosphotranfer to Lpl0329, which results in a diguanylate cyclase activity decrease whereas phosphodiesterase activity remains efficient. Altogether, these data present (i) a new histidine kinase subfamily based on the conservation of an original H box that we named HGN H box, and (ii) the first example of a bifunctional enzyme that modulates synthesis and turnover of c-di-GMP in response to phosphorylation of its receiver domain.

Characterization and site-directed mutagenesis of Wzb, an O-phosphatase from Lactobacillus rhamnosus

BackgroundReversible phosphorylation events within a polymerisation complex have been proposed to modulate capsular polysaccharide synthesis in Streptococcus pneumoniae. Similar phosphatase and kinase genes are present in the exopolysaccharide (EPS) biosynthesis loci of numerous lactic acid bacteria genomes.ResultsThe protein sequence deduced from the wzb gene in Lactobacillus rhamnosus ATCC 9595 reveals four motifs of the polymerase and histidinol phosphatase (PHP) superfamily of prokaryotic O-phosphatases. Native and modified His-tag fusion Wzb proteins were purified from Escherichia coli cultures. Extracts showed phosphatase activity towards tyrosine-containing peptides. The purified fusion protein Wzb was active on p-nitrophenyl-phosphate (p NPP), with an optimal activity in presence of bovine serum albumin (BSA 1%) at pH 7.3 and a temperature of 75°C. At 50°C, residual activity decreased to 10 %. Copper ions were essential for phosphatase activity, which was significantly increased by addition of cobalt. Mutated fusion Wzb proteins exhibited reduced phosphatase activity on p-nitrophenyl-phosphate. However, one variant (C6S) showed close to 20% increase in phosphatase activity.ConclusionThese characteristics reveal significant differences with the manganese-dependent CpsB protein tyrosine phosphatase described for Streptococcus pneumoniae as well as with the polysaccharide-related phosphatases of Gram negative bacteria.