Jean-Marie Clément

Antigenicity and immunogenicity of the HIV-1 gp41 epitope ELDKWA inserted into permissive sites of the MalE protein

The highly conserved amino acid sequence ELDKWA of HIV-1 gp41 has been inserted into Escherichia coli MalE protein which had been shown to be an adequate carrier to present foreign epitopes to the immune system. We first investigated whether eight different permissive sites of MalE are able to tolerate an insertion of 7-50 residues encoding this epitope. Secondly, antigenicity of the epitope inserted in MalE protein was estimated from monoclonal antibody 2F5 binding analysis using the BIAcore(R) technology and its immunogenicity in mice was measured as the ability of hybrid proteins to elicit antibodies against a synthetic peptide containing this epitope. This study revealed a good correlation between the antigenicity of the inserted epitope and its immunogenicity. Increasing the length of the inserted epitope, as well as inserting multicopies of this epitope increased both its antigenicity and immunogenicity. However, none of the MalE hybrid proteins tested induced anti-HIV-1 neutralizing antibodies. This study strongly suggests that the capacity of the 2F5 epitope to induce neutralizing antibodies depends on the molecular context in which it is presented.

Short palindromic repetitive DNA elements in enterobacteria: a survey

We present a survey of short palindromic repetitive elements in enterobacteria. Seven families are presented. Five were already known (RSA, IRU, 29-bp repeats, BIMEs and boxC), and their properties are updated; in particular, a new composite element is shown to include the formerly identified boxC repeats. Two repetitions, YPAL1 and YPAL2, found primarily in Yersinia, are described here for the first time.

Palindromic units: a case of highly repetitive DNA sequences in bacteria

Abstract In bacteria, there is growing evidence that highly repeated DNA sequence families are quite widespread. The palindromic unit (PU) family may represent up to 1% of the genome of E. coli and Salmonella typhimurium . The genomic localization of PUs and their consensus sequence appear to be species-specific. At least some PUs display functions at the mRNA level such as mRNA stabilization and, occasionally, mRNA processing or transcription termination. However, some of their properties, including indications that they bind a specific protein, suggest that PUs are involved in the structure of the bacterial nucleoid.

A subfamily of E. coli palindromic units implicated in transcription termination

We previously described a family of dispersed palindromic sequences highly repeated in Escherichia coli and Salmonella typhimurium genomes. These sequences, called PU (palindromic units), are located outside structural genes. Conflicting results have been reported on the effects of different PU in transcription termination. Two PU located between co-transcribed genes in S. typhimurium were found not to cause transcription termination [25]. One PU located between convergently transcribed genes in E. coli behaved as bidirectional transcription terminators [12]. In the present paper, we show that three PU located between co-transcribed genes in E. coli are not a transcription terminator. From the literature, we define a subfamily of PU, which we called PU, located between convergently transcribed genes which we implicate in bidirectional transcription termination. This plus the analysis of another PU which terminates transcription suggest that pecularities in the sequence or in the sequence environment of PU determine their role in transcription termination.

Immune responses induced by recombinant BCG strains according to level of production of a foreign antigen: malE.

A variety of viral, bacterial and parasitic antigens have been expressed in BCG and the capacity of these recombinant bacteria to induce immune responses has been well documented. However, little is known about the parameters influencing the induction of immune responses by recombinant BCG (rBCG), such as level of production and localization of the recombinant antigen. In the present study, we have constructed several rBCG strains expressing the malE gene from Escherichia coli which is either secreted or targeted to the cytoplasm or plasma membrane. Expression of malE was quantified by ELISA and localization was analyzed by flow cytometry. Even when using the same promoter, levels of cytoplasmic or membrane MalE production were far less than those from secreting strains using either mycobacterial or E. coli secretion signals. Stronger and more rapid immune responses were induced by rBCG strains with the highest levels of secreted MalE compared to cytoplasmic or membrane constructs, including both good humoral and proliferative responses in BALB/c, C57BL6 and even C3H mice, previously shown to be poor MalE responders. These results suggest that the levels of foreign antigen production play an important role in the induction of immune responses by rBCG strains.

Palindromic units from E. coli as binding sites for a chromoid-associated protein

Several hundred copies of a highly conserved extragenic palindromic sequence, 20–40 nucleotides long, exist along the chromosome of E. coli and S. typhimurium. These have been defined as palindromic units (PU) or repetitive extragenic palindromes (REP). No general function for PUs has been identified. In the present work, we provide data showing that a protein associated with a chromoid extract of E. coli protects PU DNA against exonuclease III digestion. This provides the first experimental evidence that PU constitutes binding sites for a chromoid‐associated protein. This result supports the hypothesis that PUs could play a role in the structure of the bacterial chromoid.

Analysis of λ receptor and β-lactamase synthesis and export using cloned genes in a minicell system

SummaryWe have cloned lamB, the gene for λ receptor (an outer membrane protein), on a small plasmid which also carries the gene for β-lactamase (a periplasmic protein). We have identified a promoter in the region of malK, the gene immediately preceding lamB, which is active in minicells but relatively inactive in vitro. Using a minicell system, we have found that both λ receptor and β-lactamase are made as full length precursors which are subsequently processed. We also show that the λ receptor precursor can be exported to the outer membrane before it is processed. Mature β-lactamase is found only in the periplasm, suggesting that processing may be a requirement for export to the periplasm.

Transposition of IS1397 in the Family Enterobacteriaceae and First Characterization of ISKpn1, a New Insertion Sequence Associated with Klebsiella pneumoniae Palindromic Units

IS1397 and ISKpn1 are IS3 family members which are specifically inserted into the loop of palindromic units (PUs). IS1397 is shown to transpose into PUs with sequences close or identical to the Escherichia coli consensus, even in other enterobacteria (Salmonella enterica serovar Typhimurium, Klebsiella pneumoniae, and Klebsiella oxytoca). Moreover, we show that homologous intergenic regions containing PUs constitute IS1397 transpositional hot spots, despite bacterial interspersed mosaic element structures that differ among the three species. ISKpn1, described here for the first time, is specific for PUs from K. pneumoniae, in which we discovered it. A sequence comparison between the two insertion sequences allowed us to define a motif possibly accounting for their specificity.

Immunogenicity of viral B- and T-cell epitopes expressed in recombinant bacterial proteins.

Foreign polypeptides can be expressed as genetic inserts in several permissive sites of MalE and LamB, two Escherichia coli envelope proteins. Several viral B and T-cell epitopes have been inserted in these proteins and we analyzed the role of the molecular environment on the immunogenicity of the foreign epitopes. These studies demonstrated that the antigenicity and immunogenicity of B-cell epitopes depend on their site of insertion in the carrier protein. Using bacteria expressing B-cell epitopes either at the cell surface or in the periplasm, it was also shown that the cellular location of a foreign B-cell epitope expressed by recombinant bacteria determines its T-cell dependent or independent characteristics. Analysis of in vivo immunogenicity of purified LamB or MalE hybrid proteins expressing two different T-cell epitopes established that the immunogenicity of recombinant T-cell epitopes may be strongly affected by both the insertion site and inserted adjacent residues. The in vitro analysis of specific T-cell hybridoma response to hybrid MalE proteins also showed that the molecular context of a T-cell determinant alters the diversity of its T-cell recognition.

Location of a phage binding region on an outer membrane protein

Although several of the proteins present in the outer membrane of Gram negative bacteria are known to constitute phage receptors [ 1,2], in no instance has the exact site recognized by these phages been identified. The identification of these sites should provide information not only on the nature of phage-receptor interactions, but also on the structure-function relationships in outer membrane proteins, which have biological roles in addition to being phage receptors [2,3]. Here we report on the identification of part of a phage binding site on the iamB protein of Escherichia coli. The product of gene lamB is a 50 000 Mr maltoseinducible protein which facilitates the diffusion of maltose and maltodextrins across the outer membrane 14-61. In addition it serves as a receptor for phages X, KIO and TPI 17-91. As a first attempt to locate the different functional sites on this polypeptide we have studied the binding site for phage KlO. Mutations speci~c~y affecting this site should render the cells unable to adsorb K10 without altering the other functions of the lamB protein. Such mutations were obtained and genetically mapped in [8]. The exact location of some of these mutations has now been determined by using DNA-sequencing techniques.