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International audience; Francisella tularensis is the causative agent in tularemia for which the high prevalence of treatment failure and relapse is a major concern. Directed-evolution experiments revealed that acquisition of fluoroquinolone (FQ) resistance was linked to factors in addition to mutations in DNA gyrase. Here, using F. tularensis live vaccine strain (LVS) as a model, we demonstrated that FupA/B (Fer-Utilization Protein) expression is linked to FQ susceptibility, and that the virulent strain F. tularensis subsp. tularensis SCHU S4 deleted for the homologous FupA protein exhibited even higher FQ resistance. In addition to an increased FQ minimal inhibitory concentration, LVS Delta fupA/B displayed tolerance toward bactericidal compounds including ciprofloxacin and gentamicin. Interestingly, the FupA/B deletion was found to promote increased secretion of outer membrane vesicles (OMVs). Mass spectrometry-based quantitative proteomic characterization of vesicles from LVS and LVS Delta fupA/B identified 801 proteins, including a subset of 23 proteins exhibiting differential abundance between both strains which may therefore contribute to the reduced antibiotic susceptibility of the FupA/B-deleted strain. We also demonstrated that OMVs are key structural elements of LVS Delta fupA/B biofilms providing protection against FQ. These results provide a new basis for understanding and tackling antibiotic resistance and/or persistence of Francisella and other pathogenic members of the Thiotrichales class.

International audience; Biofilms are currently considered as a predominant lifestyle of many bacteria in nature. While they promote survival of microbes, biofilms also potentially increase the threats to animal and public health in case of pathogenic species. They not only facilitate bacteria transmission and persistence, but also promote spreading of antibiotic resistance leading to chronic infections. In the case of Francisella tularensis, the causative agent of tularemia, biofilms have remained largely enigmatic. Here, applying live and static confocal microscopy, we report growth and ultrastructural organization of the biofilms formed in vitro by these microorganisms over the early transition from coccobacillary into coccoid shape during biofilm assembly. Using selective dispersing agents, we provided evidence for extracellular DNA (eDNA) being a major and conserved structural component of mature biofilms formed by both F. subsp. novicida and a human clinical isolate of F. philomiragia. We also observed a higher physical robustness of F. novicida biofilm as compared to F. philomiragia one, a feature likely promoted by specific polysaccharides. Further, F. novicida biofilms resisted significantly better to ciprofloxacin than their planktonic counterparts. Importantly, when grown in biofilms, both Francisella species survived longer in cold water as compared to free-living bacteria, a trait possibly associated with a gain in fitness in the natural aquatic environment. Overall, this study provides information on survival of Francisella when embedded with biofilms that should improve both the future management of biofilm-related infections and the design of effective strategies to tackle down the problematic issue of bacteria persistence in aquatic ecosystems.

Fluoroquinolone (FQ) resistance is a major health concern in the treatment of tularemia. Because DNA gyrase has been described as the main target of these compounds, our aim was to clarify the contributions of both GyrA and GyrB mutations found in Francisella novicida clones highly resistant to FQs. Wild-type and mutated GyrA and GyrB subunits were overexpressed so that the in vitro FQ sensitivity of functional reconstituted complexes could be evaluated. The data obtained were compared to the MICs of FQs against bacterial clones harboring the same mutations and were further validated through complementation experiments and structural modeling. Whole-genome sequencing of highly FQ-resistant lineages was also done. Supercoiling and DNA cleavage assays demonstrated that GyrA D87 is a hot spot FQ resistance target in F. novicida and pointed out the role of the GyrA P43H substitution in resistance acquisition. An unusual feature of FQ resistance acquisition in F. novicida is that the first-step mutation occurs in GyrB, with direct or indirect consequences for FQ sensitivity. Insertion of P466 into GyrB leads to a 50% inhibitory concentration (IC 50 ) comparable to that observed for a mutant gyrase carrying the GyrA D87Y substitution, while the D487E-ΔK488 mutation, while not active on its own, contributes to the high level of resistance that occurs following acquisition of the GyrA D87G substitution in double GyrA/GyrB mutants. The involvement of other putative targets is discussed, including that of a ParE mutation that was found to arise in the very late stage of antibiotic exposure. This study provides the first characterization of the molecular mechanisms responsible for FQ resistance in Francisella .

In seminiferous tubules, Sertoli cells provide structural and nutritional support for the developing germinal cells. Cell- to-cell signaling and cell adhesion require proteoglycans expressed at the cell membrane. A preliminary biochemical and structural approach indicated that cell surface proteoglycans are mostly heparan sulfate proteoglycans (HSPG). Glypican-1, syndecans-1 and -4 were identified using a molecular approach. Their differential regulation was demonstrated in immature rat Sertoli cells. Follicle-stimulating hormone (FSH) is the main regulator of Sertoli cell function. Signal transduction triggered by FSH involves both an increased intracellular cAMP synthesis and a calcium influx. This study demonstrates that FSH, through its second messengers (increase in intracellular cAMP and intracellular calcium), downregulated the glypican-1 mRNA expression in Sertoli cells from 20-day-old rats. On the other hand, syndecan-1 mRNA expression is not modulated by FSH as it would result from the antagonistic effects of increased intracellular cAMP and intracellular calcium levels. Finally, syndecan-4 mRNA expression is not regulated by this pathway. The present study was extended during Sertoli cell development. Indeed, Sertoli cells undergo extensive changes during the postnatal period both in structure and function. These important transformations are critical for the establishment of spermatogenesis and development of the adult pattern of testicular function. Our data indicated that the regulation of HSPG mRNA expression is HSPG-specific and depends on the Sertoli cell developmental stage.

An alternative N-glycosylation pathway using Glc1-3Man5GlcNAc2 as a donor to be transferred to a protein acceptor is found either in Man-P-Dol synthase deficient cells or in wild type CHO cells grown in energy deprivation conditions. Discrimination between oligomannosides of this alternative pathway and oligomannosides of the major one containing the same number of sugar residues Man6-8GlcNAc2 required structural studies. Taking advantage of the specific chromatographic behaviour of glucosylated oligomannosides, in pellicular high pH anion exchange chromatography, we developed a one-step method for the identification of the alternative N-glycosylation pathway compounds differing from those of the major one.

The N-glycosylation of proteins is accompanied by the release of soluble oligosaccharide material. Besides oligosaccharide phosphates originating from the cleavage of lipid intermediates, neutral free oligosaccharides represent the major part of this material and are heterogeneous depending on whether the reducing end has one or two N-acetylglucosamine residues. The present study focuses on the intracellular origin of neutral free oligosaccharides in a CHO cell line. Kinetic and pulse-chase experiments clearly indicate that oligosaccharides possessing a chitobiosyl unit are derived from oligosaccharide pyrophosphodolichol, whereas oligosaccharides possessing one N-acetyl-glucosamine residue are derived from newly synthesized glycoprotein. This relationship is confirmed by comparing the glycosylation pattern of lipid donors and glycoproteins with those of neutral free oligosaccharides under various incubation conditions (inhibition of protein synthesis, presence of processing inhibitors, presence or absence of glucose). Degradation of newly synthesized glycoprotein and formation of neutral oligosaccharides with one N-acetylglucosamine residue are inhibited at 16 degrees C but not affected by lysosomotropic agents such as leupeptin or NH4Cl. Together with the fact that the degradation of newly synthesized glycoproteins and the subsequent release of the glycan are recovered in permeabilized cells, these results suggest that this phenomenon occurs in the rough endoplasmic reticulum or in a closely related compartment.

Streptococcus uberis UCN 42, isolated from a case of bovine mastitis, was intermediately resistant to lincomycin (MIC = 2 μg/ml) while remaining susceptible to clindamycin (MIC = 0.06 μg/ml) and erythromycin. A 1.1-kb SacI fragment was cloned from S. uberis UCN 42 total DNA on plasmid pUC 18 and introduced into Escherichia coli AG100A, where it conferred resistance to both clindamycin and lincomycin. The sequence analysis of the fragment showed the presence of a new gene, named lnu (D), that encoded a 164-amino-acid protein with 53% identity with Lnu(C) previously reported to occur in Streptococcus agalactiae . Crude lysates of E. coli AG100A containing the cloned lnu (D) gene inactivated lincomycin and clindamycin in the presence of ATP and MgCl 2 . Mass spectrometry experiments demonstrated that the lnu (D) enzyme catalyzed adenylylation of clindamycin. A domain conserved in deduced sequences of lincosamide O -nucleotidyltransferases Lnu(A), Lnu(C), LinA N2 , and Lin(D) and in the aminoglycoside nucleotidyltransferase ANT(2′′) was identified.

Streptococcus uberis UCN60 was resistant to spiramycin (MIC = 8 μg/ml) but susceptible to erythromycin (MIC = 0.06 μg/ml), azithromycin (MIC = 0.12 μg/ml), josamycin (MIC = 0.25 μg/ml), and tylosin (MIC = 0.5 μg/ml). A 2.5-kb HindIII fragment was cloned from S. uberis UCN60 DNA on plasmid pUC18 and introduced into Escherichia coli AG100A, where it conferred resistance to spiramycin by inactivation. The sequence analysis of the fragment showed the presence of an rdmC -like gene that putatively encoded a protein belonging to the alpha/beta hydrolase family and of the first 196 nucleotides of the mph (B) gene putatively encoding a phosphotransferase known to inactivate 14-, 15-, and 16-membered macrolides in E. coli . The entire mph (B) gene was then identified in S. uberis UCN60. The two genes were expressed alone or in combination in E. coli , Staphylococcus aureus , and Enterococcus faecalis . Analysis of MICs revealed that rdmC -like alone did not confer resistance to erythromycin, tylosin, and josamycin in those three hosts. It conferred resistance to spiramycin in E. coli and E. faecalis but not in S. aureus. mph (B) conferred resistance in E. coli to erythromycin, tylosin, josamycin, and spiramycin but only low levels of resistance in E. faecalis and S. aureus to spiramycin (MIC = 8 μg/ml). The combination of mph (B) and rdmC -like genes resulted in a resistance to spiramycin and tylosin in the three hosts that significantly exceeded the mere addition of the resistance levels conferred by each resistance mechanism alone.

Streptococcus agalactiae UCN36 was resistant to lincomycin (MIC = 16 μg/ml) but susceptible to clindamycin (MIC = 0.12 μg/ml) and erythromycin (MIC = 0.06 μg/ml). A 4-kb HindIII fragment was cloned from S. agalactiae UCN36 total DNA on plasmid pUC18 and introduced into Escherichia coli AG100A, where it conferred resistance to lincomycin. The sequence analysis of the fragment showed the presence of a 1,724-bp element delineated by imperfect inverted repeats (22 of 25 bp) and inserted in the operon for capsular synthesis of S. agalactiae UCN36. This element carried two open reading frames (ORF). The deduced amino acid sequence of the upstream ORF displayed similarity with transposases from anaerobes and IS 1 . The downstream ORF, lnu (C), encoded a 164-amino-acid protein with 26% to 27% identity with the LnuA N2 , LnuA, and LnuA′ lincosamide nucleotidyltransferases reported for Bacteroides and Staphylococcus , respectively. Crude lysates of E. coli AG100A containing the cloned lnu (C) gene inactivated lincomycin and clindamycin in the presence of ATP and MgCl 2 . Mass spectrometry experiments demonstrated that the LnuC enzyme catalyzed adenylylation of lincomycin.

Our previous studies indicated that cell surface proteoglycans were mostly heparan sulfate ones (HSPG) in 20 day-old Sertoli cells [Biochim. Biophys. Acta 1510 (2001) 474]. Among these HSPG, glypican-1, syndecans-1 and -4 mRNAs were expressed and differentially regulated. Glypican-1 and syndecan-1 mRNA expression was up-regulated under PKC activation in contrast to syndecan-4 mRNA expression which was not affected [Biochim. Biophys. Acta 1474 (2000) 31]. Rat Sertoli cells undergo extensive changes during the postnatal period both in structure and function, as the hematotesticular barrier establishment occurs at around 20 day-old. The testicular PKCα expression in developing Sertoli cells results in (i) a soluble (inactive) form which is maximal at the age of 1 day and declines gradually thereafter and (ii) a particulate (active) form which is low at birth, increases six-fold on days 8–11 of age and declines thereafter. The present study focused on the glypican-1, syndecan-1 and syndecan-4 mRNA expression and regulation under PKC activation by the phorbol myristate acetate (PMA) in 10–30 day-old Sertoli cells. Our data indicated that the regulation of their expression specifically depends on the nature of HSPG and Sertoli cell developmental stage and evidenced a specific PKC regulation of HSPG mRNA expression.

In seminiferous tubules, Sertoli cells provide structural and nutritional support for the developing germinal cells. Cell- to-cell signaling and cell adhesion require proteoglycans expressed at the cell membrane. A preliminary biochemical and structural approach indicated that cell surface proteoglycans are mostly heparan sulfate proteoglycans (HSPG). Glypican-1, syndecans-1 and -4 were identified using a molecular approach. Their differential regulation was demonstrated in immature rat Sertoli cells. Follicle-stimulating hormone (FSH) is the main regulator of Sertoli cell function. Signal transduction triggered by FSH involves both an increased intracellular cAMP synthesis and a calcium influx. This study demonstrates that FSH, through its second messengers (increase in intracellular cAMP and intracellular calcium), downregulated the glypican-1 mRNA expression in Sertoli cells from 20-day-old rats. On the other hand, syndecan-1 mRNA expression is not modulated by FSH as it would result from the antagonistic effects of increased intracellular cAMP and intracellular calcium levels. Finally, syndecan-4 mRNA expression is not regulated by this pathway. The present study was extended during Sertoli cell development. Indeed, Sertoli cells undergo extensive changes during the postnatal period both in structure and function. These important transformations are critical for the establishment of spermatogenesis and development of the adult pattern of testicular function. Our data indicated that the regulation of HSPG mRNA expression is HSPG-specific and depends on the Sertoli cell developmental stage.

Basic fibroblast growth factor (bFGF) regulates diversified biological functions in rat Sertoli cells. This report demonstrates that bFGF inhibits steroidogenesis in developing rat Sertoli cells. Follicle stimulating hormone (FSH)-stimulated estradiol production was reduced by bFGF. Moreover, the amount of cytochrome P450 aromatase, responsible for the irreversible transformation of androgens into estrogens, is decreased by bFGF at the transcriptional level. The bFGF inhibitory effect was also observed in the presence of dibutyryl-cAMP, cholera toxin or RO-20-1724, all inducing high levels of cAMP, the second messenger of FSH. Heparan sulfate proteoglycans (HSPGs) were shown to be required as cofactors for bFGF signaling. Indeed, sodium chlorate, described to drastically decrease proteoglycan sulfation, abolishes the bFGF downregulation of FSH-stimulated estradiol synthesis previously observed. Glypican-1, syndecan-1 and -4, potential bFGF coreceptors, are mainly regulated at the transcriptional level. This report shows that the bFGF regulation of their expression specifically depends on the nature of HSPG and of the Sertoli cell developmental stage. In conclusion, HSPG are partners and the target of bFGF in rat Sertoli cells.

The mreA gene from Streptococcus agalactiae COH31 γ/δ, resistant to macrolides and clindamycin by active efflux, has recently been cloned in Escherichia coli , where it was reported to confer macrolide resistance (J. Clancy, F. Dib-Hajj, J. W. Petitpas, and W. Yuan, Antimicrob. Agents Chemother. 41:2719–2723, 1997). Cumulative data suggested that the mreA gene was located on the chromosome of S. agalactiae COH31 γ/δ. Analysis of the deduced amino acid sequence of mreA revealed significant homology with several bifunctional flavokinases/(flavin adenine dinucleotide (FAD) synthetases, which convert riboflavin to flavin mononucleotide (FMN) and FMN to FAD, respectively. High-performance liquid chromatography experiments showed that the mreA gene product had a monofunctional flavokinase activity, similar to that of RibR from Bacillus subtilis . Sequences identical to those of the mreA gene and of a 121-bp upstream region containing a putative promoter were detected in strains of S. agalactiae UCN4, UCN5, and UCN6 susceptible to macrolides. mreA and its allele from S. agalactiae UCN4 were cloned on the shuttle vector pAT28. Both constructs were introduced into E. coli , where they conferred a similar two- to fourfold increase in the MICs of erythromycin, spiramycin, and clindamycin. The MICs of a variety of other molecules, including crystal violet, acriflavin, sodium dodecyl sulfate, and antibiotics, such as certain cephalosporins, chloramphenicol, doxycycline, nalidixic acid, novobiocin, and rifampin, were also increased. In contrast, resistance to these compounds was not detected when the constructs were introduced into E. faecalis JH2–2. In conclusion, the mreA gene was probably resident in S. agalactiae and may encode a metabolic function. We could not provide any evidence that it was responsible for macrolide resistance in S. agalactiae COH31 γ/δ; broad-spectrum resistance conferred by the gene in E. coli could involve multidrug efflux pumps by a mechanism that remains to be elucidated.

In seminiferous tubules, Sertoli cells provide structural and nutritional support for the developing germinal cells. Cell to cell signalization and cell adhesion require proteoglycans expressed at the cell membrane. A preliminary biochemical and structural approach indicated that cell surface proteoglycans are mostly heparan sulfate (HSPG) in immature rat Sertoli cells. The present study focused on the qualitative and quantitative expression of three membrane HSPG, syndecan-1, syndecan-4 and glypican-1 in Sertoli cells of 20-day-old rat. A semi-quantitative multiplex RT-PCR strategy was developed to appreciate the effect of PKC activation on the mRNA expression of the three HSPG. Our data show that the syndecan-1 and glypican-1 mRNA expression is increased by the phorbol myristate acetate (PMA) suggesting a regulation of their expression by the phosphatidyl inositol pathway, as previously hypothesized (Fagen et al., Biochim. Biophys. Acta, 1472 (1999) 250-261). In addition, a physiological effector of the PKC as ATP gave similar effects. Thus, this over-expression could be related with paracrine factors secreted by germ cells.

A genetically engineered human IL-2 variant protein (IL-2-Mu6) expressed by BHK-21 cells was used as a model protein for studying the effect of different controlled cell culture conditions on the O- and N-glycosylation of recombinant glycoproteins. IL-2-Mu6 contains a single amino acid substitution (Glu100↔Asn) thus creating a potential N-glycosylation recognition site (Asn100-Xxx-Thr/Ser)[1, 2]. Cultivations were carried out in perfused 2-liter double membrane bioreactors. The products of cells grown in suspension and on microcarriers, both in the presence and absence of fetal calf serum, were compared [3]. Glycoprotein products obtained under different cultivation conditions showed differences in proximal α1–6 fucosylation, NeuAc content as well as in the antennarity of the N-glycans.

We have previously described that the N-glycosylation process was accompanied by the release of oligosaccharide-phosphates and neutral oligosaccharides. The relationship between oligosaccharide-P-P-dolichol and its metabolic products (glycoproteins, oligosaccharide-phosphates and neutral oligosaccharides) was investigated by analysing the structure of the oligosaccharide moieties and the kinetic behaviour of the various species in pulse and pulse/chase experiments. For these studies, a glycosylation mutant of Chinese hamster ovary cells (B3F7) which does not synthesize mannosylphosphoryldolichol was utilized. Evidence was obtained for the presence of two pools of oligosaccharide-P-P-dolichol which have different fates. One pool is not glucosylated, is rapidly labelled and immediately chased by mannose, and generates the oligosaccharide-phosphate species. The second pool is glucosylated, exhibits a lag time (5-10 min) prior to being labelled, and is utilized in the glycosylation of proteins and in the production of neutral oligosaccharides. We postulate that the cleavage of non-glycosylated lipid intermediates generating oligosaccharide-phosphates represents a 'bypass' in the dolichol cycle which allows direct regeneration of dolichyl phosphate. The other metabolic fate of non-glucosylated oligosaccharide-lipids, glucosylation, results in their use as effective substrates for the glycosylation of proteins or in the generation of neutral oligosaccharides.