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Francisella tularensis is able to invade, survive and replicate inside a variety of cell types. However, in vivo F. tularensis preferentially enters host macrophages where it rapidly escapes to the cytosol to avoid phagosomal stresses and to multiply to high numbers. We previously showed that infection of human monocytes by F. tularensis LVS triggered the deglycosylation of the glutamine transporter SLC1A5. However, this deglycosylation, specifically induced by Francisella infection, was not restricted to SLC1A5, suggesting that host protein deglycosylation processes in general might contribute to intracellular bacterial adaptation. Indeed, we later found that Francisella infection modulated the transcription of numerous glycosidase and glycosyltransferase genes in human macrophages and analysis of cell extracts revealed an important increase of N and O-protein glycosylation. In eukaryotic cells, glycosylation has significant effects on protein folding, conformation, distribution, stability and activity and dysfunction of protein glycosylation may lead to development of diseases like cancer and pathogenesis of infectious diseases. Pathogenic bacteria have also evolved dedicated glycosylation machineries and have notably been shown to use these glycoconjugates as ligands to specifically interact with the host. In this review, we will focus on Francisella and summarize our current understanding of the importance of these post-translational modifications on its intracellular niche adaptation.

Francisella tularensis is a highly infectious facultative intracellular bacterium causing the zoonotic disease tularemia. Numerous attributes required for F. tularensis intracellular multiplication have been identified recently. However, the mechanisms by which the majority of them interfere with the infected host are still poorly understood. The following review summarizes our current knowledge on the different steps of Francisella intramacrophagic life cycle and expands on the importance of nutrient acquisition.

Francisella tularensis is a highly infectious, Gram-negative bacterium responsible for the disease tularemia in a broad variety of animals, including humans. F. tularensis intracellular multiplication occurs mainly in macrophages. However, F. tularensis is able to infect many other cell types, including other phagocytic (dendritic cells, polymorphonuclear leukocytes) and nonphagocytic (alveolar epithelial cells, hepatocytes, endothelial cells and fibroblasts) cells. The ability of professional phagocytic cells to engulf and kill microbes is an essential component of innate defense. The ability of F. tularensis to impair phagocyte function and survive in the cytosol of infected cells thus constitutes a central aspect of its virulence. The F. tularensis intracellular lifecycle relies on the tightly regulated expression of a series of genes. The unraveling secrets of the regulatory cascades governing the regulation of virulence of F. tularensis will be discussed along with future challenges yet to be solved.

RB18A (TRAP220/DRIP205) is a cofactor of transcription. We herein demonstrated that RB18A downregulated p53 and upregulated MDM2 promoters. These RB18A regulations, not modified by p53wt expression, were inhibited by mutant p53 (p53mut) expression, which directly interacts with RB18A D5 domain. In addition, RB18A via its D4 domain, also interacts directly and specifically with MDM2 protein inhibiting p53mut degradation. Altogether, these mechanisms contribute to maintain a high level of p53mut expression in tumor proliferating cells. Therefore, RB18A plays a central role to control p53wt and p53mut protein content and functions in cells through a loop of regulation, which involves MDM2.

We previously demonstrated that CR2 activation on human B lymphocyte surface specifically triggered tyrosine phosphorylation of the 95-kDa nucleolin, this leading to its binding on SH2 domains of p85 sub-unit of PI 3-kinase and to activation of this enzyme. The specificity of CR2 pathway was clearly demonstrated as neither CD19 nor BCR could induce tyrosine phosphorylation of nucleolin in normal B lymphocytes. These data led us to investigate herein additional molecular events, which were triggered by CR2 activation, upstream and downstream to PI 3-kinase activation. Upstream, we demonstrated that pp60src, a tyrosine kinase of the src family, was involved in tyrosine phosphorylation of nucleolin, while syk tyrosine kinase was not. We also demonstrated a direct protein-protein interaction of pp60src with nucleolin in a CR2-dependent and CD19-independent pathway. Downstream, we demonstrated that CR2 activation also triggered Akt and GSK3 enzyme activation, this pathway being under the control of pp60src tyrosine kinase activation. These regulatory functions of activated CR2 were specific as independent of syk tyrosine kinase and of CD19 and BCR activation. Thus, CR2 activation recruits a specific mechanism to activate PI 3-kinase and its subsequent pathways, this mechanism being different to those recruited by CD19 and BCR.

We previously demonstrated that RB18A, a member of TRAP220/DRIP205/PBP family, in vivo acted as a cofactor of transcription by differently regulating p53wt transactivating activity on physiological promoters. Using p53-negative cells transfected with different constructs, we herein demonstrated that RB18A down-regulated p53wt-dependent apoptosis. This biological regulation was due to a specific diminution of p53wt protein level, as level of p53mut and GAPDH proteins was not modified. This p53wt diminution was dependent on proteasome activity, as inhibited by MG-132 inhibitor. This specific p53wt degradation was correlated with an increase in expression of MDM2, which promoted p53wt degradation into proteasome. RB18A up-regulated MDM2 expression by activating MDM2 promoter, even in absence of p53wt. Altogether, these data emphasized that RB18A could regulate p53wt function not only by direct interaction between both proteins, but also by up-regulating promoter activity of MDM2, a p53-regulating partner.

Immunological screening with the anti-p53 moAb, PAb1801 of a cDNA expression library, prepared from human B lymphoma cells, led us to identify a new human 205 kDa protein called RB18A for 'Recognized By PAb1801 moAntibody'. Immunoblotting or immunoprecipitation of fusion protein or in vitro translated protein, respectively, demonstrated that RB18A protein was recognized by several anti-p53 moAb reacting with the N or C-terminal domains of p53. Full length sequence of RB18A cDNA and computer analysis demonstrated that despite common antigenic determinants between RB18A and p53 proteins, nucleotide and deduced protein sequences did not reveal any significant homologies. RB18A mRNA was detected in all tissues tested except in kidney. In addition, RB18A protein shared identical functions with p53 protein: binding to DNA or to p53 and self-oligomerization. Furthermore, RB18A regulated p53 specific binding on his DNA consensus binding site. These functions were associated to the C-terminal domain of RB18A protein and more specifically to the PAb421 binding site present in this domain. The activation by RB18A of p53 binding on DNA was induced through an unstable interaction between both proteins. Altogether, our data demonstrated that RB18A protein shares antigenic and functional properties with p53 and regulated p53 functions.

A comparative study of tyrosine phosphorylation was performed on peripheral blood lymphocytes from systemic lupus erythematosus (SLE) patients and from healthy donors. Freshly isolated SLE lymphocytes presented an elevated tyrosine phosphorylation level when compared to healthy donors lymphocytes (p = 0.005). Among all phosphorylated proteins, those called p120, p110, p80 and p55-p60 were more phosphorylated. The level of tyrosine phosphorylation of p120 and p110 proteins discriminated significantly (p = 0.0048, respectively, p = 0.02) between SLE patients and healthy donors. Lymphocytes form SLE patients and healthy donors were then stimulated by cross-linking T cell antigens (CD3, CD4, CD8) to further distinguish the signal transduction between normal and pathologic lymphocytes. No statistical differences in the tyrosine phosphorylation pattern, following CD4 or CD8 cross-linking, were observed between SLE patients and healthy donors lymphocytes. CD3 cross-linking induced an effect on tyrosine phosphorylation different in SLE patients versus healthy donors lymphocytes. Thus, the lymphocytes of SLE patients were refractile in anti-CD3 stimulation in comparison with the healthy donors lymphocytes. Chi-square analysis demonstrated that a significantly larger number of healthy donors responded to anti-CD3 stimulation compared to SLE patients (p = 0.03). The high frequency of tyrosine phosphorylation of p110 and p80 proteins, following CD3 stimulation, in normal versus SLE lymphocytes, suggested that these proteins could be involved in abnormal signal transduction in SLE cells.

Francisella tularensis, a Gram-negative bacterium that causes the disease tularemia in a large number of animal species, is thought to reside preferentially within macrophages in vivo. F. tularensis has developed mechanisms to rapidly escape from the phagosome into the cytoplasm of infected cells, a habitat with a rich supply of nutrients, ideal for multiplication. SLC1A5 is a neutral amino acid transporter expressed by human cells, which serves, along with SLC7A5 to equilibrate cytoplasmic amino acid pools. We herein analysed whether SLC1A5 was involved in F. tularensis intracellular multiplication. We demonstrate that expression of SLC1A5 is specifically upregulated by F. tularensis in infected THP-1 human monocytes. Furthermore, we show that SLC1A5 downregulation decreases intracellular bacterial multiplication, supporting the involvement of SLC1A5 in F. tularensis infection. Notably, after entry of F. tularensis into cells and during the whole infection, the highly glycosylated form of SLC1A5 was deglycosylated only by bacteria capable of cytosolic multiplication. These data suggest that intracellular replication of F. tularensis depends on the function of host cell SLC1A5. Our results are the first, which show that Francisella intracellular multiplication in human monocyte cytoplasm is associated with a post-translational modification of a eukaryotic amino acid transporter.

We previously demonstrated that human C3d or pep16, a 16-amino acid synthetic peptide derived from human C3d, induced in vivo and in vitro tyrosine phosphorylation of pp105, an intracellular component found only in human cells that express CR2 at their surface. To determine the contribution of CR2 molecules to this enzymatic regulation, we first analyzed whether activation of CR2 by other extracellular CR2 ligands could trigger such regulation in cell extracts. Subsequently, we used cell extracts of either CR2-positive cells depleted in CR2 molecules by absorption with anti-CR2 antibodies or CR2-negative cells transfected with CR2 cDNA. We demonstrate here that pp105 phosphorylation was induced when CR2 was activated by C3d and pep16 as well as by gp350, the Epstein-Barr virus capsid protein or OKB7, an anti-CR2 monoclonal antibody (mAb). HB5, another anti-CR2 mAb, which did not activate B lymphocytes through CR2, did not induce pp105 phosphorylation. Thus, C3d, pep16, gp350, and OKB7 presented similar properties in activating CR2 to trigger pp105 phosphorylation and in regulating B lymphocyte proliferation, while HB-5 had no effect on either assays. Furthermore, our data demonstrate that the presence of CR2 activated by its extracellular ligands regulates pp105 phosphorylation through two distinct pathways: one which also requires the presence of non-activated CD19, and one which is independent of CD19. The involvement of CD19 in the first pathway was not due to the formation of putative CR2-CD19 complexes. Both pathways were TAPA-1 independent. This is the first demonstration that activated CR2 molecules can play a regulatory role in enzymatic function, such as phosphorylation, despite the absence of CD19 and TAPA-1.

gp140, previously identified as a 140-kDa C3b-binding membrane glycoprotein present on Raji cell surface, was shown to be the C3dg/C3d receptor of B lymphocytes (CR2). Specific polyclonal anti-gpl40, prepared by immunizing rabbits with this highly purified C3 receptor, blocked Raji cell rosettes with EC3b, EC3bi, EC3dg and EC3d, and also blocked normal lymphocyte rosettes with EC3dg and EC3d without affecting CR1 or CR3 activity. Moreover, a monoclonal anti-C3 (C3b/#130), described by others as reacting with the d region highly expressed on ECSbi, EC3dg and EC3d and poorly exposed on EC3b, completely inhibited EC3bi, EC3dg and EC3d rosettes with Raji cells, but had no effect on EC3b rosettes. Treatment of Raji cells with rabbit anti-gp140 blocked the uptake of three 125I-labeled monoclonal antibodies anti-B2, HB-5 and OKB7 reported to react with C3d-binding proteins, indicating that each of these monoclonal antibodies recognizes epitopes present on gp140. The neutrophil C3dg receptor was examined to determine its relationship to lymphocyte CR2. While neutrophil rosettes with EC3d were undetectable, a specificity for C3d was suggested by the inhibition of EC3dg rosettes by fluid phase C3d-complexes bearing no detectable C3dg. However, such neutrophil EC3dg and EC3bi rosettes were not inhibited by rabbit anti-gp140 nor an excess of anti-CRl, anti-CR2, and anti-CR3. In addition, neutrophils did not bind 125I-labeled anti-gp140, anti-B2, or HB-5. Thus, the neutrophil C3dg receptor is distinct from gp140, the lymphocyte CR2, and should be designated CR4.

gp140, the C3d/EBV receptor (CR2), previously isolated and characterized from human B lymphocytes, was identified on human platelets: (a) by measuring the specific binding of either polyclonal anti-gp140 IgG and monoclonal anti-C3d/EBVR antibodies, as OKB-7 and HB-5, or human C3d; (b) by isolating gp140 from solubilized platelet components with polyclonal anti-gp140 IgG or monoclonal OKB-7, using immunoprecipitation and electro-immunoblotting assays; (c) by inducing specific activation of human platelets. Cross-linking of this receptor by polyclonal anti-gp140 IgG induced aggregation of human platelets and stimulated ATP release. Absence of lactate dehydrogenase release and inhibition by EDTA and prostacyclin of anti-gp140-induced aggregation, support strongly active aggregation and absence of lysis. Platelet aggregation by anti-gp140 required metabolic activities and was modulated by fibrinogen, paf-acether or thrombin. OKB-7 triggered human platelet aggregation when cross-linked by anti-mouse second-step antibodies. In the same way, platelet activation by C3d fragment was detected, in presence of fibrinogen, only when C3d was cross-linked on the cell surface by anti-C3d F(ab′)2 fragments.

The C3d receptor is a specific marker of B lymphocytes. Recently we have shown that C3d receptor activity is carried by a gp 140 membrane antigen. A polyclonal antibody has been prepared by immunizing a rabbit with highly purified gp 140 molecule isolated from membranes of the human B lymphoblastoid cell line Raji and its high specificity was previously demonstrated. We tested the effect of this antibody to the C3d receptor on the B cell proliferative response. Purified B cells from human blood were induced to proliferate by a B cell growth factor (BCGF)-containing partially purified supernatant from activated T cells. The anti-C3d receptor F(ab')2 enhanced the BCGF-dependent B cell proliferation. This effect was dose dependent, was observed in the presence of different concentrations of BCGF and did not correspond to a change in the time course of the response. The anti-C3d receptor F(ab')2 had no mitogenic effect in the absence of T cell supernatant. In contrast the undigested anti-C3d receptor IgG suppressed the BCGF-dependent B cell proliferation. These results emphasize the potentialities of anti-gp 140 F(ab')2 to explore the involvement of the C3d receptor in the regulation of B cell response to T cell products.

gp140, the EB/C3d receptor (EBV/C3dR; CR2), is a membrane site involved in human B cell regulation. Cross-linking of this receptor on the cell surface by its specific ligands led to the enhancement of B cell proliferation in synergy with T cell factors. In vitro activation of human peripheral B lymphocytes by cross-linking membrane immunoglobulins with anti-mu antibody induced EBV/C3dR phosphorylation. These studies were pursued by analyzing cell-free phosphorylation of EBV/C3dR isolated from Raji cell fractions, and immobilized on OKB7, a monoclonal anti-EBV/C3dR antibody. Three EBV/C3dR-related antigens which could be cell-free phosphorylated were detected: gp140, the EBV/C3dR, p130 and p120. gp140, the mature form of EBV/C3dR, was isolated from plasma membrane and from purified nuclei. p130 was identified as an intracellular intermediate of EBV/C3dR glycosylation, localized in low-density microsomes. Phosphoamino acid analysis of EBV/C3dR allowed the detection of phosphotyrosine and phosphoserine residues. These data suggest that EBV/C3dR could carry an autophosphorylation activity and could be associated to serine kinases. Using polyclonal anti-p120 antibody and anti-120 kDa nuclear ribonucleoprotein monoclonal antibody (mAb), p120 was identified as a nuclear ribonucleoprotein antigenically not related to EBV/C3dR. Detection of p120 on EBV/C3dR, immobilized on OKB7, was due to interactions between both antigens, instead of anti-EBV/C3dR mAb cross-reactivity with p120. Cell-free phosphorylation of p120 was under the control of EBV/C3dR. However, it is not yet established whether other nuclear or membrane components were involved in the control of p120 cell-free phosphorylation by EBV/C3dR. From the data presented herein, we propose that phosphorylation of a 120-kDa nuclear ribonucleoprotein by EBV/C3dR-associated kinases could represent a crucial step in in vivo regulation of human B cell activation.

Gp140 is the Epstein-Barr virus receptor and the C3d receptor (EBVR/C3dR) of human B lymphocytes. Recently, we have shown that cross-linking of EBVR/C3dR on cell surface by polyclonal anti-gp140 induced B cell activation, in presence of T cell factors. Immunoregulatory abnormalities of EBV-induced B cell activation have been demonstrated in rheumatoid arthritis (RA) patients. These data prompted us to analyze the putative presence of anti-EBVR/C3dR autoantibodies in human sera. The IgG fractions from eleven RA and 10 normal sera were tested for their ability to: (a) bind to Raji cell surface; (b) inhibit the binding to cell surface of 3 anti-EBVR/C3dR monoclonal antibodies (mAb), which recognized different epitopes on gp140; (c) inhibit the binding of particle-bound C3d and (d) react with 1% Nonidet-P40-solubilized gp140 from Raji cell membranes, in immunoblotting assays. Three RA sera carry anti- EBVR/C3dR autoantibodies which react with gp140 expressed on Raji cell surface or its solubilized form. The purification of monomeric IgG fraction of selected RA sera ruled out involvement of immune complexes carrying C3 molecules, which could interfere in these assays. One of these 3 RA sera was able to inhibit the binding to cell surface of anti-EBVR/C3dR mAb and particle-bound C3d. However, the 2 other RA sera, found positive by immunoblotting, did not inhibit particle-bound C3d and presented differences in their inhibitory effect on anti-EBVR/C3dR mAb binding to Raji cell surface. These data allow us to demonstrate differences which exist in the properties of anti-EBVR/C3dR autoantibodies. These autoantibodies were not detected in all the normal and other RA sera. Anti-EBVR/C3dR autoantibodies could play a role “in vivo” in B lymphocyte activation of RA patients.

Our aim was to detect C3b receptors on human lymphoblastoid cells using a solid phase radioimmunoassay (RIA) specific for human C3b. RIA was performed by coupling rabbit antihuman C3b to acrylamide beads to make immunobeads. The specificity and sensitivity of binding of 125I-C3b to immunobeads allowed the detection of as little as 6 × 10––10M unlabeled human C3b. The cells were incubated with a C3b concentration (10––9M) giving 25 % inhibition in the RIA. The concentration of unbound C3b was then measured in the cell supernatants using RIA. Results showed that: (a) loss of C3b antigen in the cell supernatant was not due to degradation of C3b molecules, (b) C3b binding could be detected at 37 °C on the four B cell lines, but not on the two T cell lines or on the two non T-non B cell lines tested, (c) C3b bound on the B lymphoblastoid cells was not cleaved, neither into iC3b nor C3c and C3d fragments, supporting the presence of C3b receptors on the cells tested. This method allows screening of a variety of C3b receptor-positive cells.