Molecular dissection of the leukotoxin-beta2-integrin LFA-1 (CD11a/CD18) interaction, responsible for the Mannheimia haemolytica species-specificity towards ruminants

Les pneumonies bactériennes constituent un problème majeur dans l'élevage et l'engraissement des bovins, avec des répercussions très élevées en termes de morbidité et de mortalité. Dans ce contexte, les auteurs sont unanimes pour considérer que l'agent pathogène compliquant principal, voire systématique, est Mannheimia haemolytica, une bactérie qui n'affecte que les ruminants. Parmi les différents facteurs de virulence incriminables, la culpabilité de la leucotoxine s'impose, notamment parce que, ex vivo, elle n'est capable d'induire la nécrose des leucocytes qu'à la condition sine qua non qu'ils soient d'origine bovine, ovine ou caprine. Au niveau moléculaire, cette spécificité est due à une interaction entre la leucotoxine et la beta2-intégrine LFA-1, un récepteur constitué des sous-unités CD11a et CD18.Les objectifs de notre projet étaient (i) d'identifier les différences systématiques qui distinguent les sous-unités CD11a et CD18 des ruminants de celles des autres espèces, (ii) de produire une leucotoxine pure et active, (iii) de cibler la sous-unité du récepteur responsable de la spécificité d’espèce qu’exhibe la leucotoxine de M. haemolytica envers les leucocytes des ruminants et (iv) plus particulièrement, d’identifier le motif moléculaire précis impliqué.Nous avons d’abord cloné, séquencé et caractérisé les CD11a bovin, ovin et caprin, de même que les CD18 ovin et caprin, ce qui nous a permis de les contraster entre eux et avec certains de leurs homologues non ruminants, pour finalement mettre en évidence respectivement 58 et 17 sites de mutation potentiellement responsables de la virulence spécifique de M. haemolytica envers les ruminants. Nous avons ensuite mis au point un protocole de purification de la leucotoxine et vérifié la spécificité de son activité cytolytique in vitro via la réalisation de tests de cytotoxicité et de viabilité cellulaire sur diverses lignées réputées sensibles ou insensibles. Enfin, il est apparu au cours du travail que la sous-unité portant la spécificité de l’effet cytotoxique était le CD18. Nous avons alors évalué d’une part les 15 sites présents dans la région extracellulaire en tentant d’inhiber l’effet de la leucotoxine sur la lignée lymphoblastique bovine BL-3 par l’ajout de peptides dérivés de cette séquence et d’autre part les sites des portions transmembranaire et cytoplasmique en transfectant de manière transitoire la lignée lymphoblastique d’origine humaine K-562, qui n’exprime naturellement aucune beta2-intégrine, avec les récepteurs LFA-1 mutants correspondants. En conclusion, nous avons développé tous les outils nécessaires à l’identification précise du motif moléculaire du CD18 responsable de la spécificité d’espèce de M. haemolytica en excluant l’implication des sites des régions transmembranaire et cytoplasmique.Les perspectives à court terme sont (i) d’évaluer par transfection transitoire les 15 sites répertoriés dans la région extracellulaire du CD18, (ii) de tester des LFA-1 bovins chimériques dont certains domaines spécifiques du CD18 auront été remplacés par leur correspondant humain et (iii) de vérifier le maintien de la capacité de margination et de diapédèse du ou des mutants/chimères sélectionnés. A plus long terme, nous espérons (i) disséminer dans la population bovine des animaux intrinsèquement résistants aux pneumonies bactériennes à M. haemolytica par introgression (si nous parvenons à mettre en évidence un allèle correspondant au(x) mutant(s) sélectionné(s)), ou par transgenèse et/ou (ii) élaborer un jeu de peptides compétiteurs du LFA-1 pour la fixation à la leucotoxine, c’est-à-dire qui seront inhibiteurs de son activité leucotoxique in vitro et de la virulence de M. haemolytica in vivo, le but étant de proposer un peptide thérapeutique injectable à substituer aux traitements antibiotiques ne générant ni résidus ni antibiorésistance et fonctionnant à la manière d’un leurre pour la leucotoxine./Bacterial pneumonias represent the major problem in cattle breeding and fattening, with high morbidity and mortality levels. In this context, Mannheimia haemolytica plays a great role as a complicating agent in ruminant pleuropneumonias. Besides the several virulence factors incriminated, the leukotoxin is held responsible in restricting the disease to ruminants because it does not induce cytolysis of leukocytes from other species ex vivo. At the molecular level, this specificity is due to the tight interaction between leukotoxin and the beta2-integrin LFA-1, a receptor made of CD11a and CD18 subunits.The aims of this work were (i) to identify the systematic differences that distinguish ruminant CD11a and CD18 from their non-ruminant homologues, (ii) to produce an active and purified leukotoxin, (iii) to target the subunit displaying the ruminant-specificity of Mannheimia haemolytica leukotoxin and (iv) to especially identify the concerned molecular motif.First, we have cloned, sequenced and characterized bovine, ovine and caprine CD11a, as well as ovine and caprine CD18, providing the opportunity to contrast ruminant versus non-ruminant LFA-1. This analysis has finally led to the identification of respectively 58 and 17 potential mutation sites that could be the cause of the species-specific virulence of M. haemolytica. Afterwards, we have developed a purification protocol for leukotoxin and checked its specific cytotoxicity in vitro by cellular cytotoxicity and viability tests on several cell lines supposed to be sensistive or insensitive. At last, it appeared during the project that CD18 was necessary and sufficient to mediate the specificity of leukotoxin cytotoxicity. We have then evaluated (i) the extracellular region 15 sites by tempting to inhibit leukotoxin effect on bovine lymphoblastic BL-3 cell line with the addition of peptides mimicking this sequence and (ii) the transmembranous and cytoplasmic sites by transiently transfecting human lymphoblastic K-562 cell line, that naturally express no beta2-integrin, with corresponding mutated LFA-1 receptors.To conclude, we have developed all the tools required to precisely identify the CD18 molecular motif responsible for M. haemolytica species-specificity and we have excluded the implication of transmembranous and cytoplasmic regions.At short term, perspectives are (i) to evaluate by transient transfection the 15 CD18 extracellular region sites, (ii) to test chimeric bovine LFA-1 of which some specific domains would be substituted for by their human homologue and (iii) to check selected mutants or chimeras ability to perform rolling and diapedesis. At longer term, we hope (i) to disseminate in bovine population animals that would become naturally resistant to M. haemolytica pneumonias by introgression (if we could find an allele corresponding to selected mutant(s)) or by transgenesis and/or to elaborate a panel of LFA-1 competing peptides for leukotoxin binding, i.e. that inhibit in vitro leukotoxic activity and in vivo M. haemolytica virulence, with the aim to propose an injectable therapeutic peptide that could be substituted to antibiotics, generating neither residues nor antibioresistance and working as a decoy for leukotoxin

Probing of Actinobacillus pleuropneumoniae ApxIIIA toxin-dependent cytotoxicity towards mammalian peripheral blood mononucleated cells

<p>Abstract</p> <p>Background</p> <p><it>Actinobacillus pleuropneumoniae</it>, the causative bacterial agent of porcine pleuropneumonia, produces Apx toxins which belong to RTX toxin family and are recognized as the major virulence factors. So far, their target receptor(s) has not been identified and the disease cytopathogenesis remains poorly understood. Production of an active Apx toxin and characterization of its toxic activity constitute the premises necessary to the description of its interaction with a potential receptor. From this point of view, we produced an active recombinant ApxIIIA toxin in order to characterize its toxicity on peripheral blood mononucleated cells (PBMCs) isolated from several species.</p> <p>Findings</p> <p>Toxin preparation exercises a strong cytotoxic action on porcine PBMCs which is directly related to recombinant ApxIIIA since preincubation with polymyxin B does not modify the cytotoxicity rate while preincubation with a monospecific polyclonal antiserum directed against ApxIIIA does. The cell death process triggered by ApxIIIA is extremely fast, the maximum rate of toxicity being already reached after 20 minutes of incubation. Moreover, ApxIIIA cytotoxicity is species-specific because llama, human, dog, rat and mouse PBMCs are resistant. Interestingly, bovine and caprine PBMCs are slightly sensitive to ApxIIIA toxin too. Finally, ApxIIIA cytotoxicity is cell type-specific as porcine epithelial cells are resistant.</p> <p>Conclusion</p> <p>We have produced an active recombinant ApxIIIA toxin and characterized its specific cytotoxicity on porcine PBMCs which will allow us to get new insights on porcine pleuropneumonia pathogenesis in the future.</p

The wild boar (Sus scrofa) Lymphocyte function-associated antigen-1 (CD11a/CD18) receptor: cDNA sequencing, structure analysis and comparison with homologues

BACKGROUND: The most predominant beta2-integrin lymphocyte function-associated antigen-1 (LFA-1, CD11a/CD18, alphaLbeta2), expressed on all leukocytes, is essential for many adhesive functions of the immune system. Interestingly, RTX toxin-producing bacteria specifically target this leukocyte beta2-integrin which exacerbates lesions and disease development. RESULTS: This study reports the sequencing of the wild boar beta2-integrin CD11a and CD18 cDNAs. Predicted CD11a and CD18 subunits share all the main structural characteristics of their mammalian homologues, with a larger interspecies conservation for the CD18 than the CD11a. Besides these strong overall similarities, wild boar and domestic pig LFA-1 differ by 2 (CD18) and 1 or 3 (CD11a) substitutions, of which one is located in the crucial I-domain (CD11a, E168D). CONCLUSION: As most wild boars are seropositive to the RTX toxin-producing bacterium Actinobacillus pleuropneumoniae and because they have sustained continuous natural selection, future studies addressing the functional impact of these polymorphisms could bring interesting new information on the physiopathology of Actinobacillus pleuropneumoniae-associated pneumonia in domestic pigs

Porcine CD18 mediates Actinobacillus pleuropneumoniae ApxIII species-specific toxicity

Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, produces Apx toxins that are recognized as major virulence factors. Recently, we showed that ApxIIIA-cytotoxic activity specifically targets Sus scrofa leukocytes. Since both LtxA from Aggregatibacter actinomycetemcomitans (aggressive periodontitis in humans) and LktA from Mannheimia haemolytica (pneumonia in ruminants) share this characteristic, respectively towards human and ruminant leukocytes, and because both use the CD18 subunit to interact with their respective LFA-1, we hypothesized that ApxIIIA was likely to bind porcine CD18 to exercise its deleterious effects on pig leukocytes. A β 2−integrin-deficient ApxIIIA-resistant human erythroleukemic cell line was transfected either with homologous or heterologous CD11a/CD18 heterodimers using a set of plasmids coding for human (ApxIIIA-resistant), bovine (-resistant) and porcine (-susceptible) CD11a and CD18 subunits. Cell preparations that switched from ApxIIIA-resistance to -susceptibility were then sought to identify the LFA-1 subunit involved. The results showed that the ApxIIIA-resistant recipient cell line was rendered susceptible only if the CD18 partner within the LFA-1 heterodimer was that of the pig. It is concluded that porcine CD18 is necessary to mediate A. pleuropneumoniae ApxIIIA toxin-induced leukolysis

Molecular characterisation of the caprine (Capra hircus) lymphocyte function-associated antigen-1 alpha subunit-encoding cDNA

BACKGROUND: Lymphocyte function-associated antigen-1 (LFA-1, CD11a/CD18, alpha L beta 2) is required for many cellular adhesive interactions during the immune response. RESULTS: The Capra hircus CD11a-encoding cDNA was sequenced and compared with its human, murine, rat, bovine and ovine counterparts. Despite some focal differences, it shares all the main characteristics of its known mammalian homologues. CONCLUSION: Therefore, along with the caprine CD18-encoding cDNA, which has been available for a few months, the sequence data revealed here will allow the Capra hircus LFA-1 expression in vitro as a tool to explore the specificities of inflammation in the caprine species

Stability domains, substrate-induced conformational changes, and hinge-bending motions in a psychrophilic phosphoglycerate kinase: A microcalorimetric study

The cold-active phosphoglycerate kinase from the Antarctic bacterium Pseudomonas sp. TACII18 exhibits two distinct stability domains in the free, open conformation. It is shown that these stability domains do not match the structural N- and C-domains as the heat-stable domain corresponds to about 80 residues of the C-domain, including the nucleotide binding site, whereas the remaining of the protein contributes to the main heat-labile domain. This was demonstrated by spectroscopic and microcalorimetric analyses of the native enzyme, of its mutants, and of the isolated recombinant structural domains. It is proposed that the heat-stable domain provides a compact structure improving the binding affinity of the nucleotide, therefore increasing the catalytic efficiency at low temperatures. Upon substrate binding, the enzyme adopts a uniformly more stable closed conformation. Substrate-induced stability changes suggest that the free energy of ligand binding is converted into an increased conformational stability used to drive the hinge-bending motions and domain closure

The CD11a partner in Sus scrofa lymphocyte function-associated antigen-1 (LFA-1): mRNA cloning, structure analysis and comparison with mammalian homologues

BACKGROUND: Lymphocyte function-associated antigen-1 (LFA-1, CD11a/CD18, alphaLbeta2), the most abundant and widely expressed beta2-integrin, is required for many cellular adhesive interactions during the immune response. Many studies have shown that LFA-1 is centrally involved in the pathogenesis of several diseases caused by Repeats-in-toxin (RTX) -producing bacteria. RESULTS: The porcine-LFA-1 CD11a (alpha) subunit coding sequence was cloned, sequenced and compared with the available mammalian homologues in this study. Despite some focal differences, it shares all the main characteristics of these latter. Interestingly, as in sheep and humans, an allelic variant with a triplet insertion resulting in an additional Gln-744 was consistently identified, which suggests an allelic polymorphism that might be biologically relevant. CONCLUSION: Together with the pig CD18-encoding cDNA, which has been available for a long time, the sequence data provided here will allow the successful expression of porcine CD11a, thus giving the first opportunity to express the Sus scrofa beta2-integrin LFA-1 in vitro as a tool to examine the specificities of inflammation in the porcine species

A three-way comparative genomic analysis of Mannheimia haemolytica isolates

<p>Abstract</p> <p>Background</p> <p><it>Mannhemia haemolytica </it>is a Gram-negative bacterium and the principal etiological agent associated with bovine respiratory disease complex. They transform from a benign commensal to a deadly pathogen, during stress such as viral infection and transportation to feedlots and cause acute pleuropneumonia commonly known as shipping fever. The U.S beef industry alone loses more than one billion dollars annually due to shipping fever. Despite its enormous economic importance there are no specific and accurate genetic markers, which will aid in understanding the pathogenesis and epidemiology of <it>M. haemolytica </it>at molecular level and assist in devising an effective control strategy.</p> <p>Description</p> <p>During our comparative genomic sequence analysis of three <it>Mannheimia haemolytica </it>isolates, we identified a number of genes that are unique to each strain. These genes are "high value targets" for future studies that attempt to correlate the variable gene pool with phenotype. We also identified a number of high confidence single nucleotide polymorphisms (hcSNPs) spread throughout the genome and focused on non-synonymous SNPs in known virulence genes. These SNPs will be used to design new hcSNP arrays to study variation across strains, and will potentially aid in understanding gene regulation and the mode of action of various virulence factors.</p> <p>Conclusions</p> <p>During our analysis we identified previously unknown possible type III secretion effector proteins, clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated sequences (Cas). The presence of CRISPR regions is indicative of likely co-evolution with an associated phage. If proven functional, the presence of a type III secretion system in <it>M. haemolytica </it>will help us re-evaluate our approach to study host-pathogen interactions. We also identified various adhesins containing immuno-dominant domains, which may interfere with host-innate immunity and which could potentially serve as effective vaccine candidates.</p