Respiratory infections are a real threat for humans, and therefore the pig model is of interest for studies. As one of a case for studies, Actinobacillus pleuropneumoniae (APP) caused infections and still worries many pig breeders around the world. To better understand the influence of pathogenic effect of APP on a respiratory system-lungs and tracheobronchial lymph nodes (TBLN), we aimed to employ matrix-assisted laser desorption/ionization time-of-flight mass spectrometry imaging (MALDI-TOF MSI). In this study, six pigs were intranasally infected by APP and two were used as non-infected control, and 48 cryosections have been obtained. MALDI-TOF MSI and immunohistochemistry (IHC) were used to study spatial distribution of infectious markers, especially interleukins, in cryosections of porcine tissues of lungs (necrotic area, marginal zone) and tracheobronchial lymph nodes (TBLN) from pigs infected by APP. CD163, interleukin 1β (IL‑1β) and a protegrin-4 precursor were successfully detected based on their tryptic fragments. CD163 and IL‑1β were confirmed also by IHC. The protegrin-4 precursor was identified by MALDI-TOF/TOF directly on the tissue cryosections. CD163, IL‑1β and protegrin‑4 precursor were all significantly (p < 0.001) more expressed in necrotic areas of lungs infected by APP than in marginal zone, TBLN and in control lungs.
- MeSH
- Actinobacillus pleuropneumoniae patogenita MeSH
- antigeny diferenciační myelomonocytární metabolismus MeSH
- biologické markery metabolismus MeSH
- bronchy metabolismus MeSH
- CD antigeny metabolismus MeSH
- infekce bakteriemi rodu Actinobacillus metabolismus mikrobiologie MeSH
- infekce dýchací soustavy metabolismus mikrobiologie MeSH
- interleukin-1beta metabolismus MeSH
- kationické antimikrobiální peptidy metabolismus MeSH
- lymfatické uzliny metabolismus MeSH
- plíce metabolismus MeSH
- prasata MeSH
- receptory buněčného povrchu metabolismus MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice metody MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
The posttranslational Ca2+-dependent "clip-and-link" activity of large repeat-in-toxin (RTX) proteins starts by Ca2+-dependent structural rearrangement of a highly conserved self-processing module (SPM). Subsequently, an internal aspartate-proline (Asp-Pro) peptide bond at the N-terminal end of SPM breaks, and the liberated C-terminal aspartyl residue can react with a free ε-amino group of an adjacent lysine residue to form a new isopeptide bond. Here, we report a solution structure of the calcium-loaded SPM (Ca-SPM) derived from the FrpC protein of Neisseria meningitidis The Ca-SPM structure defines a unique protein architecture and provides structural insight into the autocatalytic cleavage of the Asp-Pro peptide bond through a "twisted-amide" activation. Furthermore, in-frame deletion of the SPM domain from the ApxIVA protein of Actinobacillus pleuropneumoniae attenuated the virulence of this porcine pathogen in a pig respiratory challenge model. We hypothesize that the Ca2+-dependent clip-and-link activity represents an unconventional strategy for Gram-negative pathogens to adhere to the host target cell surface.IMPORTANCE The Ca2+-dependent clip-and-link activity of large repeat-in-toxin (RTX) proteins is an exceptional posttranslational process in which an internal domain called a self-processing module (SPM) mediates Ca2+-dependent processing of a highly specific aspartate-proline (Asp-Pro) peptide bond and covalent linkage of the released aspartyl to an adjacent lysine residue through an isopeptide bond. Here, we report the solution structures of the Ca2+-loaded SPM (Ca-SPM) defining the mechanism of the autocatalytic cleavage of the Asp414-Pro415 peptide bond of the Neisseria meningitidis FrpC exoprotein. Moreover, deletion of the SPM domain in the ApxIVA protein, the FrpC homolog of Actinobacillus pleuropneumoniae, resulted in attenuation of virulence of the bacterium in a pig infection model, indicating that the Ca2+-dependent clip-and-link activity plays a role in the virulence of Gram-negative pathogens.
- MeSH
- Actinobacillus pleuropneumoniae chemie patogenita MeSH
- bakteriální proteiny chemie genetika MeSH
- bakteriální toxiny chemie MeSH
- infekce bakteriemi rodu Actinobacillus veterinární MeSH
- membránové proteiny chemie MeSH
- Neisseria meningitidis chemie MeSH
- posttranslační úpravy proteinů * MeSH
- prasata MeSH
- vápník metabolismus MeSH
- virulence MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Problems with serological cross-reactivity have led to development of a number of PCRs (individual and multiplex) for molecular typing of Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia. Most of these assays were developed for detection of specific amplicons within capsule biosynthetic genes before the availability of complete sequences for the different serovars. Here we describe comparative analysis of the complete capsular loci for all 18 serovars of A. pleuropneumoniae, and development of two multiplex PCRs for comprehensive capsule typing of this important pig pathogen.
- MeSH
- Actinobacillus pleuropneumoniae klasifikace genetika patogenita MeSH
- bakteriální polysacharidy genetika MeSH
- bakteriální pouzdra chemie klasifikace genetika MeSH
- infekce bakteriemi rodu Actinobacillus diagnóza mikrobiologie MeSH
- multiplexová polymerázová řetězová reakce metody MeSH
- nemoci prasat diagnóza mikrobiologie MeSH
- prasata MeSH
- sekvenční analýza * MeSH
- séroskupina MeSH
- sérotypizace MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
The aim of this study was to investigate isolates of Actinobacillus pleuropneumoniae previously designated serologically either as non-typable (NT) or as 'K2:07', which did not produce serovar-specific amplicons in PCR assays. We used whole genome sequencing to identify the capsule (CPS) loci of six previously designated biovar 1 NT and two biovar 1 'K2:O7' isolates of A. pleuropneumoniae from Denmark, as well as a recent biovar 2 NT isolate from Canada. All of the NT isolates have the same six-gene type I CPS locus, sharing common cpsABC genes with serovars 2, 3, 6, 7, 8, 9, 11 and 13. The two 'K2:O7' isolates contain a unique three-gene type II CPS locus, having a cpsA gene similar to that of serovars 1, 4, 12, 14 and 15. The previously NT isolates share the same O-antigen genes, found between erpA and rpsU, as serovars 3, 6, 8, and 15. Whereas the 'K2:O7' isolates, have the same O-antigen genes as serovar 7, which likely contributed to their previous mis-identification. All of the NT and 'K2:O7' isolates have only the genes required for production of ApxII (apxIICA structural genes, and apxIBD export genes). Rabbit polyclonal antisera raised against representative isolates with these new CPS loci demonstrated distinct reactivity compared to the 16 known serovars. The serological and genomic results indicate that the isolates constitute new serovars 17 (previously NT) and 18 (previously 'K2:O7'). Primers designed for amplification of specific serovar 17 and 18 sequences for molecular diagnostics will facilitate epidemiological tracking of these two new serovars of A. pleuropneumoniae.
- MeSH
- Actinobacillus pleuropneumoniae klasifikace genetika imunologie izolace a purifikace MeSH
- bakteriální pouzdra genetika MeSH
- DNA bakterií genetika MeSH
- DNA primery genetika MeSH
- genotyp * MeSH
- infekce bakteriemi rodu Actinobacillus epidemiologie veterinární MeSH
- nemoci prasat epidemiologie mikrobiologie MeSH
- polymerázová řetězová reakce metody MeSH
- prasata MeSH
- sekvenování celého genomu MeSH
- séroskupina * MeSH
- sérotypizace MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Dánsko epidemiologie MeSH
- Kanada epidemiologie MeSH
Actinobacillus pleuropneumoniae causes pleuropneumonia, an economically significant lung disease of pigs. Recently, isolates of A. pleuropneumoniae that were serologically distinct from the previously characterized 15 serovars were described, and a proposal was put forward that they comprised a new serovar, serovar 16. Here we used whole-genome sequencing of the proposed serovar 16 reference strain A-85/14 to confirm the presence of a unique capsular polysaccharide biosynthetic locus. For molecular diagnostics, primers were designed from the capsule locus of strain A-85/14, and a PCR was formulated that differentiated serovar 16 isolates from all 15 known serovars and other common respiratory pathogenic/commensal bacteria of pigs. Analysis of the capsule locus of strain A-85/14 combined with the previous serological data show the existence of a sixteenth serovar-designated serovar 16-of A. pleuropneumoniae.
- MeSH
- Actinobacillus pleuropneumoniae klasifikace genetika MeSH
- bakteriální pouzdra genetika MeSH
- diagnostické techniky molekulární metody MeSH
- DNA bakterií chemie genetika MeSH
- DNA primery genetika MeSH
- genetické lokusy * MeSH
- genom bakteriální MeSH
- infekce bakteriemi rodu Actinobacillus diagnóza veterinární MeSH
- nemoci prasat diagnóza MeSH
- pleuropneumonie mikrobiologie veterinární MeSH
- polymerázová řetězová reakce metody MeSH
- prasata MeSH
- sekvenční analýza DNA MeSH
- séroskupina * MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Susceptibility in vitro and trends in resistance to antimicrobials were determined by a dilution micromethod in a group of Actinobacillus pleuropneumoniae, Pasteurella multocida, Mannheimia haemolytica and Escherichia coli isolates from clinical cases of cattle and swine diseases in the Czech Republic from 2007 to 2011. A high susceptibility of pig and cattle respiratory pathogens to antimicrobials was found, with the exception of the moderate prevalence of M. haemolytica resistance to ampicillin. In contrast to respiratory pathogens, low susceptibility of E. coli of pig and cattle isolates to ampicillin and amoxicillin/clavulanic acid was noted. Regarding resistance trends, an increase in levels of resistance among E. coli isolates to ampicillin and amoxicillin/clavulanic acid was identified, but the resistance of respiratory isolates was low, with the exception of M. haemolytica. For the period of 2007-2011, there was a significant and almost continuous increase in sales (compared with population correction unit) of ceftiofur, cefquinome and other beta lactams for pigs. Consumption peaked in 2010. In the case of amoxicillin in combination with clavulanic acid, data showed a significant decrease in sales from 2007 to 2008, followed by a period of fluctuation. In cattle, within the groups of 3rd and 4th generation cephalosporins and for the whole group of other betalactams for the period of 2007-2011, there was a significant and almost continuous increase in sales (compared with population correction unit). Consumption peaked in 2010. In the case of ceftiofur, there was a huge increase noted from 2010. In the case of amoxicillin in combination with betalactamase inhibitor (clavulanic acid) data shows a significant decrease from 2007 to 2008, followed by a period of fluctuation in sales.
- MeSH
- Actinobacillus pleuropneumoniae účinky léků MeSH
- bakteriální léková rezistence genetika MeSH
- beta-laktamy metabolismus MeSH
- Escherichia coli účinky léků MeSH
- infekce bakteriemi čeledi Pasteurellaceae epidemiologie veterinární MeSH
- infekce vyvolané Escherichia coli epidemiologie veterinární MeSH
- inhibitory beta-laktamasy farmakologie MeSH
- Mannheimia haemolytica účinky léků MeSH
- mikrobiální testy citlivosti veterinární MeSH
- nemoci prasat epidemiologie mikrobiologie MeSH
- nemoci skotu epidemiologie mikrobiologie MeSH
- Pasteurella multocida účinky léků MeSH
- prasata MeSH
- prevalence MeSH
- skot MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika MeSH
Monocytes play an essential role in the defense against bacterial pathogens. Bone marrow (BM) and peripheral blood (PB) monocytes in pigs consist of the main "steady-state" subpopulations: CD14 hi/CD163-/SLA-DR- and CD14 low/CD163+/SLA-DR+. During inflammation, the subpopulation of "inflammatory" monocytes expressing very high levels of CD163, but lacking the SLA-DR molecule (being CD14 low/CD163+/SLA-DR-) appears in the BM and PB and replaces the CD14 low/CD163+/SLA-DR+ subpopulation. However, current knowledge of monocyte migration into inflamed tissues in pigs is limited. The aim of the present study was to evaluate the distribution of "inflammatory" CD14 low/CD163+/SLA-DR- monocytes during experimental inflammation induced by Actinobacillus pleuropneumoniae (APP) and a possible role for chemokines in attracting "inflammatory" CD14 low/CD163+/SLA-DR- monocytes into the tissues. Monocyte subpopulations were detected by flow cytometry. Chemokines and chemokine receptors were detected by RT-qPCR. The "steady-state" monocytes were found in the BM, PB, spleen and lungs of control pigs. After APP-infection, "inflammatory" monocytes replaced the "steady-state" subpopulation in BM, PB, spleen and moreover, they appeared in an unaffected area, demarcation zone and necrotic area of the lungs and in tracheobronchial lymph nodes. They did not appear in mesenteric lymph nodes. Levels of mRNA for various chemokines with their appropriate receptors were found to be elevated in BM (CCL3-CCR1/CCR5, CCL8-CCR2/CCR5, CCL19-CCR7), necrotic area of the lungs (CCL3-CCR1, CCL5-CCR1/CCR3, CCL11-CCR3, CCL22/CCR4) and tracheobronchial lymph nodes (CCL3-CCR1) and therefore they could play a role in attracting monocytes into inflamed tissues. In conclusion, "inflammatory" monocytes appear in different lymphoid tissues and the lungs after APP infection in pigs. Various chemokines could drive this process.
- MeSH
- Actinobacillus pleuropneumoniae fyziologie MeSH
- antigeny diferenciační myelomonocytární metabolismus MeSH
- CD antigeny metabolismus MeSH
- chemokiny genetika metabolismus MeSH
- infekce bakteriemi rodu Actinobacillus imunologie mikrobiologie veterinární MeSH
- lymfoidní tkáň metabolismus MeSH
- messenger RNA genetika MeSH
- monocyty cytologie metabolismus MeSH
- nemoci prasat imunologie mikrobiologie MeSH
- plíce metabolismus MeSH
- prasata MeSH
- průtoková cytometrie veterinární MeSH
- receptory buněčného povrchu metabolismus MeSH
- receptory chemokinů genetika metabolismus MeSH
- zánět mikrobiologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
A large subgroup of the repeat in toxin (RTX) family of leukotoxins of Gram-negative pathogens consists of pore-forming hemolysins. These can permeabilize mammalian erythrocytes (RBCs) and provoke their colloid osmotic lysis (hemolytic activity). Recently, ATP leakage through pannexin channels and P2X receptor-mediated opening of cellular calcium and potassium channels were implicated in cell permeabilization by pore-forming toxins. In the study described here, we examined the role played by purinergic signaling in the cytolytic action of two RTX toxins that form pores of different sizes. The cytolytic potency of ApxIA hemolysin of Actinobacillus pleuropneumoniae, which forms pores about 2.4 nm wide, was clearly reduced in the presence of P2X7 receptor antagonists or an ATP scavenger, such as pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), Brilliant Blue G, ATP oxidized sodium salt, or hexokinase. In contrast, antagonists of purinergic signaling had no impact on the hemolytic potency of the adenylate cyclase toxin-hemolysin (CyaA) of Bordetella pertussis, which forms pores of 0.6 to 0.8 nm in diameter. Moreover, the conductance of pores formed by ApxIA increased with the toxin concentration, while the conductance of the CyaA single pore units was constant at various toxin concentrations. However, the P2X7 receptor antagonist PPADS inhibited in a concentration-dependent manner the exacerbated hemolytic activity of a CyaA-ΔN489 construct (lacking 489 N-terminal residues of CyaA), which exhibited a strongly enhanced pore-forming propensity (>20-fold) and also formed severalfold larger conductance units in planar lipid bilayers than intact CyaA. These results point to a pore size threshold of purinergic amplification involvement in cell permeabilization by pore-forming RTX toxins.
- MeSH
- Actinobacillus pleuropneumoniae metabolismus MeSH
- adenylátcyklasový toxin antagonisté a inhibitory chemie metabolismus MeSH
- bakteriální proteiny antagonisté a inhibitory chemie metabolismus MeSH
- Bordetella pertussis metabolismus MeSH
- buněčná membrána metabolismus MeSH
- erytrocyty metabolismus MeSH
- hemolýza * MeSH
- hemolyziny antagonisté a inhibitory chemie metabolismus MeSH
- hexokinasa MeSH
- kultivované buňky MeSH
- lipidové dvojvrstvy metabolismus MeSH
- makrofágy MeSH
- myši MeSH
- osmotický tlak MeSH
- permeabilita buněčné membrány MeSH
- pyridoxalfosfát analogy a deriváty MeSH
- rosanilinová barviva MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Porcine pleuropneumonia caused by Actinobacillus pleuropneumoniae accounts for serious economic losses in the pig farming industry worldwide. We examined here the immunogenicity and protective efficacy of the recombinant type IV fimbrial subunit protein ApfA as a single antigen vaccine against pleuropneumonia, or as a component of a multi-antigen preparation comprising five other recombinant antigens derived from key virulence factors of A. pleuropneumoniae (ApxIA, ApxIIA, ApxIIIA, ApxIVA and TbpB). Immunization of pigs with recombinant ApfA alone induced high levels of specific serum antibodies and provided partial protection against challenge with the heterologous A. pleuropneumoniae serotype 9 strain. This protection was higher than that engendered by vaccination with rApxIVA or rTbpB alone and similar to that observed after immunization with the tri-antigen combination of rApxIA, rApxIIA and rApxIIIA. In addition, rApfA improved the vaccination potential of the penta-antigen mixture of rApxIA, rApxIIA, rApxIIIA, rApxIVA and rTbpB proteins, where the hexa-antigen vaccine containing rApfA conferred a high level of protection on pigs against the disease. Moreover, when rApfA was used for vaccination alone or in combination with other antigens, such immunization reduced the number of pigs colonized with the challenge strain. These results indicate that ApfA could be a valuable component of an efficient subunit vaccine for the prevention of porcine pleuropneumonia.
- MeSH
- Actinobacillus pleuropneumoniae genetika imunologie patogenita MeSH
- bakteriální adheziny genetika imunologie MeSH
- bakteriální fimbrie genetika imunologie MeSH
- bakteriální vakcíny genetika imunologie MeSH
- Escherichia coli genetika MeSH
- exotoxiny genetika metabolismus MeSH
- faktory virulence genetika imunologie MeSH
- infekce bakteriemi rodu Actinobacillus imunologie prevence a kontrola veterinární MeSH
- molekulární sekvence - údaje MeSH
- nemoci prasat imunologie prevence a kontrola MeSH
- pleuropneumonie imunologie prevence a kontrola veterinární MeSH
- polymerázová řetězová reakce veterinární MeSH
- prasata MeSH
- rekombinantní proteiny genetika imunologie MeSH
- sekvenční analýza DNA veterinární MeSH
- vakcinace veterinární MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- randomizované kontrolované studie MeSH
Limited data regarding the susceptibility of Actinobacillus pleuropneumoniae to antimicrobials has been published during recent years. Accordingly, the aim of the present study was to investigate the distribution of MICs for the isolates of A. pleuropneumoniae from diseased pigs in the Czech Republic between 2007 and 2009. A total of 242 isolates were tested for susceptibility to 16 antimicrobial agents by a broth microdilution method. A low degree of resistance was observed for florfenicol (0.8%), amoxicillin and clavulanic acid (0.8%), tilmicosin (1.2%), tiamulin (1.7%) and ampicillin (3.3%), whereas resistance to tetracycline was detected more frequently, 23.9% of isolates. Interestingly, resistance to florfenicol has not yet been reported in any study investigating antimicrobial resistance of A. pleuropneumoniae. By PCR the presence of the floR gene was confirmed in all florfenicol resistant isolates.
- MeSH
- Actinobacillus pleuropneumoniae účinky léků izolace a purifikace MeSH
- amoxicilin farmakologie MeSH
- ampicilin farmakologie MeSH
- antibakteriální látky farmakologie MeSH
- bakteriální geny MeSH
- diterpeny farmakologie MeSH
- kyselina klavulanová farmakologie MeSH
- mikrobiální testy citlivosti MeSH
- nemoci prasat epidemiologie mikrobiologie MeSH
- prasata mikrobiologie MeSH
- tetracyklin farmakologie MeSH
- thiamfenikol analogy a deriváty farmakologie MeSH
- tylosin analogy a deriváty farmakologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika MeSH
