Gallibacterium anatis-secreted metalloproteases degrade chicken IgG

Abstract

Gallibacterium anatis (previously named Pasteurella haemolytica-like) is considered a normal inhabitant of genital and upper respiratory tracts of healthy chickens, but it is also associated with different pathological conditions. Secreted metalloproteases from field and reference G. anatis cultures were obtained by methanol precipitation and were characterized. Proteins of molecular mass higher than 100 kDa showing proteolytic activity were observed in 10% polyacrylamide gels copolymerized with 1% bovine casein. They were active at alkaline pH, and inhibited by ethylenediamine tetraacetic acid. Their activity was stable at 50°C, but partially inhibited at 60°C, and totally inhibited at higher temperatures. Secreted proteins were able to degrade chicken IgG after 24 h of incubation, and cross-reacted with a polyclonal antibody against purified protease from Actinobacillus pleuropneumoniae. Secreted metalloproteases could play a role in infections caused by G. anatis.

Les métalloprotéases secrétées par Gallibacterium anatis dégradent les IgG de poulet

Gallibacterium anatis (auparavant nommé Pasteurella haemolytica-like) est considéré comme un habitant normal du tractus génital et du tractus respiratoire supérieur des poulets sains, mais ce germe est aussi associé à différentes conditions pathologiques. Les métalloprotéases secrétées par les cultures de G. anatis, réalisées à partir de souches de référence ou de terrain, ont été obtenues par précipitation au méthanol puis ont été caractérisées. Des protéines de masse supérieure à 100 kDa montrant une activité protéolytique ont été observées dans 10% des gels de polyacrilamide copolymérisés avec 1% de caséine bovine. Elles étaient actives à un pH alcalin et inhibées par l'EDTA. Leur activité était stable à 50°C, mais partiellement inhibée à 60°C, et totalement inhibée à des températures supérieures. Les protéines secrétées sont capables de dégrader les IgG de poulet après 24 h d'incubation et réagissent avec un anticorps polyclonal anti protéase purifiée d' Actinobacillus pleuropneumoniae. Les metalloprotéases pourraient jouer un rôle dans les infections causées par G. anatis.

Von Gallibacterium anatis sezernierte Metalloproteasen bauen Hühner-IgG ab.

Gallibacterium anatis (früher als Pasteurella haemolytica-ähnlich bezeichnet) wird als normaler Bewohner des Genital-und Respirationstrakt von gesunden Hühnern angesehen, ist aber auch mit verschiedenen pathologischen Veränderungen assoziiert. Sezernierte Metalloproteasen von G.anatis-Kulturen von Feld- und Referenzstämmen wurden durch Methanolfällung gewonnen und charakterisiert. Proteine mit einem Molekulargewicht von über 100 kDa wiesen proteolytische Aktivität in 10%igen Polyacrylamidgelen auf, die mit 1% Rinderkasein ko-polymerisiert waren. Sie waren aktiv im basischen pH-Bereich und wurden durch EDTA gehemmt. Ihre Aktivität war bei 50°C stabil, wurde aber partiell inhibiert bei 60°C und vollkommen inaktiviert bei noch höheren Temperaturen. Die sezernierten Proteine waren in der Lage, 24 Stunden nach der Inkubation Hühner-IgG abzubauen und zeigten eine Kreuzreaktion mit einem polyklonalen Antikörper gegen gereinigte Protease von Actinobazillus pleuropneumoniae. Sezernierte Metalloproteasen könnten bei durch G. anatis verursachten Infektionen eine Rolle spielen.

Las metaloproteasas secretadas por Gallibacterium anatis degradan las IgG de pollo

Gallibacterium anatis (previamente llamado Pasteurella haemolytica-like) se considera un habitante normal del tracto genital y respiratorio superior de pollos sanos, pero también se asocia con diferentes condiciones patológicas. Se obtuvieron y caracterizaron diferentes metaloproteasas del cultivo de cepas de campo y de referencia de G. anatis mediante la precipitación con metanol. Se detectaron proteínas con un peso molecular de más de 100 kDa que mostraban actividad proteolítica en geles de poliacrilamida al 10 % copolimerizados con caseína bovina al 1%. Eran activas a pH alcalino y se inhibían con EDTA. Su actividad era estable a 50 °C, pero se inhibía parcialmente a 60 °C, y totalmente a temperaturas más elevadas. Las proteínas secretadas eran capaces de degradar IgG de pollo tras 24 h de incubación, y presentaron reactividad cruzada con un anticuerpo policlonal contra proteasa purificada de Actinobacillus pleuropneumoniae. Las metaloproteasas secretadas podrían jugar algún papel en la infección causada por G. anatis.

Introduction

Bacteria formerly classified as Actinobacillus salpingitidis, avian Pasteurella haemolytica and Pasteurella anatis presently belong to genus Gallibacterium, a recently established new member in the Pasteurellaceae family (Christensen et al., 2003). These bacteria form part of the normal upper-respiratory and lower genital tracts of different domestic and non-domestic healthy birds (Mushin et al., 1980; Bojesen et al., 2003), with chickens being the preferred host (Bisgaard, 1977) They are also associated with pathological lesions in laying hens, including salpingitis, septicaemia, hepatitis, respiratory tract lesions (Bojesen et al., 2004), increased mortality and abnormality in egg production, and increased mortality in pullets (Hacking & Pettit, 1974; Rhoades & Rimler, 1984; Shaw et al., 1990).

The importance of Gallibacterium spp. as a pathogen is not clear. However, different reports indicate that these species are potential pathogens because they have been isolated in pure culture from a range of pathological lesions in poultry, including septicaemia and respiratory tract lesions, among others (Addo & Mohan, 1985; Shaw et al., 1990).

As a mucosal pathogen, Gallibacterium must have different virulence factors that enhance colonization, invasion and toxicity, avoid the immune response and promote obtaining nutriment.

Humans and different animals have developed intricate immune systems as an evolutionary response to protect themselves from micro-organism invasion. However, bacteria have a number of strategies to attack them; one of these is protease production. Proteases are present in all living organisms. They are enzymes that catalyse the hydrolysis of peptide bonds in proteins or peptides. They include serine proteases, cysteine proteases, aspartate proteases and metalloproteases. Most bacterial metalloproteases from pathogenic organisms contain zinc, are extracellular and have been either demonstrated to or suggested to play an important role in virulence (Häse & Finkelstein, 1993; Miyhosi & Shinoda, 2000). For example, bacterial toxins responsible for tetanus, botulism and anthrax are zinc-containing metalloproteases (Rosseto et al., 2003). Metalloproteases have been described in several members of the Pasteurellaceae family: Actinobacillus pleupneumoniae, Pasteurella multocida, Actinobacillus suis (Negrete-Abascal et al., 1998 1999 2004) and Haemophilus paragallinarum (Negrete-Abascal, unpublished data). However, their roles in virulence has not been established, although it has been demonstrated that the metalloprotease of A. pleuropneumoniae was produced in vivo in lung tissues of pigs suffering of porcine pleuropneumonia (García-González et al., 2004).

In this work we describe the biochemical characteristics of secreted metalloproteases from two haemolytic strains isolated from chicken, previously identified as avian P. haemolytica (Gallibacterium anatis bv. haemolytica, biovar 4) and one G. anatis reference strain F 149^T. Secreted proteases could be participating in the pathogenesis of Gallibacterium infections.

Materials and Methods

Bacteria

Two haemolytic bacterial strains isolated from chicken (250900 and 249101 strains), biochemically characterized as G. anatis biovar 4 according to Christensen et al. (2003), were used. Strain 250900 was isolated from the trachea and infraorbital sinuses, while strain 249101 was isolated from the oviduct, magnum, follicles and different internal organs from laying hens and considered an invasive bacterium. The reference strain of G. anatis F 149^T (ATCC 43329^T) was kindly donated by Dr H. Christensen (The Royal Veterinary and Agricultural University, Denmark).

Bacteria growth conditions, isolation of proteases and proteolytic activity

Bacteria were grown overnight on brain heart infusion (BHI) agar plates. For protease production, all Gallibacterium (field and reference) strains were inoculated in 10 ml BHI and incubated for 6 h at 37°C with shaking. Next, the culture was inoculated in 200 ml BHI and grown overnight under the same conditions. Cells were separated by centrifugation (12 000×g, 4°C, 20 min). Cell-free culture supernatant proteins were concentrated using cold methanol (two volumes) for overnight precipitation. They were centrifuged under the same conditions for 1 h, and the pellet was resuspended in 50 mM Tris–HCl buffer (pH 8). Methanol precipitated samples were considered a crude preparation (CP). The protein concentration was measured as described by Bradford (1976). To detect proteolytic activity, 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis copolymerized with bovine casein (1%) were performed (Negrete-Abascal et al., 1999 2004) at room temperature. Gels were stained with Coomassie-blue R250.

Optimal pH

In order to know the optimal pH for protease activity, gel wells were loaded with 10 to 15 µg CP protein mixed with sample buffer (Laemmli, 1970). Samples were not boiled nor treated with reducing agents. After electrophoresis, gels were incubated with either 50 mM acetate (pH 5 and 6), or 50 mM Tris–HCl (pH 7 and 8), or 50 mM Glycine–NaOH (pH 9 to 10) buffers.

Protease inhibitors

Inhibitors at the following concentrations were added 30 min before sample buffer addition: 10 or 20 mM ethylenediamine tetraacetic acid (EDTA), a metal ion chelating; 10 mM p-hydroxymercuribenzoate, a cysteine protease inhibitor; or 5 mM phenylmethylsulfonylfluoride, a serine protease inhibitor. After electrophoresis, inhibitors were also added at the same concentration to the incubation buffer (pH 8). To confirm the effect of EDTA, gels were incubated in buffers with 10 mM CaCl₂ to permit substrate degradation.

Temperature stability

Samples of CP proteins were incubated at 37, 40, 50, 60, 70, and 80°C for 10 min and then electrophoresed. Proteolytic activity was tested as already described.

Immune recognition

CP proteins were separated by electrophoresis and transferred to a nitrocellulose membrane. The membrane was blocked with 5% skim milk in phosphate buffer saline–Tween 20, and incubated with a 1:500-diluted polyclonal serum against a high molecular mass protease secreted by A. pleuropneumoniae (Negrete-Abascal et al., 1998). Rabbit pre-immune serum was used as the negative control. The immune reaction was revealed with peroxidase-labelled goat IgG anti-rabbit antibody using diaminobenzidine and H₂O₂ (Negrete-Abascal et al., 1999).

IgG chicken degradation

In order to know whether CP proteins were able to degrade chicken IgG, 10 µg chicken IgG (Sigma) was mixed with 15 to 20 µg CP from the 249101 and 250900 field isolates and incubated at 37°C during 24, 48 or 72 h. After each period of time, samples were mixed with buffer sample without 2-mercaptoethanol and boiled for 5 min. Mixes containing both chicken IgG and CP proteins were separated by electrophoresis in a 10% polyacrylamide gel and stained with Coomassie blue R250. Chicken IgG without CP proteins was incubated during 72 h as a negative control.

Results and Discussion

The role of Gallibacterium spp. as pathogens has not been clearly demonstrated. However, different reports indicate that Gallibacterium members might be pathogenic as they have been isolated from a range of pathological lesions in poultry (Hacking & Pettit, 1974; Rhoades & Rimler, 1984; Shaw et al., 1990; Bojesen et al., 2004). G. anatis field and reference isolates produced proteases when cultured in vitro.

CP proteins from G. anatis bv. haemolytica field isolates (Figure 1a: lane 1, 250900 strain; lane 2, 249101 strain) and G. anatis reference strain (lane 3) showed proteolytic activity. Proteins at a molecular mass >100 kDa demonstrated the highest activity, but proteolytic activity of proteins with less molecular mass (approximately 50 kDa) was observed in CP from both field isolates (Figure 1b), suggesting that they produce more than one metalloprotease or that they form part of a high molecular mass oligomer as has been described for A. pleuropneumoniae. (Negrete-Abascal et al., 1998; García González et al., 2004).

Figure 1.  Proteolytic activity in 10% polyacrylamide gels copolymerized with 1% bovine casein. Cell-free culture supernatants of G. anatis strains were precipitated with 1 volume of methanol and 20 µg dissolved protein was loaded into each well. Lane 1, 250900 isolate; lane 2, 249101 isolate; lane 3, F149^T G. anatis reference strain. 1b: Effect of pH on proteolytic activity. Gels were incubated at pH 6 to 10 (lanes 1, 3, 5, 7, 9, 249101 isolate; lanes 2, 4, 6, 8, 10, 250900 isolate).

Protease from the 249101 field isolate was active in a broad pH range (6 to 10), with pH 9.0 being optimal (Figure 1b, lane 7), whereas 250900 protease was active only at pH 8 and 9 (Figure 1b, lanes 6 and 8). As such, pH 9.0 was used in all the characterization tests. Proteases active in a wide range of pH have been described in different pathogenic micro-organisms including A. pleuropneumoniae, A. suis, and P. multocida (Negrete-Abascal et al., 1998 1999 2004), all of them members of the Pasteurellaceae family to which G. anatis belongs. It is possible that G. anatis proteases are closely related with proteases of those Pasteurellaceae members.

When CP proteins of the two field isolates were incubated at different temperatures, proteolytic activity was observed after incubation at 50°C (Figure 2a,b, lanes 5) or lower temperatures. Activity diminished at 60°C for both CP proteins (lane 6 for each gel), and was totally inhibited at higher temperatures. Heat-stable metalloproteases similar to those of G. anatis have been described in P. multocida, A. pleuropneumoniae or A. suis, suggesting a common related source among them (Negrete-Abascal et al., 1998 1999 2004).

Figure 2.  Temperature effect. The same conditions, isolates and sample amount as in Figure 1b. The first sample (lane 1) was not incubated while the other samples were incubated at (lane 2) room temperature, (lane 3) 37°C, (lane 4) 40°C, (lane 5) 50°C, (lane 6) 60°C, (lane 7) 70°C and (lane 8) 80°C during 10 min prior to electrophoresis. 2a: 250900 isolate; 2b: 249101 isolate.

G. anatis-secreted proteolytic activity was totally inhibited by chelating agent EDTA but it was not affected by serine or cysteine protease inhibitors (Figure 3a). Therefore, these secreted enzymes are considered metalloproteases. Proteolytic activity from the 250900 and reference strains was totally inhibited with 10 mM EDTA, while the 249101 isolate was inhibited only with 20 or 30 mM EDTA. As CP concentration was the same, this could indicate that 249101-secreted proteases and metal ions are more tightly associated than those of the other strains and, as a result, harder to inhibit. It is also possible that this bacterium produces a higher amount or a different number of proteases, which would equally make them more difficult to inhibit.

Figure 3.  Inhibitory effect on the proteolytic activity. The same conditions, isolates and sample amount as in Figure 1b. Gels were incubated in 50 mM Tris–HCl buffer (pH 9.0) with protease inhibitors: 10 mM EDTA (lanes 3, 4), 5 mM p-hydroxymercuribenzoate (lanes 5, 6) and 5 mM phenylmethylsulfonylfluoride (lanes 7, 8). A sample without inhibitor was used as control (lanes 1, 2). Lanes 1, 3, 5, 7, 250900 isolate; lanes 2, 4, 6, 8, 249101 isolate. 3b: Immunoblot analysis of CP from G. anatis reference strain (lanes 1 and 3) and A. pleuropneumoniae (lanes 2 and 4) reacted with the polyclonal serum raised against the purified protease of A. pleuropneumoniae serotype 1 (lanes 1 and 2) or with preimmune rabbit serum (3 and 4).

By immunoblotting, recognition bands of high molecular mass were observed when CP of the G. anatis reference strain was incubated with a polyclonal serum against a high molecular mass purified protease secreted by A. pleuropneumoniae serotype 1 (Figure 3b. lane 1). This recognition was observed at a similar molecular mass as CP from A. pleuropneumoniae serotype 1 used as the positive control (lane 2). No recognition was observed when rabbit pre-immune serum was used (lanes 3 and 4), indicating a specific recognition. This result indicates the presence of common epitopes among the G. anatis protease and proteases secreted by A. pleuropneumoniae and other Pasteurellaceae members (Negrete-Abascal et al., 1998 1999 2004), suggesting a common source.

Proteases from both field and reference isolates were able to partially cleave chicken IgG after 48 to 72 h of incubation at 37°C (Figure 4). At 72 h post incubation, CP proteins from 249101 degraded the immunoglobulin completely (Figure 4, line 7). Immunoglobulin degradation by pathogenic bacterial-secreted proteases is a well-known mechanism of immune system evasion (Mushin et al., 1980; Kornfeld & Plaut, 1991), as well as an amino acid source for different micro organisms (Jansen et al., 1995).

Figure 4.  Chicken IgG degradation by CP from G. anatis. Twenty micrograms of CP from field isolates or reference strain were mixed with 10 µg chicken IgG and incubated at 37°C during 24 h (lanes 2, 5 and 8), 48 h (lanes 3, 6 and 9) and 72 h (lanes 4, 7 and 10). Lane 1, control chicken IgG incubated during 72 h; lanes 2 to 4, G. anatis reference strain; lanes 5 to 7, 249101 isolate; lanes 8 to 10, 250900 isolate.

The differences in proteolytic activity among G. anatis field isolates (e.g. major degradation of chicken IgG, and a higher amount of EDTA needed to inhibit its activity) suggest differences between the strains able to invading host tissues and those isolated from healthy chickens. It could be interesting to investigate the differences between G. anatis invasive and indigenous strains and to test whether these differences are due to an environmentally controlled gene expression or to the presence of more than one protease in the 249101 isolate. In order to test this, it will be necessary to look for proteolytic activities in a higher number of invasive and commensal G. anatis isolates. The role of G. anatis-secreted proteases degrading chicken IgG in pathogenesis remains to be uncovered.

Translations of the abstract in French, Germany and Spanish are available on the Avian Pathology website.

This work was supported by DGAPA-UNAM. Project PAPIIT: IN219203 and CONACYT, project: G38590-B. The English version was revised by M. en C. Isabelle Blanckaert.