Identification, Biochemical Characterization, and In Vivo Detection of a Zn-Metalloprotease with Collagenase Activity from Mannheimia haemolytica A2.

Respiratory diseases in ruminants are a main cause of economic losses to farmers worldwide. Approximately 25% of ruminants experience at least one episode of respiratory disease during the first year of life. Mannheimia haemolytica is the main etiological bacterial agent in the ruminant respiratory disease complex. M. haemolytica can secrete several virulence factors, such as leukotoxin, lipopolysaccharide, and proteases, that can be targeted to treat infections. At present, little information has been reported on the secretion of M. haemolytica A2 proteases and their host protein targets. Here, we obtained evidence that M. haemolytica A2 proteases promote the degradation of hemoglobin, holo-lactoferrin, albumin, and fibrinogen. Additionally, we performed biochemical characterization for a specific 110 kDa Zn-dependent metalloprotease (110-Mh metalloprotease). This metalloprotease was purified through ion exchange chromatography and characterized using denaturing and chaotropic agents and through zymography assays. Furthermore, mass spectrometry identification and 3D modeling were performed. Then, antibodies against the 110 kDa-Mh metalloprotease were produced, which achieved great inhibition of proteolytic activity. Finally, the antibodies were used to perform immunohistochemical tests on postmortem lung samples from sheep with suggestive histology data of pneumonic mannheimiosis. Taken together, our results strongly suggest that the 110-Mh metalloprotease participates as a virulence mechanism that promotes damage to host tissues.

Changes in digestive enzyme activities during the early ontogeny of the South American cichlid (Cichlasoma dimerus).

Cichlasoma dimerus is a neotropical cichlid that has been used as a biological model for neuroendocrinology studies. However, its culture is problematic in terms of larval feeding to allow having enough fry quantity and quality. Larviculture requires full knowledge about the digestive system and nutrition; therefore, this study was intended to assess the digestive enzymes' changes at different ages during the early ontogeny. Acid protease activity was detectable from the first day after hatching (dah), increasing to its maximum peaks on 9 dah. In contrast, alkaline proteases had low activity in the first days of life but reached their maximum activity on 17 dah. Chymotrypsin, L-aminopeptidase, and carboxypeptidase A activities increased at 6 dah, while trypsin activity was first detected on 13 dah and reached its maximum activity on 17 dah. Lipase and α-amylase activity were detectable at low levels in the first days of life, but the activity fluctuated and reaching its maximum activity at 21 dah. Alkaline phosphatase continued to oscillate and had two maximum activity peaks, the first at 6 dah and the second at 19 dah. Zymograms of alkaline proteases on day 6 dah six revealed four activity bands with molecular weights from 16.1 to 77.7 kDa. On 13 dah, two more activity bands of 24.4 and 121.9 kDa were detected, having a total of six proteases. The enzymatic activity analyzes indicate the digestive system shows the low activity of some enzymes in the first days after hatching, registering significant increases on 6 dah and the maximum peaks of activities around at 17 dah. Therefore, we recommend replacing live food with dry feed and only providing dry feed after day 17 dah.

Comparison of several non-specific skin mucus immune defences in three piscine species of aquaculture interest.

Fish skin mucus is a viscous and semipermeable barrier made mainly of water, glycoproteins and soluble proteins. It represents an important defence against the environment and previous studies have reported the presence of different substances involved in immune defence responses in it. The aim of the present work was to characterize skin mucus protease activity by zymography and esterase activity of the subfamily of carboxylesterases in three species of interest for aquaculture: gilthead sea bream, sea bass and meagre. Mucus antioxidant power was also determined by adapting ferric reducing antioxidant power (FRAP) analysis. As a result of these non-specific immune defence parameters, we compared the antibacterial capacity of skin mucus in these species via in vitro dual bacteria strains-skin mucus co-culture growths. We used Pseudomonas anguilliseptica and Vibrio anguillarum as marine pathogenic bacteria and Escherichia coli as non-pathogenic. For each fish species, in the respective zymograms, we determined a pattern of proteolytic digestion bands. A high-molecular-weight band (around 200 kDa; H-band) was evident in sea bream and sea bass, and showed chymotrypsin activity. One or two intermediate-molecular-weight bands (around 75 kDa; I-bands) with non-trypsin and non-chymotrypsin activity, and putatively with metalloprotease activity, were evident in all species. Finally, low-molecular-weight bands (between 14 and 30 kDa; L-bands) showed distinct patterns for each species and matched trypsin activity. Despite the conservative pattern of digestion bands, the levels of total proteolytic activity (TPA) were 5 and 10 times higher in meagre than in sea bass and sea bream, respectively. In parallel, three carboxylesterase activities were detected in the mucus of the three fish species, using myristate (pNPM-CE activity), butyrate (pNPB-CE activity) and acetate (pNPA-CE activity) as substrates. Both pNPB-CE and pNPA-CE were the most abundant in fish mucus, and meagre was again the species with the highest levels. In contrast, the antioxidant power of meagre skin mucus was the lowest. We established the capacity of skin mucus to block or limit bacterial growth (lytic activity) using 24 h growth curves. The log-growth phase of V. anguillarum was strongly blocked by sea bream and meagre mucus for a few hours; but not by sea bass mucus. However, if mucus was not renewed, log-growth was at the end of 24 h studied period. For its part, P. anguilliseptica growth curve was delayed by the three mucus types during the entire growth period. Only meagre achieved lytic activity against E. coli growth. All parameters studied here will be of a great interest as non-invasive bioindicators of non-specific immune defences in fish skin mucus.

Mannheimia haemolytica A2 secretes different proteases into the culture medium and in outer membrane vesicles.

Respiratory diseases in ruminants have a significantly negative impact on the worldwide economy. The bacterium Mannheimia haemolytica is involved in pneumonic infections in bovine and ovine. In gram-negative bacteria, six secretion systems related to the colonization process and host tissue damage have been reported. In addition, in the last two decades, the production of outer membrane vesicles has been studied as a different bacterial strategy to release virulence factors, such as exotoxins, lipopolysaccharides, and proteases. However, in M. haemolytica serotype A2, protease secretion and release in vesicles have not been reported as virulence mechanisms. The aim of this work was to identify proteases released into the culture supernatant and in vesicles of M. haemolytica A2. Our results showed evident differences in the molecular mass and activity of proteases present in culture supernatants and outer membrane vesicles based on zymography assays. The biochemical characterization of M. haemolytica proteases revealed that the main types were cysteine and metalloproteases. A specific metalloprotease of 100 kDa was active in the culture supernatants, but it was not active and was found in low quantities in vesicles. Proteases could be an important virulence factor during the infectious pneumonic process led by M. haemolytica.

Complex Formation between the Transcription Factor WRKY53 and Antioxidative Enzymes Leads to Reciprocal Inhibition.

The transcription factor WRKY53 of the model plant Arabidopsis thaliana is an important regulator of leaf senescence. Its expression, activity and degradation are tightly controlled by various mechanisms and feedback loops. Hydrogen peroxide is one of the inducing agents for WRKY53 expression, and a long-lasting intracellular increase in H2O2 content accompanies the upregulation of WRKY53 at the onset of leaf senescence. We have identified different antioxidative enzymes, including catalases (CATs), superoxide dismutases (SODs) and ascorbate peroxidases (APXs), as protein interaction partners of WRKY53 in a WRKY53-pulldown experiment at different developmental stages. The interaction of WRKY53 with these enzymes was confirmed in vivo by bimolecular fluorescence complementation assays (BiFC) in Arabidopsis protoplasts and transiently transformed tobacco leaves. The interaction with WRKY53 inhibited the activity of the enzyme isoforms CAT2, CAT3, APX1, Cu/ZuSOD1 and FeSOD1 (and vice versa), while the function of WRKY53 as a transcription factor was also inhibited by these complex formations. Other WRKY factors like WRKY18 or WRKY25 had no or only mild inhibitory effects on the enzyme activities, indicating that WRKY53 has a central position in this crosstalk. Taken together, we identified a new additional and unexpected feedback regulation between H2O2, the antioxidative enzymes and the transcription factor WRKY53.