Alkaline Protease AprA Research Articles

Discovery of a temperature-dependent protease spoiling meat from Pseudomonas fragi: Target to myofibrillar and sarcoplasmic proteins rather than collagen

Chilled meat frequently suffered microbial spoilage because bacteria can secrete various proteases that break down the proteins. In this study, Pseudomonas fragi NMC 206 exhibited a temperature-dependent secretion pattern, with the ability to release the specific protease only below 25 °C. It was identified as alkaline protease AprA by LC-MS/MS, with the molecular weight of 50.4 kDa, belonging to the Serralysin family metalloprotease. Its significant potential for meat spoilage in situ resulted in alterations in meat color and sensory evaluation, as well as elevated pH, total volatile basic nitrogen (TVB-N) and the formation of volatile organic compounds (VOCs). The hydrolysis of meat proteins in vitro showed that AprA possessed a considerable proteolysis activity and degradation preferences on meat proteins, especially its ability to degrade myofibrillar and sarcoplasmic proteins, rather than collagen. These observations demonstrated temperatures regulated the secretion of AprA, which was closely related to chilled chicken spoilage caused by bacteria. These will provide a new basis for the preservation of meat products at low temperatures.

Carbapenem-Resistant Pseudomonas aeruginosa Strains-Distribution of the Essential Enzymatic Virulence Factors Genes.

Pseudomonas aeruginosa is one of the most commonly isolated bacteria from clinical specimens, with increasing isolation frequency in nosocomial infections. Herein, we investigated whether antimicrobial-resistant P. aeruginosa strains, e.g., metallo-beta-lactamase (MBL)-producing isolates, may possess a reduced number of virulence genes, resulting from appropriate genome management to adapt to a changing hospital environment. Hospital conditions, such as selective pressure, may lead to the replacement of virulence genes by antimicrobial resistance genes that are crucial to survive under current conditions. The study aimed to compare, using PCR, the frequency of the chosen enzymatic virulence factor genes (alkaline protease-aprA, elastase B-lasB, neuraminidases-nan1 and nan2, and both variants of phospholipase C-plcH and plcN) to MBL distribution among 107 non-duplicated carbapenem-resistant P. aeruginosa isolates. The gene encoding alkaline protease was noted with the highest frequency (100%), while the neuraminidase-1 gene was observed in 37.4% of the examined strains. The difference in lasB and nan1 prevalence amongst the MBL-positive and MBL-negative strains, was statistically significant. Although P. aeruginosa virulence is generally more likely determined by the complex regulation of the virulence gene expression, herein, we found differences in the prevalence of various virulence genes in MBL-producers.

Synthetic Peptides to Target Stringent Response-Controlled Virulence in a Pseudomonas aeruginosa Murine Cutaneous Infection Model.

Microorganisms continuously monitor their surroundings and adaptively respond to environmental cues. One way to cope with various stress-related situations is through the activation of the stringent stress response pathway. In Pseudomonas aeruginosa this pathway is controlled and coordinated by the activity of the RelA and SpoT enzymes that metabolize the small nucleotide secondary messenger molecule (p)ppGpp. Intracellular ppGpp concentrations are crucial in mediating adaptive responses and virulence. Targeting this cellular stress response has recently been the focus of an alternative approach to fight antibiotic resistant bacteria. Here, we examined the role of the stringent response in the virulence of P. aeruginosa PAO1 and the Liverpool epidemic strain LESB58. A ΔrelA/ΔspoT double mutant showed decreased cytotoxicity toward human epithelial cells, exhibited reduced hemolytic activity, and caused down-regulation of the expression of the alkaline protease aprA gene in stringent response mutants grown on blood agar plates. Promoter fusions of relA or spoT to a bioluminescence reporter gene revealed that both genes were expressed during the formation of cutaneous abscesses in mice. Intriguingly, virulence was attenuated in vivo by the ΔrelA/ΔspoT double mutant, but not the relA mutant nor the ΔrelA/ΔspoT complemented with either gene. Treatment of a cutaneous P. aeruginosa PAO1 infection with anti-biofilm peptides increased animal welfare, decreased dermonecrotic lesion sizes, and reduced bacterial numbers recovered from abscesses, resembling the phenotype of the ΔrelA/ΔspoT infection. It was previously demonstrated by our lab that ppGpp could be targeted by synthetic peptides; here we demonstrated that spoT promoter activity was suppressed during cutaneous abscess formation by treatment with peptides DJK-5 and 1018, and that a peptide-treated relA complemented stringent response double mutant strain exhibited reduced peptide susceptibility. Overall these data strongly indicated that synthetic peptides target the P. aeruginosa stringent response in vivo and thus offer a promising novel therapeutic approach.

The LasB Elastase of Pseudomonas aeruginosa Acts in Concert with Alkaline Protease AprA To Prevent Flagellin-Mediated Immune Recognition.

The opportunistic pathogen Pseudomonas aeruginosa is capable of establishing severe and persistent infections in various eukaryotic hosts. It encodes a wide array of virulence factors and employs several strategies to evade immune detection. In the present study, we screened the Harvard Medical School transposon mutant library of P. aeruginosa PA14 for bacterial factors that modulate interleukin-8 responses in A549 human airway epithelial cells. We found that in addition to the previously identified alkaline protease AprA, the elastase LasB is capable of degrading exogenous flagellin under calcium-replete conditions and prevents flagellin-mediated immune recognition. Our results indicate that the production of two proteases with anti-flagellin activity provides a failsafe mechanism for P. aeruginosa to ensure the maintenance of protease-dependent immune-modulating functions.

Pseudomonas syringae Evades Host Immunity by Degrading Flagellin Monomers with Alkaline Protease AprA

Bacterial flagellin molecules are strong inducers of innate immune responses in both mammals and plants. The opportunistic pathogen Pseudomonas aeruginosa secretes an alkaline protease called AprA that degrades flagellin monomers. Here, we show that AprA is widespread among a wide variety of bacterial species. In addition, we investigated the role of AprA in virulence of the bacterial plant pathogen P. syringae pv. tomato DC3000. The AprA-deficient DC3000 ΔaprA knockout mutant was significantly less virulent on both tomato and Arabidopsis thaliana. Moreover, infiltration of A. thaliana Col-0 leaves with DC3000 ΔaprA evoked a significantly higher level of expression of the defense-related genes FRK1 and PR-1 than did wild-type DC3000. In the flagellin receptor mutant fls2, pathogen virulence and defense-related gene activation did not differ between DC3000 and DC3000 ΔaprA. Together, these results suggest that AprA of DC3000 is important for evasion of recognition by the FLS2 receptor, allowing wild-type DC3000 to be more virulent on its host plant than AprA-deficient DC3000 ΔaprA. To provide further evidence for the role of DC3000 AprA in host immune evasion, we overexpressed the AprA inhibitory peptide AprI of DC3000 in A. thaliana to counteract the immune evasive capacity of DC3000 AprA. Ectopic expression of aprI in A. thaliana resulted in an enhanced level of resistance against wild-type DC3000, while the already elevated level of resistance against DC3000 ΔaprA remained unchanged. Together, these results indicate that evasion of host immunity by the alkaline protease AprA is important for full virulence of strain DC3000 and likely acts by preventing flagellin monomers from being recognized by its cognate immune receptor.

Pseudomonas aeruginosa Alkaline Protease Can Facilitate Siderophore-Mediated Iron-Uptake via the Proteolytic Cleavage of Transferrins

In order to determine whether Pseudomonas aeruginosa alkaline protease AprA is involved in facilitating siderophore-mediated iron-acquisition from human transferrins, we measured bacterial growth, the production of siderophore and AprA, iron-acquisition from transferrins, and the proteolytic cleavage of transferrins in an alkaline minimal medium (pH 8.3) containing human transferrins as an iron source and compared these on a time scale. The growth of P. aeruginosa was found to be stimulated in proportion to the iron-saturation levels of transferrins. AprA production and the proteolytic cleavage of transferrins began concomitantly with siderophore production from the early growth phase when P. aeruginosa was actively growing and consuming most iron for growth. However, the AprA-free, but siderophore-containing, culture ultrafiltrates could also remove iron from transferrin. These results indicate that alkaline protease AprA can facilitate the siderophore-mediated iron-uptake of P. aeruginosa via the proteolytic cleavage of transferrins. However, the proteolytic cleavage by AprA is not essentially required for iron-acquisition from transferrins.

Enhanced alkaline protease production in addition to alpha-amylase via constructing a Bacillus subtilis strain.

Bacillus subtilis Bios11 strain was previously isolated and identified. This strain naturally produces a high level of alpha-amylase. The multicopy (pS1) plasmid that carries the complete alkaline protease aprA gene was introduced to this host strain by transformation. The newly constructed strain was found to express the aprA gene and produces a high level of alkaline protease. The level of alpha-amylase production was not affected compared with the parent strain. The pS1 plasmid in the new host was proved to be segregationally and structurally stable, and the multicopy aprA gene was expressed at the stationary phase. This expression did not affect growth rate and sporulation frequency. Moreover, the level of alpha-amylase was maintained. Both alkaline protease and alpha-amylase enzymes were purified using a single-step affinity chromatography column. The use of the newly constructed strain would be valuable to the enzyme industry and would promote recycling of some food-processing wastes.