[Biochemical properties of Scedosporium aurantiacum extracellular elastase-like protease].

Extracellular elastase-like protease is one of the key virulence proteases of Scedosporium aurantiacum. To date, little is known about this enzyme in terms of genetic information, structure, properties and virulence mechanism due to the difficulties in purification caused by its low secretion amount, high specific activity, uncompleted genome sequencing and annotation. This work investigated the gene, structure and enzymatic properties of this enzyme. The S. aurantiacum elastase-like protease from the fungal culture supernatant was analyzed through tandem mass spectrometry (MS/MS) approach, illustrating its primary structure. Bioinformatics tools were employed to predict the conserved domain and tertiary structure, the enzymatic properties were also studied. It turned out that S. aurantiacum extracellular elastase-like protease demonstrated well hydrolysis towards elastin and bovine achilles tendon collagen, with Vmax of 18.14 µg/s and 17.57 µg/s respectively, better than fish scale gelatin, with the lowest hydrolysis effect on casein. Its activity towards elastin was lower than that of the elastase from porcine pancreas, with values of Kcat/Km of 3.541 (µg/s) and 4.091 (µg/s), respectively. It was an alkaline protease, with optimal pH 8.2 and temperature 37 oC. Zn2+ promoted the enzymatic activity while Ca2+, Mg2+, Na+, elastatinal and PMSF inhibited its activity. Its sequence was similar to Paecilomyces lilacinus secreted serine protease (PDB Entry: c3f7oB_) with multiple conserved fractions each containing more than 7 amino acids, thus suitable for design of PCR primer. This study increased our knowledge on S. aurantiacum extracellular elastase-like protease in terms of structure and enzymatic properties, and may facilitate later studies on protein expression and virulence mechanism.

Splicing transcriptome-wide association study to identify splicing events for pancreatic cancer risk.

A large proportion of the heritability of pancreatic cancer risk remains elusive, and the contribution of specific mRNA splicing events to pancreatic cancer susceptibility has not been systematically evaluated. In this study, we performed a large splicing transcriptome-wide association study (spTWAS) using three modeling strategies (Enet, LASSO and MCP) to develop alternative splicing genetic prediction models for identifying novel susceptibility loci and splicing introns for pancreatic cancer risk by assessing 8275 pancreatic cancer cases and 6723 controls of European ancestry. Data from 305 subjects of whom the majority are of European descent in the Genotype-Tissue Expression Project (GTEx) were used and both cis-acting and promoter-enhancer interaction regions were considered to build these models. We identified nine splicing events of seven genes (ABO, UQCRC1, STARD3, ETAA1, CELA3B, LGR4 and SFT2D1) that showed an association of genetically predicted expression with pancreatic cancer risk at a false discovery rate ≤0.05. Of these genes, UQCRC1 and LGR4 have not yet been reported to be associated with pancreatic cancer risk. Fine-mapping analyses supported likely causal associations corresponding to six splicing events of three genes (P4HTM, ABO and PGAP3). Our study identified novel genes and splicing events associated with pancreatic cancer risk, which can improve our understanding of the etiology of this deadly malignancy.

Biochemical properties of Scedosporium aurantiacum extracellular elastase-like protease / 生物工程学报

Extracellular elastase-like protease is one of the key virulence proteases of Scedosporium aurantiacum. To date, little is known about this enzyme in terms of genetic information, structure, properties and virulence mechanism due to the difficulties in purification caused by its low secretion amount, high specific activity, uncompleted genome sequencing and annotation. This work investigated the gene, structure and enzymatic properties of this enzyme. The S. aurantiacum elastase-like protease from the fungal culture supernatant was analyzed through tandem mass spectrometry (MS/MS) approach, illustrating its primary structure. Bioinformatics tools were employed to predict the conserved domain and tertiary structure, the enzymatic properties were also studied. It turned out that S. aurantiacum extracellular elastase-like protease demonstrated well hydrolysis towards elastin and bovine achilles tendon collagen, with Vmax of 18.14 μg/s and 17.57 μg/s respectively, better than fish scale gelatin, with the lowest hydrolysis effect on casein. Its activity towards elastin was lower than that of the elastase from porcine pancreas, with values of Kcat/Km of 3.541 (μg/s) and 4.091 (μg/s), respectively. It was an alkaline protease, with optimal pH 8.2 and temperature 37 oC. Zn2+ promoted the enzymatic activity while Ca2+, Mg2+, Na+, elastatinal and PMSF inhibited its activity. Its sequence was similar to Paecilomyces lilacinus secreted serine protease (PDB Entry: c3f7oB_) with multiple conserved fractions each containing more than 7 amino acids, thus suitable for design of PCR primer. This study increased our knowledge on S. aurantiacum extracellular elastase-like protease in terms of structure and enzymatic properties, and may facilitate later studies on protein expression and virulence mechanism.

Localization of trypsin, chymotrypsin and elastase in the digestive tract of amphioxus Branchiostoma japonicum with implications to the origin of vertebrate pancreas.

The organ pancreas is characteristic of all vertebrates, but its evolutionary origin remains largely unknown. The serine proteinases trypsin, chymotrypsin and elastase are produced primarily in the pancreas in vertebrates. Here we clearly demonstrate by enzymatic activity assay, histochemical staining and in situ hybridization that trypsin, chymotrypsin and elastase are widely distributed in the digestive tract of the amphioxus Branchiostoma japonicum, especially in the hepatic caecum and mid-gut. This suggests that an extensive region of the amphioxus digestive tract including the hepatic caecum is homologous to the vertebrate exocrine pancreatic cells, providing a new angle for the study of the origin and evolution of vertebrate pancreas.

Identification and expression profiling of serine protease-related genes in Tenebrio molitor.

In insects, serine proteases and serine protease homologs (SPs/SPHs) are involved in a variety of physiological processes including digestion, development, and immunity. Here, we identified 112 SP and 88 SPH genes in the genome of the yellow mealworm, Tenebrio molitor. Based on the features of domain structure, they were divided into "S" group containing single Tryp-SPc or Tryp-SPHc domain, "C" group containing 1-4 CLIP domain (CLIPA-D) and "M" group containing the CBD, CUB, EGF, Fz, Gd, LDLa, PAN, SEA, SR, Sushi, and TSP domains, and have 115, 48, and 37 gene members, respectively. According to the active sites in the catalytic triad, the putative trypsin, chymotrypsin, or elastase-like enzyme specificity of the identified SPs/SPHs were predicted. Phylogenetic and genomic location analyses revealed that gene duplication exists in the large amount of SPs/SPHs. Gene expression profiling using RNA-seq data along with real time reverse transcription-polymerase chain reaction analysis showed that most SP/SPH genes display life stage specific expression patterns, indicating their important roles in development. Many SP/SPH genes are specifically or highly expressed in the gut, salivary gland, fat body, hemocyte, ovary, and testis, suggesting that they participate in digestion, immunity, and reproduction. The findings lay the foundation for further functional characterization of SPs/SPHs in T. molitor.

Loss-of-function variant in chymotrypsin like elastase 3B (CELA3B) is associated with non-alcoholic chronic pancreatitis.

BACKGROUND: Genetic alterations in digestive enzymes have been associated with chronic pancreatitis (CP). Recently, chymotrypsin like elastase 3B (CELA3B) emerged as a novel risk gene. Thus, we evaluated CELA3B in two European cohorts with CP. METHODS: We analyzed all 8 CELA3B exons in 550 German non-alcoholic CP (NACP) patients and in 241 German controls by targeted DNA sequencing. In addition, we analyzed exons 6 and 7 by Sanger sequencing and the c.129+1G>A variant by melting curve analysis in 1078 further German controls. As replication cohort, we investigated up to 243 non-German European NACP patients and up to 1665 controls originating from Poland, Hungary, and Sweden. We assessed the cellular secretion and the elastase activity of recombinant CELA3B variants. RESULTS: In the German discovery cohort, we detected a splice-site variant in intron 2, c.129+1G>A, in 9/550 (1.64%) CP patients and in 5/1319 (0.38%) controls (P=0.007, OR=4.4, 95% CI=1.5-13.0). In the European replication cohort, this variant was also enriched in patients (9/178 [5.06%]) versus controls (13/1247 [1.04%]) (P=0.001, OR=5.1, 95% CI=2.1-12.0). We did not find the two previously reported codon 90 variants, p.R90C and p.R90L. CONCLUSIONS: Our data indicate that CELA3B is a susceptibility gene for CP. In contrast to previous reports suggesting that increased CELA3B activity is associated with CP risk, the splice-site variant identified here is predicted to cause diminished CELA3B expression. How reduced CELA3B function predisposes to pancreatitis remains to be elucidated.

Antimicrobial resistance, virulence factors, and genotypes of Pseudomonas aeruginosa clinical isolates from Gorgan, northern Iran.

Pseudomonas aeruginosa is an important nosocomial pathogen with a capacity of resistance to multiple antibiotics and production of various extracellular and cell-associated virulence factors that clearly contribute to its pathogenicity. The objective of this study was to investigate the antibiotic susceptibility, virulence factors, and clonal relationship among clinical isolates of P. aeruginosa. Different clinical specimens from hospitalized patients were investigated for P. aeruginosa. Susceptibility of the isolates was evaluated by disc diffusion and broth microdilution methods, as described by the Clinical and Laboratory Standards Institute (CLSI) guideline. A total of 97 P. aeruginosa isolates were recovered from clinical specimens. The percentage of isolates resistant to antimicrobials was imipenem 25.77%, meropenem 15.46%, gentamicin 16.49%, tobramycin 15.46%, amikacin 16.49%, ciprofloxacin 20.61%, levofloxacin 24.74, ceftazidime 20.61%, piperacillin 15.46%, piperacillin/tazobactam 12.37%, colistin 9.27%, and polymyxin B 11.34%. Of isolates, 87.62% possessed ß-hemolytic activity, 78.35% lecithinase, 59.8% elastase, 37.11% DNase, and 28.86% twitching motility. The frequency of virulence genes in isolates was lasB 82.47%, plcH 82.47%, exoA 58.76%, exoS 56.7%, and pilA 10.3%. ERIC-PCR typing clustered P. aeruginosa isolates to 19 common types (CT1-CT19) containing isolates from different hospitals and 43 single types (ST1-ST43). Colistin and polymyxin B were the most effective agents against the majority of P. aeruginosa isolates, emphasizing the effort to maintain their antibacterial activity as last-line therapy. The frequency of some virulence factors and genes was noticeably high, which is alarming. In addition, more effective strategies and surveillance are necessary to confine and prevent the inter-hospital and/or intra-hospital dissemination of P. aeruginosa between therapeutic centers.

Mutational analysis on stable expression and LasB inhibition of LasB propeptide in Pseudomonas aeruginosa.

Three major proteases, elastase B (LasB), protease IV (PIV), and elastase A (LasA) expressed in Pseudomonas aeruginosa play important roles in infections and pathogeneses. These are activated by a proteolytic cascade initiated by the activation of LasB. In this study, we investigated whether LasB could be inhibited using its propeptide (LasBpp). Although LasA and PIV were inhibited by their propeptides, LasB was not inhibited by purified LasBpp because LasB degraded LasBpp. To address this problem, mutant LasBpp variants were constructed to obtain a mutant LasBpp resistant to LasB degradation. A C-terminal deletion series of LasBpp was tested in vivo, and two positive candidates, T2 and T2-1, were selected. However, both caused growth retardation and were unstably expressed in vivo. Since deleting the C-terminal end of LasBpp significantly affected its stable expression, substitution mutations were introduced at the two amino acids near the truncation site of T2-1. The resulting mutants, LasBppE172D, LasBppG173A, and LasBppE172DG173A, significantly diminished LasB activity when overexpressed in vivo and were stably expressed in MW1, a quorum sensing mutant that does not produce LasB. In vitro analysis showed that purified LasBppE172DG173A inhibited LasB activity to a small extent. Summarizing, C-terminal modification of LasBpp profoundly affected the stable expression of LasBpp, and little enhanced the ability of LasBpp to resist degradation by LasB.

The Small RNA AmiL Regulates Quorum Sensing-Mediated Virulence in Pseudomonas aeruginosa PAO1.

Pseudomonas aeruginosa is an opportunistic and nosocomial pathogen of humans with hundreds of its virulence factors regulated by quorum sensing (QS) system. Small noncoding RNAs (sRNAs) are also key regulators of bacterial virulence. However, the QS regulatory sRNAs (Qrrs) that have been characterized in P. aeruginosa are still largely unknown. Here, sRNA AmiL (PA3366.1) in the amiEBCRS operon of PAO1 was identified as a novel Qrr by transcriptome sequencing (RNA-Seq). The expression of AmiL was negatively regulated by the las or rhl system, of which RhlR probably inhibited its transcription. AmiL deletion mutant and overexpressing strains were constructed in PAO1. Broad phenotypic changes were found, including reduced pyocyanin synthesis, elastase activity, biofilm formation, hemolytic activity, and cytotoxicity, as well as increased rhamnolipid production and swarming motility. AmiL appears to be a new regulator that influences diverse QS-mediated virulence. Furthermore, AmiL directly targeted PhzC, a key member of pyocyanin synthesis. AmiL also negatively regulated lasI expression in the early growth of PAO1, but predominantly increased rhlI expression and C4-HSL production in the middle and late stages. Therefore, a novel QS-sRNA signaling cascade of las/rhl (RhlR)-AmiL-PhzC/las/rhl was demonstrated, and it will help to shed new light on the virulence regulatory network of P. aeruginosa PAO1. IMPORTANCE P. aeruginosa is a common nosocomial pathogen that causes diverse opportunistic infections in humans. The virulence crucial for infection is mainly regulated by QS. Small noncoding RNAs (sRNAs) involved in virulence regulation have also been identified in many bacteria. Recently, there is a growing interest in the new sRNA species, QS regulatory sRNAs (Qrrs). Understanding Qrrs-mediated regulation in P. aeruginosa virulence is therefore important to combat infection. In this study, a previously uncharacterized sRNA AmiL in PAO1 has been identified as a novel Qrr. It has been found to influence diverse QS-mediated virulence factors including pyocyanin, elastase, rhamnolipid, and hemolysin, as well as biofilm formation, swarming motility, and cytotoxicity. Furthermore, PhzC essential for pyocyanin synthesis was a direct target of AmiL. QS gene expression and C4-HSL production were also regulated by AmiL. This study provides insights into the roles of Qrr AmiL in modulating P. aeruginosa virulence.

Lung emphysema and impaired macrophage elastase clearance in mucolipin 3 deficient mice.

Lung emphysema and chronic bronchitis are the two most common causes of chronic obstructive pulmonary disease. Excess macrophage elastase MMP-12, which is predominantly secreted from alveolar macrophages, is known to mediate the development of lung injury and emphysema. Here, we discovered the endolysosomal cation channel mucolipin 3 (TRPML3) as a regulator of MMP-12 reuptake from broncho-alveolar fluid, driving in two independently generated Trpml3-/- mouse models enlarged lung injury, which is further exacerbated after elastase or tobacco smoke treatment. Mechanistically, using a Trpml3IRES-Cre/eR26-τGFP reporter mouse model, transcriptomics, and endolysosomal patch-clamp experiments, we show that in the lung TRPML3 is almost exclusively expressed in alveolar macrophages, where its loss leads to defects in early endosomal trafficking and endocytosis of MMP-12. Our findings suggest that TRPML3 represents a key regulator of MMP-12 clearance by alveolar macrophages and may serve as therapeutic target for emphysema and chronic obstructive pulmonary disease.

[Correlation of seminal plasma elastase with sperm reactive oxygen species and semen parameters in infertile men].

OBJECTIVE: To investigate the relationship of seminal plasma elastase (SPE) with sperm reactive oxygen species (ROS) and semen parameters in infertile men. METHODS: Between July 2021 and December 2021, a total of 145 subjects aged 20-51 years with male infertility were enrolled. SPE, seminal leukocytes, sperm ROS, DNA fragmentation index (DFI) and other semen parameters were detected. We divided patients into an inflammation group (SPE ≥ 290 ng/ml, n = 48) and a non-inflammation group (SPE < 290 ng/ml, n = 97), analyzed the relationship of SPE with seminal leukocytes, sperm ROS, semen volume, sperm concentration, total sperm motility, the percentages of progressively motile sperm (PMS) , morphologically normal sperm (MNS), and DFI. RESULTS: The concentration of seminal leukocytes, sperm ROS level and DFI were significantly higher in inflammation group than in non-inflammation group (P < 0.05). No statistically significant differences were observed in semen volume, sperm concentration, total sperm motility, PMS, MNS or sperm deformity index (SDI) between two groups of patients (P > 0.05). Spearman correlation analysis showed that the SPE level was correlated positively with the concentration of seminal leukocytes (r = 0.658, P < 0.01), sperm ROS level (r = 0.229, P = 0.006) and DFI (r = 0.192, P = 0.021), but not correlated with semen volume, sperm concentration, total sperm motility, PMS, MNS or SDI (P > 0.05). CONCLUSION: The level of seminal plasma elastase is related with the concentration of seminal leukocytes, sperm ROS level and DFI, and has some reference value in the diagnosis of male infertility.

Apelin expression deficiency in mice contributes to vascular stiffening by extracellular matrix remodeling of the aortic wall.

Numerous recent studies have shown that in the continuum of cardiovascular diseases, the measurement of arterial stiffness has powerful predictive value in cardiovascular risk and mortality and that this value is independent of other conventional risk factors, such as age, cholesterol levels, diabetes, smoking, or average blood pressure. Vascular stiffening is often the main cause of arterial hypertension (AHT), which is common in the presence of obesity. However, the mechanisms leading to vascular stiffening, as well as preventive factors, remain unclear. The aim of the present study was to investigate the consequences of apelin deficiency on the vascular stiffening and wall remodeling of aorta in mice. This factor freed by visceral adipose tissue, is known for its homeostasic role in lipid and vascular metabolisms, or again in inflammation. We compared the level of metabolic markers, inflammation of white adipose tissue (WAT), and aortic wall remodeling from functional and structural approaches in apelin-deficient and wild-type (WT) mice. Apelin-deficient mice were generated by knockout of the apelin gene (APL-KO). From 8 mice by groups, aortic stiffness was analyzed by pulse wave velocity measurements and by characterizations of collagen and elastic fibers. Mann-Whitney statistical test determined the significant data (p < 5%) between groups. The APL-KO mice developed inflammation, which was associated with significant remodeling of visceral WAT, such as neutrophil elastase and cathepsin S expressions. In vitro, cathepsin S activity was detected in conditioned medium prepared from adipose tissue of the APL-KO mice, and cathepsin S activity induced high fragmentations of elastic fiber of wild-type aorta, suggesting that the WAT secretome could play a major role in vascular stiffening. In vivo, remodeling of the extracellular matrix (ECM), such as collagen accumulation and elastolysis, was observed in the aortic walls of the APL-KO mice, with the latter associated with high cathepsin S activity. In addition, pulse wave velocity (PWV) and AHT were increased in the APL-KO mice. The latter could explain aortic wall remodeling in the APL-KO mice. The absence of apelin expression, particularly in WAT, modified the adipocyte secretome and facilitated remodeling of the ECM of the aortic wall. Thus, elastolysis of elastic fibers and collagen accumulation contributed to vascular stiffening and AHT. Therefore, apelin expression could be a major element to preserve vascular homeostasis.

Apelin-13 in septic shock: effective in supporting hemodynamics in sheep but compromised by enzymatic breakdown in patients.

Sepsis is a prevalent life-threatening condition related to a systemic infection, and with unresolved issues including refractory septic shock and organ failures. Endogenously released catecholamines are often inefficient to maintain blood pressure, and low reactivity to exogenous catecholamines with risk of sympathetic overstimulation is well documented in septic shock. In this context, apelinergics are efficient and safe inotrope and vasoregulator in rodents. However, their utility in a larger animal model as well as the limitations with regards to the enzymatic breakdown during sepsis, need to be investigated. The therapeutic potential and degradation of apelinergics in sepsis were tested experimentally and in a cohort of patients. (1) 36 sheep with or without fecal peritonitis-induced septic shock (a large animal experimental design aimed to mimic the human septic shock paradigm) were evaluated for hemodynamic and renal responsiveness to incremental doses of two dominant apelinergics: apelin-13 (APLN-13) or Elabela (ELA), and (2) 52 subjects (33 patients with sepsis/septic shock and 19 healthy volunteers) were investigated for early levels of endogenous apelinergics in the blood, the related enzymatic degradation profile, and data regarding sepsis outcome. APLN-13 was the only one apelinergic which efficiently improved hemodynamics in both healthy and septic sheep. Endogenous apelinergic levels early rose, and specific enzymatic breakdown activities potentially threatened endogenous apelin system reactivity and negatively impacted the outcome in human sepsis. Short-term exogenous APLN-13 infusion is helpful in stabilizing cardiorenal functions in ovine septic shock; however, this ability might be impaired by specific enzymatic systems triggered during the early time course of human sepsis. Strategies to improve resistance of APLN-13 to degradation and/or to overcome sepsis-induced enzymatic breakdown environment should guide future works.

Hereditary pancreatitis in a young adult: Acute to chronic.

This report investigates an unusual case of recurrent pancreatitis. A 22-year-old female was admitted to the emergency room for severe abdominal pain, nausea, and weight loss. She reported having these symptoms since she was a toddler. The clinician ordered fecal pancreatic elastase-1, fat-soluble vitamins, molecular studies, and imaging of the pancreas by computed tomography. The screening test result for fecal pancreatic elastase-1 revealed severe pancreatic exocrine insufficiency, and the concentrations of fat-soluble vitamins were also low. Imaging showed scattered calcifications in the pancreas. These findings supported a diagnosis of chronic pancreatitis. Due to the rarity of chronic pancreatitis in young adults, molecular studies were performed. The patient was found to be homozygous for a mutation in the SPINK1 gene, which is associated with hereditary pancreatitis. This case report discusses hereditary pancreatitis and highlights data on the utilization of fecal pancreatic elastase-1 to assess pancreatic exocrine insufficiency.

Extracellular proteolytic activation of Pseudomonas aeruginosa aminopeptidase (PaAP) and insight into the role of its non-catalytic N-terminal domain.

Pseudomonas aeruginosa secretes several endopeptidases, including elastase, alkaline proteinase (Apr), a lysine-specific endopeptidase (LysC), and an aminopeptidase (PaAP), all of which are important virulence factors. Activation of the endopeptidases requires removal of an inhibitory N-terminal propeptide. Activation of pro-PaAP, in contrast, requires C-terminal processing. The activating proteases of pro-PaAP and their cleavage site(s) have not yet been defined. Studying pro-PaAP processing in a wild type P. aeruginosa strain and strains lacking either elastase or both elastase and Apr, we detected three processing variants, each ~56 kDa in size (AP56). Activity assays and N- and C-terminal sequence analyses of these variants pointed at LysC as the principal activating protease, cleaving a Lys512-Ala513 peptide bond at the C-terminal end of pro-PaAP. Elastase and/or Apr are required for activation of LysC, suggesting both are indirectly involved in activation of PaAP. To shed light on the function(s) of the N-terminal domain of AP56, we purified recombinant AP56 and generated from it the 28 kDa catalytic domain (AP28). The kinetic constants (Km and Kcat) for hydrolysis of Leu-, Lys-, Arg- and Met-p-nitroanilide (pNA) derivatives by AP56 and AP28 were then determined. The catalytic coefficients (Kcat/Km) for hydrolysis of all four substrates by AP28 and AP56 were comparable, indicating that the non-catalytic domain is not involved in hydrolysis of small substrates. It may, however, regulate hydrolysis of natural peptides/proteins. Lys-pNA was hydrolyzed 2 to 3-fold more rapidly than Leu-pNA and ~8-fold faster than Arg- or Met-pNA, indicating that Lys-pNA was the preferred substrate.

Molecular Analysis of the Contribution of Alkaline Protease A and Elastase B to the Virulence of Pseudomonas aeruginosa Bloodstream Infections.

Pseudomonas aeruginosa is a major cause of nosocomial bloodstream infections. This microorganism secretes two major proteases, alkaline protease A (AprA) and elastase B (LasB). Despite several in vitro studies having demonstrated that both purified proteases cleave a number of components of the immune system, their contribution to P. aeruginosa bloodstream infections in vivo remains poorly investigated. In this study, we used a set of isogenic mutants deficient in AprA, LasB or both to demonstrate that these exoproteases are sufficient to cleave the complement component C3, either soluble or deposited on the bacteria. Nonetheless, exoprotease-deficient mutants were as virulent as the wild-type strain in a murine model of systemic infection, in Caenorhabditis elegans and in Galleria mellonella. Consistently, the effect of the exoproteases on the opsonization of P. aeruginosa by C3 became evident four hours after the initial interaction of the complement with the microorganism and was not crucial to survival in blood. These results indicate that exoproteases AprA and LasB, although conferring the capacity to cleave C3, are not essential for the virulence of P. aeruginosa bloodstream infections.

Elastase-mediated membrane fusion of highly pathogenic porcine reproductive and respiratory syndrome virus at host cell surface.

Infection by enveloped viruses includes endocytosis and/or membrane fusion at the plasma membrane, where host cell proteases play an essential role. Among them, elastase-mediated infection has been documented for several enveloped viruses. Porcine reproductive and respiratory syndrome virus (PRRSV), an economically critical factor in global swine industry, is previously reported to infect host cells via low pH-dependent clathrin-mediated endocytosis (CME) and undergo membrane fusion in recycling endosomes. In the current study, we identified that elastase was significantly elevated in the lung tissues of highly pathogenic PRRSV (HP-PRRSV)-infected pigs compared to the mock-infected ones. We subsequently demonstrated that elastase contributed to HP-PRRSV infection in both MARC-145 cells and porcine alveolar macrophages (PAMs). Mechanistically, HP-PRRSV entered host cells at the cell surface via elastase-mediated membrane fusion, independent of low pH and CME, and its glycoprotein 5 (GP5) was cleaved by the protease during this process. All these findings deepen our understanding of HP-PRRSV infection, and are beneficial for prevention and control of the disease.

Overexpression of the celA1 gene in BCG modifies surface pellicle, glucosamine content in biofilms, and affects in vivo replication.

Biofilm formed in vitro by mycobacteria has been associated with increased antibiotic tolerance as compared with planktonic cells. Cellulose has been identified as a component of DTT-exposed biofilms formed by M. tuberculosis. The celA1 gene of M. tuberculosis encodes a cellulase, which could affect the formation of biofilm by slow-growing mycobacteria. In this work, the celA1 gene of M. tuberculosis was cloned into the integrative pMV361 plasmid and then transformed into M. bovis BCG Pasteur to produce BCG:celA1, to have celA1 expressed from the strong promoter hsp60. We compared planktonic and biofilm growth, possible presence of CelA1 in whole protein extracts, quantitated biofilm, presence of monosaccharides, and bacillary burden in lungs after aerosol infection in BALB/c mice. Differences in the appearance of the surface pellicle and of the biofilm attached to the substrate were observed. In biofilms, we observed a significant decrease of glucosamine in BCG:celA1 compared with BCG:pMV361. Finally, BCG:celA1 had lower viable bacteria than the BCG:pMV361 strain after 24 h and 3 weeks post-infection, but no difference was found at 9 weeks post-infection.

Hypertonic glucose inhibits growth and attenuates virulence factors of multidrug-resistant Pseudomonas aeruginosa.

BACKGROUND: Pseudomonas aeruginosa is the most common Gram-negative pathogen responsible for chronic wound infections, such as diabetic foot infections, and further exacerbates the treatment options and cost of such conditions. Hypertonic glucose, a commonly used prolotherapy solution, can accelerate the proliferation of granulation tissue and improve microcirculation in wounds. However, the action of hypertonic glucose on bacterial pathogens that infect wounds is unclear. In this study, we investigated the inhibitory effects of hypertonic glucose on multidrug-resistant P. aeruginosa strains isolated from diabetic foot infections. Hypertonic glucose represents a novel approach to control chronic wound infections caused by P. aeruginosa. RESULTS: Four multidrug-resistant P. aeruginosa clinical strains isolated from diabetic foot ulcers from a tertiary hospital in China and the reference P. aeruginosa PAO1 strain were studied. Hypertonic glucose significantly inhibited the growth, biofilm formation, and swimming motility of P. aeruginosa clinical strains and PAO1. Furthermore, hypertonic glucose significantly reduced the production of pyocyanin and elastase virulence factors in P. aeruginosa. The expression of major quorum sensing genes (lasI, lasR, rhlI, and rhlR) in P. aeruginosa were all downregulated in response to hypertonic glucose treatment. In a Galleria mellonella larvae infection model, the administration of hypertonic glucose was shown to increase the survival rates of larvae infected by P. aeruginosa strains (3/5). CONCLUSIONS: Hypertonic glucose inhibited the growth, biofilm formation, and swimming motility of P. aeruginosa, as well as reduced the production of virulence factors and quorum sensing gene expression. Further studies that investigate hypertonic glucose therapy should be considered in treating chronic wound infections.

Binding of elastase-1 and enterocytes facilitates Trichinella spiralis larval intrusion of the host's intestinal epithelium.

Elastase-1 is one member of serine protease family, distributes in organisms widely and plays a crucial role in the invasion and development of Trichinella spiralis. In order to identify the binding of T. spiralis elastase-1 (TsEla) with host's intestinal epithelial cells (IECs) and its role in Trichinella larval intrusion, TsEla gene was cloned and expressed in our previous study. The recombinant TsEla (rTsEla) has the enzymatic activity to degrade specific peptide substrate. A specific binding between rTsEla and IECs was detected by Far Western blot and ELISA. In an in vitro invasion assay, rTsEla promoted the larval intrusion, whereas anti-rTsEla serum inhibited the larval penetration. The larval intrusion was also suppressed after the silencing of TsEla by siRNA. Silencing of TsEla gene by siRNA-291 meditated RNA interference suppressed TsEla protein expression, reduced the worm infectivity, development and reproductive capacity. These results indicated that TsEla plays an important role in the T. spiralis intrusion of host's intestinal epithelia, and it could be a prospective vaccine molecular target against T. spiralis infection.