PAP1 activates the prophenoloxidase system against bacterial infection in Musca domestica.

We previously identified three putative prophenoloxidase-activating proteinase (mdPAP1, mdPAP2, and mdPAP3) genes from housefly Musca domestica by transcriptomic analysis. In this study, mdPAP1 cDNA was cloned, and the function of its encoded protein was analyzed. The cDNA of mdPAP1 was 1358 bp, and it contained a single open reading frame of 1122 bp encoding a predicted MdPAP1 protein of 373 amino acids. The estimated molecular weight of MdPAP1 was 41267.08 Da with an isoelectric point of 6.25. The deduced amino acid sequence of MdPAP1 exhibited high similarity to known PAPs of insects. mdPAP1 was detected in larvae, pupae, and adult housefly, and the expression level of mdPAP1 was upregulated in bacterial challenged larvae. The recombinant protein of MdPAP1 expressed in Escherichia coli could cleave the prophenoloxidase into phenoloxidase in M. domestica hemolymph infected by bacteria and result in a significant increase of the total phenoloxidase activity. In addition, RNA interference-mediated gene silencing of mdPAP1 significantly increased the mortality of M. domestica larvae. Results indicated that mdPAP1 was involved in the activation of the prophenoloxidase against bacterial infection in M. domestica.

Comparative studies of the expression of creatine kinase isoforms under immune stress in Pelodiscus sinensis.

The expression and localization of different isoforms of creatine kinase in Pelodiscus sinensis (PSCK) were studied to reveal the role of PSCK isozymes (PSCK-B, PSCK-M, PSCK-S) under bacterial infection-induced immunologic stress. The computational molecular dynamics simulations predicted that PSCK-S would mostly possess a kinase function in a structural aspect when compared to PSCK-B and PSCK-M. The assay of biochemical parameters such as total superoxide dismutase (T-SOD), lactate dehydrogenase (LDH), malondialdehyde (MDA), catalase (CAT), and the content of ATP were measured along with total PSCK activity in different tissue samples under bacterial infection. The expression detections of PSCK isozymes in vitro and in vivo were overall well-matched where PSCK isozymes were expressed differently in P. sinensis tissues. The results showed that PSCK-B mostly contributes to the spleen, followed by the liver and myocardium; PSCK-M mostly contributes to the liver, followed by the myocardium and skeletal muscle, while PSCK-S contributes to the spleen and is uniquely expressed in skeletal muscle. Our study suggests that the various alterations of PSCK isozymes in tissues of P. sinensis are prone to defense the bacterial infection and blocking energetic imbalance before severe pathogenesis turned on in P. sinensis.

Inhibitors of DNA Glycosylases as Prospective Drugs.

DNA glycosylases are enzymes that initiate the base excision repair pathway, a major biochemical process that protects the genomes of all living organisms from intrinsically and environmentally inflicted damage. Recently, base excision repair inhibition proved to be a viable strategy for the therapy of tumors that have lost alternative repair pathways, such as BRCA-deficient cancers sensitive to poly(ADP-ribose)polymerase inhibition. However, drugs targeting DNA glycosylases are still in development and so far have not advanced to clinical trials. In this review, we cover the attempts to validate DNA glycosylases as suitable targets for inhibition in the pharmacological treatment of cancer, neurodegenerative diseases, chronic inflammation, bacterial and viral infections. We discuss the glycosylase inhibitors described so far and survey the advances in the assays for DNA glycosylase reactions that may be used to screen pharmacological libraries for new active compounds.

Host poly(ADP-ribose) polymerases (PARPs) in acute and chronic bacterial infections.

Protein ADP-ribosylation is a reversible post-translational modification, which alters protein activity, localization, interactome or stability, leading to perturbation of cell signaling. This review summarizes the emerging data indicating that host cell ADP-ribosylating enzymes, poly(ADP-ribose) polymerases (PARPs), influence the course of a bacterial infection, in parallel to ADP-ribosylating bacterial toxins. Host cell PARP targeting could be an efficient therapeutic approach to treat certain bacterial infections, possibly by repurposing the approved or clinical trial PARP inhibitors developed for cancer therapy.

Mitochondria-Associated Matrix Metalloproteinases 2 and 9 in Acute Renal Pathologies.

Activities of MMP-2 and MMP-9 in the cytoplasm and mitochondria of kidney cells were evaluated on the models of acute renal pathologies: pyelonephritis, rhabdomyolysis, and ischemia/reperfusion of the kidney. In acute pyelonephritis, a significant increase in the level of MMP-2 and MMP-9 in kidney cells and the appearance of mitochondrial MMP-2 isoform with a lower molecular weight, but still exhibiting proteolytic activity were observed. A direct correlation between the level of MMP-2 and MMP-9 in the kidney and the severity of inflammation in pyelonephritis was revealed. Obviously, the appearance of active protease in the mitochondria of the kidney cells could have an impact on their functioning and, generally, on the fate of renal cells in this pathology.

Protease activity sensors noninvasively classify bacterial infections and antibiotic responses.

BACKGROUND: Respiratory tract infections represent a significant public health risk, and timely and accurate detection of bacterial infections facilitates rapid therapeutic intervention. Furthermore, monitoring the progression of infections after intervention enables 'course correction' in cases where initial treatments are ineffective, avoiding unnecessary drug dosing that can contribute to antibiotic resistance. However, current diagnostic and monitoring techniques rely on non-specific or slow readouts, such as radiographic imaging and sputum cultures, which fail to specifically identify bacterial infections and take several days to identify optimal antibiotic treatments. METHODS: Here we describe a nanoparticle system that detects P. aeruginosa lung infections by sensing host and bacterial protease activity in vivo, and that delivers a urinary detection readout. One protease sensor is comprised of a peptide substrate for the P. aeruginosa protease LasA. A second sensor designed to detect elastases is responsive to recombinant neutrophil elastase and secreted proteases from bacterial strains. FINDINGS: In mice infected with P. aeruginosa, nanoparticle formulations of these protease sensors-termed activity-based nanosensors (ABNs)-detect infections and monitor bacterial clearance from the lungs over time. Additionally, ABNs differentiate between appropriate and ineffective antibiotic treatments acutely, within hours after the initiation of therapy. INTERPRETATION: These findings demonstrate how activity measurements of disease-associated proteases can provide a noninvasive window into the dynamic process of bacterial infection and resolution, offering an opportunity for detecting, monitoring, and characterizing lung infections. FUND: National Cancer Institute, National Institute of Environmental Health Sciences, National Institutes of Health, National Science Foundation Graduate Research Fellowship Program, and Howard Hughes Medical Institute.

From Host Heme To Iron: The Expanding Spectrum of Heme Degrading Enzymes Used by Pathogenic Bacteria.

Iron is an essential nutrient for many bacteria. Since the metal is highly sequestered in host tissues, bound predominantly to heme, pathogenic bacteria often take advantage of heme uptake and degradation mechanisms to acquire iron during infection. The most common mechanism of releasing iron from heme is through oxidative degradation by heme oxygenases (HOs). In addition, an increasing number of proteins that belong to two distinct structural families have been implicated in aerobic heme catabolism. Finally, an enzyme that degrades heme anaerobically was recently uncovered, further expanding the mechanisms for bacterial heme degradation. In this analysis, we cover the spectrum and recent advances in heme degradation by infectious bacteria. We briefly explain heme oxidation by the two groups of recognized HOs to ground readers before focusing on two new types of proteins that are reported to be involved in utilization of heme iron. We discuss the structure and enzymatic function of proteins representing these groups, their biological context, and how they are regulated to provide a more complete look at their cellular role.

Associations of periparturient plasma biochemical parameters, endometrial leukocyte esterase and myeloperoxidase, and bacterial detection with clinical and subclinical endometritis in postpartum dairy cows.

This study was aimed at demonstrating associations between peripheral biochemical parameters, endometrial leukocyte esterase (LE) and myeloperoxidase (MPO), and bacterial detection with the degree of endometrial inflammation, and determining the best time postpartum for diagnosing endometritis to predict subsequent fertility in dairy cows. Plasma albumin, blood urea nitrogen (BUN), total cholesterol (T-cho), NEFA, and BHBA concentrations were analyzed in 43 Holstein cows at 3, 5 and 7 weeks postpartum (W3, W5 and W7). Endometrial samples were collected at W3, W5 and W7 to examine LE and MPO activities, bacterial detection rates, and PMN% profiles. The 43 cows were divided into healthy (HE), subclinical endometritis (SE), and clinical endometritis (CE) groups, classified differently at W3, W5 and W7 based on the definitions of SE and CE for each of the three weeks pp. LE level had an association with PMN% in all weeks pp (P<0.05). Albumin and BUN levels had weak negative associations with endometrial PMN% at W3. Pathogenic bacterial detection rates were higher in the cows with endometritis at W3 and W5. Conception rate at first artificial insemination tended to be lower (P=0.057) in the cows diagnosed with endometritis at W3 than in the healthy cows. In conclusion, associations were found between endometrial LE and endometritis, but not for MPO and endometritis. Diagnosing endometritis in W3 may be the best moment to predict subsequent fertility.

Extracellular ADP facilitates monocyte recruitment in bacterial infection via ERK signaling.

As the most prominent clinical drug targets for the inhibition of platelet aggregation, P2Y12 and P2Y13 have been found to be highly expressed in both platelets and macrophages. However, the roles and function of P2Y12/13 in the regulation of macrophage-mediated innate immune responses remain unclear. Here, we demonstrate that adenosine 5'-diphosphate (ADP), the endogenous ligand of P2Y1, P2Y12 and P2Y13, was released both in E. coli-infected mice and from macrophages treated with either lipopolysaccharide (LPS) or Pam3CSK4. Furthermore, the expression of P2Y13 was clearly increased in both LPS-treated macrophages and tuberculosis patients. ADP protected mice from E. coli 0111-induced peritonitis by recruiting more macrophages to the infected sites. Consistent with this, ADP and ADP-treated cell culture medium attracted more macrophages in the transwell assay by enhancing the expression of MCP-1. Nevertheless, P2Y1 is dispensable for ADP-mediated protection against bacterial infection. However, either P2Y12/P2Y13 deficiency or blocking the downstream signaling of P2Y12/P2Y13 blocked the ADP-mediated immune response and allowed more bacteria to persist in the infected mice. Furthermore, extracellular signal-regulated kinase (ERK) phosphorylation was clearly increased by ADP, and this type of activation could be blocked by either forskolin or analogs of cyclic AMP (cAMP) (for example, 8-bromo-cAMP). Accordingly, ADP-induced MCP-1 production and protection against bacterial infection could also be reduced by U0126, forskolin and 8-bromo-cAMP. Overall, our study reveals a relationship between danger signals and innate immune responses, which suggests the potential therapeutic significance of ADP-mediated purinergic signaling in infectious diseases.

Protein kinase A determines platelet life span and survival by regulating apoptosis.

Apoptosis delimits platelet life span in the circulation and leads to storage lesion, which severely limits the shelf life of stored platelets. Moreover, accumulating evidence indicates that platelet apoptosis provoked by various pathological stimuli results in thrombocytopenia in many common diseases. However, little is known about how platelet apoptosis is initiated or regulated. Here, we show that PKA activity is markedly reduced in platelets aged in vitro, stored platelets, and platelets from patients with immune thrombocytopenia (ITP), diabetes, and bacterial infections. Inhibition or genetic ablation of PKA provoked intrinsic programmed platelet apoptosis in vitro and rapid platelet clearance in vivo. PKA inhibition resulted in dephosphorylation of the proapoptotic protein BAD at Ser155, resulting in sequestration of prosurvival protein BCL-XL in mitochondria and subsequent apoptosis. Notably, PKA activation protected platelets from apoptosis induced by storage or pathological stimuli and elevated peripheral platelet levels in normal mice and in a murine model of ITP. Therefore, these findings identify PKA as a homeostatic regulator of platelet apoptosis that determines platelet life span and survival. Furthermore, these results suggest that regulation of PKA activity represents a promising strategy for extending platelet shelf life and has profound implications for the treatment of platelet number-related diseases and disorders.

The dihydroorotate dehydrogenases: Past and present.

The flavoenzyme dihydroorotate dehydrogenase catalyzes the stereoselective oxidation of (S)-dihydroorotate to orotate in the fourth of the six conserved enzymatic reactions involved in the de novo pyrimidine biosynthetic pathway. Inhibition of pyrimidine metabolism by selectively targeting DHODHs has been exploited in the development of new therapies against cancer, immunological disorders, bacterial and viral infections, and parasitic diseases. Through a chronological narrative, this review summarizes the efforts of the scientific community to achieve our current understanding of structural and biochemical properties of DHODHs. It also attempts to describe the latest advances in medicinal chemistry for therapeutic development based on the selective inhibition of DHODH, including an overview of the experimental techniques used for ligand screening during the process of drug discovery.

Phosphatidylinositol 3-Kinase p110δ Isoform Regulates CD8+ T Cell Responses during Acute Viral and Intracellular Bacterial Infections.

The p110δ isoform of PI3K is known to play an important role in immunity, yet its contribution to CTL responses has not been fully elucidated. Using murine p110δ-deficient CD8(+) T cells, we demonstrated a critical role for the p110δ subunit in the generation of optimal primary and memory CD8(+) T cell responses. This was demonstrated in both acute viral and intracellular bacterial infections in mice. We show that p110δ signaling is required for CD8(+) T cell activation, proliferation and effector cytokine production. We provide evidence that the effects of p110δ signaling are mediated via Akt activation and through the regulation of TCR-activated oxidative phosphorylation and aerobic glycolysis. In light of recent clinical trials that employ drugs targeting p110δ in certain cancers and other diseases, our study suggests caution in using these drugs in patients, as they could potentially increase susceptibility to infectious diseases. These studies therefore reveal a novel and direct role for p110δ signaling in in vivo CD8(+) T cell immunity to microbial pathogens.

Serum chitotriosidase levels in cancer patients undergoing high dose chemotherapy and stem cell transplantation.

OBJECTIVE: Human chitotriosidase (ChT) is an active chitinase expressed by activated phagocytes. Increased ChT activity has been reported in systemic Candida albicans infections and in Gram-negative and Gram-positive bacterial infections, indicating that an increase in ChT activity reflects phagocyte activation. The aim of this study was to determine the changes in serum ChT activity in patients who underwent high dose chemotherapy (HDC) and stem cell transplantation (SCT), who are at an increased risk for fungal and bacterial infections due to depression of the immune system during the neutropenic period. PATIENTS AND METHODS: A total of 55 SCT patients were included in the study. Serum ChT activity was determined before the initiation of HDC and during the neutropenic period after hematopoietic stem cell reinfusion on post-transplant first, fifth and tenth days. RESULTS: Chitotriosidase levels before transplantation were significantly lower than the results at first, fifth and tenth days post-hematopoietic stem cell reinfusion. CONCLUSIONS: Although the number of neutrophils was low, ChT enzyme activity was high in newly produced granules of neutrophils. Chitotriosidase may be supplemented as a drug for preventing and treating infections in the near future.

Increased ß-glucuronidase activity in bronchoalveolar lavage fluid of children with bacterial lung infection: A case-control study.

BACKGROUND AND OBJECTIVE: ß-Glucuronidase is a lysosomal enzyme released into the extracellular fluid during inflammation. Increased ß-glucuronidase activity in the cerebrospinal and peritoneal fluid has been shown to be a useful marker of bacterial inflammation. We explored the role of ß-glucuronidase in the detection of bacterial infection in bronchoalveolar lavage fluid (BALF) of paediatric patients. METHODS: In this case-control study, % polymorphonuclear cell count (PMN%), ß-glucuronidase activity, interleukin-8 (IL-8), tumour necrosis factor-α (TNF-α) and elastase were measured in culture-positive (≥10(4) cfu/mL, C+) and -negative (C-) BALF samples obtained from children. RESULTS: A total of 92 BALF samples were analysed. The median ß-glucuronidase activity (measured in nanomoles of 4-methylumbelliferone (4-MU)/mL BALF/h) was 246.4 in C+ (interquartile range: 71.2-751) and 21.9 in C- (4.0-40.8) (P < 0.001). The levels of TNF-α and IL-8 were increased in C+ as compared with C- (5.4 (1.7-12.6) vs 0.7 (0.2-6.2) pg/mL, P < 0.001 and 288 (76-4300) vs 287 (89-1566) pg/mL, P = 0.042, respectively). Elastase level and PMN% did not differ significantly (50 (21-149) vs 26 (15-59) ng/mL, P = 0.051 and 20 (9-40) vs 18 (9-34) %, P = 0.674, respectively). The area under the curve of ß-glucuronidase activity (0.856, 95% confidence interval (CI): 0.767-0.920) was higher than that of TNF-α (0.718; 95% CI: 0.614-0.806; P = 0.040), IL-8 (0.623; 95% CI: 0.516-0.722; P = 0.001), elastase (0.645; 95% CI: 0.514-0.761; P = 0.008) and PMN% (0.526; 95 % CI: 0.418-0.632; P < 0.001). CONCLUSIONS: This study demonstrates a significant increase of ß-glucuronidase activity in BALF of children with culture-positive bacterial inflammation. In our population ß-glucuronidase activity showed superior predictive ability for bacterial lung infection than other markers of inflammation.

Caspases as the key effectors of inflammatory responses against bacterial infection.

Caspase cysteine proteases are factors widely recognized for their role in the induction of apoptotic cell death. Caspases induce apoptosis during the inflammatory response to pathogen infection; in addition, caspases such as caspase-1 and caspase-11 are known to be involved in the production of inflammatory cytokines in response to bacterial infections. Caspase-1 is activated in the inflammasome, an intracellular protein complex that is formed by the recognition of intracellular ligands or cellular stresses by sensor molecules such as NOD-like receptors. Under certain conditions, caspase-11 is required for the activation of the caspase-1 inflammasome, referred to as the non-canonical inflammasome. In addition to these caspases, accumulating evidence indicates that caspase-8 also contributes to the production of inflammatory cytokines. In contrast to caspase-1, caspase-8 is activated by receptors located on the plasma membrane including dectin-1, TLR-3/4, and Fas. Recently, Fas-mediated caspase-8 activation and inflammatory cytokine production have been shown to play a significant role in the regulation of bacterial infections. This review highlights the functional roles and activation mechanisms of caspase-1/-11 in innate immune responses against bacterial infection. In addition, we discuss the novel aspects of caspase-8 function in comparison with caspase-1/-11 during innate inflammatory responses.

Expression and biochemical characterization of recombinant human epididymis protein 4.

Whey acidic proteins (WAP) belong to a large gene family of antibacterial peptides that perform critical immune system functions. The function of human epididymis protein 4 (HE4), a 124-amino acid long polypeptide that has two whey acidic protein four-disulfide core (WFDC) domains, is not well studied. Here, a fusion gene encoding the HE4 protein fused to an IgG1 Fc domain was constructed. The recombinant HE4 protein was expressed as a secretory protein in Pichia pastoris and mammalian HEK293-F cells and was subsequently purified. Our data suggested that the HE4 protein produced by these two expression systems bound to both gram-negative and gram-positive bacteria, but demonstrated slightly inhibitory activity towards the growth of Staphylococcus aureus. Moreover, HE4 exhibited proteinase inhibitory activity towards trypsin, elastase, matrix metallopeptidase 9, and the secretory proteinases from Bacillus subtilis. The effects of glycosylation on the biochemical characterization of HE4 were also investigated. LC-ESI-MS glycosylation analysis showed that the high-mannose glycosylated form of HE4 expressed by P. pastoris has lower biological activity when compared to its complex-glycosylated form produced from HEK293-F cells. The implications of this are discussed, which may be provide theoretical basis for its important role in the development of cancer and innate immune system.

Evaluation of microbial enzymes in normal and abnormal cervicovaginal fluids of cervical dysplasia: a case control study.

The aim of the present study was to evaluate the role of microbial enzymes in normal and abnormal cervicovaginal fluids of cervical dysplasia. The cervicovaginal infections were evaluated through the estimation of microbial enzymes in patients with and without abnormal cervical cytology like bacterial and fungal infections. The patients were categorized based on infection caused by organism and stages of dysplasia. The pH, Whiff test, and Pap smear tests were conducted for normal and abnormal cervical swabs based on standard protocols. Microbial enzymes include mucinase, sialidases, and proteases of the cervical swabs and are estimated according to standard methods. The results of abnormal cervical cytological smears showed increased pH and the presence of amines with different levels of Pap smear test. Increased levels of microbial enzymes were observed in patients with abnormal cytology than normal cytology. Three microbial enzymes mucinase, sialidase, and protease were significantly (P < 0.01) more elevated in patients with bacterial infections (8.97 ± 0.64, 10.39 ± 0.28, 8.12 ± 0.64) than without dysplasia (2.02 ± 0.8, 1.98 ± 0.3, 1.96 ± 0.8). The results reinforce that the microbial infection seems to be more prone to cervical dysplasia and may act as risk-factor for the development of cervical cancer along with HPV infection.

Bacteria induce prolonged PMN survival via a phosphatidylcholine-specific phospholipase C- and protein kinase C-dependent mechanism.

Polymorphonuclear leukocytes (PMNs) are essential for the human innate immune defense, limiting expansion of invading microorganisms. PMN turnover is controlled by apoptosis, but the regulating signaling pathways remain elusive, largely due to inherent differences between mice and humans that undermine use of mouse models for understanding human PMN biology. Here, we aim to elucidate signal transduction mediating survival of human peripheral blood PMNs in response to bacteria, such as Yersinia pseudotuberculosis, an enteropathogen that causes the gastro-intestinal disease yersiniosis, as well as Escherichia coli and Staphylococcus aureus. Determinations of cell death reveal that uninfected control cells undergo apoptosis, while PMNs infected with either Gram-positive or -negative bacteria show profoundly increased survival. Infected cells exhibit decreased caspase 3 and 8 activities, increased mitochondrial integrity and are resistant to apoptosis induced by a death receptor ligand. This bacteria-induced response is accompanied by pro-inflammatory cytokine production including interleukin-8 and tumor necrosis factor-α competent to attract additional PMNs. Using agonists and pharmacological inhibitors, we show participation of Toll-like receptor 2 and 4, and interestingly, that protein kinase C (PKC) and phosphatidylcholine-specific phospholipase C (PC-PLC), but not tyrosine kinases or phosphatidylinositol-specific phospholipase C (PI-PLC) are key players in this dual PMN response. Our findings indicate the importance of prolonged PMN survival in response to bacteria, where general signaling pathways ensure complete exploitation of PMN anti-microbial capacity.

Evolutionary dynamics of the human NADPH oxidase genes CYBB, CYBA, NCF2, and NCF4: functional implications.

The phagocyte NADPH oxidase catalyzes the reduction of O2 to reactive oxygen species with microbicidal activity. It is composed of two membrane-spanning subunits, gp91-phox and p22-phox (encoded by CYBB and CYBA, respectively), and three cytoplasmic subunits, p40-phox, p47-phox, and p67-phox (encoded by NCF4, NCF1, and NCF2, respectively). Mutations in any of these genes can result in chronic granulomatous disease, a primary immunodeficiency characterized by recurrent infections. Using evolutionary mapping, we determined that episodes of adaptive natural selection have shaped the extracellular portion of gp91-phox during the evolution of mammals, which suggests that this region may have a function in host-pathogen interactions. On the basis of a resequencing analysis of approximately 35 kb of CYBB, CYBA, NCF2, and NCF4 in 102 ethnically diverse individuals (24 of African ancestry, 31 of European ancestry, 24 of Asian/Oceanians, and 23 US Hispanics), we show that the pattern of CYBA diversity is compatible with balancing natural selection, perhaps mediated by catalase-positive pathogens. NCF2 in Asian populations shows a pattern of diversity characterized by a differentiated haplotype structure. Our study provides insight into the role of pathogen-driven natural selection in an innate immune pathway and sheds light on the role of CYBA in endothelial, nonphagocytic NADPH oxidases, which are relevant in the pathogenesis of cardiovascular and other complex diseases.