Characterization of antibody response in patients with acute and chronic chikungunya virus disease.

BACKGROUND: Chikungunya virus (CHIKV) is a re-emerging arbovirus capable of causing chronic arthralgia, which can last for months to years. Although neutralizing antibodies have been shown to be important for viral clearance, is it not clear whether the quantitative and qualitative nature of antibodies play a role in progression to chronic disease. OBJECTIVES: To characterize and compare the antibody responses in acute and chronic patients in a prospective observational CHIKV study in Curaçao during the 2014-2015 outbreak. STUDY DESIGN: We performed virus neutralization tests and ELISA on plasma samples collected from a prospective observational chikungunya study in Curaçao to compare the complement-dependent and -independent neutralization capacity, as well as the antibody avidity index of acute and chronic patients. RESULTS: We found that there was no significant difference in the virus neutralization titers between patients with acute and chronic chikungunya infection. Furthermore, we found that complement increased the neutralization capacity when large amounts of virus was used. Moreover, we found that patients with acute chikungunya disease had a significantly higher antibody avidity index compared to those with chronic disease. CONCLUSIONS: This study suggests that virus neutralization titers in late convalescent sera do not play a role in chronic chikungunya. However, the median antibody avidity was lower in these patients and may therefore suggest a role for antibody avidity in the development of chronic disease.

De novo peroxisome biogenesis in Penicillium chrysogenum is not dependent on the Pex11 family members or Pex16.

We have analyzed the role of the three members of the Pex11 protein family in peroxisome formation in the filamentous fungus Penicillium chrysogenum. Two of these, Pex11 and Pex11C, are components of the peroxisomal membrane, while Pex11B is present at the endoplasmic reticulum. We show that Pex11 is a major factor involved in peroxisome proliferation. We also demonstrate that P. chrysogenum cells deleted for known peroxisome fission factors (all Pex11 family proteins and Vps1) still contain peroxisomes. Interestingly, we find that, unlike in mammals, Pex16 is not essential for peroxisome biogenesis in P. chrysogenum, as partially functional peroxisomes are present in a pex16 deletion strain. We also show that Pex16 is not involved in de novo biogenesis of peroxisomes, as peroxisomes were still present in quadruple Δpex11 Δpex11B Δpex11C Δpex16 mutant cells. By contrast, pex3 deletion in P. chrysogenum led to cells devoid of peroxisomes, suggesting that Pex3 may function independently of Pex16. Finally, we demonstrate that the presence of intact peroxisomes is important for the efficiency of ß-lactam antibiotics production by P. chrysogenum. Remarkably, distinct from earlier results with low penicillin producing laboratory strains, upregulation of peroxisome numbers in a high producing P. chrysogenum strain had no significant effect on penicillin production.

Peroxisomes are required for efficient penicillin biosynthesis in Penicillium chrysogenum.

In the fungus Penicillium chrysogenum, penicillin (PEN) production is compartmentalized in the cytosol and in peroxisomes. Here we show that intact peroxisomes that contain the two final enzymes of PEN biosynthesis, acyl coenzyme A (CoA):6-amino penicillanic acid acyltransferase (AT) as well as the side-chain precursor activation enzyme phenylacetyl CoA ligase (PCL), are crucial for efficient PEN synthesis. Moreover, increasing PEN titers are associated with increasing peroxisome numbers. However, not all conditions that result in enhanced peroxisome numbers simultaneously stimulate PEN production. We find that conditions that lead to peroxisome proliferation but simultaneously interfere with the normal physiology of the cell may be detrimental to antibiotic production. We furthermore show that peroxisomes develop in germinating conidiospores from reticule-like structures. During subsequent hyphal growth, peroxisome proliferation occurs at the tip of the growing hyphae, after which the organelles are distributed over newly formed subapical cells. We observed that the organelle proliferation machinery requires the dynamin-like protein Dnm1.

The Penicillium chrysogenum aclA gene encodes a broad-substrate-specificity acyl-coenzyme A ligase involved in activation of adipic acid, a side-chain precursor for cephem antibiotics.

Activation of the cephalosporin side-chain precursor to the corresponding CoA-thioester is an essential step for its incorporation into the beta-lactam backbone. To identify an acyl-CoA ligase involved in activation of adipate, we searched in the genome database of Penicillium chrysogenum for putative structural genes encoding acyl-CoA ligases. Chemostat-based transcriptome analysis was used to identify the one presenting the highest expression level when cells were grown in the presence of adipate. Deletion of the gene renamed aclA, led to a 32% decreased specific rate of adipate consumption and a threefold reduction of adipoyl-6-aminopenicillanic acid levels, but did not affect penicillin V production. After overexpression in Escherichia coli, the purified protein was shown to have a broad substrate range including adipate. Finally, protein-fusion with cyan-fluorescent protein showed co-localization with microbody-borne acyl-transferase. Identification and functional characterization of aclA may aid in developing future metabolic engineering strategies for improving the production of different cephalosporins.

Post-translational modification of therapeutic peptides by NisB, the dehydratase of the lantibiotic nisin.

Post-translationally introduced dehydroamino acids often play an important role in the activity and receptor specificity of biologically active peptides. In addition, a dehydroamino acid can be coupled to a cysteine to yield a cyclized peptide with increased biostability and resistance against proteolytic degradation and/or modified specificity. The lantibiotic nisin is an antimicrobial peptide produced by Lactococcus lactis. Its post-translational enzymatic modification involves NisB-mediated dehydration of serines and threonines and NisC-catalyzed coupling of cysteines to dehydroresidues, followed by NisT-mediated secretion. Here, we demonstrate that a L. lactis strain containing the nisBTC genes effectively dehydrates and secretes a wide range of medically relevant nonlantibiotic peptides among which variants of adrenocorticotropic hormone, vasopressin, an inhibitor of tripeptidyl peptidase II, enkephalin, luteinizing hormone-releasing hormone, angiotensin, and erythropoietin. For most of these peptides, ring formation was demonstrated. These data show that lantibiotic enzymes can be applied for the modification of peptides, thereby enabling the biotechnological production of dehydroresidue-containing and/or thioether-bridged therapeutic peptides with enhanced stability and/or modulated activities.

Effect of brain death on gene expression and tissue activation in human donor kidneys.

BACKGROUND: After kidney transplantation, decreased graft survival is seen in grafts from brain dead (BD) donors compared with living donors. This might result partly from a progressive nonspecific inflammation in the graft. In this study, we focused on the effects of BD on inflammatory response (adhesion molecules, leukocyte invasion, gene expression) and stress-related heat shock proteins in the human kidney. Research outcomes and clinical donor parameters were then linked to outcome data after transplantation. METHODS: Kidney biopsy specimens and serum were obtained during organ retrieval from BD and living organ donor controls. Immunohistochemistry and semiquantitative reverse transcriptase-polymerase chain reaction were performed on the biopsy specimens. Clinical and laboratory parameters from BD donors were recorded and connected to outcome data of the recipients of the kidneys studied. RESULTS: After brain death, immunohistochemistry showed an increase of E-selectin (P<0.01) and interstitial leukocyte invasion (P<0.05) compared with controls. Also, reverse transcriptase-polymerase chain reaction showed a threefold increased heme oxygenase-1 (P<0.05) and Hsp70 (P<0.01) gene expression after BD. Levels of monocyte chemotactic protein-1 and transforming growth factor-beta were twice as high after brain death but did not reach significance. Transplantation outcome was influenced by several donor variables: positively most notably by donor treatment with desmopressin and negatively by high serum urea levels during brain death and by high intercellular adhesion molecule and vascular cell adhesion molecule expression in the kidney. Heme oxygenase-1 proved to have a protective function, but only in kidneys from living donors. CONCLUSIONS: The presence of interstitial leukocytes and the early adhesion molecule E-selectin in BD donor kidneys indicates an early-phase inflammatory process during organ retrieval. Elevated levels of monocyte chemotactic protein-1 and transforming growth factor-beta suggest a role for monocytes/macrophages in this phase. We suggest that BD causes a stress-related response against which protective heat shock proteins are formed in the future graft. This stress response may be too severe to be fully counteracted by elevated heat shock proteins. Which systemic and/or local factors trigger brain death-related graft injury is currently under investigation.

NisT, the transporter of the lantibiotic nisin, can transport fully modified, dehydrated, and unmodified prenisin and fusions of the leader peptide with non-lantibiotic peptides.

Lantibiotics are lanthionine-containing peptide antibiotics. Nisin, encoded by nisA, is a pentacyclic lantibiotic produced by some Lactococcus lactis strains. Its thioether rings are posttranslationally introduced by a membrane-bound enzyme complex. This complex is composed of three enzymes: NisB, which dehydrates serines and threonines; NisC, which couples these dehydrated residues to cysteines, thus forming thioether rings; and the transporter NisT. We followed the activity of various combinations of the nisin enzymes by measuring export of secreted peptides using antibodies against the leader peptide and mass spectroscopy for detection. L. lactis expressing the nisABTC genes efficiently produced fully posttranslationally modified prenisin. Strikingly, L. lactis expressing the nisBT genes could produce dehydrated prenisin without thioether rings and a dehydrated form of a non-lantibiotic peptide. In the absence of the biosynthetic NisBC enzymes, the NisT transporter was capable of excreting unmodified prenisin and fusions of the leader peptide with non-lantibiotic peptides. Our data show that NisT specifies a broad spectrum (poly)peptide transporter that can function either in conjunction with or independently from the biosynthetic genes. NisT secretes both unmodified and partially or fully posttranslationally modified forms of prenisin and non-lantibiotic peptides. These results open the way for efficient production of a wide range of peptides with increased stability or novel bioactivities.

Effective removal of alginate-poly-L-lysine microcapsules from pancreatic islets by use of trypsin-EDTA.

Although the transplantation of alginate-poly-L-lysine-alginate encapsulated islets of Langerhans usually is successful, graft survival is still limited. Molecular analysis by RT-PCR of the encapsulated islets may provide insight into the mechanisms that affect islets during graft failure. However, RT-PCR on encapsulated islets is not possible because the poly-L-lysine of the capsule interferes with both cDNA synthesis and PCR amplification. We applied a method that mechanically removes the microcapsules from the islets after a short trypsin-EDTA treatment (decapsulation), thereby enabling RT-PCR analysis. The results of this study show that the decapsulation procedure does not affect islet vitality and has only minor effects on islet function and morphology. The decapsulation does not affect GAPDH, beta-actin, Bcl-2, or Bax gene expression. This method is an improvement over the time-consuming manual dissection of microcapsules because it allows for the rapid and relatively harmless removal of capsules on a larger scale. Decapsulation offers the possibility of applying RT-PCR, as well as other methods, which cannot be performed on encapsulated islets.