The ESX-1 Substrate PPE68 Has a Key Function in ESX-1-Mediated Secretion in Mycobacterium marinum.

Mycobacteria use specialized type VII secretion systems (T7SSs) to secrete proteins across their diderm cell envelope. One of the T7SS subtypes, named ESX-1, is a major virulence determinant in pathogenic species such as Mycobacterium tuberculosis and the fish pathogen Mycobacterium marinum. ESX-1 secretes a variety of substrates, called Esx, PE, PPE, and Esp proteins, at least some of which are folded heterodimers. Investigation into the functions of these substrates is problematic, because of the intricate network of codependent secretion between several ESX-1 substrates. Here, we describe the ESX-1 substrate PPE68 as essential for secretion of the highly immunogenic substrates EsxA and EspE via the ESX-1 system in M. marinum. While secreted PPE68 is processed on the cell surface, the majority of cell-associated PPE68 of M. marinum and M. tuberculosis is present in a cytosolic complex with its PE partner and the EspG1 chaperone. Interfering with the binding of EspG1 to PPE68 blocked its export and the secretion of EsxA and EspE. In contrast, esxA was not required for the secretion of PPE68, revealing a hierarchy in codependent secretion. Remarkably, the final 10 residues of PPE68, a negatively charged domain, seem essential for EspE secretion, but not for the secretion of EsxA and of PPE68 itself. This indicates that distinctive domains of PPE68 are involved in secretion of the different ESX-1 substrates. Based on these findings, we propose a mechanistic model for the central role of PPE68 in ESX-1-mediated secretion and substrate codependence. IMPORTANCE Pathogenic mycobacteria, such Mycobacterium tuberculosis and Mycobacterium marinum, use a type VII secretion system (T7SS) subtype, called ESX-1, to mediate intracellular survival via phagosomal rupture and subsequent translocation of the mycobacterium to the host cytosol. Identifying the ESX-1 substrate that is responsible for this process is problematic because of the intricate network of codependent secretion between ESX-1 substrates. Here, we show the central role of the ESX-1 substrate PPE68 for the secretion of ESX-1 substrates in Mycobacterium marinum. Unravelling the mechanism of codependent secretion will aid the functional understanding of T7SSs and will allow the analysis of the individual roles of ESX-1 substrates in the virulence caused by the significant human pathogen Mycobacterium tuberculosis.

Modification of a PE/PPE substrate pair reroutes an Esx substrate pair from the mycobacterial ESX-1 type VII secretion system to the ESX-5 system.

Bacterial type VII secretion systems secrete a wide range of extracellular proteins that play important roles in bacterial viability and in interactions of pathogenic mycobacteria with their hosts. Mycobacterial type VII secretion systems consist of five subtypes, ESX-1-5, and have four substrate classes, namely, Esx, PE, PPE, and Esp proteins. At least some of these substrates are secreted as heterodimers. Each ESX system mediates the secretion of a specific set of Esx, PE, and PPE proteins, raising the question of how these substrates are recognized in a system-specific fashion. For the PE/PPE heterodimers, it has been shown that they interact with their cognate EspG chaperone and that this chaperone determines the designated secretion pathway. However, both structural and pulldown analyses have suggested that EspG cannot interact with the Esx proteins. Therefore, the determining factor for system specificity of the Esx proteins remains unknown. Here, we investigated the secretion specificity of the ESX-1 substrate pair EsxB_1/EsxA_1 in Mycobacterium marinum Although this substrate pair was hardly secreted when homologously expressed, it was secreted when co-expressed together with the PE35/PPE68_1 pair, indicating that this pair could stimulate secretion of the EsxB_1/EsxA_1 pair. Surprisingly, co-expression of EsxB_1/EsxA_1 with a modified PE35/PPE68_1 version that carried the EspG5 chaperone-binding domain, previously shown to redirect this substrate pair to the ESX-5 system, also resulted in redirection and co-secretion of the Esx pair via ESX-5. Our results suggest a secretion model in which PE35/PPE68_1 determines the system-specific secretion of EsxB_1/EsxA_1.

Protease domain and transmembrane domain of the type VII secretion mycosin protease determine system-specific functioning in mycobacteria.

Mycobacteria use type VII secretion systems to secrete proteins across their highly hydrophobic diderm cell envelope. Pathogenic mycobacteria, such as Mycobacterium tuberculosis and Mycobacterium marinum, have up to five of these systems, named ESX-1 to ESX-5. Most of these systems contain a set of five conserved membrane components, of which the four Ecc proteins form the core membrane-embedded secretion complex. The fifth conserved membrane protein, mycosin protease (MycP), is not part of the core complex but is essential for secretion, as it stabilizes this membrane complex. Here we investigated which MycP domains are required for this stabilization by producing hybrid constructs between MycP1 and MycP5 in M. marinum and analyzed their effect on ESX-1 and ESX-5 secretion. We found that both the protease and transmembrane domain are required for the ESX system-specific function of mycosins. In addition, we observed that the transmembrane domain strongly affects MycP protein levels. We also show that the extended loops 1 and 2 in the protease domain are probably primarily involved in MycP stability, whereas loop 3 and the MycP5-specific loop 5 are dispensable. The atypical propeptide, or N-terminal extension, is required only for MycP stability. Finally, we show that the protease domain of MycPP1, encoded by the esx-P1 locus on the pRAW plasmid, is functionally redundant to the protease domain of MycP5 These results provide the first insight into the regions of mycosins involved in interaction with and stabilization of their respective ESX complexes.

RD5-mediated lack of PE_PGRS and PPE-MPTR export in BCG vaccine strains results in strong reduction of antigenic repertoire but little impact on protection.

Tuberculosis is the deadliest infectious disease worldwide. Although the BCG vaccine is widely used, it does not efficiently protect against pulmonary tuberculosis and an improved tuberculosis vaccine is therefore urgently needed. Mycobacterium tuberculosis uses different ESX/Type VII secretion (T7S) systems to transport proteins important for virulence and host immune responses. We recently reported that secretion of T7S substrates belonging to the mycobacteria-specific Pro-Glu (PE) and Pro-Pro-Glu (PPE) proteins of the PGRS (polymorphic GC-rich sequences) and MPTR (major polymorphic tandem repeat) subfamilies required both a functional ESX-5 system and a functional PPE38/71 protein for secretion. Inactivation of ppe38/71 and the resulting loss of PE_PGRS/PPE-MPTR secretion were linked to increased virulence of M. tuberculosis strains. Here, we show that a predicted total of 89 PE_PGRS/PPE-MPTR surface proteins are not exported by certain animal-adapted strains of the M. tuberculosis complex including M. bovis. This Δppe38/71-associated secretion defect therefore also occurs in the M. bovis-derived tuberculosis vaccine BCG and could be partially restored by introduction of the M. tuberculosis ppe38-locus. Epitope mapping of the PPE-MPTR protein PPE10, further allowed us to monitor T-cell responses in splenocytes from BCG/M. tuberculosis immunized mice, confirming the dependence of PPE10-specific immune-induction on ESX-5/PPE38-mediated secretion. Restoration of PE_PGRS/PPE-MPTR secretion in recombinant BCG neither altered global antigenic presentation or activation of innate immune cells, nor protective efficacy in two different mouse vaccination-infection models. This unexpected finding stimulates a reassessment of the immunomodulatory properties of PE_PGRS/PPE-MPTR proteins, some of which are contained in vaccine formulations currently in clinical evaluation.

Arginine and citrulline and the immune response in sepsis.

Arginine, a semi-essential amino acid is an important initiator of the immune response. Arginine serves as a precursor in several metabolic pathways in different organs. In the immune response, arginine metabolism and availability is determined by the nitric oxide synthases and the arginase enzymes, which convert arginine into nitric oxide (NO) and ornithine, respectively. Limitations in arginine availability during inflammatory conditions regulate macrophages and T-lymfocyte activation. Furthermore, over the past years more evidence has been gathered which showed that arginine and citrulline deficiencies may underlie the detrimental outcome of inflammatory conditions, such as sepsis and endotoxemia. Not only does the immune response contribute to the arginine deficiency, also the impaired arginine de novo synthesis in the kidney has a key role in the eventual observed arginine deficiency. The complex interplay between the immune response and the arginine-NO metabolism is further underscored by recent data of our group. In this review we give an overview of physiological arginine and citrulline metabolism and we address the experimental and clinical studies in which the arginine-citrulline NO pathway plays an essential role in the immune response, as initiator and therapeutic target.

Comparison of capillary electrophoresis sequencing with the new CEQ 2000 DNA Analysis System to conventional gel based systems for HIV drug resistance analysis.

To date the majority of sequencing technologies have been based on use of gel plates. In this study sequencing by capillary electrophoresis for HIV-1 genotyping on the CEQ 2000 sequencer (Beckman Coulter Inc.) has been investigated and compared to an 'in house' protocol on the Prism-377 sequencer (Applied Biosystems) and to the HIV-1 TruGene kit (Visible Genetics Inc.), two gel plate-based systems. Plasma from 20 HAART-treated patients with virological failure were analyzed for protease (PR) and reverse transcriptase (RT) genes. A total of 520 RT codons (26/patient) and 360 PR codons (18/patient) related to antiretroviral drug resistance were evaluated. The overall agreement between CEQ 2000 and Prism-377 results was 100% for the RT and PR primary and secondary mutations. The overall agreement between CEQ 2000 and TruGene was 100% for primary and > or =97% for secondary mutations. Discrepant results would have never led to errors in genotype interpretation. Performances for a 24 patients/week/one technician genotyping throughput were analyzed. For Prism-377, TruGene and CEQ 2000, manual processing required 5, 4 and 2,5 days, sequence data analysis needed additional 3, 1 and 2 days and cost/patient was approximately 49, 214 and 39 $, respectively. The CEQ 2000 sequencer offers a reliable alternative for fast and cost effective HIV drug resistance analysis.

TT virus infection during chronic hepatitis C.

OBJECTIVE: The pathogenic role of TT virus (TTV) is not well known, especially during chronic hepatitis C virus (HCV) infection. We retrospectively investigated the presence of TTV DNA in the plasma of patients with chronic HCV infection and compared the characteristics of TTV-DNA-positive and -negative groups. METHODS: Between November 1996 and November 1998, 234 patients were included. Inclusion criteria were persistently elevated serum alanine aminotransferase (ALT) levels, anti-HCV and HCV-RNA positivity, and seronegativity for hepatitis B virus and human immunodeficiency virus markers. TTV DNA was amplified in nested polymerase chain reaction with TTV-specific primers, and products were analyzed by agarose-gel electrophoresis. Data were analyzed using the chi2, Fisher's exact test, or Mann-Whitney test, as appropriate. RESULTS: TTV DNA was detected in 19 (8.1%; 95% confidence interval: 4.6-11.6%) patients. TTV-DNA-positive and TTV-DNA-negative patients did not differ statistically for age, gender ratio, source of HCV infection, HCV disease duration, biological parameters, histological grade, HCV-RNA load, or HCV genotype. Although nonsignificant (p = 0.21), there was a trend for a higher prevalence of TTV DNA in patients with an unknown cause of HCV infection (4/22, 18.2%) than in intravenous drug users (4/84; 4.8%), in those exposed to potential risk factors (4/49; 8.2%), or in those having received blood transfusion (7/79; 8.9%). CONCLUSIONS: Because the rates of HCV replication and the severity of liver lesions in TTV-DNA-negative and -positive patients were similar, the hepatic pathogenicity of TTV in chronic hepatitis C patients is questionable.

New molecular assays to predict occurrence of cytomegalovirus disease in renal transplant recipients.

Thirty renal transplant recipients, after transplantation, were tested weekly with the following assays: cytomegalovirus (CMV) antigenemia (pp65 Ag), plasma qualitative Amplicor CMV (P-AMP), plasma and peripheral blood leukocyte quantitative Amplicor CMV monitor (P- and PBL-CMM), peripheral blood leukocyte (PBL) quantitative Quantiplex bDNA CMV, version 2.0 (bDNA), and whole-blood Nuclisens pp67 CMV (pp67). Eleven patients developed symptomatic CMV disease, and 7 developed asymptomatic CMV infection. For prediction of CMV disease, the sensitivity, specificity, and positive and negative predictive values, respectively, were as follows: 100%, 63%, 61%, and 100% for pp65 Ag; 100%, 42%, 50%, and 100% for bDNA; 91%, 47%, 50%, and 90% for PBL-CMM; 55%, 74%, 55%, and 74% for P-AMP; 55%, 74%, 55%, and 74% for P-CMM; and 64%, 79%, 64%, and 79% for pp67. First positive results in PBL were obtained 9-10 days before symptoms of CMV disease, compared with 5-6 days in plasma and 0 days in whole blood. PBL assays appear to be more appropriate than plasma assays when pre-emptive therapy is required to prevent the rapid progression from the first detection of the virus to CMV disease.

Prevalence of GBV C/HGV RNA and GBV C/HGV antibodies in French volunteer blood donors: results of a collaborative study.

BACKGROUND AND OBJECTIVES: Posttransfusion hepatitis still occurs at an incidence of about 1 in 118,000 for HBV and 1 in 220,000 for HCV. This collaborative study aimed to determine the prevalence of a novel flavivirus, GBV-C/HGV, even though its role in transfusion-associated hepatitis is uncertain. MATERIALS AND METHODS: GBV-C/HGV RNA was detected by PCR using either the Boehringer detection kit or by primers previously described. HGV antibodies were detected by a serological assay from Boehringer. RESULTS: The observed GBV-C/HGV RNA frequency was 3.4%. HGV antibodies occurred in 9.5% of donors. CONCLUSION: In our study, 12. 9% of the donors had been in contact with the GBV-C/HGV virus.

Schwann cell hyperplasia and tumors in transgenic mice expressing a naturally occurring mutant NF2 protein.

Specific mutations in some tumor suppressor genes such as p53 can act in a dominant fashion. We tested whether this mechanism may also apply for the neurofibromatosis type-2 gene (NF2) which, when mutated, leads to schwannoma development. Transgenic mice were generated that express, in Schwann cells, mutant NF2 proteins prototypic of natural mutants observed in humans. Mice expressing a NF2 protein with an interstitial deletion in the amino-terminal domain showed high prevalence of Schwann cell-derived tumors and Schwann cell hyperplasia, whereas those expressing a carboxy-terminally truncated protein were normal. Our results indicate that a subset of mutant NF2 alleles observed in patients may encode products with dominant properties when overexpressed in specific cell lineages.

Stable mixed chimerism without relapse after related allogeneic umbilical cord blood transplantation in a child with severe aplastic anemia.

Umbilical cord blood (UCB) cells from HLA-matched donors are used as an alternative to bone marrow for allogeneic transplantation and reports of successful UCB transplantation in patients with severe aplastic anemia (SAA) are scarce. SAA was discovered in a 4-year-old girl in February 1990. Transfusion support started in August 1990 and standard treatments were unsuccessful. The birth of an HLA-compatible brother in October 1993 permitted the cryopreservation of UCB. In December 1994 UCB transplantation was decided upon. No toxicity occurred. G-CSF was started at day 28. WBC and PMN reached 0.5 x 10(9)/l at days 33 and 37. RBC and platelet transfusion independence were reached at days 50 and 52. Mixed chimerism was demonstrated in blood cells at 1.5, 4 and 6 months after UCBT by molecular biology (VNTR). FISH studies yielded similar results at 15 and 18 months. Twenty months after UCBT, molecular biology showed full donor chimerism. Clinical follow-up (last follow-up: 32 months post transplant) is unremarkable. We suggest that CY and ATG may be a suitable regimen for related HLA-compatible UCBT in patients with SAA. Residual recipient cells can disappear even very late after UCBT, permitting the establishment of complete donor chimerism.

Impaired interaction of naturally occurring mutant NF2 protein with actin-based cytoskeleton and membrane.

Although schwannomin, the product of the neurofibromatosis type 2 gene, shares homology with three cytoskeleton-to-membrane protein linkers defining the ERM family, the mechanism by which it exerts a tumor suppressive activity remains elusive. Based on the knowledge of naturally occurring mutations, a functional study of schwannomin was initiated. Constructs encoding the two wild-type isoforms and nine mutant forms were transfected into HeLa cells. Transiently expressed wild-type isoforms were both observed underneath the plasma membrane. At this location they were detergent insoluble and redistributed by a cytochalasin D treatment, suggesting interaction with actin-based cytoskeletal structures. Proteins with single amino acid substitutions at positions 219 and 220 demonstrated identical properties. Three different truncated schwannomins, that are prototypic for most naturally occurring NF2 mutations, were affected neither in their location nor in their cytochalasin D sensitivity. However, they were revealed to be detergent soluble, indicating a relaxed interaction with the actin-based structures. An increased solubility was also observed for a mutant with a single amino acid substitution at position 360 in the C-terminal half of the protein. Mutant proteins with either a single amino acid deletion at position 118 or an 83 amino acid deletion within the N-terminal domain had lost the submembraneous localization and tended to accumulate in perinuclear patches that were unaffected by cytochalasin D treatment. A similar behavior was observed when the N-terminal domain was entirely deleted. Taken together these observations suggest that the N-terminal domain is the main determinant that localizes the protein at the membrane where it interacts weakly with actin-based cytoskeletal structures. The C-terminal domain potentiates this interaction. With rare exceptions, most naturally occurring mutant schwannomins that have lost their tumor suppressive activity are impaired in an interaction involving actin-based structures and are no longer firmly maintained at the membrane.

Cystic smooth-muscle tumor of the liver and spleen associated with Epstein-Barr virus after renal transplantation.

Immunosuppression is known to favor the development of various types of tumors. After organ transplantation, the risk of lymphoproliferative disease, whether clonal or not, is particularly increased and clearly associated with Epstein-Barr virus infection. We report a case of an unusual large cystic tumor of the liver with satellite hepatic and splenic nodules occurring 4 years after renal transplantation. Radiologic examination showed a rich vascularization of the tumor. Light and electron microscopy of a surgical liver biopsy, completed by an immunohistochemical study, demonstrated a well-differentiated tumor of smooth-muscle origin. Using in situ hybridization, we showed large amounts of Epstein-Barr virus messenger RNAs within the tumor cells. In addition, Southern blot analysis revealed that viral DNA was present in the form of a single monoclonal episome within the tumor. The polymerase chain reaction analysis of the genomic DNA of tumoral cells also indicated a monoclonal pattern. At last, the tumor was shown to be of host origin. Six months later, and despite three courses of chemotherapy, the tumoral lesions were unchanged. This case underlines the role of Epstein-Barr virus infection in the development of unusual and clonal smooth-muscle tumors after organ transplantation. The evolution of these rare tumors is uncertain.

Molecular analysis of genetic changes in ependymomas.

Ependymomas are glial cell-derived tumors. They are, in contrast to other gliomas (astrocytomas, oligodendrogliomas, and oligoastrocytomas), ill-defined with respect to the genes and chromosomal segments important in their tumorigenesis. In this study, we extensively screened 17 ependymomas for genetic changes characteristic of other gliomas. Allelic loss was detected on chromosome arm 22q in three tumors; on chromosome 10 in two tumors; on chromosome arm 17p in two tumors; and on chromosome arms 6q, 9p, 13q, and 19q, each in one tumor. No allelic losses were found on chromosome arms 1p and 16q. None of the tumors had EGFR gene amplification. In each case, the chromosomal segment affected by the deletion included the region known to harbor a tumor suppressor gene important in glioma tumorigenesis. We conclude that ependymomas resemble the other glial neoplasms with respect to type and location of the chromosomal changes involved. Given the relatively infrequent occurrence of these genetic changes, ependymomas should be considered genetically as low-grade gliomas.

Family with neurofibromatosis type 2 and autosomal dominant hearing loss: identification of carriers of the mutated NF2 gene.

A family is presented in which neurofibromatosis type 2 (NF2) and autosomal dominant hearing loss segregate in an apparently independent way. The presence of the latter condition caused anxiety in all family members at risk for NF2 in whom hearing loss became apparent. Previously, we identified a G-->A transition in the donor splice site of exon 5 of the NF2 gene in a family member with proven NF2. As expected, the mutation was present in two other family members who fulfilled the diagnostic criteria for NF2. Four out of five family members at risk for NF2 developed hearing loss. Two of these had the G-->A transition. The mutation was absent in the two other individuals with hearing loss and in the fifth family member without hearing loss or other clinical symptoms. In this family, the identification of the underlying NF2 gene mutation excluded NF2 as the cause of hearing loss in two potential carriers of the mutated gene. On the other hand, it enabled the identification of two carriers of the NF2 gene mutation who did not fulfill the diagnostic criteria for NF2. They will have to be monitored very carefully for the development of NF2-associated tumors. The consistent association within this family of a relatively mild clinical phenotype with the NF2 mutation, supports earlier suggestions that intrafamilial variability is small in NF2.

Predominant occurrence of somatic mutations of the NF2 gene in meningiomas and schwannomas.

The NF2 gene is a putative tumor-suppressor gene that, when it is altered in the germline, causes neurofibromatosis type 2, a tumor-susceptibility disease that mainly predisposes to schwannomas and meningiomas. The recent isolation of the NF2 gene on chromosome 22 allows the identification of somatic mutations in human tumors. We have searched for mutations of the NF2 gene in 331 primary human tumors using a screening method based on denaturing gradient gel electrophoresis, which allows the detection of mutations in 95% of the coding sequence. Mutations were observed in 17 of 57 meningiomas and in 30 of 89 schwannomas. No mutations were observed for 17 ependymomas, 70 gliomas, 23 primary melanomas, 24 pheochromocytomas, 15 neuroblastomas, 6 medulloblastomas, 15 colon cancers, and 15 breast cancers. All meningiomas and one-half of the schwannomas with identified NF2 mutations demonstrated chromosome 22 allelic losses. We conclude that the involvement of the NF2 gene in human tumorigenesis may be restricted to schwannomas and meningiomas, where it is frequently inactivated by a two-hit process.

Analysis of the NF2 tumor-suppressor gene and of chromosome 22 deletions in gliomas.

Recurrent deletions of chromosome fragments observed in neoplasms are thought to participate in tumor development through the inactivation of tumor-suppressor genes. In gliomas, the most frequent deletions involve chromosome arms 9p, 10q, 17p, 19q and 22q. We have analysed deletions of chromosome 22 in gliomas by studying loss of heterozygosity (LOH) at 8 microsatellite loci. LOH for this chromosome fragment was observed in 17/70 (24%) cases, most of them encompassing the region which encodes the gene altered in neurofibromatosis 2 (NF2), an inherited disease which predisposes to tumors of the nervous system. To investigate the possible involvement of the NF2 tumor-suppressor gene in the tumorigenesis of gliomas, we searched for alterations in its genomic structure and in its mature transcript. Northern-blot and reverse transcriptase-PCR experiments showed that the NF2 transcript is expressed and does not demonstrate obvious structural alterations. Moreover, analysis, at the genomic level, of the 16 coding exons of the NF2 gene by denaturing gradient gel electrophoresis failed to detect any somatically acquired point mutations. Altogether, these data strongly suggest that, although gliomas demonstrate recurrent chromosome 22 deletions most frequently encompassing the NF2 region, the NF2 gene is not altered in these tumors.